ArticlePDF Available

Bioavailability of iron to Pseudomonas fluorescens strain A506 on flowers of pear

Authors:

Abstract and Figures

Pseudomonas fluorescens strain A506 (A506) produces an antibiotic toxic to Erwinia amylovora in defined culture media containing at least 0.1 mM FeCl3. To estimate the relative availability of iron on flowers, A506 was transformed with an 'iron biosensor', which consisted of an iron-regulated promoter (pvd) fused to an ice nucleation reporter gene (inaZ). A506 (pvd-inaZ) established high populations on blossoms, ranging from 105 to 107 colony forming units on pear. In two trials on screenhouse grown pear trees, A506 (pvd-inaZ) expressed high ice nucleation activity on blossoms indicating limited iron bioavailability, or a low-iron environment unlikely to induce antibiosis by A506. A506 (pvd-inaZ) also colonized blossoms when mixed with the iron chelate, ferric ethylenediaminedi-(o-hydroxyphenylacetic) acid (FeEDDHA). Co-treatment of flowers with a mixture of A506 (pvd-inaZ) and 3 mM FeEDDHA significantly decreased ice nucleation activity compared to flowers treated with A506 (pvd-inaZ) in water. Lower concentrations (i.e. 0.3 mM FeEDDHA) did not consistently increase iron available to A506 on flowers sufficient to suppress expression of the iron regulated reporter gene construct. These results indicate that pear flowers represent an iron-limited environment to A506 and that co-treatment with at least 3 mM FeEDDHA significantly increases the level of iron biologically available to this bacterium.
Content may be subject to copyright.
This article is from the
December 2004 issue of
published by
The American Phytopathological Society
For more information on this and other topics
related to plant pathology,
we invite you to visit APSnet at
www.apsnet.org
1286 PHYTOPATHO LOGY
Biological Control
Bioavailability of Iron to Pseudomonas fluorescens Strain A506
on Flowers of Pear and Apple
Todd N. Temple, Virginia O. Stockwell, Joyce E. Loper, and Kenneth B. Johnson
First to fourth authors: Department of Botany and Plant Pathology, Oregon State University, Corvallis 97330; and third author: U.S. Depart-
ment of Agriculture–Agricultural Research Service, Horticultural Crops Research Laboratory, 3420 NW Orchard Avenue, Corvallis
97330.
Accepted for publication 16 August 2004.
ABSTRACT
Temple, T. N., Stockwell, V. O., Loper, J. E., and Johnson, K. B. 2004.
Bioavailability of iron to Pseudomonas fluorescens strain A506 on flow-
ers of pear and apple. Phytopathology 94:1286-1294.
The addition of 0.1 mM FeCl3 to a defined culture medium induces the
bacterial epiphyte Pseudomonas fluorescens strain A506 (A506) to pro-
duce an antibiotic toxic to the fire blight pathogen, Erwinia amylovora.
Consequently, because A506 is registered and applied as a commercial
product to suppress E. amylovora before floral infection of pear and ap-
ple, the relative availability of iron to A506 on surfaces of pear and apple
flowers is of potential significance. An ‘iron biosensor’ construct of A506
was developed by transformation with an iron-regulated promoter (pvd)
fused to a promoterless ice nucleation reporter gene (inaZ). This con-
struct, A506 (pvd-inaZ), established high populations on pear and apple
flowers, ranging from 104 to 106 CFU/flower. In seven trials on pear and
apple trees, A506 (pvd-inaZ) expressed high ice nucleation activity (INA)
on flowers, indicating limited iron bioavailability or a low-iron environ-
ment unlikely to induce antibiotic production by A506. A506 (pvd-inaZ)
also colonized flowers when mixed with chemicals containing iron:
FeSO4 or the iron chelates ferric ethylenediaminedi-(o-hydroxyphenyl-
acetic) acid (FeEDDHA) and ferric diethylenetriamine pentaacetate
(FeDTPA). These compounds represent an array of commercial iron formu-
lations applied to foliage to avert iron chlorosis. Treatment of flowers
with a mixture of A506 (pvd-inaZ) and 3 mM FeEDDHA or FeDTPA
significantly decreased INA compared with flowers treated with A506
(pvd-inaZ) in water. Lower concentrations (0.3 mM) of FeEDDHA, how-
ever, did not consistently suppress INA. These results indicate that apple
and pear flowers represent an iron-limited environment to A506 and that
treatment with 3 mM FeEDDHA is needed to increase significantly the
level of iron available to this bacterium.
Pseudomonas fluorescens strain A506 (A506) is a commer-
cially available biological control agent (BlightBan A506; Nufarm
Americas Inc., Sugar Land, TX) used for the suppression of fire
blight on pear and apple trees. Effective biocontrol with A506 re-
quires that the bacterium preemptively colonize and establish
large populations on flower surfaces prior to colonization by the
fire blight pathogen, Erwinia amylovora (6,10,24,26,30). Disease
suppression by A506 has been proposed to occur by the mechanism
of “competitive exclusion,” whereby A506 outcompetes E. amy-
lovora for sites and nutrients essential for epiphytic growth and
subsequent infection of the host plant (27,29,30).
Recently, Stockwell et al. (25) observed that A506 produced a
large zone of inhibition to E. amylovora when cultured on defined
media amended with 0.1 mM FeCl3. Because the zone of inhibi-
tion on iron-amended media is indicative of antibiotic production,
questions arise with regard to the potential significance of antibio-
sis in the biocontrol interaction and the relative bioavailability of
iron to A506 in the microhabitats located on surfaces of pear and
apple flowers. If sufficient iron is available to A506 in situ,
production of the antibiotic may occur, suggesting that this strain
may utilize an additional mechanism to attain an epiphytic advan-
tage over E. amylovora. Conversely, if little iron is available on
floral surfaces, competitive exclusion may be the principal
mechanism of suppression, raising the question as to whether bio-
control can be enhanced by the addition of an exogenous source
of iron to the flower environment.
Iron is the fourth most abundant element on earth; however, at
neutral or basic pH in aerated environments, it exists principally
as insoluble iron oxides (12,23). Prior studies (7,15) have demon-
strated that the amount of bioavailable iron on aerial plant sur-
faces can be limiting to microbes. In response, bacteria that reside
on plant surfaces produce iron-sequestering compounds called
siderophores (1,20,22,23). Fluorescent pseudomonads produce
pyoverdines, which are distinct yellow-green, water-soluble, low
molecular weight siderophores (1). Typically, pyoverdine is pro-
duced and secreted into the environment when iron bioavailability
is low and its production is suppressed when iron is abundant
(22,23).
Phyllosphere environments are relatively uncharacterized with
regard to the bioavailability of nutrients to microbial epiphytes.
For iron specifically, Loper and Lindow (15) determined that this
nutrient was limiting, at least partially, to epiphytic populations of
P. syringae on bean leaf surfaces. Subsequently, Joyner and
Lindow (7) reported that iron bioavailability to P. syringae was
heterogeneous on leaves and that only 10% of bacterial cells ex-
perienced iron-limiting conditions. Floral surfaces as microbial
habitats are less characterized than leaves. We recognize that stig-
matic surfaces of pear and apple flowers provide a protected,
nutrient-rich, hydrated environment, which bacteria exploit and
where they amass large populations (5). The diversity and relative
abundance of nutrients such as iron that support microbial growth
on stigmas is not known.
For Pseudomonas spp., a reporter gene for iron bioavailability
has been developed that consists of a transcriptional fusion be-
tween the iron-regulated promoter of a pyoverdine biosynthesis
and uptake region (pvd, from P. syringae) and a promoterless ice
nucleation gene (inaZ, also from P. syringae) (15,16). The inaZ
gene product is an outer-membrane protein (InaZ) that catalyzes
Corresponding author: V. O. Stockwell
E-mail address: stockwev@science.oregonstate.edu
Publication no. P-2004-1011-01R
© 2004 The American Phytopathological Society
Vol. 94, No. 12, 2004 1287
(nucleates) the formation of ice when suspended in supercooled
water at temperatures ranging from –2 to –10°C (11); in the ab-
sence of ice nuclei, water can supercool to temperatures ap-
proaching –40°C before freezing (11,15,16). The transcriptional
activity of the reporter gene is quantified after establishment of
the host bacterium in the environment of interest (16). Abundant
bioavailable iron results in low ice nucleation activity (INA) (i.e.,
the frequency of ice nuclei per CFU), whereas limited bioavail-
able iron results in high INA (13,15,16,18).
The purpose of this study was to estimate the relative availabil-
ity of iron to P. fluorescens strain A506 on pistillate surfaces of
pear and apple flowers by using an iron biosensor construct of
this bacterium. Moreover, exogenous sources of iron were applied
to flowers along with the iron biosensor construct of A506 to de-
termine if they enhance bioavailable iron in the microhabitats
occupied by the bacterium on floral surfaces.
MATERIALS AND METHODS
Bacterial strains and plasmids. The bacterium P. fluorescens
strain A506 was used in all experiments. It was first isolated from
a pear leaf in California by S. Lindow (University of California,
Berkeley), and is resistant to streptomycin and rifampicin (10).
Constructs of A506 included (i) the biosensor strain, A506 (pvd-
inaZ), which contained an iron-regulated promoter for pyoverdine
production (pvd) fused to promoterless inaZ (15); (ii) A506 IceC,
a positive control strain where inaZ is transcribed constitutively
from its native iron-independent promoter; and (iii) A506 Ice, a
negative control strain consisting of promoterless inaZ cloned in
opposite orientation to the lac promoter in the cloning vector,
plasmid VSP61a (such that inaZ is not transcribed). pVSP61a was
the cloning vector for all constructs; this stable plasmid was used
originally in P. syringae (15) and confers resistance to kanamycin
(15). The iron biosensor and the control constructs have been used
in P. putida strain N1R and P. fluorescens strain Pf-5 (13,15).
Effects of iron on pyoverdine production and antibiosis by
A506 in culture. The relationship of iron concentration to
pyoverdine production and antibiosis by A506 was evaluated on
solidified 925 minimal medium with 1.5% potassium gluconate as
the carbon source (8). A 15-µl suspension of A506 was spotted
onto petri dishes containing solidified 925 medium amended with
ferric citrate at concentrations ranging from 0.001 to 1 mM. After
72 h of incubation at 20°C, plates were examined visually for the
characteristic yellow-green fluorescence associated with pyover-
dine under long wavelength (404 nm) UV irradiation. A506 was
removed from the agar surface with a cotton swab and the remain-
ing cells were killed by exposure to chloroform vapor or a germi-
cidal UV lamp. Plates were lightly sprayed with a 107 CFU/ml
aqueous suspension of E. amylovora strain Ea153 (Ea153) (6).
After 48 to 72 h, plates were examined for the presence of zones
inhibitory to the growth of Ea153.
Semi-quantitative methods to measure pyoverdine and antibio-
sis were used for broth cultures of A506. In broth cultures, A506
was grown at 20°C with agitation (200 rpm) in triplicate 5-ml vol-
umes of both 925 medium and modified RSM (2). For 1 liter of
modified RSM medium, 0.75 g of Ca(NO3)2 · 4H2O, 0.25 g of
MgSO4 · 7 H2O, 18.22 g of ACES [N-(2-acetamido)-2-amino-
ethanesulfonic acid], and 2.00 g of NaOH were dissolved in
879 ml of deionized water, and the pH was adjusted to 7.0. After
autoclaving, 1 ml of 1 M KH2PO4 (pH 7.0), 20 ml of 50%
(vol/vol) glycerol, and 100 ml of 10% (wt/vol) Casamino acids
were added. RSM has been used previously for evaluation of iron-
regulated transcription of pvd-inaZ and for pyoverdine production
by pseudomonads (15); however, the antibiotic of A506 cannot be
detected by bioassay in RSM. Ferric citrate was added to 925 me-
dium or RSM to final concentrations ranging from 0.001 to
1 mM. After 48 and 72 h of incubation, the absorbance at 600 nm
(A600) of cultures was measured to estimate cell density. To esti-
mate pyoverdine concentration, cells were removed from a 1-ml
broth culture sample by centrifugation (5 min, 2,500 × g) and
FeCl3 was added to the supernatant to a final concentration of
1 mM. After gentle agitation for 30 min, samples were centri-
fuged for 5 min to remove precipitates. A405 of the clarified super-
natant was measured with an Ultrospec 3100 Pro (Amersham
Biosciences Corp., Piscataway, NJ). Pyoverdine production was
calculated by dividing the ferric-pyoverdine complex A405 value
by the cell density A600 value (15).
Relative antibiotic production, measured with a dilution end-
point method, was determined for the 72-h broth cultures in 925
medium. As above, cells were removed from cultures by centrifu-
gation and the supernatant was sterilized by filtration (0.2 µm).
For each culture, 200 µl of culture filtrate, serial dilutions (1:2,
1:4, 1:8, 1:16, 1:32, and 1:64) of the filtrate made with 10 mM
potassium phosphate buffer, pH 7.0, or phosphate buffer alone
were placed into individual wells of a 96-well Falcon microtest
plate (Becton-Dickinson Labware, Franklin Lakes, NJ). A 20-µl
sample of a 106 CFU/ml suspension of Ea153 was added to each
well of the microtiter plate. After 24 h of incubation at room tem-
perature, a 8.5-µl sample was removed from each well and placed
on solidified 925 medium amended with 1.5% potassium glucon-
ate, 0.1 µM thiamine, and 0.1 µM nicotinic acid. Plates were air
dried in a laminar airflow hood until the visible liquid was ab-
sorbed and then incubated at 20°C for 3 days. Plates were ob-
served for the greatest dilution of culture filtrate that inhibited
growth of Ea153 compared with the control, and a relative anti-
biosis value was assigned to the endpoint dilution (e.g., if growth
of Ea153 was inhibited with 1:16 dilution of the culture filtrate,
but not 1:32, then the sample was assigned the relative antibiosis
value of 16). All experiments conducted with solidified and broth
media were repeated once.
Transcriptional activity of pvd-inaZ in culture. The effect of
iron on INA expression by P. fluorescens A506 (pvd-inaZ) and
P. putida N1R containing pvd-inaZ was evaluated in 5-ml broth
cultures of modified RSM medium (described above) in 15-mm
test tubes. The medium was supplemented with ferric citrate or a
chelated form of iron ferric ethylenediaminedi-(o-hydroxyphenyl-
acetic (FeEDDHA) acid (Sequestrene 138; Becker Underwood,
Ames, IA) to final concentrations ranging from 1 to 0.001 mM.
Bacterial strains from overnight broth cultures were added to
broth at a concentration of 1 × 106 CFU/ml. After 24 h at 20°C
on a rotary shaker (200 rpm), samples were evaluated for INA
with the droplet freezing assay (described below) and for CFU by
dilution plating on Pseudomonas agar F (PAF) medium (Difco
Laboratories, Detroit, MI) amended with kanamycin at 50 µg/ml.
Reported values are the means of three replicate cultures. The re-
sults from duplicate experiments were similar, and results of a
representative experiment are presented.
Screenhouse experiment. Seven experiments to measure INA
in constructs of A506 applied to flowers of pear or apple cultivars
were conducted during March to May 2001 to 2003 (Table 1).
The pear and apple trees ranged from 5 to 10 years old, and were
located in a screenhouse facility at the Oregon State University,
Department of Botany and Plant Pathology Field Laboratory near
Corvallis. Trees in the screenhouse were protected from rain and
ultraviolet radiation by a translucent, fiberglass roof, and from in-
sect visitations by 2-by-2-mm steel screen walls. Flowers of pear
(Pyrus calleryana cv. Aristocrat in 2001 to 2003 experiments and
P. communis cv. Bartlett in 2002 to 2003 experiments) and apple
(Malus × domestica cv. Golden Delicious and crabapple Malus ×
Snowdrift in 2001 experiments) were spray inoculated with aque-
ous suspensions of A506 (pvd-inaZ), A506 Ice, or A506 IceC.
Constructs of A506 were cultured for 2 to 4 days at 27°C on
nutrient agar (Difco Laboratories) containing 0.4% wt/vol glyc-
erol, 0.5% wt/vol sucrose, and 0.1 mM ferric citrate. Ferric citrate
was added to the medium to suppress INA in the inoculum. Bacte-
ria were scraped from the surface of the medium and suspended
1288 PHYTOPAT HOLOGY
in 10 mM potassium phosphate buffer (pH 7.0). Bacterial
suspensions were adjusted to 1 × 108 CFU/ml (optical density at
600 nm 0.1) with the aid of a spectrophotometer (Spectronic 20;
Bausch and Lomb, Rochester, NY). All bacterial suspensions
were sprayed to near runoff with hand-held trigger sprayers
(800-ml capacity). In all experiments, each A506 construct was
applied onto several flower clusters on a branch of a tree. Each set
of construct inoculations was replicated on three trees per cultivar.
Iron treatments. Additional treatments involved inoculum of
A506 (pvd-inaZ) applied to flowers with commercial formula-
tions of iron (Table 1). All seven experiments included A506
(pvd-inaZ) mixed at the time of inoculation with 3 mM FeED-
DHA (Sequestrene 138, 100% FeEDDHA; Becker Underwood).
This concentration of FeEDDHA (1 lb. of Sequestrene 138 per
100 gallons of water) is the manufacturer’s recommended rate of
Sequestrene 138 for a foliar spray to alleviate iron chlorosis of
pome fruit trees. A506 (pvd-inaZ) also was combined with Se-
questrene 138 at concentrations ranging from 30 to 0.03 mM. A
control treatment in 2002 included A506 (pvd-inaZ) mixed with
0.1 mM EDDHA (no iron). We also evaluated a treatment where
flowers were inoculated with A506 (pvd-inaZ) in water and then
oversprayed with 3 mM FeEDDHA at 48 h after inoculation.
Additional formulations of iron included as co-treatments with
A506 (pvd-inaZ) in several experiments were 0.1 and 18 mM
FeSO4 (Sigma-Aldrich, St. Louis), 2.7 mM ferric diethylenetria-
mine pentaacetate (FeDTPA) (Sequestrene 330, 100% FeDTPA;
Becker Underwood), and 0.3% (vol/vol) metalosate iron (5%
[wt/vol] total iron as amino acid chelates; Albion Laboratories,
Inc., Clearfield, UT) (Table 1). The concentrations represent the
manufacturer’s recommended rate as foliar sprays to alleviate iron
chlorosis of tree crops.
Estimation of population size of A506 constructs. For each
treatment, flowers were sampled three to four times during bloom
to estimate population size of each construct of A506. Sample
sizes were 10 flowers/treatment except for the Golden Delicious
apple trial in 2001, where only 6 flowers were sampled. Sampled
flowers were transported to the laboratory in individual wells of
sterile, 24-well microtiter plates, and then processed individually
for dilution plating. The pistil and hypanthium from pear flowers
or pistils only from apple flowers were dissected using sterile for-
ceps and sonicated (Sonix IV, Inglewood, CA) in 1 ml of sterile
10 mM potassium phosphate buffer (pH 7.0) for 3 min. After
sonication and vortex mixing, a 10-µl drop of the wash and two
100-fold serial dilutions were spread onto PAF medium amended
with rifampicin at 50 µg/ml, kanamycin at 50 µg/ml, and cyclo-
heximide at 50 µg/ml. The detection limit was 1 × 102 CFU/flower.
Colonies were counted after 3 days.
Ice nucleation assay. INA was determined in a most probable
number, droplet-freezing assay (10). Forty 10-µl drops from the
flower wash and from appropriate dilutions were dispensed on
aluminum foil boats covered with a thin layer of dried wax (SC
Johnson paste wax, Racine, WI). The foil boats were floated on
ethanol maintained at –5°C (11,15). The number of drops that
froze at each dilution was recorded.
Data analysis. For each treatment, CFU/flower and INA were
estimated from the count data obtained in the dilution plating and
freezing-droplet assays. Population size data were transformed to
log10 (CFU/flower). Calculation of INA was based on the method
presented by Vali (28), N = Vt · ln[1/(1 – Pf)]/Vd · D, where N is
the concentration of ice nuclei per sample, Vt and Vd represent
volumes of the dilution tube and the droplet, respectively, D is the
serial dilution (100, 10–1, 10–2, 10–3, 10–4, or 10–5), and Pf is the
proportion of frozen drops at the selected dilution. The INA was
calculated by dividing N by the CFU/flower and transforming to
log10 (N/CFU).
For each treatment, the mean log10 (CFU/flower) and INA
(mean log10 [ice nuclei/CFU]) were plotted as a function of hours
after inoculation. For individual sampling dates, analysis of vari-
ance (ANOVA) was conducted to test equality of means among
constructs of A506 inoculated in water and, separately, among
rates of iron co-treatments applied with A506 (pvd-inaZ) (PROC
GLM, Statistical Analysis System; SAS Institute, Cary, NC).
Similarly, within a replicate, average population size or INA for a
construct or treatment was calculated for all samples taken from
24 to 120 h after inoculation, and these averages were subjected
to ANOVA. Fischer’s protected least significant difference (P
0.05) was used to evaluate differences among means.
RESULTS
Effect of iron concentration on pyoverdine production and
antibiosis by A506 in culture. On solidified 925 medium
containing 0.001 mM ferric citrate, A506 was highly fluorescent
when exposed to long UV and did not exhibit antibiosis against
TABLE 1. Cultivar, inoculation date, and treatments
Cultivar and inoculation dateu
Aristocrat pear Bartlett pear Snowdrift Golden
Strain, treatment 20 Mar 2001 2 Apr 2002 15 Mar 2003 13 Apr 2002 2 Apr 2003 21 Apr 2001 10 May 2001
A506 Ice in water X X X X X X X
A506 IceC in water X X X X X X X
A506 (pvd-inaZ) in:
Water X X X X X X X
0.03 mM FeEDDHAv X X … X … … …
0.3 mM FeEDDHA X X X X X X
3 mM FeEDDHA X X X X X X X
30 mM FeEDDHA X X … X … … …
Water, then flowers sprayed with
3 mM FeEDDHA after 48 h
X
...
X
X
0.1 mM EDDHAw … … … X … … …
0.1 mM FeSO4x … … … … … X X
18 mM FeSO4 … … … … … ... X
2.7 mM FeDTPAy … X … X … …
0.3% (vol/vol) metalosate ironz … X … X … …
u Snowdrift = Snowdrift crabapple, Golden = Golden Delicious apple, X = tested, and … = not tested.
v Final concentration of ferric ethylenediaminedi-(o-hydroxyphenylacetic) acid (FeEDDHA) in solution from commercial product Sequestrene 138, Becker
Underwood, Ames, IA.
w EDDHA obtained from Sigma-Aldrich, St. Louis.
x Ferrous sulfate from Sigma-Aldrich.
y Final concentration of FeDTPA in solution from commercial product Sequestrene 330, Becker Underwood.
z Commercial agricultural mixture of iron amino acid chelates from Albion Labs, Clearfield UT.
Vol. 94, No. 12, 2004 1289
Ea153 (data not shown). On 925 medium amended with 0.1 mM
ferric citrate, A506 was not fluorescent under UV radiation and
inhibited the growth of Ea153. Thus, similar concentrations of
ferric citrate that suppressed pyoverdine-associated fluorescence
of A506 on solidified 925 medium also induced antibiotic produc-
tion and the inhibition of growth of Ea153.
In semi-quantitative assays conducted in 925 and RSM broth
media, pyoverdine production by A506 was not detected in media
containing 0.01 to 0.1 mM ferric citrate (Fig. 1), and compara-
tively less pyoverdine was produced in 925 than in RSM. In con-
trast, antibiotic production by A506 grown in 925 medium in-
creased with greater concentrations of ferric citrate in the medium,
which confirmed the inverse relationship between pyoverdine
production and antibiotic production by this bacterium (Fig. 1).
INA expressed by A506 (pvd-inaZ) in culture. After 24 h of
growth in RSM, the density of A506 (pvd-inaZ) increased 2,000-
fold to 2 × 109 CFU/ml; iron amendments to this medium did not
affect the final cell density. The transcriptional activity of this
construct was responsive to the concentration of iron in the
growth medium with a change of five to six log units of activity
occurring over a three-log-unit range of iron concentrations (Fig.
2). For both FeEDDHA and ferric citrate, the greatest rates of re-
duction of INA occurred as the iron concentration increased from
0.01 to 0.1 mM of iron (Fig. 2).
Population size of A506 constructs applied to flowers in wa-
ter. A506 (pvd-inaZ), A506 Ice, and A506 IceC were recovered
in similar numbers from pear and apple flowers sampled over the
time course of the experiment (Fig. 3; apple data not shown). In
general, measured population sizes of the A506 constructs ranged
from 104 to 106 CFU/flower. ANOVA for mean log10 CFU popula-
tions averaged over sampling times from 24 to 120 h postinocula-
tion showed no significant differences (P > 0.05) among the con-
structs of A506 in all seven experiments (data not shown).
Ice nucleation activity of A506 constructs applied to flowers
in water. As expected, INA for cells treated with A506 Ice was
very low (near the detection limit of 1 × 10–6 log10 [ice nu-
clei/CFU]) on most colonized flowers (Fig. 4). In contrast, and
also as expected, A506 IceC yielded high INA with values for
log10 (ice nuclei/CFU) ranging from –0.2 to –2.0 over the seven
experiments (Table 2; Fig. 4) The INA of A506 Ice and A506
IceC were significantly different from each other at each time
point in all experiments.
The inoculum suspensions and the flowers sampled immedi-
ately after inoculation with A506 (pvd-inaZ) expressed low INA,
indicating that the addition of ferric citrate to the culture media
suppressed the pvd promoter and production of InaZ in this con-
struct. The values for log10 (ice nuclei/CFU) from initial flower
samples treated with A506 (pvd-inaZ) ranged from –5.4 to –4.3
Fig. 2. Ice nucleation activity (log [ice nuclei/CFU]) of
P
seudomonas
f
luorescens A506 (pvd-inaZ) (#) and P. p u t i d a N1R (X) after 24 h of incu-
bation in RSM medium (2) as influenced by concentration of A, ferric citrate
or B, ferric ethylenediaminedi-(o-hydroxyphenylacetic) acid. Points are means
of three replicate cultures; vertical bars drawn through points represent ±one
standard error of the mean.
Fig. 3. Population size (log) of Pseudomonas fluorescens constructs A506
(pvd-inaZ) (#), A506 IceC (X), or A506 Ice (V) on individual flowers in
relation to hours after inoculation in suspensions of water. Panels represent
experiments conducted on screenhouse-grown trees of A, Aristocrat pear
2002, B, Aristocrat pear 2003, C, Bartlett pear 2002, and D, Bartlett pear 2003.
Vertical bars drawn through points represent ± one standard error of the mean.
Fig. 1. Pyoverdine production of Pseudomonas fluorescens A506 after 72 h o
f
incubation at 20°C in RSM medium (X) (2) and 925 medium (#) (8) supple-
mented with ferric citrate at concentrations between 0.001 and 1 mM. Pyover-
dine production was estimated from the absorbance at 405 nm (A405) of the
ferric-pyoverdine complex in culture media divided by A600 measurement o
f
cell density (13). Antibiotic production (Q) of A506 in the 925 medium
amended with ferric citrate is presented as the greatest dilution of culture
media with detectable antibiotic activity against Erwinia amylovora. The
values represent means of three replicate cultures and the vertical bars are
± one standard error of the mean.
1290 PHYTOPAT HOLOGY
(Fig. 4), which was similar to INA of cells treated with A506 Ice
in six of seven experiments (data not shown). By 48 h after inocu-
lation, log10 (ice nuclei/CFU) for flowers treated with A506 (pvd-
inaZ) had increased to a range of values from –1.5 to –0.3 (Fig.
4), and this increase in activity was maintained over the time
course of the experiments (Fig. 4).
Average INA for A506 IceC and A506 (pvd-inaZ) did not differ
significantly (P 0.05) in five of seven experiments (Table 2). In
all experiments, averages for both A506 IceC and A506 (pvd-
inaZ) were significantly larger (P 0.05) than those obtained for
A506 Ice (Table 2). For Aristocrat and Bartlett pear in 2003, the
average INA response for A506 (pvd-inaZ) was intermediate to
the responses measured for A506 IceC and A506 Ice (Table 2).
From analysis of pooled data, similar INA was observed for A506
IceC and A506 (pvd-inaZ) on apple and pear flowers but the INA
of A506 Ice from flowers was significantly (P 0.05) smaller
(Table 2).
Effect of FeEDDHA on population size and INA of A506
IceC and A506 (pvd-inaZ). Populations of A506 (pvd-inaZ)
mixed with FeEDDHA ranged from 104 to 106 CFU/flower
among the experiments (Fig. 5), similar to the measured popula-
tion sizes of A506 (pvd-inaZ) applied to flowers in water. In six of
seven experiments, ANOVA of the population size of A506 (pvd-
inaZ) averaged from samples taken 24 to 120 h after inoculation
revealed no significant effects (P > 0.05) of co-treatment with any
tested concentration of FeEDDHA (Fig. 5).
In two preliminary experiments in 2000, 3 mM FeEDDHA did
not affect the INA of the construct A506 IceC on pear or crabap-
ple flowers (data not shown), demonstrating that the presence of
FeEDDHA did not interfere with measured INA. The INA of
A506 (pvd-inaZ) recovered from flowers was affected signifi-
cantly (P < 0.05) by the concentration of FeEDDHA in the inocu-
lum suspension. The INA of A506 (pvd-inaZ) on flowers treated
with 0.03 mM FeEDDHA was similar to mixing the construct
with water (Table 3). Mixing A506 (pvd-inaZ) with 0.3 mM
FeEDDHA significantly reduced the average INA of cells com-
pared with treatment with water in three of six experiments (Table
3; Fig. 6A, B, and C). In all experiments, the INA of A506 (pvd-
inaZ) on flowers mixed in 3 mM FeEDDHA was suppressed sig-
nificantly (P 0.05) compared with treatment with this construct
TABLE 2. Log10 (ice nucleation activity/CFU) on flowers of pear and apple treated with constructs of Pseudomonas fluorescens A506 suspended in waterz
Year and cultivar
2001 2002 2003
A506 construct Snowdrift Golden Aristocrat pear Aristocrat pear Bartlett pear Aristocrat pear Bartlett pear Pooled
IceC –2.0 a –1.1 a –0.5 a –1.5 a –2.0 a –0.5 a –1.1 a –1.1 a
pvd-inaZ –1.4 a –0.8 a –0.2 a –1.3 a –1.9 a –1.0 b –1.8 b –1.2 a
Ice –5.7 b –3.1 b –4.0 b –4.8 b –4.6 b –5.1 c –5.2 c –4.6 b
z Ice nucleation activity was measured with the droplet freezing assay; CFU were estimated by dilution plating individual flowers onto a selective medium. Mean
shown is the average of two to five flower samples taken 24 to 120 h after treatment. An aqueous suspension of constructs of A506 at 1 × 108CFU/ml was
sprayed onto flowers to near runoff. Snowdrift = Snowdrift crabapple and Golden = Golden Delicious apple. Means within a column followed by the same letter
are not significantly different according to Fisher’s protected least significant difference at P = 0.05.
Fig. 4. Ice nucleation activity (log [ice nuclei/CFU]) of Pseudomonas fluorescens constructs A506 (pvd-inaZ) (#), A506 IceC (X), or A506 Ice (V) on individual
flowers in relation to hours after inoculation in suspensions of water. Panels represent experiments conducted on screenhouse-grown trees of A, Aristocrat pear
2002, B, Aristocrat pear 2003, C, Bartlett pear 2002, and D, Bartlett pear 2003. Vertical bars drawn through points represent ± one standard error of the mean.
Vol. 94, No. 12, 2004 1291
in 0.3 mM FeEDDHA or in water (Table 3; Fig. 6). Greater con-
centrations of FeEDDHA did not further suppress INA, because
the average INA of A506 (pvd-inaZ) on flowers suspended in
30 mM FeEDDHA was similar to flowers treated with the con-
struct in 3 mM FeEDDHA (Table 3).
In two experiments in 2003, an overspray of 3 mM FeEDDHA
on flowers 48 h after inoculation with A506 (pvd-inaZ) sup-
pressed INA compared with flowers treated with A506 (pvd-inaZ)
in water (Fig. 6B and D). For Bartlett pear in 2002, the additional
control treatment A506 (pvd-inaZ) mixed with 0.1 mM EDDHA
(nonferrated chelate) yielded responses similar to those observed
for A506 (pvd-inaZ) in water (Table 3).
Effect of additional agricultural iron sources on A506 (pvd-
inaZ). In 2003, mean population sizes of A506 (pvd-inaZ) mixed
with FeDTPA or metalosate-iron were not significantly different
(P > 0.05) from those of A506 (pvd-inaZ) mixed in water (data
not shown). In 2001, mean population sizes of A506 (pvd-inaZ)
mixed with 0.1 mM FeSO4 on Golden Delicious apple were
significantly smaller (P < 0.05) than when the construct was
mixed in water; however, this treatment did not suppress popula-
tions of A506 (pvd-inaZ) on crabapple (data not shown). Combin-
ing A506 (pvd-inaZ) with 18 mM FeSO4 in 2001 resulted in
significantly smaller (P < 0.05) population sizes of A506 (pvd-
inaZ) of 3,000-fold fewer cells when compared with the water-
treated control (data not shown).
From 24 to 120 h after inoculation, the average INA of A506
(pvd-inaZ) on treated flowers mixed with FeSO4, FeDTPA, or
metalosate-iron ranged from –2.4 to –4.5, which was significantly
(P < 0.05) lower than the INA measured with A506 (pvd-inaZ) on
flowers in water (Table 3). Although 0.1 and 18 mM FeSO4 re-
duced INA compared with the water control, the level of suppres-
sion of activity was significantly less than that obtained from
treatment with 3 mM FeEDDHA (Table 3). The relative degree of
suppression of INA with FeDTPA or metalosate-iron was statisti-
cally similar to the degree of suppression obtained with 3 mM
FeEDDHA (Table 3).
DISCUSSION
This study used an iron biosensor to examine the relative
biological availability of iron to A506 (pvd-inaZ) on surfaces of
pear and apple flowers through the suppression or expression of
an iron-regulated promoter at the transcriptional level. This ques-
tion is of interest because A506, a biological control agent for fire
blight disease of pear and apple flowers, produces an antibiotic
inhibitory to the target pathogen when cultured on defined media
that contain iron at concentrations of 0.1 mM (25). Thus,
whether or not iron is available to A506 on flower surfaces may
potentially influence the effectiveness of the biocontrol interac-
tion. To our knowledge, this is the first time that a biosensor has
been used to guide the development of a disease control tactic for
an agricultural crop. Based on the data collected in this study,
field studies are ongoing to determine if the addition of iron to the
floral habitat directly influences the quality of the biocontrol
interaction.
The antibiotic of A506 has not been characterized, but it can be
detected readily with bioassays conducted on chemically defined
culture media. We confirmed that addition of 0.1 mM ferric cit-
rate to broth cultures or solidified media resulted in production of
detectable concentrations of the antibiotic by A506. Like many
fluorescent pseudomonads, A506 produces a pyoverdine sidero-
phore on iron-deplete media, but not in media amended with
0.1 mM ferric chloride. We consistently observed that concentra-
tions of iron that suppressed pyoverdine production also stimu-
TABLE 3. Log10 (ice nucleation activity/CFU) on flowers of pear and apple treated with Pseudomonas fluorescens A506 (pvd-inaZ) suspended in water or an iron
solutiony
Year and cultivar
2001 2002 2003
Trea tme ntz Snowdrift Golden Bartlett pear Aristocrat pear Bartlett pear Aristocrat pear Bartlett pear Pooled
Water –1.4 a –0.8 a –2.8 a –1.3 a –1.9 a –1.0 a –1.8 a –1.4 a
0.03 mM FeEDDHA –1.2 a –2.1 a –1.3 a
0.3 mM FeEDDHA –2.8 b –1.9 ab –3.0 a –1.4 a –2.6 b –2.0 b –2.0 a
3 mM FeEDDHA –4.1 c –5.0 e –4.5 b –4.8 b –4.5 c –4.6 d –4.8 c –4.2 c
30 mM FeEDDHA –4.6 b –4.1 c –3.5 bc
0.1 mM EDDHA –1.6 a –1.6 a
0.1 mM FeSO4 –2.4 b –3.5 cd … … … … … –2.9 b
18 mM FeSO4 –3.0 bc … … … … … –3.0 b
2.7 mM FeDTPA –3.6 c –4.2 b –3.9 c
0.3% (vol/vol) metalosate iron –3.6 c –4.5 bc –4.0 c
y Ice nucleation activity was measured with the droplet freezing assay; CFU were estimated by dilution plating individual flowers onto a selective medium. Mean
shown is the average of two to five flower samples taken 24 to 120 h after treatment. A506 (pvd-inaZ) at 1 × 108CFU/ml was sprayed onto flowers to near runof
f
in water or an iron solution. Snowdrift = Snowdrift crabapple and Golden = Golden Delicious apple. Means within a column followed by the same letter are not
significantly different according to Fisher’s protected least significant difference at P = 0.05; … = not tested.
z FeEDDHA = ferric ethylenediaminedi-(o-hydroxyphenylacetic) acid.
Fig. 5. Population size (log) of Pseudomonas fluorescens strain A506 (pvd-
inaZ) on individual flowers in relation to hours after inoculation in suspen-
sions of water (#), 0.3 mM ferric ethylenediaminedi-(o-hydroxyphenylacetic)
acid (FeEDDHA) (M, A to C only), 3 mM FeEDDHA (Q), or 3 mM
FeEDDHA at 48 h after inoculation (M, 2003 only). Panels represent experi-
ments conducted on screenhouse-grown trees of A, Aristocrat pear 2002, B,
Aristocrat pear 2003, C, Bartlett pear 2002, and D, Bartlett pear 2003. Vertical
bars drawn through points represent ± one standard error of the mean.
1292 PHYTOPAT HOLOGY
lated antibiosis of A506 (Fig. 1). Without information on the
structure of the antibiotic of A506 or the biosynthetic genes, this
inverse relationship between pyoverdine production and antibiosis
should be considered correlative, not causative. Nonetheless, this
correlation indicates that, if iron is present in a habitat at suffi-
cient concentrations to suppress pyoverdine production, then ade-
quate iron should be present to induce antibiosis by A506.
Measuring the transcriptional activity of pvd-inaZ provides a tool
to determine if iron is available in a habitat at concentrations ade-
quate to suppress the iron-regulated promoter involved in pyover-
dine production (15), which we construe may be adequate for
stimulation of antibiosis.
Broth culture experiments confirmed that the transcriptional ac-
tivity of pvd-inaZ in A506 was responsive to iron concentration.
In RSM medium containing 0.1 mM ferric citrate, transcrip-
tional activity of pvd-inaZ in A506 was suppressed and pyover-
dine was not detected in culture supernatants (Figs. 1 and 2).
Loper and Lindow (15) also observed that iron-replete media that
suppressed transcription of pvd-inaZ in Pseudomonas syringae
and production of pyoverdine. P. putida N1R (pvd-inaZ), whose
transcriptional activity is known under varying concentrations of
ferric citrate, was used as a comparative control (14). In addition,
the population size of A506 (pvd-inaZ) was similar to that of
P. putida N1R (pvd-inaZ) (Fig. 2). The transcriptional pvd activity
expressed by A506 (pvd-inaZ) was similar to N1R (pvd-inaZ) in
response to ferric citrate or FeEDDHA at the concentrations
tested. In the screenhouse, the amount of FeEDDHA (3 mM) re-
quired to suppress the transcriptional activity of the biosensor in
A506 (pvd-inaZ) was at least one log unit higher on flowers than
in broth culture (0.1 mM). This may be due, in part, to unique fea-
tures of floral habitats occupied by A506 (pvd-inaZ). We conclude
that results of the influence of iron on transcriptional activity of
pvd-inaZ in the broth culture experiments underestimated the con-
centration of exogenous iron required to suppress transcription of
pvd-inaZ in A506 on flowers.
After inoculation on flowers, population size determinations
were made to compare relative reproductive fitness among con-
structs of A506 and to express ice nucleation frequency in terms
of ice nuclei/CFU. With specific consideration to fitness, all three
constructs (A506 (pvd-inaZ), A506 Ice, and A506 IceC) main-
tained populations sizes of 104 to 106 CFU/flower (Fig. 3). There
was no indication that a specific construct (A506 (pvd-inaZ),
A506 Ice, and A506 IceC) was detrimental to the reproductive
fitness of A506 on flowers. Similarly, P. syringae 31R1, P. putida
N1R, and P. fluorescens Pf-5 containing pvd-inaZ, IceC, or Ice
showed no apparent differences in epiphytic fitness among con-
structs in the rhizosphere or phyllosphere of several plants (13–
15,17).
The floral stigma represents a unique microbial habitat differ-
ent from other aerial plant surfaces in that it offers an abundant,
but transient, supply of nutrients (5). Leveau and Lindow (9)
noted that bacterial epiphytes of bean leaves are commonly clus-
tered near surface features where nutrient leakage would be likely
occur (cracks, ruptures, stomates, and so on). Using a pvd-gfp
biosensor as well as pvd-inaZ in P. syringae, Joyner and Lindow
(7) found that most (90%) epiphytic cells on bean leaves sensed
relatively abundant iron in the phyllosphere, with only a minority
of cells experiencing limiting conditions. In contrast, we found
that bioavailable iron is in limited supply on stigmatic surfaces of
pear and apple flowers. This conclusion is supported by the ob-
served shift in INA of A506 (pvd-inaZ) from initially low levels
(i.e., similar to A506 Ice) to higher activities by 48 h after
inoculation. In most of the experiments, these higher INAs ap-
proached those of A506 IceC. A major difference between bean
leaves and rosaceous stigmas is that bacterial populations are
typically a 1,000-fold greater (per gram of tissue basis) on the flo-
Fig. 6. Ice nucleation activity (log [ice nuclei/CFU]) of Pseudomonas fluorescens strain A506 (pvd-inaZ) on individual flowers in relation to hours after
inoculation in suspensions of water (#), 0.3 mM ferric ethylenediaminedi-(o-hydroxyphenylacetic) acid (FeEDDHA) (M, A to C only), 3 mM FeEDDHA (Q), or
3 mM FeEDDHA at 48 h after inoculation (M, 2003 only). Panels represent experiments conducted on screenhouse-grown trees of A, Aristocrat pear 2002, B,
Aristocrat pear 2003, C, Bartlett pear 2002, and D, Bartlett pear 2003. Vertical bars drawn through points represent ± one standard error of the mean.
Vol. 94, No. 12, 2004 1293
ral habitat (5). The high density of bacterial cells coupled with
rapid growth that occurs on stigmatic tissues may result in high
demand for iron and a rapid depletion of any existing iron
sources. Marschner et al. (18) suggested that iron demand by
bacteria should be highest when the population is growing rapidly
and other nutrients (e.g., carbon) are readily available. Working
with white lupine, this group observed that iron-limiting condi-
tions to a pvd-inaZ construct of P. fluorescens Pf-5 were more
strongly associated with the root tips where bacterial populations
increase rapidly compared with older root surfaces. Similarly,
Loper and Henkels (13) found that the rhizosphere of bean was
iron limiting to Pf-5 (pvd-inaZ) in the 2 days following inocula-
tion of this strain, but became less limiting as time progressed.
We evaluated several iron chelates and a ferrous compound for
their ability to provide iron to A506 on floral surfaces. Most of
our efforts focused on the use of the iron chelate FeEDDHA as a
source of exogenous iron for A506. The addition of FeEDDHA to
the inoculum had no significant effect on the measured population
size of A506 (pvd-inaZ) on flowers (Fig. 5). Significantly,
FeEDDHA, when applied at concentrations of 3 mM, sup-
pressed the shift from initially low to high frequencies of ice
nucleation (Fig. 6). These data indicate the 3 mM FeEDDHA
treatment was at or somewhat above the minimal concentration of
iron required to suppress the transcriptional activity of pvd-inaZ.
In 2003, flowers sprayed with 3 mM FeEDDHA delayed for 48 h
after inoculation with A506 (pvd-inaZ) in water also resulted in a
significant decrease in INA (Fig. 6). We conclude that chelated
forms of iron can be oversprayed on flowers colonized with A506
or mixed with the inoculum to increase iron bioavailability to the
bacterium on the surface of pear or apple flowers.
Ferrous sulfate (FeSO4), FeDTPA, and metalosate-iron were in-
cluded to determine if other formulations of iron used commer-
cially in agriculture could suppress INA of A506 (pvd-inaZ) on
flowers. The chelated forms of iron, applied at concentrations rec-
ommended by manufacturers for iron chlorosis, suppressed the
INA of A506 (pvd-inaZ) to a degree that was similar to that ob-
served in the treatment amended with 3 mM FeEDDHA (Table 3).
FeSO4 reduced INA of A506 (pvd-inaZ) compared with the water
treatment, but not to the same degree as the iron chelates (Table
3). The high rate of FeSO4 (18 mM) which is recommended for
alleviation of iron chlorosis caused a significant reduction in the
recovered population size of A506 (pvd-inaZ) on flowers when
compared with treatments of FeEDDHA, EDDHA (chelate only),
or water. Flowers treated with 18 mM FeSO4 appeared severely
blackened, especially on petals and the tips of pistils. Severe
phytotoxicity and marked reduction in populations of A506 (pvd-
inaZ) were not observed with 0.1 mM FeSO4 or with the other
iron chelates at concentrations tested.
Compared with the other iron-containing compounds tested,
FeEDDHA may show the most promise as an additive with A506
in field applications for fire blight suppression. Of the compounds
evaluated, FeEDDHA has the highest affinity for ferric iron with a
stability (iron binding) constant of 33.9 (3). From a microbial
perspective, in iron-poor environments, siderophores are produced
and secreted to sequester iron for growth (19). Consequently, the
affinity by which iron is held by a chelate could allow iron to be
collected by one organism’s siderophore but not by the others.
The stability constant for the pyoverdine produced by A506 is un-
known, but it is likely to be similar to that for the pyoverdine of
P. aeuruginosa, at 32.0 (21). Because FeEDDHA and pyoverdines
have similar iron-stability constants, we expect that A506 (pvd-
inaZ) would utilize iron bound to this chelate. The observed shift
in INA by A506 (pvd-inaZ) in culture and in planta in the pres-
ence of FeEDDHA supports the expectation that A506 can utilize
iron chelated by EDDHA. The siderophore of E. amylovora, des-
ferrioxamine, which is a hydroxamate, has a considerably lower
stability constant of 20.0 (4) and grows poorly in culture media
amended with FeEDDHA. Thus, the high affinity of EDDHA for
iron may provide more selectivity (i.e., iron available to A506 but
not to E. amylovora) than FeDTPA or metalosate-iron (stability
constants of 28.0 and estimated between 4.0 to 12.1, respectively)
(19).
In conclusion, the findings of this study provide evidence that a
construct of P. fluorescens strain A506 containing the iron biosen-
sor pvd-inaZ showed levels of INA consistent with the hypothesis
that iron has limited bioavailability on surfaces of pear and apple
flowers. Moreover, amendments of the iron chelate, FeEDDHA,
at concentrations of 3 mM suppressed INA, indicating that A506
(pvd-inaZ) acquired sufficient iron to suppress this iron-regulated
promoter. The implications of these results are that production of
the iron-induced antibiotic of A506 is unlikely to occur in field
applications without the addition of exogenous iron to flower sur-
faces. Applications of FeEDDHA, however, may modify the habi-
tat on pear and apple flowers and, thus, may positively influence
antibiotic production by A506 and fire blight suppression.
ACKNOWLEDGMENTS
This study was supported in part by grants from the United States De-
partment of Agriculture Western Region Integrated Pest Management
WRIPM-00R14 and the Winter Pear Control Committee. We thank
Becker-Underwood for the gift of Sequestrene 138 and Sequestrene 330,
Albion Laboratories for the gift of Metalosate Iron, and M. D. Henkels
and other members of J. Loper’s group for insightful comments and
technical assistance.
LITERATURE CITED
1. Abdallah, M. A. 1991. Pyoverdins and pseudobactins. Pages 139-154 in:
CRC Handbook of Microbial Iron Chelates. G. Winkelmann, ed. CRC
Press, Boca Raton, FL.
2. Buyer, J. S., Sikora, L. J., and Chaney, R. L. 1989. A new growth medium
for the study of siderophore-mediated interactions. Biol. Fertil. Soils
8:98-101.
3. Chaney, R. L. 1988. Plants can utilize iron from Fe-N,N
-di-(2-hy-
droxybenzoyl)-ethylenediamine-N,N
-diacetic acid, a ferric chelate with
106 greater formation constant than Fe-EDDHA. J. Plant Nutr. 11:
1033-1050.
4. Expert, D., Dellagi, A., and Kachadourian, R. 2000. Iron and fire blight:
Role in pathogenicity of desferrioxamine E, the main siderophore of
Erwinia amylovora. Pages 179-195 in: Fire Blight, The Disease and Its
Causative Agent, Erwinia amylovora. J. L. Vanneste, ed. CABI Publish-
ing, New York.
5. Johnson, K. B., and Stockwell, V. O. 1998. Management of fire blight: A
case study in microbial ecology. Annu. Rev. Phytopathol. 36:227-248.
6. Johnson, K. B., Stockwell, V. O., McLaughlin, R. J., Sugar, D., Loper, J.
E., and Roberts, R. G. 1993. Effect of antagonistic bacteria on estab-
lishment of honey bee-dispersed Erwinia amylovora in pear flowers and
on fire blight control. Phytopathology 83:995-1002.
7. Joyner, D. C., and Lindow, S. E. 2000. Heterogeneity of iron bioavailabil-
ity on plants assessed with a whole-cell GFP-based bacterial biosensor.
Microbiology 146:2435-2445.
8. Langley, R. A., and Kado, C. I. 1972. Studies on Agrobacterium
tumefaciens. Conditions for mutagenesis by N-methyl-N-nitro-N-nitroso-
guanidine and relationships of A. tumefaciens to crown-gall tumor
induction. Mutat. Res. 14:277-286.
9. Leveau, J. H. J., and Lindow, S. E. 2001. Appetite of an epiphyte: Quanti-
tative monitoring of bacterial sugar consumption in the phyllosphere.
Proc. Nat. Acad. Sci. USA 98:3446-3453.
10. Lindow, S. E. 1985. Integrated control and role of antibiosis in biological
control of fire blight and frost injury. Pages 83-115 in: Biological Control
on the Phylloplane. C. Windels and S. E. Lindow, eds. The American
Phytopathological Society, St. Paul, MN.
11. Lindow, S. E. 1990. Bacterial ice nucleation activity. Pages 185-198 in:
Methods in Phytobacteriology. S. Klement, K. Rudolg, and D. C. Sands,
eds. Akademiai Kiado, Budapest.
12. Lindsay, W. L., and Schwab, A. P. 1982. The chemistry of iron in soils
and its availability to plants. J. Plant Nutr. 5:821-840.
13. Loper, J. E., and Henkels, M. D. 1997. Availability of iron to Pseudo-
monas fluorescens in rhizosphere and bulk soil evaluated with an ice
nucleation reporter gene. Appl. Environ. Microbiol. 63:99-105.
14. Loper, J. E., and Henkels, M. D. 1999. Utilization of heterologous
siderophores enhances levels of iron available to Pseudomonas putida in
the rhizosphere. Appl. Environ. Microbiol. 65:5357-5363.
1294 PHYTOPAT HOLOGY
15. Loper, J. E., and Lindow, S. E. 1994. A biological sensor for iron available
to bacteria in their habitats on plant surfaces. Appl. Environ. Microbiol.
60:1934-1941.
16. Loper, J. E., and Lindow, S. E. 2001. Reporter gene systems useful in
evaluating in situ gene expression by soil- and plant-associated bacteria.
Pages 627-637 in: Manual of Environmental Microbiology, 2nd ed. C. J.
Hurst, G. R. Knudsen, M. J. McInerney, L. D. Stetzenbach, and M. V.
Walter, eds. American Society for Microbiology Press, Washington DC.
17. Marschner, P., and Crowley, D. E. 1997. Iron stress and pyoverdine
production by fluorescent pseudomonad in the rhizosphere of white
lupine (Lupinus albus L.) and barley (Hordeum vulgare L.). Appl.
Environ. Microbiol. 63:277-281.
18. Marschner, P., Crowley, D. E., and Sattelmacher, B. 1997. Root coloni-
zation and iron nutritional status of Pseudomonas fluorescens in different
plant species. Plant Soil 196:311-316.
19. Martell, A. E., and Smith, R. M. 1974. Critical Stability Constants, Vol. 1:
Amino Acids. Plenum Press, New York.
20. Matzanke, B. F. 2000. Structures, coordination chemistry and functions of
microbial iron chelates. Pages 15-64 in: CRC Handbook of Microbial Iron
Chelates. G. Winkelmann, ed. CRC Press, Boca Raton, FL.
21. Meyer, J. M., and Abdallah, M. A. 1978. The fluorescent pigment of
Pseudomonas fluorescens: Biosynthesis, purification, and physico-
chemical properties. J. Gen. Microbiol. 107:319-328.
22. Meyer, J. M., and Stintzi, A. 1998. Iron metabolism and siderophores in
Pseudomonas and related species. Pages 201-239 in: Pseudomonas.
Biotechnology Handbooks, Vol. 10. T. C. Montie, ed. Plenum Press, New
Yo rk .
23. Neilands, J. B. 1984. Siderophores of bacteria and fungi. Microbiol. Sci.
1:9-14.
24. Nuclo, R. L., Johnson, K. B., Stockwell, V. O., and Sugar, D. 1998.
Secondary colonization of pear flowers by two bacterial antagonists of the
fire blight pathogen. Plant Dis. 82:661-668.
25. Stockwell, V. O., Johnson, K. B., and Loper, J. E. 2001. Enhancement of
biocontrol of fire blight with an iron chelate. (Abstr.) Phytopathology
91(suppl.):S86.
26. Stockwell, V. O., Johnson, K. B., and Loper, J. E. 2002. Biological control
of fire blight: Understanding interactions among introduced and
indigenous microbial communities. Pages 225-239 in: Phyllosphere
Microbiology. S. E. Lindow, E. I. Hecht-Poinar, and V. J. Elliott, eds. The
American Phytopathological Society, St. Paul, MN.
27. Thomson, S. V. 1986. The role of the stigma in fire blight infections.
Phytopathology 76:476-482.
28. Vali, G. 1989. Principles of ice nucleation. Pages 1-28 in: Biological Ice
Nucleation and Its Applications. R. E. Lee, Jr., G. J. Warren, and L. V.
Gusta, eds. The American Phytopathological Society, St. Paul, MN.
29. Wilson, M., Epton, H. A. S., and Sigee, D. C. 1989. Erwinia amylovora
infection of hawthorn flower II. The stigma. J. Phytopathol. 127:15-28.
30. Wilson, M., and Lindow, S. E. 1993. Interactions between the biological
control agent Pseudomonas fluorescens strain A506 and Erwinia amy-
lovora in pear flowers. Phytopathology 83:117-123.
... Bare root dip treatment or soil drench with Pseudomonas fluorescens CHA0 significantly suppressed Rhizoctonia solani of tomato (Siddiqui and Shaukat, 2002). Pseudomonas fluorescens strain A 506 is a commercially available biological control agent (Available in the name of blight Ban 506; N. farm Americas Inco., Sugar Land, TX) used for the suppression of fire blight on pear and apple trees (Temple et al., 2004). ...
Article
Crop protection is an important area of agriculture which needs attention because of most of the hazardous inputs added into the agricultural system are in the form of chemicals. Production of the crop is, however, constrained by several disease infections including fungal diseases. The present study, was isolate twelve Pseudomonas fluorescens isolates from rhizospheric soil of faba bean and were tested for their antagonistic activity against Botrytis fabae that is known to attack faba bean crops. All Pseudomonas fluorescens isolates are shown successfully employed in controlling chocolate spot diseases of plant. Pseudomonas fluorescens isolates 10 (88.1%) showed highest antagonistic activity against Botrytis fabae . All isolate of Pseudomonas fluorescens are indicated successfully employed in controlling chocolate spot diseases of plant due to their antifungal metabolites. Therefore, these isolates can be used as potential of biocontrol agents.
... (Punja andUtkhede 2003, Montesinos 2007). Examples of antibiotic agents are: Pseudomonas fluorescens ("Blight Ban"), which is used against fire blight on apple and pear and produces an antibiotic toxin against Erwinia amylovora (Temple et al. 2006); and Bacillus amyloliquefaciens FZB42 ("RhizoVital"), claimed to have a plant-strengthening effect due to antagonistic efficacy against soil-born pathogens. In the latter case, more than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores, including the polyketides bacillaene and difficidin, respectively (Chen et al. 2007). ...
Chapter
The use of agrochemicals in animal husbandry and crop cultivation is well established, but the public acceptance is generally low and in some cases, substances have already been legally banned because their application poses risks for public health. Microbes that are able to suppress the growth of pathogens have been shown to be an effective alternative to maintain animal or plant health. Isolation and screening of potent strains as well as the characterization of their mode of action and the assessment of potential risks play an important role in order to obtain a safe and acceptable biological product. The development of a commercial production process, product formulation, and the requirements for the registration process are further critical items, which will determine over the commercial success of the final product.
... In addition to fire blight control, the biocontrol agent also reduces the severity of frost damage caused by ice-nucleation-active bacteria (10). Beyond its commercial applications, A506 has been used as a biosensor of the chemical nature of microbial habitats (12)(13)(14)(15)) and as a model organism for studies evaluating resource competition (16,17), survival and growth of bacteria on aerial plant surfaces (8,9,18,19), spatial patterns of bacterial cell aggregates on plants (20,21), and the viable but nonculturable condition that can occur in bacteria (22,23). ...
Article
Full-text available
Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of Pseudomonas spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of Pseudomonas fluorescens A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOBP6 family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of Pseudomonas spp. or the Enterobacteriaceae. The origin of vegetative replication (oriV) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of Pseudomonas syringae, but pA506 lacks repA, a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne rulAB genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces.
... The issue of Pseudomonas bacteria incorporation into biosensor systems was investigated considering environmental sources of microorganisms. Temple et al., [2] have designed an iron biosensor (namely an iron-regulated promoter) following engineering Pseudomonas cells that colonize tree tissues in order to estimate the relative abundance of iron on some tree blossoms. Joyner and Lindow [3] developed an iron biosensor based on a Pseudomonas syringae mutant that exhibited irondependent fluorescence when iron was supplied to the growth media. ...
Article
Fluorescence emission of pyoverdine - the siderophore synthesized by iron scavenger bacteria - was studied using in vitro cultures of Pseudomonas aeruginosa with the aim to design a biosensor system for liquid sample iron loading. Diluted suspensions of colloidal magnetite nanoparticles were supplied in the culture medium (10 microl/l and 100 microl/l) to simulate magnetic loading with iron oxides of either environmental waters or human body fluids. The electromagnetic exposure to radiofrequency waves of bacterial samples grown in the presence of magnetic nanoparticles was also carried out. Cell density diminution but fluorescence stimulation following 10 microl/l ferrofluid addition and simultaneous exposure to radiofrequency waves was evidenced. The inhibitory influence of 100 microl/l ferrofluid combined with RF exposure was evidenced by fluorescence data. Mathematical model was proposed to approach quantitatively the dynamics of cell density and fluorescence emission in relation with the consumption of magnetite nanoparticle supplied medium. The biosensor scheme was shaped based on the response to iron loading of bacterial sample fluorescence.
... Erwinia carotovora (now Pectobacterium carotovorum) produces the high-affinity hydroxamate siderophore, aerobactin (Ishimaru & Loper, 1992), and a high-affinity class of siderophores, desferriodoxamines, is conserved among Erwinia and Pantoea species (Smits & Duffy, 2011). Erwinia amylovora infects the blossoms of pear and apple trees, which are known to provide an iron-limited environment (Temple et al., 2004), a fact that supplies a possible role for these conserved high-affinity siderophores. ...
Article
Metals play essential roles in many biological processes, but are toxic when present in excess. This makes their transport and homeostatic control of particular importance to living organisms. Within the context of plant-pathogen interactions the availability and toxicity of transition metals can have a substantial impact on disease development. Metals are essential for defensive generation of reactive oxygen species (ROS) and other plant defences, and can be used directly to limit pathogen growth. Metal-based antimicrobials are used in agriculture to control plant disease and there is increasing evidence that metal hyperaccumulating plants use accumulated metal to limit pathogen growth. Pathogens and hosts compete for available metals, with plants possessing mechanisms to withhold essential metals from invading microbes. Pathogens, meanwhile, use low metal conditions as a signal to recognise and respond to the host environment. Consequently, metal sensing systems such as fur (iron) and zur (zinc) regulate the expression of pathogenicity and virulence genes; and pathogens have developed sophisticated strategies to acquire metal during growth in plant tissues, including the production of multiple siderophores. This review explores the impact of transition metals on the processes that determine the outcome of bacterial infection in plants, with a particular emphasis on zinc, iron and copper. © 2012 Federation of European Microbiological Societies. Published by Blackwell PublishingLtd. All rights reserved.
Article
Full-text available
The goal of our study was to select bacterial isolates effective for control of fire blight and to run preliminary tests to elucidate their mechanism of action. Protective sprays of 'Idared' apple flowers on trees growing in the greenhouse with Pseudomonas graminis 49M strain at 2 concentrations (107 or 108 cfu/ml) showed that its efficacy against blossom blight was greater if a higher cell concentration was applied (89.0 versus 71.2% determined 5 days after inoculation with E. amylovora, strain Ea659). Strain 49M showed similar control efficacy to Pseudomonas fluorescens strain A506 and BlightBan A506 against blossom blight and Miedzian 50 WP (copper oxichloride) and Aliette 80 WG (fosetyl-Al) appeared to be less effective than 49M. Efficacy of 49M to reduce severity of fire blight of shoots of M.26 apple rootstock 7 and 17 days after inoculation with Ea659 ranged from 99.2 to 86.3%, respectively. Study on the biotic relationship between 49M and Ea659 on 3 artificial media (NAS, King's B, LB) indicated that 49M inhibited growth of the pathogen on King's medium B only. Strain 49M produced siderophores and biofilms, but not N-acyl homoserine lactones (AHL).
Article
Full-text available
In the greenhouse, Pseudomonas fluorescens strain A506 effectively colonized the pistils of pear blossoms. P. fluorescens A506 significantly reduced colonization of pear pistils by Erwinia amylovora when the biological control agent was inoculated 72 h in advance of the pathogen, but not when it was coinoculated with E. amylovora. P. fluorescens A506 probably excluded E. amylovora by preemptively utilizing a growth limiting resource required by the pathogen. A506 also colonized the nectaries of pear blossoms, in which it maintained high populations for 60-72 h []
Article
Full-text available
Dispersal of the bacteria Pseudomonas fluorescens strain A506 and Erwinia herbicola strain C9-1S from treated to nontreated pear blossoms, and the effect of their spread on fire blight, were investigated in an orchard block of 10 rows containing 4 trees per row. Center rows of trees were sprayed with a mixture of P. fluorescens A506 and E. herbicola C9-1S at 30, 15, and 50% bloom in 1994, 1995, and 1996, respectively. Immediately after spraying, antagonists were detected only on treated blossoms. In 1994 and 1996, as bloom progressed, both P. fluorescens A506 and E. herbicola C9-1S were detected on nontreated blossoms located up to 4 rows (10 m) from the treated rows. In 1995, establishment of the antagonists on treated blossoms was poor and spread to nontreated trees was limited, apparently because of cold temperatures. Each year, honey bees were used to inoculate all trees with E. amylovora at 80% bloom. After full bloom in 1994 and 1996, the proportion of blossoms with E. amylovora populations >10(5) CFU per flower were highest in the outermost rows, and decreased linearly (P < 0.05) with proximity to treated rows. In 1994, diseased blossom clusters decreased significantly (P < 0.05) from the outermost rows to the treated rows, but there was no significant effect of distance on disease incidence in 1995 or 1996. Secondary colonization of blossoms by P. fluorescens A506 and E. herbicola C9-1S can play a role in disease suppression, but, among seasons, rates of secondary colonization by P. fluorescens A506 and E. herbicola C9-1S were variable, indicating that multiple applications of antagonists may be necessary to optimize biological control.
Article
Pseudomonas spp. have the capacity to utilize siderophores produced by diverse species of bacteria and fungi, and the present study was initiated to determine if siderophores produced by rhizosphere microorganisms enhance the levels of iron available to a strain of Pseudomonas putida in this natural habitat. We used a previously described transcriptional fusion (pvd-inaZ) between an iron-regulated promoter (pvd) and the ice nucleation reporter gene (inaZ) to detect alterations in iron availability to P. putida. Ice nucleation activity (INA) expressed from the pvd-inaZ fusion by P. putida N1R or N1R Pvd(-), a derivative deficient in the production of a pyoverdine siderophore, was inversely related to the concentration of ferric citrate in a culture medium. In culture, INA expressed by NIR Pvd(-) (pvd-inaZ) was reduced in the presence of the ferric complex of pseudobactin-358, a pyoverdine siderophore produced by P. putida WCS358 that can be utilized as a source of iron by NIR Pvd(-). In the rhizosphere of cucumbers grown in sterilized soil, N1R Pvd(-) (pvd-inaZ) expressed INA, indicating that iron availability was sufficiently low in that habitat to allow transcription of the iron-regulated pvd promoter. Coinoculation with WCS358 or N1R significantly decreased INA expressed by N1R Pvd(-) (pvd-inaZ) in the rhizosphere, whereas coinoculation with a pyoverdine-deficient mutant of WCS358 did not reduce INA expressed by N1R Pvd(-) (pvd-inaZ), These results indicate that iron availability to N1R Pvd(-) (pvd-inaZ) in the rhizosphere was enhanced by the presence of another strain of P. putida that produces a pyoverdine that N1R Pvd(-) (pvd-inaZ) was able to utilize as a source of iron, In culture, strain N1R Pvd(-) also utilized ferric complexes of the siderophores enterobactin and aerobactin as sources of iron. In the rhizosphere of cucumbers grown in sterilized soil, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of strains of Enterobacter cloacae that produced enterobactin, aerobactin, or both siderophores, but INA expressed by N1R Pvd(-) (pvd-inaZ) was not altered in the presence of a mutant of E. cloacae deficient in both enterobactin and aerobactin production, Therefore, the iron status of P. putida was altered by siderophores produced by an unrelated bacterium coinhabiting the rhizosphere, Finally, we demonstrated that INA expressed by N1R containing pvd-inaZ in the rhizosphere differed between plants grown in sterilized versus nonsterilized field soil. The results of this study demonstrate that (i) P. putida expresses genes for pyoverdine production and uptake in the rhizosphere, but the level of gene expression is influenced by other bacteria that coexist with P. putida in this habitat, and (ii) diverse groups of microorganisms can alter the availability of chemical resources in microbial habitats on root surfaces.
Article
In field trials conducted in 1991 and 1992 at Medford, OR, and in 1992 at Wenatchee, WA, Pseudomonas fluorescens strain A506 and Erwinia herbicola strain C9-1 established epiphytic populations on pear blossoms and were effective antagonists for the biological control of fire blight. Both bacterial antagonists, water, or streptomycin sulfate were applied to trees at 30% and full bloom. Pear trees were challenged-inoculated with freeze-dried cells of E. amylovora vectored to blossoms by honey bees. One week after full bloom, the antagonists were established in more than 95% of treated blossoms in Oregon in 1991 and Washington in 1992, but in less than 50% of blossoms in Oregon in 1992 [...]
Article
The solubility of iron in soils is controlled by Fe(OH)3(soil)in well‐oxidized soils, by Fe3(OH)s(ferrosic hydroxide) in moderately oxidized soils, and by FeCO3(siderite) in highly reduced soils. The Fe(III) hydrolysis species Fe(OH)2 , and Fe(OH)3° are the major solution species of inorganic Fe, but they are maintained too low to supply available iron to plants. Iron is absorbed by plants as Fe and must be in the general range >10–7.7 M to avoid iron deficiency. The redox of soil‐root environments must be
Article
Optimum mutagenesis of Agrobacterium tumefaciens by N-methyl-N′-nitro-N-nitrosoguanidine occurred at pH 6.5 using 250 μg/ml of the mutagen for 3 h at 30°. Antibiotic-resistant mutants and amino acid auxotrophs were selected and scored for crown-gall tumor-inducing ability on Helianthus annuus (sunflower). Mutants resistant to neomycin, kanamycin or rifampicin were not directly affected in their tumor-inducing ability. Mutants that were resistant to neomycin were also resistant to kanamycin and vice versa. Various amino acid auxotrophs varied in virulence. Some of the auxotrophs that required histidine, leucine or tryptophan had simultaneously lost their virulence. The alteration of virulence of the organism is not dependent on its growth since the avirulent auxotrophs when supplemented with the amino acid requirement grew in vivo almost as well as the prototrophic strains and yet remained avirulent.
Article
The biosynthesis of a yellow-green, fluorescent, water-soluble pigment by Pseudomonas jluorescens occurred only when the bacteria were iron-deficient and was not directly influenced by the nature of the organic carbon source. The pigment formed a very stable Fe3+ complex and was purified in this form. Pseudomonasfluorescens produced only one molecular species of fluorescent pigment; however, its lability under mild alkaline conditions led to the formation of several pigmented decomposition products. The spectral properties of the pure pigment, its molecular weight (1500 rf: 75) and its stability constant for Fe3+ (of the order of were determined. Both its biosynthesis and its chemical properties (formation of a stable Fe3+ complex) suggest that the fluorescent pigment is a desferrisiderophore.