Content uploaded by Ann M. Hirsch
Author content
All content in this area was uploaded by Ann M. Hirsch
Content may be subject to copyright.
Content uploaded by Ann M. Hirsch
Author content
All content in this area was uploaded by Ann M. Hirsch
Content may be subject to copyright.
Content uploaded by Paulina Estrada-de los Santos
Author content
All content in this area was uploaded by Paulina Estrada-de los Santos
Content may be subject to copyright.
REGULAR ARTICLE
Nodulation and effective nitrogen fixation of Macroptilium
atropurpureum (siratro) by Burkholderia tuberum,
anodulatingandplantgrowthpromotingbeta-proteobacterium,
are influenced by environmental factors
Annette A. Angus &Andrew Lee &
Michelle R. Lum &Maya Shehayeb &Reza Hessabi &
Nancy A. Fujishige &Shailaja Yerrapragada &
Stephanie Kano &Nannie Song &Paul Yang &
Paulina Estrada de los Santos &Sergio M. de Faria &
Felix D. Dakora &George Weinstock &Ann M. Hirsch
Received: 9 October 2012 /Accepted: 7 January 2013
#Springer Science+Business Media Dordrecht 2013
Abstract
Background and aims Burkholderia tuberum STM678
T
was isolated from a South African legume, but did not
renodulate this plant. Until a reliable host is found,
studies on this and other interesting beta-rhizobia cannot
advance. We investigated B. tuberum STM678
T
’s abil-
ity to induce Fix
+
nodules on a small-seeded, easy-to-
propagate legume (Macroptilium atropurpureum).
Previous studies demonstrated that B. tuberum elicited
either Fix
-
or Fix
+
nodules on siratro, but the reasons for
this difference were unexplored.
Methods Experiments to promote effective siratro
nodule formation under different environmental con-
ditions were performed. B. tuberum STM678
T
’s abil-
ity to withstand high temperatures and desiccation was
checked as well as its potential for promoting plant
growth via mechanisms in addition to nitrogen fixa-
tion, e.g., phosphate solubilization and siderophore
Plant Soil
DOI 10.1007/s11104-013-1590-7
Responsible Editor: Hans Lambers.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-013-1590-7) contains
supplementary material, which is available to authorized users.
A. A. Angus :M. Shehayeb :R. Hessabi :N. A. Fujishige :
S. Kano :N. Song :P. Yang :A. M. Hirsch
Department of Molecular, Cell, and Developmental
Biology, University of California,
Los Angeles, CA 90095, USA
A. Lee
Department of Microbiology, Immunology, and Molecular
Genetics, University of California,
Los Angeles, CA 90095, USA
A. M. Hirsch
Molecular Biology Institute, University of California,
Los Angeles, CA 90095, USA
M. R. Lum
Biology Department, Loyola Marymount University,
Los Angeles, CA 90045, USA
S. Yerrapragada
Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
P. Estrada de los Santos
Departamento de Microbiología, Escuela Nacional de
Ciencias Biológicas, Instituto Politécnico Nacional,
Prolongación de Carpio y Plan de Ayala,
México, DF 11340, Mexico
production. Potential genes for these activities were
found in the sequenced genomes.
Results Higher temperatures and reduced watering
resulted in reliable, effective nodulation on siratro.
Burkholderia spp. solubilize phosphate and produce
siderophores. Genes encoding proteins potentially in-
volved in these growth-promoting activities were
detected and are described.
Conclusions Siratro is an excellent model plant for B.
tuberum STM678
T
. We identified genes that might be
involved in the ability of diazotrophic Burkholderia
species to survive harsh conditions, solubilize phos-
phate, and produce siderophores.
Keywords Siratro .Burkholderia .Abiotic stress .
Phosphate solubilization .Siderophore
Abbreviations
hpi hours post-inoculation
dpi days post-inoculation
CAS chrome azurol S
Introduction
Burkholderia tuberum STM678
T
, the major focus of this
study, and other nitrogen-fixing members of the
Burkholderiaceae have been isolated not only from a
broad range of plant species, but also from various parts
of the world—especially South Africa, Brazil, and
Australia, three major centers of biological diversity
(Gyaneshwar et al. 2011). The three other
Burkholderia strains included in this study are B. una-
mae MTl-641
T
,B. silvatlantica SRMrh20
T
, and B. sil-
vatlantica PVA5. They were isolated from both legume
and non-legume plants, and all fix atmospheric nitrogen
into ammonia (Table 1). Of these four, only B. tuberum
nodulates legumes, but so far a model plant for
the detailed study of this nitrogen-fixing, beta-
proteobacterial species has not been developed. Other
legume-nodulating Burkholderia species include B.
phymatum STM815
T
(Vandamme et al. 2002), B.
nodosa Br3437
T
, Br3641, Br2470 (Chen et al. 2007),
and a number of B. mimosarum strains (Chen et al.
2006). The latter three species nodulate Mimosa spp.
effectively and have closely related nodulation (nodA)
and nitrogenase-encoding (nifH) genes. In contrast, the
B. tuberum STM678
T
nodA (<75 %) and nifH genes are
distantly related (90 % gene identity) to the comparable
genes from the Mimosa-nodulating strain B. phymatum
STM815
T
(Gyaneshwar et al. 2011).
Vand a mme e t al. ( 2002)werethefirsttonameand
describe the taxonomic position of B. tuberum STM678
T
,
which was isolated from the South African legume
Aspalathus carnosa (tribe: Crotalarieae). However, this
species has not yet been shown to nodulate its original
host (see history in Elliott et al. 2007a and Gyaneshwar et
al. 2011). Thus, Moulin et al. (2001)usedsiratro
(Macroptilium atropurpureum, Phaseoleae), which is
nodulated by a large number of alpha-rhizobial strains
(Vincent 1970;PueppkeandBroughton1999)todem-
onstrate that B. tuberum STM678
T
could nodulate
legumes. However, the siratro nodules that formed were
reported as Fix
-
(ineffective in nitrogen fixation) and only
partially infected. At the same time, B. tuberum
STM678
T
was shown to have the nodulation gene
nodA, as well as two copies of nodC (Moulin et al.
2001), indicating that this strain had the potential to
nodulate legumes. Later, Elliott et al. (2007a)reported
that B. tuberum STM678
T
developed Fix
+
(capable of
nitrogen fixation) nodules not only on four species of
Cyclopia (Podalyrieae), another genus of South African
legumes that grows in the acidic soils of the fynbos, but
also on siratro plants grown in glass tubes at 26 °C. These
higher temperatures may be closer to the conditions
encountered in the wild by the South African hosts and
this legume. Siratro normally grows in moist, subtropical
to tropical regions, but numerous studies have demon-
strated that it is much more tolerant of dryness than other
S. M. de Faria
Embrapa Agrobiologia,
Seropédica 23890-000 RJ, Brazil
F. D. Dakora
Chemistry Department, Tshwane University of Technology,
Arcadia Campus, 175 Nelson Mandela,
Drive Private Bag X680,
Pretoria 0001, South Africa
G. Weinstock
Department of Genetics,
Washington University School of Medicine,
St. Louis, MO 63110, USA
A. M. Hirsch (*)
University of California-Los Angeles (UCLA),
621 Charles E. Young Drive South,
Los Angeles, CA 90095-1606, USA
e-mail: ahirsch@ucla.edu
Plant Soil
legumes or various grass species (Ahmed and Quilt 1980;
Sheriff and Ludlow 1984;Ohashietal.2000). Siratro
survives drought because of its long taproot, which rea-
ches distant water sources (Sheriff and Ludlow 1984). It
also tolerates both acid (pH4.5) and alkaline soils (pH
8.5) (http://www.tropicalforages.info/key/Forages/
Media/Html/Macroptilium_atropurpureum.htm).
Although Rhizobium-induced siratro nodulation and
growth was improved at higher temperatures (26 °C or
greater), these traits were nonetheless still dependent on
the inoculum employed (Herridge and Roughley 1976).
Because we observed that effective nodulation of
siratro by B. tuberum STM678
T
occurred sporadically
at 21/22 °C, our objective was to establish whether
temperature and other environmental factors could
influence the ability of this bacterial species to estab-
lish Fix
+
nodules in a predictable way, with the ulti-
mate goal of using this legume as a model system for
studying host responses and for future mutant screen-
ing experiments. Lima et al. (2009) in a survey of
nitrogen-fixing bacteria from various soils under dif-
ferent land uses in the Western Amazon region exam-
ined edaphic factors such as Ca
2+
, Mg
2+
, Cu
2+
, base
saturation, exchangeable bases, as well as pH on sira-
tro nodulation. Similarly, soil pH, phosphate, and
CaCO
3
content and granulation were found to influ-
ence rhizobial nodulation of siratro and Mimosa
(Mishra et al. 2012). Both studies concluded that this
small-seeded legume is a valuable plant for trapping
both alpha- and beta-rhizobia.
We compared siratro nodulation by B. tuberum with
that induced by Rhizobium tropici CIAT899, which is
reported to be tolerant of acid soils and high concen-
trations of aluminum (Graham et al. 1982). In this
study, we investigated temperature, desiccation, and
artificial substrate type, each of which potentially
could dictate effective nodulation of siratro by B.
tuberum STM678
T
under laboratory conditions. The
fact that Elliott et al. (2007a) grew seedlings in 30 mL
glass tubes suggests that siratro root nodulation was
not negatively affected by light as has been observed
for some legumes, e.g., Pisum sativum (van Brussel et
al. 1982) and Lotus japonicus (Suzuki et al. 2011), so
we did not examine this parameter in our studies.
In addition to agar-based cultures, we employed
mixtures of perlite, vermiculite, and sand in both open
containers and in closed Magenta jars, the latter with
small holes drilled into the tops because closed con-
tainers often accumulate ethylene, which inhibits nod-
ulation (Lee and LaRue 1992). We also utilized Vigna
unguiculata (cowpea) as a host because it is reported
to be nodulated by both B. tuberum (F.D. Dakora,
personal communication) and R. tropici (Hernandez-
Lucas et al. 1995). Also, because many bacteria pro-
mote growth via mechanisms other than nitrogen fix-
ation, we examined whether B. tuberum STM678
T
and
three additional non-nodulating, but nitrogen-fixing
Burkholderia species were able to secrete sidero-
phores and to solubilize phosphate, two common
mechanisms of plant growth promotion. Although B.
unamae has already been shown to have many plant
growth-promoting activities (Caballero-Mellado et al.
2007; Castro-González et al. 2011), we included it in
our analysis for comparison.
Lastly, we looked for DNA sequences in the recent-
ly sequenced genomes of four plant-associated
Burkholderia species (Table 1) that might be respon-
sible for their performance under environmental stress.
These include such traits as the ability to: 1) synthesize
trehalose, a disaccharide with an unusual α,α1-1
Table 1 Strains and plasmids used in this study
Strains Relevant characteristics Source or reference
Rhizobium tropici UMR1899
(CIAT899)
Acid tolerant. Graham et al. 1982,1994
Burkholderia tuberum STM678
T
Wild-type. Isolated from Aspalathus carnosa nodules
in South Africa; nodulates Cyclopia spp. effectively.
Moulin et al. 2001; Vandamme
et al. 2002; Chen et al. 2003.
Burkholderia tuberum TnGFP GFP
+
, Tet
R
derivative of STM678. Elliott et al. 2007a
B. unamae MTI-641
T
Wild-type. Isolated from maize and sugarcane in Mexico. Caballero-Mellado et al. 2004
B. silvatlantica PVA5 Wild-type. Isolated from roots of Gleditisia tricanthos in Brazil. de Faria et al. 1999.
B. silvatlantica SRMrh20
T
Wild-type. Isolated from maize and sugarcane in Brazil. Perin et al. 2006.
Plasmids
pHC60 GFP-plasmid, Tet
R
Cheng and Walker 1998.
Plant Soil
linkage between two glucose molecules, which is syn-
thesized following desiccation or temperature stress by
several alpha-rhizobia including Bradyrhizobium
japonicum and Sinorhizobium meliloti (Streeter and
Gomez 2006); 2) solubilize phosphate from forms that
have limited solubility (Rodríguez et al. 2006); and 3)
produce siderophores for obtaining iron from soil
environments (Andrews et al. 2003). The presence of
these traits as well as the ability to induce nitrogen-
fixing nodules make the siratro-B. tuberum system an
important new model system to study various facets of
the interaction between plants and symbiotic
microbes, particularly the beta-rhizobia.
Materials and methods
Culture of bacteria and plants
The bacteria used in this study are listed in Table 1.
The plasmid pHC60 was mobilized into R. tropici
CIAT899 using a triparental mating. For routine cul-
ture, the Rhizobium strains were grown either on TY or
RDM (Vincent 1970) containing 10 μg/mL tetracy-
cline (tet) to select for the plasmids, whereas the
Burkholderia strains were grown on LB without salt
or on YEM with or without 10 μg/mL tet. All bacteria
were grown at 30 °C.
Seeds of M. atropurpureum were scarified for 1–
2 min. using a scarifying cup (Brigham and Hoover
1956) prior to surface-sterilization. V. unguiculata
subsp. unguiculata (PI339603) seeds were not scarified.
Siratro seeds were first briefly washed with 95 % etha-
nol for 5 min. and then sterilized in a 50 mL conical
centrifuge tube with full-strength commercial bleach on
a rotating platform for 35–45 min. Five rinses of sterile
water followed the bleaching step with the last rinse
overnight. Cowpea seeds were placed in 10 % bleach
for 10 min after the initial 95 % ethanol step, and also
copiously rinsed with sterile water after sterilization.
The sterilized seeds were placed on water agar (1 %)
for 3 days in the dark to assess germination and sterility
before planting. For seeds to be sown in Magenta jars,
autoclaved perlite:vermiculite (1:1 by volume) or per-
lite:vermiculite:sand (1:1:1 by volume) watered with ¼
strength Hoagland’s solution minus N was utilized.
Magenta jar tops had holes drilled into them, and the
holes were covered with sterile rayon adhesive film
(AeraSeal, Excel Scientific, Wrightwood, CA) to permit
airflow. The entire apparatus was autoclaved before seed
planting. When plants touched the top of the Magenta
jar, an autoclaved extender consisting of a Magenta jar
unit with one end sawed off was added to the system to
allow further shoot elongation.
Seeds were also sown directly after sterilization in
autoclaved containers half-filled with either perlite:
vermiculite (1:1 by volume) or a mixture of sand:
perlite:vermiculite (1:1:1 by volume) watered with ¼
strength Hoagland’s solution minus N. The moisture
in the dishpans was monitored with a soil moisture
meter (General Specialty Tools & Instruments; Model
DSMM500) to give an initial reading of 10–15 %, the
pans were weighed, and once a week, ¼strength
Hoagland’s solution minus N was added to the dish-
pans to the same weight to restore the moisture levels.
For the studies in 30 mL culture tubes, either
Jensen’s or ¼strength Hoagland’s solution minus N
media were used for agar slants (1 % Plant Agar,
PhytoTechnology Laboratories, Shawnee Mission,
KS), and the bottoms of the tubes were covered with
aluminum foil. Two siratro seeds were planted after
the sterilization procedure per tube and inoculated
immediately with 1 mL of 10
6
/mL bacteria re-
suspended in sterile water or phosphate-buffered sa-
line after centrifugation. For the dishpans, 100–
150 mL of inoculum were added, whereas 20 mL were
used for the Magenta jars and the polypropylene pots.
Microscopy
For confocal microscopy, GFP-labeled bacteria in as-
sociation with plant roots and nodules were visualized
using a Zeiss LSM 5 Exciter. Images were obtained
with the Zeiss ZEN acquisition/imaging software us-
ing a 5×or 10×objective and excitation at 488 nm for
observation of GFP, and 545 nm for observation of
root autofluorescence.
Light microscopic analysis using epifluorescence
was performed as described in Fujishige et al.
(2008).
Temperature influence on bacteria and desiccation
effects on bacteria and siratro
For the temperature studies, 10 μL spots of lag/early
log (13 h), log (17 h), and stationary (40 h) phase
cultures of B. tuberum and R. tropici were diluted to
a starting concentration of OD
600
=0.1 and then
Plant Soil
spotted in triplicate onto LB agar plates and incubated
at temperatures of 30 °C, 37 °C, or 40 °C for 24 h.
For the desiccation studies, assays were set up
according to the method described by Hugenholtz et
al. (1995). Briefly, nitrocellulose filters (0.45 μm pore)
were sterilized under UV light for 24 h before spotting
the center in triplicate with 100 μL of each overnight
bacterial culture (17 or 40 h) diluted in fresh LB to
OD
600
=0.1. Filters were placed inside standard-sized
sterile petri dishes and were allowed to air-dry, then
placed inside a desiccation chamber filled with fresh
silica desiccant (Sigma) and sealed using a vacuum
line. The filters remained undisturbed within the
chamber for 24 h, after which they were transferred
with a sterile forceps onto fresh LB agar plates and
incubated at 30 °C for 4 days to recover desiccation-
resistant strains.
For the in vivo studies on water-stress effects, siratro
plants were grown in sterilized perlite:vermiculte
(dishpans) or perlite:vermiculte:sand (pots and Magenta
jars) and watered with ¼strength Hoagland’s solution
minus N. After 2 weeks of watering twice weekly, one
set of B. tuberum-inoculated siratro plants kept the initial
regime whereas the second set was watered every other
week with the moisture content restored to ca. 15 % at
this time. Treatments representing the uninoculated as
well as inoculated plants were observed for signs of
desiccation stress after another 4–6 weeks.
Phosphate and siderophore assays
Phosphate solubilization by strains B. tuberum
STM678
T
,B. unamae MTl-641
T
,B. silvatlantica
strain PVA5, and B. silvatlantica strain SRMrh20
T
was performed using both solid and liquid
Pikovskaya (1948) (PVK) medium as modified by
Xie et al. (2009). The liquid medium contained
36.7 mM CaHPO
4
. The bacterial strains were grown
in liquid TY until stationary phase (24–30 h) at which
time the cells were harvested by centrifugation (8,000
×g, 10 min), and the pellets washed with sterile water
three times to remove traces of the TY medium. The
cell pellets were diluted to OD
600
=0.2 in sterile water.
For solid cultures, 10 μL droplets were spotted onto
plates, which were incubated at 30 °C for up to
10 days. The size of the colony and the clearing zone
around the colony were measured. Quantification of
the phosphate solubilized in liquid cultures was per-
formed by use of the QuantiChrom Phosphate Assay
kit (BioAssay systems; cat # DIPI-500), recording the
absorption at 620 nm.
Both liquid and solid PVK medium were set to an
initial pH of 7.55, 6.53, and 5.28, and then inoculated
in triplicate. For the inoculated cultures, a 1 mL ali-
quot of a single culture of each strain was selected at
random at 24 and 48 h intervals, centrifuged for 3 min
to pellet the cells, and the pH of the supernatant was
recorded. As a control, tubes of uninoculated medium
of the same starting pH as the inoculated tube were
prepared and treated the same as the test samples.
All four strains were also tested on solid PVK
medium supplemented with 0.035 % bromocresol pur-
ple, which has been shown to be an effective pH
indicator ranging in color from purple at pH7.0 to
yellow at pH5.0 (Yao and Byrne 2001). A triplicate
set of the plates were inoculated with 10 μL of 48-
h grown cultures and incubated at 30 °C. Colony size,
zones of clearance, and zones of color diffusion were
measured after 72 h, and the ratio of zone of clearance
to colony size calculated. The ratio was used to ac-
count for any difference in growth rate that occurred
on media of different pH levels.
Chrome azurol S (CAS) agar medium devoid of
nutrients was used as an indicator of the presence of
siderophores using 0.9 % (w/v) agar as a gelling agent.
The medium for a liter of the overlay medium includ-
ed: CAS, 60.5 mg; hexadecyltrimetyl ammonium bro-
mide (HDTMA or CTAB), 72.9 mg; piperazine-1,4-
bis(2-ethanesulfonic acid) (PIPES), 30.24 g; and
10 mL of 1 mM FeCl
3
·6H
2
Oin10mMHCl
(Schwyn and Neilands 1987). Bacteria were first
grown in liquid TY until OD
600
=0.1, at which time
10 μL droplets were spotted onto TY agar plates,
allowed to dry, and incubated at 30 °C for 2 days.
Molten CAS agar was poured over the bacterial spots
grown on solid TY. The overlaid plates were observed
for yellow to orange halos around the bacterial spots.
Results
Nodulation tests Earlier, we inoculated siratro plants
that had been grown in dishpans containing perlite and
vermiculite and watered twice weekly with nitrogen-
free medium with B. tuberum STM678
T
, and found that
at a day/night temperature of 22 °C/18 °C only ineffec-
tive nodules formed on the roots (data not shown).
Because Elliott et al. (2007a) reported the presence of
Plant Soil
Fix
+
nodules on siratro when the temperature was in-
creased to 26 °C day/22 °C night, we tested siratro
nodulation not only at a higher temperature, but also
under restricted water availability and in various sub-
strates to see whether reliable nodulation and nitrogen
fixation could be achieved with B. tuberum.
When dry weights and shoot lengths were mea-
sured after 6 weeks of growth in dishpans at higher
temperatures and restricted water availability, a signif-
icant increase in the B. tuberum-inoculated plants
compared to the uninoculated controls was observed
using a one-way ANOVA with Tukey’s post-hoc test
(Fig. 1a). Nevertheless, under these conditions, plants
were slow to show evidence of nitrogen fixation. It
took approximately 4–6weeksbeforethefoliage
turned a dark green. The shoot lengths of the B.
tuberum-inoculated plants (Fig. 1b) in particular were
highly variable because not all plants developed the
elongated internodes seen in Fig. 2a at the same time.
Surprisingly, siratro plants inoculated with R. tropici
and grown under the same conditions were small, had
yellow leaves, short internodes, and did not develop
nitrogen-fixing nodules (Figs. 1b and 2b). Their aver-
age dry weight and stem length were also lower than
those of the B. tuberum-inoculated siratro plants, but
not statistically different than the values obtained for
the uninoculated controls (Fig. 1).
Siratro nodules are determinate in that they lack a
persistent nodule meristem (Hirsch 1992). A compari-
son of nodule development between GFP-labeled R.
tropici CIAT899 and B. tuberum STM678
T
demonstrat-
ed very little difference in the early nodulation stages.
Both rhizobia elicited root hair deformation and entered
the root hairs via infection threads (Fig. 2d and e). B.
tuberum-elicited nitrogen-fixing nodules were estab-
lished on a well-developed siratro root system
(Fig. 2c), and the shoots also expanded, producing
climbing stems at about 6–8weeks(Fig.2a).
Examination of the internal structure of siratro nodules
showed that the nodule cells consisted of two types:
interstitial cells devoid of bacteria and cells filled with
green-fluorescing bacteria (Fig. 2f). This type of cell
arrangement is characteristic of nodules that develop in
response to infection thread penetration (Sprent 2007).
Plants in Magenta jars were nodulated within
3 weeks, and nitrogen fixation was also delayed.
Under the same conditions, R. tropici CIAT899 again
induced only ineffective nodules. We repeated the
experiment in glass tubes and found that although
the B. tuberum-inoculated plants formed Fix
+
nodules,
the R. tropici-inoculated plants did not (data not
shown). However, the B. tuberum-inoculated plants
became water-stressed after 8 weeks in the glass tubes
due to the drying of the agar. They also occasionally
formed bacteria-free callus-like structures on the roots,
even on the uninoculated plants, indicating that an
agar-based screen in glass tubes might not be suitable
for making meaningful conclusions about nodulation
and nitrogen fixation ability.
Temperature and desiccation stress Because siratro is
tolerant of dry conditions and nodulation by B.
tuberum is enhanced at higher temperatures, we rea-
soned that the bacteria used to inoculate siratro might
tolerate these abiotic stresses. B. tuberum and R. tro-
pici were thus compared for their potential to tolerate
Fig. 1 Burkholderia tuberum STM678
T
enhances plant biomass
and siratro shoot length better than R. tropici CIAT899. The plants
were harvested 6 weeks post inoculation, their shoot lengths
measured, and then the plants were dried in a 65 °C oven for
3 days to obtain biomass measurements. B. tuberum STM678
T
- or
R. tropici CIAT899-inoculated plants showed an increase in dry
weight (a) and shoot lengths (b) compared to uninoculated plants.
Overall, B. tuberum-inoculated plants were more robust with
respect to height, weight, and greenness compared to R. tropici-
inoculated plants. One-way ANOVA with Tukey’s post hoc test
was done for comparison of the means. Different letters represent
values that differ significantly, P<0.01
Plant Soil
temperature and desiccation stress in culture. R. tropici
had been shown earlier to acquire thermo-tolerance in
response to high temperature exposure, although un-
der such conditions, the bacteria did not fix nitrogen
symbiotically (Michiels et al. 1994). We hypothesized
that tolerance to severe stress could be one mechanism
whereby B. tuberum could effectively nodulate siratro
under adverse conditions. A number of possible mech-
anisms, such as trehalose synthesis, involvement of
heat shock proteins, or glycine betaine/proline accu-
mulation as well as exopolysaccharide production,
may confer temperature and/or desiccation tolerance
to Burkholderia.
Experimental analysis Incubation at temperatures of
30 °C, 37 °C, and 40 °C, demonstrated that B. tuberum
inoculated onto plates during the early growth phases,
both lag and log, exhibited limited growth at 30 °C
and no growth at the higher temperatures. In contrast,
R. tropici at the same stages of growth grew at both
37 °C and 40 °C, confirming this species’tolerance to
high temperatures. On the other hand, for cultures
incubated at stationary phase, B. tuberum grew at all
temperatures whereas R. tropici stationary phase cells
survived only at 30 °C.
Because the B. tuberum-inoculated plants formed
effective nodules at higher temperatures, we reasoned
that inoculated host plants might be more desiccation-
tolerant than the uninoculated plants. R. tropici was not
included in these experiments because it induced a Fix
-
nodule phenotype on siratro at 26 °C. Following an
additional 6–8 weeks of desiccation-simulating
Fig. 2 Effects of
B. tuberum STM678
T
and R.
tropici CIAT899 on siratro
nodulation. aHealthy, sira-
tro plant with elongated
internodes and root nodules
(arrows) had been inoculat-
ed with B. tuberum
STM678
T
and harvested
7.5 weeks post-inoculation.
Bar= 2 cm. bStunted, yel-
low siratro plant with nu-
merous nodules (arrows)
that had been inoculated
with R. tropici CIAT899 and
harvested 7.5 weeks post-
inoculation. Bar= 2 cm. c
Pink nodules from a B.
tuberum STM678
T
-inoculat-
ed siratro 6 weeks post-
inoculation. Bar=5 mm. d
Infection thread formed
within a siratro root hair by
GFP-labeled R. tropici
CIAT899. Bar=50 μm. e
Infection thread formed
within a siratro root hair by
GFP-labeled B. tuberum
STM678
T
. Bar= 50 μm. f
Young siratro nodule filled
with GFP-labeled B.
tuberum STM678
T
. Intersti-
tial cells (arrows) are devoid
of STM678
T
. Bar= 1 mm
Plant Soil
conditions (see Materials and Methods), the B. tuberum-
inoculated siratro plants were smaller than the un-
stressed B. tuberum-inoculated plants, but nonetheless
were green and developed pink Fix
+
nodules, strongly
suggesting that the bacteria were fixing nitrogen
(Supplementary Information Fig. 1). Although many
of the well-watered, inoculated plants produced vining
internodes, fewer of the water-stressed inoculated plants
did. By contrast, the uninoculated plants in both treat-
ments were stunted, yellow, and lacked nodules.
In vitro studies investigating the desiccation toler-
ance of B. tuberum and R. tropici showed that both
species were viable following simulated dry condi-
tions (data not shown). Although R. tropici tolerated
desiccation stress in early (exponential) growth phase
and late (stationary) phase cultures, B. tuberum
exhibited the most robust recovery from desiccation
stress when in the stationary growth phase. One ex-
planation for both the thermal and desiccation toler-
ance results reported here may be the overall slower
growth of B. tuberum STM678
T
compared to R. tro-
pici CIAT899 and hence longer recovery time.
Genome analysis Because only a draft genome of R.
tropici CIAT899 exists (Pinto et al. 2009), we could not
make comparisons between it and the Burkholderia
species with regard to specific gene sequences. In some
cases, we utilized other Rhizobium genomes to make the
comparison between alpha- and beta-rhizobia.
We hypothesized that trehalose, two glucose mole-
cules held together by a glycosidic α-(1, 1) bond and
important in temperature and desiccation tolerance in
alpha-rhizobia, may also be significant for stress ad-
aptation in B. tuberum.Bradyrhizobium japonicum
possesses three different trehalose biosynthetic path-
ways: 1) from UDP-glucose and glucose-6-phosphate
via the enzyme trehalose 6-phosphate synthase (TPS;
EC:2.4.1.15; otsA); 2) from the conversion of maltoo-
ligosaccharides to maltooligosyl trehalose via the en-
zyme maltooligosyl trehalose synthase (MOTS;
EC:5.4.99.15; treY); and 3) from the direct catalysis
of maltose to trehalose by way of trehalose synthase
(TS; EC:5.4.99.16; treS) (Streeter and Gomez 2006).
The four beta-rhizobia in Table 1have genes for the
same three pathways as Br. japonicum as well as multi-
ple copies of them. For the first pathway, the B. tuberum
genome contains one operon consisting of otsA (TPS)
and otsB (trehalose 6-phosphatase; EC:3.1.3.1.2; Fig. 3)
as well as three additional copies of otsA, two of which
are paralogs (data not shown). A similar situation exists
for the other three Burkholderia strains (Fig. 3), except
that none of the otsA genes have paralogs as seen in the
B. tuberum genome.
Genes encoding MOTS and maltooligosyl trehalose
hydrolase (EC: 3.2.1.14; treZ) were also found in the
genomes of all four Burkholderia species. The gene
neighborhoods are also very well conserved among
the four Burkholderia species, and are similar in terms
of gene organization to that of Rhizobium and
Bradyrhizobium species, especially Br. japonicum
USDA110 (Fig. 4). The gene sequences in the TS
operon—glycogen branching enzyme (glgX;also
known as treX), and alpha-1,4-glucan:alpha-1,4-glu-
can 6-glycosyltransferase (glgB)—were also detected
in both the beta- and alpha-rhizobial genomes.
Lastly, the genomes of the plant-associated Burkholderia
strains, except for B. unamae,possessgenesequences
encoding a neutral trehalase (EC:3.2.1.28; treF). This
DNA sequence was not detected in the alpha-rhizobial
genomes used in our analysis (data not shown).
Phosphate solubilization Available phosphate (P) is
generally in short supply in soils. Most of it is tied
up in insoluble forms in either organic or inorganic
complexes, which are unavailable to plants. Plant
growth-promoting bacteria have the ability to hydro-
lyze organic P via a variety of mechanisms including:
1) nonspecific acid phosphatases (NSAPs), 2) phy-
tases, and 3) phosphonatases and C-P lyases
(Rodríguez et al. 2006). By contrast, inorganic phos-
phate is usually solubilized via the production of or-
ganic acids (Rodríguez and Fraga 1999).
Experimental analysis Inorganic P-solubilizing activity
was observed for each of the four plant-associated
Burkholderia species when PVK agar medium was sup-
plemented with glucose as a carbon source, but B. tuberum
was not as effective at solubilizing P compared to non-
nodulating Burkholderia spp. (Fig. 5a). R. tropici
CIAT899 solubilized P at a level similar to B. tuberum
but statistically lower compared to B. unamae (Fig. 5a).
Additionally, only PVA5 and SRMrh20
T
were positive for
solubilization of P when sucrose instead of glucose was
added as a carbon source (Supplementary Information
Fig. 2). Results from the QuantiChrom
TM
assay kit, which
detects insoluble P confirm that the four Burkholderia spp.
solubilize P after 24 h in liquid PVK medium (Fig. 5b). R.
tropici CIAT899 was not tested in this assay.
Plant Soil
Because organic acid secretion is one of the principle
ways that bacteria accomplish inorganic P solubilization
(Rodríguez and Fraga 1999), we examined whether the
ability of the four plant-associated Burkholderia strains
to solubilize phosphate was correlated with an alteration
in pH. With the exception of B. tuberum STM678
T
, all
the media in which the Burkholderia strains grew
showed a pH drop close to 3.30 after 20 h of incubation
regardless of the initial pH of the medium (Table 2). By
44 hpi, B. tuberum also elicited a significant pH drop in
the medium. All four Burkholderia spp. solubilized
CaHPO
4
to varying degrees on solid PVK plates by 72
hpi (Supplementary information Fig. 2).
When grown in PVK medium containing bromocresol
purple at pH7.55, 6.53, and 5.28, the two B. silvatlantica
strains demonstrated the greatest amount of phosphate
solubilization at all the pH values tested whereas B.
tuberum exhibited the least (Table 3). The sizes of the
clearance zones and of the diameters of the color diffusion
zones, related to the change from neutral to acidic pH, are
presented in Table 3.Acorrelationbetweenthesizesofthe
diffusion zone and the zone of P solubilization is observed.
Fig. 3 Gene maps of the
otsA/otsB operon. The gene
neighborhoods (shaded) of
the four Burkholderia spp.
are conserved with that of
Bradyrhizobium japonicum
USDA110
Fig. 4 MOTS and maltooli-
gosyl trehalose hydrolase
gene maps. The gene neigh-
borhoods (shaded) of the
four Burkholderia spp. are
conserved with that of
Bradyrhizobium japonicum
USDA110
Plant Soil
Genome analysis Inorganic phosphate solubilization
activity that is related to the production of organic
acids (Rodríguez and Fraga 1999)isapropertyof
several enzymes, particularly glucose dehydroge-
nase and the cofactor pyrroloquinoline quinone
(PQQ). We searched for amino acid sequences of
similar proteins in the Burkholderia genomes by
querying with a Sinorhizobium meliloti sequence
annotated as a probable glucose dehydrogenase
(pyrroloquinoline-quinone) protein predicted to be
involved in 6-phosphogluconate synthesis via glu-
conate (Fig. 6a). Each Burkholderia species has at
least one coding sequence, and B. silvatlantica
SRMrh20
T
and PVA5 each have two copies
(Table 4). Overall, the B. tuberum genome has
the fewest number of gene sequences that could
be involved in phosphate solubilization, which
may explain the difference in activity of this strain
on the PVK plates versus the other three.
Although we did not test the four Burkholderia
strains for their ability to solubilize organic phos-
phate experimentally, we found gene sequences for
NSAPs and similar enzymes, but not genes encod-
ing phytases or phosphonatases and C-P lyases
(Table 4). The genomes of all four Burkholderia
spp. examined possess DNA sequences that encode
apurpleacidphosphatase-likeprotein(Table4),
which in B. cenocepacia has a pH optimum of 8.5
(Yeung et al. 2009). Purple acid phosphatase
(PAP) generally breaks down phosphoric acid
esters and phosphoric acid anhydrides, but the B.
cenocepacia PAP wa s re por te d to be a ct ive t o-
wards phosphotyrosine, phosphoserine, and phos-
phoenolpyruvate (Yeung et al. 2009). The gene
neighborhoods of the diazotrophic Burkholderia
strains are almost identical while that of B.
tuberum STM678
T
differs.
A gene for a Burkholderia-type acid phosphatase
(AcpA), generally used for the breakdown of organic
P complexes, is also present in the genomes of all
four of the species studied herein and in some alpha-
rhizobia (Fig. 6b). Because B. tuberum exhibited
lower phosphate solubilization compared to the
non-nodulating strains, we also compared the
genomes of two of the sequenced nodulating strains
with the genomes of B. unamae,B. silvatlantica
PVA5, and B. silvatlantica SRMrh20
T
.TheacpA
gene neighborhoods of both B. tuberum and B.
phymatum STM815
T
differed not only from each
other, but also from the comparable neighborhoods
in B. unamae and in the two B. silvatlantica species.
Interestingly, for the genomes of two sequenced B.
mimosarum strains, the acpA neighborhoods
matched those of the non-nodulating strains, sug-
gesting that the arrangement of genes putatively
involved in organic phosphate solubilization is not
correlated with nodulation ability per se. Also, two
Fig. 5 Burkholderia species solubilize inorganic phosphate. a
P-solubilizing activity for the four plant-associated Burkholde-
ria species and R. tropici CIAT899 under the same conditions
with glucose as a carbon source.Measurements indicate the
zone of clearance (in mm), calculated by measuring the sur-
rounding halo after 48 h after equal OD readings of bacteria had
been inoculated onto plates. bQuantification of P-solubilization
activity in broth culture (the medium alone contains 36 μM P)
using the QuantiChrom™assay kit, which detects insolubilized
phosphate. B. tuberum and B. unamae solubilize P at levels
close to that of the standard (30 μM) after 24 h, whereas B.
silvatlantica PVA5 and SRMrh20 solubilize significantly more
inorganic P. One-way ANOVA with Tukey’s post hoc test was
done for comparison of the means. Different letters represent
values that differ significantly, P<0.01
Plant Soil
AcpA-encoding genes were detected in the genomes
of the non-nodulating strains, with no orthologs in
B. tuberum STM678
T
,whichhadonlyoneacpA
(Table 4).
Siderophore secretion
Alpha-rhizobia are known to bind iron through the
secretion of siderophores, and several plant-associated
Burkholderia species have already been shown to be
capable of this activity (Caballero-Mellado et al.
2007; Suárez-Moreno et al. 2012; Weisskofp et al.
2011). Similar to phosphate solubilization, these ac-
tivities are beneficial because they support the growth
and development of plants, especially in marginal-
ized soils, by making unattainable mineral nutrients
available.
Experimental analysis Iron acquisition via siderophore
secretion was detected using the CAS overlay agar plate
assay on all four Burkholderia species. The presence of
yellow/orange halos around the bacterial colonies indi-
cates the presence of iron-binding siderophores. B.
tuberum consistently formed the smallest halos whereas
B. unamae established the largest (Supplementary infor-
mation Fig. 3). To determine whether a connection could
be made between halo size and genes involved in side-
rophore production, we analyzed the sequenced
genomes of the four Burkholderia species.
Genome analysis Genome analysis revealed that sev-
eral genes described as siderophore receptors, sidero-
phore transport system components, and siderophore-
interacting proteins are found in the genomes of the four
plant-associated species. Interestingly, the B. unamae
Table 2 pH of PVK medium 20 and 44 h post-inoculation (hpi)
Strain pH7.55
a
pH6.53
a
pH5.28
a
20 hpi 44 hpi 20 hpi 44 hpi 20 hpi 44 hpi
Uninoculated
b
6.01 6.21 5.38 5.54 5.03 4.86
B. tuberum STM678
T
7.35± 0.04 3.93± 0.02 6.45± 0.03 3.71± 0.16 4.33± 0.14 3.65± 0.08
B. unamae MTI-641
T
3.33± 0.05 3.17± 0.01 3.38± 0.06 3.09± 0.03 3.28± 0.09 3.14± 0.02
B. silvatlantica PVA5 3.41± 0.04 3.18± 0.01 3.28± 0.05 3.20± 0.12 3.27± 0.06 3.07± 0.01
B. silvatlantica SRMrh20
T
3.35± 0.01 3.16± 0.03 3.32± 0.02 3.02± 0.02 3.27± 0.07 3.07± 0.01
a
Original pH of medium and subsequent measurements in triplicate are displayed
b
The uninoculated control was incubated under the same conditions as the inoculated cultures. The values indicate only a single reading
recorded per time point
Table 3 Zones of clearance and color change on PVK agar plates containing bromocresol purple 72 hpi
Strain pH7.55
a
pH6.53
a
pH5.28
a
Halo:colony
average
b
Diameter of color
diffusion
c
(mm)
Halo:colony
average
b
Diameter of color
diffusion
c
(mm)
Halo:colony
average
b
Diameter of color
diffusion
c
(mm)
B.tuberum STM678
T
1.33 11 1.29 11 1.43 n.r.
B.unamae MTI-641
T
1.53 26 1.67 21 1.63 n.r.
B. silvatlantica PVA5 2.00 31 1.79 28 1.81 n.r.
B.silvatlantica SRMrh20
T
1.92 29 1.80 33 1.69 n.r.
a
Original pH of medium and subsequent measurements in triplicate are displayed
b
The ratio of the zone of clearance to the colony size is reported. A value >1 indicates solubilization of CaHPO
4
c
The average diameter of the diffusion of color is shown. A change in color from purple to yellow indicates a decrease in the pH of the
medium
n.r., not recorded. Measurements for pH5.28 are not shown due to a complete diffusion of yellow throughout the plates
Plant Soil
genome contains 16 genes potentially encoding TonB-
dependent siderophore receptors whereas each B. silvat-
lantica strain has four (Table 5). B. tuberum has three
genes annotated as TonB siderophore receptors (Table 5),
one of which may be non-functional (see below). Some
of the TonB-dependent siderophore receptors listed in
Tab le 5are in an operon (see consecutive gene numbers
and similar superscripted letters) with 1) an ABC-type
cobalamin/Fe
3+
-siderophore transport system, 2) an
ATPase component (EC:3.6.3.34), 3) an ABC-type
cobalamin/Fe
3+
-siderophore transport system, permease
component, and 4) an ABC-type Fe
3+
-hydroxamate
transport system, periplasmic component genes
(Table 5). In addition, several ABC-type Fe
+3
-sidero-
phore transport system, and ATPase and permease com-
ponent genes were found in the genomes, but were not
associated with siderophore protein-encoding genes.
Genes encoding a potential periplasmic component of a
Fig. 6 P solubilization gene
maps. aThe PQQ gene
neighborhoods of four Bur-
kholderia spp. compared to
that of S. meliloti.bThe
orientation and surrounding
neighborhoods for the acpA
gene in the nodulating spe-
cies B. tuberum and B. phy-
matum are different from the
non-nodulating Burkholde-
ria species. However, the
nodulating B. mimosarum
strain gene neighborhoods
are identical to those of B.
unamae and the two B. sil-
vatlantica strains
Plant Soil
hydroxamate transport system were also present
(Table 5). Such a transport system had been detected
earlier in B. unamae (Caballero-Mellado et al. 2007).
The first of three B. tuberum siderophore receptors,
GCWU001488_01779, is adjacent to a gene putatively
encoding a protein involved in ABC-type cobalamin/
Fe
3+
-siderophore transport (GCWU001488_01778)
(Table 5). GCWU001488_01779 is orthologous and
69–70 % identical to genes encoding putative side-
rophore receptors in B. glumae (bglu_2g14800)
and B. gladioli (bgla_2g10490). The immediate
gene neighborhoods of B. glumae and B. gladioli
are almost identical to that of B. tuberum, but a
putative transposon adjacent to GCWU001488_01778
suggests that this insertion in the B. tuberum operon
could result in the truncation of a putative ABC-type
Fe
3+
-hydroxamate transport system, periplasmic com-
ponent (GCWU001488_01777). On the other hand,
homologs to either the ATPase or periplasmic compo-
nent genes observed in B. unamae and B. tuberum were
not detected in the two strains of nitrogen-fixing B.
silvatlantica examined in this study (Table 5).
The second putative TonB siderophore receptor
(GCWU001488_04562) in B. tuberum is orthologous
to a gene of the same name in several Burkholderia
species and also in B. unamae (64 % identity;
GCWU001489_04829), but not to genes in either of
the two B. silvatlantica strains (Table 5). Like the B.
unamae siderophore receptor, this gene is not adjacent
to other iron transporter-encoding genes. The B. tuberum
siderophore receptor is predicted to be in COG1629 (Fe
receptor), whereas the B. unamae ortholog is annotated
as a protein in COG4774 (catecholate receptor).
However, so far no experimental evidence exists for the
latter type of receptor in B. unamae.
The third gene annotated as a B. tuberum TonB
siderophore receptor (GCWU001488_05039) is
orthologous to a comparable sequence in a number
of Burkholderia species, including the ones studied
here (from 74 to 76.3 % sequence identity), and also
to genes in the B. xenovorans (84.2 %) and B. phyto-
firmans (84 %) genomes. A gene that putatively enc-
odes 2OG-Fe(II) oxygenase (GCWU001488_05038)
is adjacent to the TonB siderophore receptor as well as
to its ortholog siderophore receptors in the other three
nitrogen-fixing Burkholderia spp. (Table 5).
In B. unamae, a gene annotated as lysine/ornithine N-
monooxygenase (GCWU001489_07200) shows 59–
60 % identity to a Bordetella pertussis gene encoding
a biosynthesis enzyme (alcA) for alcaligin, a dihydrox-
amate siderophore. Although similarities existed be-
tween the B. unamae sequence and a B. tuberum gene,
only 31 % identity on the protein level was observed
between them. Moreover, no other alc-like genes were
found in B. tuberum or in the two B. silvatlantica strains.
Adjacent to the B. unamae alcA-like gene lies a se-
quence encoding an arabinose efflux protein and adja-
cent to that is an alcB-like gene with 70.9 % identity to a
protein annotated in Burkholderia sp. CCGE1002 as a
siderophore biosynthesis protein (YP_003610151.1),
and with ca. 58 % identity to an alcaligin biosynthesis
protein in two strains of Achromobacter xylosoxidans
(EGP43454.1, YP_003979104.1), and 55 % identity to
an alcB gene product in Bordetella pertussis Tohama I
Table 4 List of locus tags for gene sequences encoding proteins that are likely to be involved in phosphate solubilization
Gene function and name Locus tag
B. tuberum STM678
T
B. unamae MTI641
T
B. silvatlantica PVA5 B. silvatlantica SRMrh20
T
Inorganic Phosphate Solubilization
Membrane-bound PQQ-dependent dehydrogenase glucose/quinate/shikimate family
GCWU001488_06170 GCWU001489_05348 GCWU001490_06856 GCWU001491_04527
- - GCWU001490_04976 GCWU001491_06776
Organic Phosphate Solubilization
Putative purple alkaline phosphatase
GCWU001488_04845 GCWU001489_02415 GCWU001490_05774 GCWU001491_07093
Acid phosphatase AcpA
GCWU001488_05111 GCWU001489_03077 GCWU001490_05252 GCWU001491_06457
- GCWU001489_01734 GCWU001490_01837 GCWU001491_02862
Plant Soil
Table 5 Number (and locus tags) of genes in Burkholderia spp. genomes that are likely to encode proteins for siderophore-mediated iron uptake
B. tuberum STM678T B. unamae MTI-641 T B. silvatlantica PVA5 B. silvatlantica SRMrh20T
TonB-dependent siderophore receptor 3 16 4 4
GCWU001488_01779
z
,
a
GCWU001489_01847
x
GCWU001490_04037
x,m
GCWU001491_03470
y
GCWU001488_04562
z
’GCWU001489_02174
x,l
GCWU001490_05013
x
GCWU001491_06738
x
GCWU001488_05039
x,k
GCWU001489_02646
y
GCWU001490_05887
y
GCWU001491_06980
y
GCWU001489_03097
b,y
GCWU001490_05954
y
GCWU001491_07451
x, n
GCWU001489_04829
x
GCWU001489_05097
y
GCWU001489_05762
c,y
GCWU001489_05953
d,y
GCWU001489_05997
e,z
GCWU001489_06000
z
GCWU001489_06507
f.y
GCWU001489_07702
g,y
GCWU001489_08020
x
GCWU001489_08176
h,z
GCWU001489_08634
i,y
GCWU001489_08731
y
ABC-type cobalamin/Fe
3+
-siderophore
transport systems, ATPase components
(EC:3.6.3.34)
151 1
GCWU001488_01778
a
GCWU001489_03782
p
GCWU001490_02093
q
GCWU001491_00020
r
GCWU001488_03141
o
GCWU001489_05763,
c
GCWU001489_05949
d
GCWU001489_06508
f
GCWU001489_07448
ABC-type Fe
3+
-siderophore transport
system, permease component
251 1
GCWU001488_01777
a*
GCWU001489_03783
p
GCWU001490_02094
q
GCWU001491_00021
r
GCWU001488_03140
o
GCWU001489_05765
c
GCWU001489_05951
d
GCWU001489_06510
f
GCWU001489_07447
ABC-type siderophore export system, fused
ATPase and permease components
040 0
GCWU001489_05766,
c
GCWU001489_05950
d
Plant Soil
Table 5 (continued)
B. tuberum STM678T B. unamae MTI-641 T B. silvatlantica PVA5 B. silvatlantica SRMrh20T
GCWU001489_06511
f
,
GCWU001489_08630
ABC-type Fe
3+
-hydroxamate transport system,
periplasmic component
151 1
GCWU001488_01777
a*
GCWU001489_03778 GCWU001490_02089 GCWU001491_00016
GCWU001488_03145 GCWU001489_05764
c
GCWU001489_05952
d
GCWU001489_06509
e
GCWU001489_07446
2OG-Fe(II) oxygenase 1 1 1 1
GCWU001488_05038
k
GCWU001489_02175
l
GCWU001490_04038
m
GCWU001491_07450
n
Ferric dicitrate sensor; FecR family 0 4 0 0
GCWU001489_05998
d
GCWU001489_07701
f
GCWU001489_08177
h
GCWU001489_08636
Lysine/ornithine N-monooxygenase (alcA-like) 0 GCWU001489_07200
o
00
Siderophore biosynthesis protein (alcB-like) 0 GCWU001489_07202
o
00
Siderophore synthetase component (alcC-like) 0 1 0 0
GCWU001489_07203
o
Siderophore ferric iron reductase (alcD-like) 0 1 0 0
GCWU001489_07204
o
Uncharacterized protein related to arylsulfate
sulfotransferase involved in siderophore
biosynthesis
110 0
GCWU001488_05679 GCWU001489_06523
FAD-binding 9, siderophore-interacting
domain protein
111 1
GCWU001488_05828 GCWU001489_02672 GCWU001490_04846 GCWU001491_06907
a-r
Genes with the same superscripted letter are within the same operon;
x
COG4774, outer membrane receptor for monomeric catechol;
y
COG4773, outer membrane receptor for ferric
coprogen and ferric-rhodotorulic acid;
z
COG1629, outer membrane receptor proteins, mostly Fe transport;
zz
COG4206, outer membrane cobalamin receptor protein;
*
truncated
Plant Soil
(NP_881084.1). Immediately adjacent to the alcB-like
sequence is a gene coding for a siderophore synthetase
component (GCWU001489_07205), with 63 % amino
acid sequence identity to alcC in A. xylosoxidans
(EFV887439.1), 61 % identity to the alcC gene in the
various Bordetella species (NP_881085.1, CAA3891.1,
and NP_885606.1), and 59 % to alcC in Pseudomonas
stutzeri (YP_004716031.1). Lastly, a siderophore ferric
iron reductase for alcaligin synthesis (alcD),
(GCWU001489_07204), which is annotated in
Burkholderia spp. CCGE1002 as a hypothetical protein
(YP_003610153.1; 61 % identity), is found in B. una-
mae (Table 5, Fig. 7). The gene call was made on 42 %
identity to the alcD gene in Bordetella pertussis,B.
bronchiseptica, and B. parapertussis (NP_881086.1,
CAA3891.1, and NP_885607.1). These and other pro-
teins, such as arylsulfate sulfotransferase (Mathew et al.
2001), may be involved in siderophore synthesis and are
listed in Table 5.
Discussion
B. tuberum STM678
T
was originally identified as a
Bradyrhizobium species (Muofhe and Dakora 1998)
because of its slow growth on plates upon isolation
from nodules, and was named Bradyrhizobium aspa-
lati because it was isolated from Aspalathus carnosa
(see Elliott et al. 2007a and Gyaneshwar et al. 2011).
However, it induced effective nodulation on siratro
roots (Elliott et al. 2007a), although Moulin et al.
(2001) had reported the phenotype as Fix
-
.
We wanted to obtain a deeper understanding of the
conditions that modulated whether Fix
+
or Fix
-
nod-
ules are produced on siratro so that effective nodula-
tion could be obtained on a reliable and predictable
basis. We found that a porous soil mixture with infre-
quent watering resulted in B. tuberum STM678
T
-in-
duced Fix
+
nodules on siratro. However, when R.
tropici CIAT899, a strain that is also tolerant of acid
soils and thus expected to be a good reference strain,
was used as an inoculum under the conditions tested,
Fix
-
nodules developed on siratro. In contrast, cowpea
plants included in the same dishpans as the siratro
plants nodulated and fixed nitrogen (data not shown).
An analysis of the literature concerning R. tropici
CIAT899 does not provide definitive answers.
Hernandez-Lucas et al. (1995) reported that this strain
induced two types of nodules on siratro—those that had
leghemoglobin and those that lacked it and contained
senescent, darkened cells. It was not clear from this
report whether the nodules that contained leghemoglo-
bin were actually fixing nitrogen. Collavino et al. (2005)
tested the guaA mutant of R. tropici CIAT899 on siratro
and found that it elicited ineffective nodules, but a
mature, wild-type, Fix
+
nodule was not illustrated in this
study. However, unpublished data from one of the
authors (O.M. Aquilar, pers. com.) indicates that Fix
+
nodules were elicited by this strain. The difference in
phenotypes described in our results and the previously
published work cannot be explained at this time, in part
due to the differences in growth conditions. Changes in
temperature and water availability could make a signif-
icant difference in nodule outcome. Moreover, it is also
possible that R. tropici CIAT899 only marginally nod-
ulates siratro as suggested by the results of Hernandez-
Lucas et al. (1995).
R. tropici CIAT899 was originally described as R.
leguminosarum bv. phaseoli and nodulates bean and
Leucaena sp. effectively (Martínez-Romero et al.
1991; Riccillo et al. 2000) at high temperatures al-
though acetylene reduction activity was reduced in
bean (Michiels et al. 1994). This strain’s ability to
nodulate a diverse number of legumes and under dif-
ferent environmental conditions is most likely condi-
tioned by the large variety of Nod factors produced by
CIAT899. R. tropici Nod factors have typical back-
bones consisting of four or five β-1,4 linked N-ace-
tylglucosamine residues, but the number and types of
substitutions on the lipo-chitooligosaccharide varies
significantly depending on the environmental condi-
tions. For example, under low pH, 59 Nod factors
were produced whereas 29 different structures were
synthesized at neutral pH (Móron et al. 2005).
Additional and different Nod factors were also
detected following salt stress (Estévez et al. 2009). In
contrast, Boone et al. (1999) identified only two major
Nod factors of B. tuberum STM678
T
, then known as
Bradyrhizobium aspalati. These factors have either a
tetrameric or pentameric N-acetylglucosamine back-
bone, but instead of having substitutions on the reduc-
ing end as observed in alpha-rhizobia, these Nod
factors are substituted only on the non-reducing end
of the molecule. In any case, whether or not different
Nod factors arise following abiotic stress has not been
tested for the beta-rhizobia. Other than for B. tuberum
STM678
T
, no other Nod factor structures have been
determined for the nodulating Burkholderia species.
Plant Soil
B. tuberum STM678
T
-inoculated siratro plants
remained green and developed pink, Fix
+
nodules even
under desiccating conditions, which differs from
Rhizobium strains in general (Michiels et al. 1994).
Both B. phymatum STM815
T
and B. tuberum
STM678
T
have been shown to fix nitrogen ex planta,
although not to the levels of B. vietnamiensis TVV75
(Elliott et al. 2007b). This ability may be the reason that
B. tuberum is able to fix nitrogen symbiotically under
stressful conditions.
Besides nitrogen fixation, rhizosphere bacteria en-
hance plant growth in a number of diverse ways,
among them protecting plants from abiotic stress,
and improving phosphate or iron nutrition. Various
environmental stresses such as reduced water avail-
ability and increased temperature also significantly
influence nodulation effectiveness. We found that sir-
atro nodulation was improved by higher temperatures
and was effective even under conditions of reduced
water availability. In contrast, R. tropici elicited Fix
-
nodules on siratro when grown under these conditions.
Trehalose is synthesized by a broad range of organ-
isms including plants, bacteria, archaea, and insects
(Iturriaga et al. 2009) as a means of dealing with either
desiccation or thermal stress. The beta-rhizobial
genomes, except for B. unamae, possessed an addi-
tional trehalose biosynthetic pathway compared to
alpha-rhizobial genomes. A partial otsA gene sequence
(AFH35528.1) from R. tropici exhibited only 46 %
amino acid sequence identity to the otsA sequence in
the Burkholderia spp. However, we cannot make a
more direct comparison with R. tropici because only
a draft genome analysis of this species has been pub-
lished (Pinto et al. 2009). Information about the num-
ber of trehalose biosynthetic pathways in this species
is thus not available at this time.
Of the four Burkholderia spp. investigated, B. silvat-
lantica PVA5 and SRMrh20
T
exhibited the largest
amount of phosphate solubilization activity and also
possessed the most gene sequences potentially involved
in inorganic and organic phosphate breakdown based on
analysis of the sequenced genomes. The drop in pH
upon mineralizing CaHPO
4
in the PVK plates strongly
suggests that production of glucuronic or another organ-
ic acid may be involved in inorganic P solubilization,
and that the plant-associated Burkholderia species are
likely inhabitants of soils that are low in soluble phos-
phate. Burkholderia species have been described previ-
ously as having a preference for acidic soils (Garau et al.
2009;DosReisetal.2010). A recent study of
Burkholderia in French Guinea suggests that beta-
rhizobia exclusive of Cupriavidus taiwanensis, which
prefers alkaline soils, were dominant in low pH soils
(Mishra et al. 2012). In a similar vein, Estrada-de los
Santos et al. (2011) isolated only a few Burkholderia
species associated with agricultural plants growing in
the alkaline soils of northern Mexico in contrast to
Cupriavidus species, which were isolated more fre-
quently. Isolation of Burkholderia species from alkaline
soils in Australia has been reported (see Gyaneshwar et
al. 2011), but more studies are needed.
Siderophores, which make iron available, are impor-
tant for host colonization by bacteria (Mietzner and
Morse 1994). Of the four nitrogen-fixing Burkholderia
strains, B. unamae produced the largest halos using the
CAS-overlay medium whereas B. tuberum exhibited the
least activity. Genome analysis showed that B. unamae
contains more gene sequences for TonB-dependent as-
sociated siderophore receptors and their associated
genes than did the other three species, which may ex-
plain the increased activity. Although B. unamae does
not nodulate plants, it was originally isolated from the
roots of maize and sugarcane (Table 1) and thus can be
classified as a plant-associated diazotroph. The genome
of B. tuberum, which contains nod genes, has three
siderophore receptors and the genomes of the other
Fig. 7 Genes potentially encoding a hydroxamate siderophore.
Of the four Burkholderia genomes surveyed, only the genome
of B. unamae has genes coding for alcaligin, a hydroxamate
siderophore. The B. unamae gene neighborhood is compared to
the orthologous operon in Achromobacter xylosoxidans AB
Plant Soil
two B. silvatlantica strains each have four. Both B.
silvatlantica SRMrh20
T
(Perin et al. 2006) and PVA5
(de Faria et al. 1999) were isolated from the interstices
of plant roots and fix nitrogen, but are not capable of
nodulating roots.
Earlier reports indicated that siratro’s small size,
ability to grow under controlled conditions, prolonged
seed viability, and promiscuity with respect to nodu-
lation (Vincent 1970; Pueppke and Broughton 1999)
supported its usefulness as a plant for studying
responses to rhizobia. Recently, Lima et al. (2009)
and Mishra et al. (2012) endorsed its value as a trap
plant for both alpha- and beta-rhizobia. Based on our
studies, we believe that this small-seeded legume is
also an excellent model system for analyzing plant
responses to B. tuberum inoculation under a wide
range of environmental conditions.
Acknowledgments This research was supported in part by a
grant (IOB-0537497) from the National Science Foundation
(USA) to GW and AMH and a Shanbrom Family Foundation
grant to AMH. A University of California Office of The Presi-
dent, President’s Postdoctoral Fellowship, supported AA. We
thank the National Germplasm Collection of the USDA-ARS
for seeds of various cowpea varieties that were tested and the
Joint Genome Institute/Department of Energy for the annotation
platform. LMU’s Seaver College of Science and Engineering
M.A.N.E. laboratory is thanked for the use of the confocal
microscope.
Liamara Perin and Veronica M. Reis of EMBRAPA are
acknowledged for their previous research on Burkholderia spe-
cies and for providing helpful information regarding B. silvat-
lantica SRMrh20
T
. J. Peter Young is thanked for providing the
B. tuberum/gfp strain. Members of the Hirsch laboratory, espe-
cially Drs. Drora Kaplan and Nisha Tak are thanked for review-
ing the manuscript.
This paper is dedicated to the memory of Jesus Cabellero-
Mellado, one of the pioneers in the study of the plant-associated
Burkholderia species.
References
Ahmed B, Quilt P (1980) Effect of soil moisture stress on yield,
nodulation and nitrogenase activity of Macroptilium atro-
purpureum cv. Siratro and Desmodium intortum cv.
Greenleaf. Plant Soil 57:187–194
Andrews SC, Robinson AK, Rodriguez-Quiñones F (2003)
Bacterial iron homeostasis. FEMS Microbiol Rev 27:215–
237
Boone CM, Olsthoorn MMM, Dakora FD, Spaink HP, Thomas-
Oates JE (1999) Structural characterization of lipo-
oligosaccharides isolated from Bradyrhizobium aspalati,
microsymbionts of commercially important South African
legumes. Carbohyd Res 317:155–163
Brigham RD, Hoover MM (1956) A scarifying cup for small
lots of legume seed. Agron J 48:531–532
Caballero-Mellado J, Martínez-Aquilar L, Paredes-Valdez G,
Estrada-de los Santos P (2004) Burkholderia unamae sp.
nov., an N
2
-fixing rhizospheric and endophytic species. Int
J System Evol Microbiol 54:1165–1172
Caballero-Mellado J, Onofre-Lemus J, Estrada-de Los Santos P,
Martínez-Aguilar L (2007) The tomato rhizosphere, an
environment rich in nitrogen-fixing Burkholderia species
with capabilities of interest for agriculture and bioremedi-
ation. Appl Environ Microbiol 73:5308–5319
Castro-González R, Martínez-Aquilar L, Ramírez-Trujillo A,
Estrada-de los Santos P, Caballero-Mellado J (2011) High
diversity of culturable Burkholderia species associated
with sugarcane. Plant Soil. doi:10.1007/s11104-011-
07680-0
Chen W-M, Moulin L, Bontemps C, Vandamme P, Béna G,
Boivin-Masson C (2003) Legume symbiotic nitrogen by
β-proteobacteria is widespread in nature. J Bacteriol
185:7266–7272
Chen W-M, James EK, Coenye T, Chou J-H, Barrios E, de Faria
SM, Elliott GN, Sheu SY, Sprent JI, Vandamme P (2006)
Burkholderia mimosarum sp. nov., isolated from root nod-
ules of Mimosa spp. from Taiwan and South America. Int J
System Evol Microbiol 56:1847–1851
Chen W-M, de Faria SM, James EK, Elliott GN, Lin KY, Chou
J-H, Sheu SY, Cnockaert M, Sprent JI, Vandamme P
(2007) Burkholderia nodosa sp. nov., isolated from root
nodules of the woody Brazilian legumes Mimosa bimucro-
nata and Mimosa scabrella. Int J System Evol Microbiol
57:1055–1059
Cheng H-P, Walker GC (1998) Succinoglycan is required for
initiation and elongation of infection threads during nodu-
lation of alfalfa by Rhizobium meliloti. J Bacteriol
180:5183–5191
Collavino M, Riccillo PM, Grasso DH, Crespi M, Aguilar OM
(2005) GuaB activity is required in Rhizobium tropici
during the early stages of nodulation of determinate nod-
ules but is dispensable for the Sinorhizobium meliloti-al-
falfa symbiotic interaction. Mol Plant-Microbe Inter
18:742–750
de Faria SM, de Lima HC, Olivares FL, Melo RB, Xavier RP
(1999) Nodulação em espécies florestais, especificidade hos-
pedeira e implicações na sistemática de leguminosae. In:
Siqueira JO, Moreira FMS, Lopes AS, Guiherme LRG,
Faquin V, Furtinia Neto AE, Carvalho JG (eds) Soil Fertility,
Soil Biology, and PlantNutrition Interrelationships. Soc Brasil
Ciên Univ Fed Lavras. pp. 667–686
Dos Reis FB Jr, Simon MF, Gross E, Boddey RM, Elliott GN,
Neto NE, Loureiro MF, de Queiroz LP, Scotti MR, Chen
W-M, Norén A, Rubio MC, de Faria SM, Bontemps C, Goi
SR, Young JP, Sprent JI, James EK (2010) Nodulation and
nitrogen fixation by Mimosa spp. in the Cerrado and
Caatinga biomes of Brazil. New Phytol 186:934–946
Elliott GN, Chen W-M, Bontemps C, Chou J-H, Young JPW,
Sprent JI, James EK (2007a) Nodulation of Cyclopia spp.
(Leguminosae, Papilionoideae) by Burkholderia tuberum.
Ann Bot 100:1403–1411
Elliott GN, Chen W-M, Chou J-H, Wang H-C, Sheu SY, Perin
L, Reis VM, Moulin L, Simon MF, Bontemps C,
Sutherland JM, Bessi R, de Faria SM, Trinick MJ,
Plant Soil
Prescott AR, Sprent JI, James EK (2007b) Burkholderia
phymatum is a highly effective nitrogen-fixing symbiont of
Mimosa spp. and fixes nitrogen ex planta. New Phytol
173:168–180
Estévez J, Soria Díaz ME, Fernández de Córdoba F, Móron B,
Manyan H, Gil A, Thomas-Oates J, van Brussel AAN,
Dardanelli MS, Sousa C, Megías M (2009) Different and
new Nod factors produced by Rhizobium tropici CIAT899
following Na
+
stress. FEMS Microbiol Lett 293:220–231
Estrada-de los Santos P, Vacaseydel-Aceves NB, Martínez-
Aquilar L, Cruz-Hernández MA, Mendoza-Herrera A,
Caballero-Mellado J (2011) Cupriavidus and Burkholderia
species associated with agricultural plants that grow in alka-
line soils. J Microbiol 49:867–876
Fujishige NA, Lum MR, De Hoff PL, Whitelegge JP, Faull KF,
Hirsch AM (2008) Rhizobium common nod genes are
required for biofilm formation Mol. Microbiology
67:504–515
Garau G, Yates RJ, Deiana P, Howieson JG (2009) Novel strains
of nodulating Burkholderia have a role in nitrogen fixation
with papilionoid herbaceous legumes adapted to acid, in-
fertile soils. Soil Biol Biochem 41:125–134
Graham PH, Viteri SE, Mackie F, Vargas AAT, Palacios A
(1982) Variation in acid soil tolerance among strains of
Rhizobium phaseoli. Field Crops Res 5:121–128
Graham PH, Draeger KJ, Ferrey ML, Conroy MJ, Hammer BE,
Martínez E, Arons SR, Quinto C (1994) Acid pH tolerance
in strains of Rhizobium and Bradyrhizobium and initial
studies on the basis for acid tolerance of Rhizobium tropici
UMR1899. Can J Microbiol 40:189–207
Gyaneshwar P, Hirsch AM, Moulin L, Chen WM, Elliott GN,
Bontemps C, Estrada-de los Santos P, Gross E, dos Reis
Junior FB, Sp rent JI, Young JPW, James EK (2011)
Legume nodulating β-proteobacteria: diversity, host range
and future prospects. Mol Plant-Microbe Inter 24:1276–1288
Hernandez-Lucas L, Segovia L, Martinez-Romero E, Pueppke
SG (1995) Phylogenetic relationships and host range of
Rhizobium spp. that nodulate Phaseolus vulgaris L. Appl
Environ Microbiol 61:2775–2779
Herridge DF, Roughley RJ (1976) Influence of temperature and
Rhizobium strain on nodulation and growth of two tropical
legumes. Trop Grass 10:21–23
Hirsch AM (1992) Tansley review No. 40. Developmental biol-
ogy of legume nodulation. New Phytol 122:211–237
Hugenholtz P, Cunningham MA, Hendrlkz JK, Fuerst JA (1995)
Desiccation resistance of bacteria isolated from an air-
handling system biofilm determined using a simple quan-
titative membrane filter method. Lett Appl Micro 21:41–46
Iturriaga G, Suárez R, Nova-Franco B (2009) Trehalose metab-
olism: from osmoprotection to signaling. Int J Mol Sci
10:3793–3810
Lee HK, LaRue TA (1992) Exogenous ethylene inhibits nodu-
lation of Pisum sativum L. cv Sparkle. Plant Physiol
100:1759–1763
Lima AS, Nobrega RSA, Barberi A, da Silva K, Ferreira DF,
Moreira FMS (2009) Nitrogen-fixing bacteria communities
occurring in soils under different uses in the Western
Amazon Region as indicated by nodulation of siratro
(Macroptilium atropurpureum). Plant Soil 319:127–145
Martínez-Romero E, Segovia L, Mercant FM, Franco AA,
Graham P, Pardon MA (1991) Rhizobium tropici, a novel
species nodulating Phaseolus vulgaris L. beans and
Leucaena sp. trees. Int J Syst Evol Microbiol 41:417–426
Mathew JA, Tan YP, Srinivasa Rao PS, Lim TM, Leung KY
(2001) Edwardisella tarda mutants defective in sidero-
phore production, motility, serum resistance and catalase
activity. Microbiology 149:449–457
Michiels J, Verreth C, Vanderleyden J (1994) Effect of temper-
ature stress on bean-nodulating Rhizobium strains. Appl
Environ Microbiol 60:1206–1212
Mietzner TA, Morse SA (1994) The role of iron-binding pro-
teins in the survival of pathogenic bacteria. Annu Rev Nutr
14:471–493
Mishra RP, Tisseyre P, Melkonian R, Chaintreuil C, Miché L,
Klonowska A, Gonzalez S, Bena G, Laguerre G, Moulin L
(2012) Genetic diversity of Mimosa pudica rhizobial sym-
bionts in soils of French Guiana: investigating the origin
and diversity of Burkholderia phymatum and other beta-
rhizobia. FEMS Microbiol Ecol 79:487–503
Móron B, SoriaDíaz ME, Ault J, Verroios G, Noreen S,
Rodríguez-Navarro DN, Gil-Serrano A, Thomas-Oates J,
Megías M, Sousa C (2005) Low pH changes the profile of
nodulation factors produced by Rhizobium tropici
CIAT899. Chem Biol 12:1020–1040
Moulin L, Munive A, Dreyfus B, Boivin-Masson C (2001)
Nodulation of legumes by members of the beta-subclass
of proteobacteria. Nature 411:948–950
Muofhe ML, Dakora FD (1998) Bradyrhizobium species isolat-
ed from indigenous legumes of the Western Cape exhibit
high tolerance of low pH. In: Elmerich C, Kondorosi A,
Newton WE (eds) Biological nitrogen fixation for the 21st
century. Kluwer Academic Publishers, Dordrecht, p 519
Ohashi Y, Saneoka H, Fujita K (2000) Effect of water stress of
growth, photosynthesis, and photoassimilate translocation
in soybean and tropical pasture legume siratro. Soil Sci
Plant Nutr 46:417–425
Perin L, Martinez-Aquilar L, Paredes-Valdez G, Baldani JI,
Estrada-de Los Santos P, Reis VM, Cabellero-Mellado J
(2006) Burkholderia silvatlantica sp. nov., a bacterium
associated with field-grown sugarcane and maize. Int J
Syst Evol Microbiol 56:1931–1937
Pikovskaya RI (1948) Mobilization of phosphorous soil in con-
nection with the vital activity of some microbial species.
Microbiol (Mikrobiol) 17:362–370
Pinto FGS, Chueire LMO, Vasconcelos ATR, Nicolás MF,
Almeida LGP, Souza RC, Menna P, Barcellos FG,
Megías N, Hungria M (2009) Novel genes related to nod-
ulation, secretion systems, and surface structures revealed
by a genome draft of Rhizobium tropici strain PRF 81.
Funct Integr Genom 9:263–270
Pueppke SG, Broughton WJ (1999) Rhizobium sp. strain
NGR234 and R. fredii USDA257 share exceptionally
broad, nested host ranges. Mol. Plant-Microbe Inter
12:293–318
Riccillo PM, Collavino MM, Grasso DH, England R, de Bruijn
FJ, Aguilar OM (2000) A guaB mutant strain of Rhizobium
tropici CIAT899 pleiotropically defective in thermal toler-
ance and symbiosis. Mol Plant-Microbe Inter 13:1228–
1236
Rodríguez H, Fraga R (1999) Phosphate solubilizing bacteria
and their role in plant growth promotion. Biotechnol Adv
17:319–339
Plant Soil
Rodríguez H, Fraga R, Gonzalez T, Bashan Y (2006) Genetics
of phosphate solubilization and its potential applications
for improving plant growth-promoting bacteria. Plant Soil
287:15–21
Schwyn B, Neilands JB (1987) Universal chemical assay for the
detection and determination of siderophores. Anal
Biochem 160:47–56
Sheriff DW, Ludlow MM (1984) Physiological reactions to an
imposed drought by Macroptilium atropurpureum and
Cenchrus ciliaris in a mixed sward. Aust J Plant Physiol
11:23–34
Sprent JI (2007) Evolving ideas of legume evolution and diver-
sity: a taxonomic perspective on the occurrence of nodula-
tion. New Phytol 186:934–946
Streeter JG, Gomez ML (2006) Three enzymes for trehalose
synthesis in Bradyrhizobium cultured bacteria and in bac-
teroids from soybean nodules. Appl Environ Microbiol
72:4250–4255
Suárez-Moreno AR, Cabellero-Mellado J, Coutinho BG,
Mendonça-Previato L, James EK, Venturi V (2012)
Common features of environmental and potentially beneficial
plant-associated Burkholderia. Microb Ecol 63:249–266
Suzuki A, Suriyagoda L, Shigeyama T, Tominaga A, Sasaki M,
Hiratsuka Y, Yoshinaga A, Arima S, Agarie S, Sakai T, Inada
S, Jikumaru Y, Kamiya Y, Uchiumi T, Abe M, Hashiguchi M,
Akashi T, Sato S, Kaneko T, Tabata S, Hirsch AM (2011)
Lotus japonicus nodulation is photomorphogenetically
controlled by sensing the R/FR ratio through JA signaling.
Proc Natl Acad Sci USA 108:16837–16842
Van Brussel AAN, Tak T, Wetselaar A, Pees E, Wijffelman CA
(1982) Small Leguminosae as test plants for nodulation of
Rhizobium leguminosarum and other rhizobia an agrobac-
teria harbouring a leguminosarum sym-plasmid. Plant Sci
Lett 27:317–325
Vandamme P, Goris J, Chen W-M, de Vos P, Willems A (2002)
Burkholderia tuberum sp. nov. and Burkholderia phyma-
tum sp. nov., nodulate the roots of tropical legumes. Syst
Appl Microbiol 25:507–512
Vincent JM (1970) A manual for the practical study of root-
nodule bacteria. Blackwell Scientific Publications, London
Weisskofp L, Heller S, Eberl L (2011) Burkholderia species are
major inhabitants of white lupin cluster roots. Appl
Environ Microbiol 77:7715–7720
Xie J, Knight D, Legget M (2009) Comparison of media used to
evaluate Rhizobium leguminosarum biovar viciae for
phosphate-solubilizing ability. Can J Microbiol 55:910–915
Yao W, Byrne R (2001) Spectrophotometric determination of
freshwater pH using bromocresol purple and phenol red.
Environ Sci Technol 35:1197–1201
Yeung S-L, Cheng C, Lui TKO, Tsang JSH, Chan W-T, Lim BL
(2009) Purple acid phosphastase-like sequences in pro-
karyotic genomes and the characterization of an atypical
purple alkaline phosphatase from Burkholderia cenocepa-
cia J2315. Gene 440:1–8
Plant Soil
