MICROBIAL
ECOLOGY
Microb Ecol (1998) 36:101–110
© 1998 Springer-Verlag New York Inc.
Factors Affecting Attachment of Enterobacter cloacae to
Germinating Cotton Seed
M.A. Hood,1 K.V. van Dijk,2 E.B. Nelson2
1
2
University of West Florida, Department of Biology, Pensacola, FL 32514, USA
Cornell University, Department of Plant Pathology, Ithaca, NY 14850, USA
Received: 24 April 1997; Accepted 29 October 1997
A
B S T R A C T
Attachment of Enterobacter cloacae EcCT-50,—a biological seed protectant used to control the
seed-rotting fungi, Pythium ultimum—to cotton seed was examined using conventional fluorescent
microscopy (CFM), scanning electron microscopy (SEM), and laser scanning microscopy (LSM). In
sand microcosms, E. cloacae quickly attached to the seed coat, with maximum attachment, 3 to 5
h after inoculation at 24°C. In contrast, initial attachment of non-bacterized seed by Pythium
ultimum was not observed until 6 h (and not until 8 h on bacterized seeds). Comparison of the
movement of E. cloacae and P. ultimum in seed exudate gradient semi-soft agar showed faster
movement by the bacterium within the first 6 h, and reduction of P. ultimum hyphal and germ tube
growth in the presence of the bacterium. Microscopic observation of the seed coat revealed an early,
intimate association, mediated, in part, by fimbriae, and confirmed a loose association of E. cloacae
with the seed coat previously reported. Spatially, the attached E. cloacae cells were distributed over
the entire surface of the seed coat, but were especially abundant in the groves and near cracks where
water imbibition and seed exudate release may occur. As the seed germinated and exposed various
seed tissues, the bacterium rapidly attached to these tissues. Attachment of the bacterium to the
surface of intact germinating seeds, excised seed coat, polystyrene, and glass was 300, 110, 51, and
<1 cell field−1 3 h−1, respectively, suggesting that attachment is enhanced by seed germination.
Attachment of E. cloacae to the seed coat was optimum in sands with high water concentrations, at
temperatures of 18 to 30°C, and at times that corresponded with optimum water imbibition during
germination. Using several assays, attachment was shown to be enhanced by seed exudate, and
compounds such as methanol, fructose, and calcium. The results suggest that the release of certain
nutrients and water imbibition during germination may play a role in the rapid attachment to the
seed by E. cloacae. The ability of E. cloacae to rapidly move and attach to the seed coat may be related
to its ability to function as a biocontrol agent.
Introduction
Pythium seed rot and preemergence damping-off are common fungal diseases of numerous economically important
Correspondence to: M.A. Hood
plants. Biocontrol of these diseases by certain bacteria has
been the subject of extensive studies [22, 35, 41]. The rhizobacterium, Enterobacter cloacae strain EcCT-501, has been
shown to be an especially effective seed protectant against
seed and seedling diseases of many plants caused by Pythium
102
ultimum [11, 20, 25]. Although the mechanism of Pythium
suppression is not yet completely known, bacterial interference with seed exudate may be part of the mechanisms of
biological control [6, 17, 19, 20, 21, 23, 24, 25, 38]. Reduction of seed exudate–stimulant molecule(s), i.e., long-chain
unsaturated fatty acids, especially linoleic, by E. cloacae
EcCT-501, was shown to correlate with loss of biocontrol
[24, 31, 38].
To understand the mechanism of biocontrol by E. cloacae, spatial-temporal relationships among host, pathogen
and biocontrol agent may be important. Although P. ultimum is not an especially aggressive or invasive pathogen, it
can colonize seeds within 6 to 12 h after planting [25]. In
one report [26], fungal colonization was observed as early as
4 h. Because disease (seed rot and damping-off) is directly
correlated with fungal colonization [26], bacteria, to be effective biocontrol agents, may be required to rapidly and
extensively colonize seed, and quickly become active, to protect seeds from fungal colonization and infection. This short
time period before infection may be a critical period for
successful seed protection. Thus, early events in seed colonization by the bacterium seem especially relevant in understanding the biocontrol mechanisms [30]. It was, therefore,
the purpose of this study to observe the early interactions of
E. cloacae EcCT-501 and cotton seeds, to understand how
the bacterium attaches to the seed, and what factors play a
role in attachment.
Materials and Methods
Bacterial and Fungal Strains
Methods used to prepare and maintain cultures of P. ultimum P-4
and E. cloacae EcCT-501 have been described by van Dijk and
Nelson [38]. Fresh cultures of E. cloacae were grown in trypticase
soy broth (TSB, Difco) or in cotton seed exudate (CSE), pH = 7.0,
at 27°C for 15 h; harvested by centrifugation at 9750 g, for 10 min,
at 15°C; washed twice in 0.02 M phosphate buffer saline, pH 7.0;
and resuspended to a turbidity equal to an optical density of 0.7 at
600 nm (approximately 108 cells ml−1). Also, strains of Staphylococcus epidermidis and Escherichia coli (from Cornell’s Microbiology Department Culture Collection) and E. coli ATCC 25922 were
used in the initial attachment experiments for comparisons.
Sand Microcosms
Sand microcosms consisted of sterile (autoclaved) sand (particle
size 0.5–1.0 mm) and Pythium-infected sand. Pythium-infected
sand was prepared as follows: Pythium-infected wheat leaves were
transferred to 2% water agar amended with 50 µg ml−1 of penicillin
G and rifampicin (WARP medium). After 4 days on WARP, a
M.A. Hood et al.
mycelial disk was transferred to a defined mineral salts medium
containing soy lecithin (SM+L medium) [25]. Four days later, disks
of SM+L medium were leached in Chen and Zentmeyer’s buffer
[3] for two consecutive 10-min intervals. They were then rinsed in
sterile, deionized, distilled water and incubated in the dark at 24°C
for 2 days. These disks, containing sporangia, were placed in autoclaved sand. Cotton seeds were placed directly above the disks,
covered with sand, incubated at 24°C in growth chambers with a 16
h photoperiod, and watered daily with sterile, distilled water. After
4 days, infected plant and seed tissue were ground and mixed with
the sand. Seeds were added again. The procedure was repeated for
one month, until P. ultimum levels were high enough for infection
of all seeds. Although the number of viable fungal cells was not
determined, Pythium levels in these sands were such that all seeds
planted either rotted or the emerging radicle became infected
within 24 h.
Liquid Microcosms
Liquid microcosms consisted of sterile, foil-covered beakers containing a bacterial suspension (108 cells ml−1) in phosphate saline
buffer. Controls consisted of buffer alone.
Seed Treatment
Numerous types of seeds (including cotton, tomato, cucumber,
cantaloupe, bentgrass, ryegrass, and wheat) were examined for both
autofluoresence and background fluorescence after staining with
acridine orange (AO). Although cotton seed coat exhibited some
fluorescence with and without staining, bacterial cells could be seen
and counted on cotton seeds better than with any other seed type
examined. Therefore, cotton seeds were chosen for attachment
studies.
All cotton seeds (Gossypium hirsutum L. ‘Acala SJ-28) selected
had no cracks or other visible deformities. They were surfacedisinfected for five minutes with 0.5% sodium hypochlorite, then
thoroughly rinsed three times with sterile, deionized, distilled water. Seeds were planted in sand microcosms by placing them into a
1 cm deep indention in the sand. One ml of the bacterial suspension in phosphate saline buffer (108 cells ml−1) was placed on top
of, and around, the seed. Control treatments consisted of 1.0 ml
buffer or no treatment. Seeds were covered with ∼1 cm of sand, and
incubated at 24°C for various time intervals. Seeds were removed
and gently dipped in buffer once to remove excess sand particles. A
second treatment consisted of dipping the seed in buffer, followed
by washing in a stream of buffer for 30 sec. This was used to
determine tightly associated cells. The rinse was generated from a
500-ml Nalgene bottle, to give a total rinse volume of 30 ml in 30
sec. The seed coat was removed, fixed for scanning electron microscopy (SEM) or stained for conventional fluorescent microscopy (CFM) and laser scanning microscopy (LSM) with acid fuchsin (0.03% in 85% lactic acid) to visualize fungi, or acridine orange
(0.02% in phosphate saline buffer, pH 7.0, for 30 sec) to visualize
bacteria. Samples were then dipped in buffer to remove excess
stain, and air dried. Three seeds were removed at each time interval.
The seed coats were cut (∼0.25 × 0.25 cm in size) from the micro-
Attachment by Enterobacter cloacae
pilar end near the emerged radicle. Ten fields per seed coat, or a
total of 30 fields, were counted.
Seeds were also removed at various times during germination,
and, as tissues were exposed, each seed coat layer and each tissue
were removed, rinsed, stained with AO, and examined, as described
above.
Seeds placed in liquid microcosms (1 seed ml−1) were incubated
at 24°C, and, in temperature effects experiments, at various temperatures of 5 through 37°C. They were removed at various times,
rinsed, stained, and examined, as described.
In addition to whole seed, attachments to other surfaces (excised seed coat, glass, and polystrene) were conducted in liquid
microcosms. Seed coat was removed, mounted in silicon on glass
slides, and dried overnight. Glass slides and polystrene panels were
prepared, as previously described [15]. Slides were submerged in
the standard bacterial suspension, removed at various times,
stained, and examined, as described.
Water uptake by seeds was determined by weighing seeds before
and after submersion in sterile, deionized, distilled water, at various
times, at 24°C.
Microscopy
Conventional fluorescent microscopy (CFM) was done using a
Zeiss (Model 18) microscope equipped with epifluorescent accessories (filter set 450-490, FT 510, LP 520). Laser scanning microscopy (LSM) was performed, as described by Ghiorse et al. [9], using
a Zeiss LSM-210 laser scanning confocal microscope equipped with
an external argon laser (488 and 514 nm lines) and an internal
helium-neon laser (633 nm), as well as a conventional light source
for epifluorescence with filter sets appropriate for acridine orange.
Software programs for z sectioning were used to scan up and down
the focal planes of the seed coat. For scanning electron microscopy
(SEM), seed coats were immediately fixed in 3% glutaraldehyde
and 1% tannic acid in 0.05 phosphate buffer, pH = 7, for 3 h in the
cold, and then dehydrated in a graded ethanol series (75, 95,
100%). The O-T-O method, as described by Malick and Wilson
[16], was used for critical point drying. Samples were mounted,
dried, sputter coated with Au/Pb, and examined under SEM
(Model Hitachi 4500 FESEM). The work was conducted at the
Cornell Integrated Microscopy Center.
Motility Assay
Cotton seed exudate (CSE), prepared as described by van Dijk and
Nelson [38], was filter-sterilized through a thoroughly-washed 0.2
µm membrane filter, and added to sterile Noble agar (Difco) to give
a final agar concentration of 0.4%. Gradient agar plates were made
with a distilled water agar base and a seed exudate overlay, and
inoculated with fresh cultures of E. cloacae, P. ultimum, and a
mixture of both. Plates were observed at various time intervals,
using a dissecting microscope. Movement was measured as the
distance from initial line of inoculum to the point of new cells.
Fungal germ tube length was measured in the presence of CSE
or linoleic acid, using the method for germination assays described
103
by van Dijk and Nelson [38]. CSE and linoleic acid (0.2 mg ml−1
optimum concentrations for sporulation) were added to agar plugs
of the fungi after 2 and 4 h, at 24°C. The plugs were then examined.
Ten µl of the standard concentration of bacteria were added to
other agar plugs. Germ tube lengths were determined using acid
fuschin, light microscopy and an ocular micrometer. Ten fields
were examined, and 30 germ tubes measured.
Effect of Seed Exudates and Various Components
on Attachment
Cotton seed exudate and the following components were examined: sugars (glucose, mannose, fructose) at 1000 µg ml−1; amino
acids (leucine, arginine, lysine) at 1000 µg ml−1; volatiles (ethanol,
methanol, and acetylaldehyde) at 100 µg ml−1; electrolytes (NaCl,
KCl, and CaCl2) at 2000 µg ml−1; and fatty acids (linoleic, linolenic,
and oleic acid) at 1 µg ml−1.
Seed-Liquid Assay (SL). Whole seeds were added to buffer containing the standard concentration of bacteria, plus the seed exudate or
compound at the appropriate concentration, and incubated at
24°C. Seeds were removed after 4 h, rinsed, stained, and examined,
as previously described.
Seed Coat-Slide Assay (SCS). Seed coats were removed, mounted
on glass slides with silicon, and allowed to dry overnight. The
various compounds (in 10 µl aliquots) were added directly to the
seed coats, followed by 10 µl of the bacterial suspension. The slides
were incubated at 24°C in a moisture chamber consisting of a petri
dish and a Whatman No. 1 paper filter saturated with water. After
4 h, slides were rinsed, stained with AO, and observed under CFM,
as described.
Agar Assay and GFP Strains (A-GFP). PdRA157, a pUC19 derivative, which carries the gene encoding wildtype green fluorescent
protein (GFP) [4], was electroporated in E. cloacae EcCt-501, using
a BioRad gene pulser, as recommended by the manufacturers. After
incubation at 37°C for 1 h in SOC medium, E. cloacae cells were
plated on LB agar [18] + ampicillin (100 mg l−1) and incubated at
27°C overnight. Colonies were selected, streaked on LB medium,
and incubated overnight. Simple smears of the cultures were made
and examined under CFM to ensure that cells produced enough
GFP to be visualized.
Agar slides were prepared using 3% Noble agar (Difco) in buffer
and agar plus the various compounds. Molten agar, at ∼48°C, was
placed on the surface of a scratched glass slide and allowed to
solidify and dry for 30 min at room temperature. The agar slides
were placed into the bacterial suspension (optical density of 1.0 at
600 nm or 1010 cells ml−1) for 2 h at 24°C. Slides were removed,
rinsed by dipping in and out of buffer 10 times for 10 sec, and cells
counted under CFM using the same filter sets as with acridine
orange.
Experimental Design
All experiments were repeated independently three times (except
for the motility experiments which were repeated twice). Attach-
104
M.A. Hood et al.
Fig. 1. SEM micrographs of attached E. cloacae cells on cotton seed. Seeds incubated for 5 h at 24°C in sands. Panels show seed coat with
no bacterium (A, bar = 30 µm); seed coat with bacteria (B, bar = 30 µm); bacterial clusters in grooves with seed cracks often present (C,
bar = 6.7 µm); bacteria with fimbriae (D, bar = 1.5 µm).
ment experiments examining the effects of seed exudate and other
compounds were established as paired treatments, with and without substrate. An adhesion index was calculated, as described by
Hood and Winter [15], representing the mean number of cells
attached in the presence of compound divided by the mean number of cells attached without compound. Mean and standard deviations were calculated for all data sets. Regression analysis and
analysis of variance were done using standard procedures. Coefficients of variance (standard deviation divided by mean times 100)
were calculated for attachment assay data.
Results
E. cloacae on Cotton Seeds in Sand
Spatial arrangements of E. cloacae EcCT-501 on cotton seeds
in sand microcosms using CFM revealed occasional, patchy,
loose aggregations of bacteria on the uppermost surface of
unrinsed seeds. In these aggregates, individual cells were
difficult to distinguish, making quantitative analyses impossible. On the rinsed seeds, the more intimately associated
bacterial cells could be easily visualized and counted. Figure
1 shows the spatial arrangement of these cells on cotton seed
using SEM. Although the intimately associated cells were
distributed over the entire surface of the seed coat, they
showed a propensity for the troughs of grooves, especially
where cracks were evident. The presence of thick (∼25 nm)
fimbriae on many, but not all, cells was observed.
Using LSM, the same pattern of spatial distribution was
observed. Numerous clusters of attached cells could be observed in or near the bottom of the troughs, as well as along
the side of troughs, confirming the preference of cells for
Attachment by Enterobacter cloacae
Fig. 2. Attachment of E. cloacae to cotton seeds in sands at 24°C.
TSB, E. cloacae grown on TSB. CSE, E. cloacae grown on cotton
seed exudate.
these crevices. Three other bacterial strains examined, S. epidermidis and two strains of E. coli, exhibited little attachment
to seed coat under these conditions (data not shown). This
suggests that attachment to the seed coat is not a universal
trait.
Using CFM, attachment of E. cloacae and P. ultimum to
seeds in sands was directly determined. Attached bacterial
cells could be observed within 1 h after application. The
general pattern of bacterial attachment at 24°C was an increase in cell number until 4 to 8 h followed by a decline
(Fig. 2). Attachment by E. cloacae was far greater when the
bacterium was grown on CSE, although the same pattern of
increase and decline was noted. In contrast, fungal hyphae
could not be observed on the seed coat until after 6 h (Fig.
3). The abundance of fungal hyphae on cotton seed coat was
reduced considerably in the presence of the bacterium, although the time of first observation was the same. No difference in fungal attachment was noted in the presence of
bacteria grown in either TSB or in CSE prior to treating
seeds.
Attachment of E. cloacae to seeds in the liquid microcosm
showed the same general pattern of increase and decline as in
sands, with optimum attachment at 4 h at 24°C, and a more
gradual decline in adherent cells over the next 8 h (Fig. 4).
As germination of the seed progressed, various layers and
tissues were exposed to the bacteria. The seed coat, for example, consists of several layers: the outer most layer or
epidermis, a second outer brown coat, a layer of colorless
cells, and a layer of palisade cells. Below the seed coat, the
endosperm tissue also consists of several layers. Finally, the
cotyledon, which consists of several tissues including the
radicle [13]. As each layer was exposed and the radicle
105
Fig. 3. Attachment of cotton seed by P. ultimum in Pythiuminfected soils. P, P. ultimum alone; P+E(TSB), P. ultimum and E.
cloacae grown on TSB; P+E(CSE), P. ultimum and E. cloacae grown
on cotton seed exduate.
emerged, they were stained and examined for the presence of
the bacterium. Although the bacteria rapidly attached to
these tissues, considerable background fluorescence, due to
both tissue auto-fluorescence and acridine orange staining,
made quantitative measurements impossible.
Movement of P. ultimum and E. cloacae in Response to
Cotton Seed Exudate
Using gradient plates of CSE agar, the movements of E.
cloacae and P. ultimum were compared. Bacterial movement
(flagella mediated) was detected as early as 1 h. P. ultimum
movement (hyphal growth) lagged far behind and could not
be observed until 6 h (Fig. 5). In the presence of the bacterium, P. ultimum movement (hyphae growth) was not observed until 8 h. After that time, however, the rate of hyphal
growth began to approach that of bacterial motility.
Germ tube growth was also measured (data not shown).
Fig. 4.
Attachment of E. cloacae to cotton seed in liquid.
106
M.A. Hood et al.
Fig. 5. Movement of E. cloacae and P. ultimum in cotton seed
exudate gradient agar. E, E. cloacae; P, P. ultimum; and P(+E), P.
ultimum in the presence of E. cloacae.
P. ultimum sporangia, in the presence of optimum levels of
CSE and linoleic acid, germinated 1.5 to 2 h after exposure
at 24°C, similar to that reported previously [25]. The length
of germ tube growth in the presence of CSE and linoleic acid
was approximately 300 µm and 55 µm, respectively, in 4 h at
24°C (somewhat shorter than another study [26] that reported 400 µm). In the presence of bacteria, germination
and germ tube growth were reduced by as much as an order
of magnitude. The distance of germ tube growth in the presence of these simulants represents nearly an order of magnitude less than the distance E. cloacae was capable of moving in the first 4 h after exposure to nutrients.
Attachment of E. cloacae to Various Surfaces
A comparison of attachment of E. cloacae to various surfaces,
including an intact germinating seed, excised seed coat,
polystyrene, and glass showed that E. cloacae had a propensity for the surface of intact germinating seed (Fig. 6). Attachment was 3-fold greater to the surface of germinating
seeds than to excised seed coats, one-half that observed on
excised seed coat on polystrene, and there was little or no
attachment to glass.
Effects of Various Substrates on Attachment of E. cloacae
Three attachment assays, i.e., whole seed in liquid (SL), excised seed coat on slide (SCS), and agar as the substrate
(A-GFP), were used to determine the effects of seed exudate
and other compounds on bacterial attachment. The compounds selected represented exudate molecules reported in
the literature [10, 14, 21, 34, 39, 40]. The data, expressed as
Fig. 6.
Attachment of E. cloacae to various surfaces.
an adhesion index (AI) [15], represented either no effect of
the treatment on adhesion (AI = approximately 1.0); enhanced adhesion, as compared with the control (AI = >1.0);
or inhibition of adhesion (AI = <1.0). In all three methods,
attachment was enhanced most dramatically by cotton seed
exudate (Table 1). Likewise, fructose, methanol, calcium
and, in some assays, the three amino acids leucine, lysine,
and arginine, also enhanced attachment. Other compounds
showed either no influence on attachment or slightly reduced attachment. Trends across assay methods were not
consistent.
The three attachment assays differed in the pattern of
variation. Statistical variation in the A-GFP method (which
measures attachment to the homogenous surface of agar
with compound) was considerably narrower than the other
two methods. The range of coefficients of variance for this
method was from 2.1 to 3.4. In contrast, the SL method
(which measures attachment of bacteria to whole seeds with
compound dissolved in liquid) and the SCS method (which
measures attachment to inert seed coat plus compound) had
a range of coefficents of variance of 12.5 to 13.5 and 11.2 to
10.8, respectively.
Effects of Temperature, Water Content of Sand, and Water
Uptake by Seeds on Attachment by E. cloacae to Cotton Seed
The effect of water content in sands on bacterial attachment
is shown in Fig. 7. A strong positive correlation (R = 0.93)
was observed between numbers of attached cells on seeds
Attachment by Enterobacter cloacae
107
Table 1. Attachment of E. cloacae EcCT-501 using three attachment assays in the presence of various compounds
Assaysa
Substrate
SL
SCS
A-GFP
Cotton seed exudate
Sugars
Glucose
Mannose
Fructose
Amino acids
Leucine
Lysine
Arginine
Volatiles
Ethanol
Methanol
Acetylaldehyde
Electrolytes
Na+
K+
Ca++
Fatty acids
Linolenic acid
Linoleic acid
Oleic acid
10.1b
5.1
5.7
0.8
0.9
5.0
1.1
0.9
2.5
0.6
0.6
4.3
10.2
7.0
3.3
2.1
1.0
0.25
0.1
0.9
1.5
0.8
10.0
1.0
0.9
2.0
1.0
0.1
5.1
0.3
1.2
1.0
3.3
1.5
1.0
2.3
1.0
1.0
10.0
1.0
0.9
0.3
1.0
0.8
0.5
ND
0.8
ND
a
Assays: LS, Liquid seed assay; SCS, Seed coat-slide assay; A-GFP, agar plus
compound and cells transformed with green fluorescent protein assay
b
Data expressed as Adhesion index (AI) = ratio of attachment with and
without substance (mean number of cells attached in presence of substance/
mean number of cells attached without substance)
and water concentration. Although the saturation point of
the sand used in these studies was 35%, the 100% concentration could be approached using the liquid microcosm.
A strong correlation (R = 0.955) was also noted between
numbers of attached cells on seeds over time and the
Fig. 7. Effect of water concentration in sands on the attachment
of E. cloacae to cotton seed (100 percent represents attachment
value at maximum water concentration).
Fig. 8. Effect of temperature on the attachment of E. cloacae to
cotton seed (100 percent attachment at 30°C).
amount of water uptake by seeds during germination (data
not shown). Highest levels of water uptake by seed occurred
at 4 to 5.5 h at 24°C, corresponding to optimum attachment.
Attachment of E. cloacae to seed coat occurred optimumly at a temperature range between 18 and 30°C (Fig. 8).
Whether this pattern of attachment is related to fimbriae
production, which are produced most abundantly at this
temperature range [1, 5] is unknown.
Discussion
The associations of bacteria with plant tissue involve complicated and dynamic interactions. Although the complexities of the bacteria-root interactions have been described
[37], the nature of bacteria-seed interactions is less understood. Results from this study indicate that attachment of E.
cloacae EcCT-501 to cotton seed is a complex, dynamic process probably influenced by temporal changes accompanying
seed germination.
E. cloacae exhibited both a loose association with cotton
seed, which has been previously reported [30] and referred
to as colonization, and an intimate association. This intimate
association, or direct attachment to the surface of the seed,
appeared to be mediated, in part, by fimbriae. The fimbriae,
thinner than those described for E. coli type-1, are probably
similar to type-1 described for E. aerogenes [1]. Spatial arrangement of the bacteria on the surface of the seed reflected
a somewhat patchy distribution, a general pattern reported
for bacteria on seed surfaces [7], although there seemed to
be more cells in and along the sides of the troughs of the seed
coat. Clusters of cells were especially abundant around areas
where cracks in the coat were observed. These areas are
known to be important sites of nutrient leakage during water
108
imbibition and germination [32, 33, 34]. Such spatial distribution suggests that E. cloacae may be responding to these
nutrients.
The decline of cell numbers and the presence of the bacteria on newly exposed tissue suggest that the bacteria are
also able to detach from the seed surface, move, and attach
to the new tissue and emerging radicle. Quantitative measurements of bacteria on germinated seed tissue were difficult to make with the methods employed here, because of
tissue autoflourescence and fluorescence due to AO staining.
However, with newer technologies [29], it may be possible to
visualize and quantitate adhesion to various seed tissues.
Although we attempted to transform E. cloacae cells with
green fluorescent protein (GFP) and use CFM to visualize
attachment of the bacteria to these tissues, cells were difficult
to see and count. It may be possible, in future studies, to
block background tissue autofluorescence and enhance the
level of GFP produced by the bacterium, allowing quantitative measurements to be made.
The fact that E. cloacae attached to whole seeds in greater
numbers than excised seed coat further supports the idea
that attachment is influenced by germination. During germination, there are chemical changes in the surface properties of seed coat, water is taken up, and nutrients are released. The surface of cotton seeds becomes more wettable
with time, which may affect attachment. The strong correlation between seed water uptake and bacterial numbers on
the surface of the seed suggests some relationship between
this germination event and bacterial attachment.
The observation that cotton seed exudate and exudate
components (most notably fructose, methanol, leucine, lysine, and calcium ion) enhanced adherence of E. cloacae
further implicates the role of seed exduate in bacterial attachment. Why some compounds (especially the amino acids) showed such variation in effect among methods is unknown, but may reflect inherent differences among the
methods. Leucine, the most hydrophobic of the amino acids
examined, enhanced attachment to the whole seed and the
excised coat, but inhibited attachment to the hydrophilic
agar surface. The fatty acids had little effect, except for oleic
acid, which inhibited attachment to whole seed and excised
seed. The sugars, glucose and mannose, inhibited attachment
using the agar method, but had little effect on the whole seed
or coat. Because type 1 fimbriae produced by E. coli are
mannose sensitive [5], this might explain the reduced attachment in the presence of this sugar. The reason some
compounds enhanced attachment while others had little ef-
M.A. Hood et al.
fect or reduced attachment is unknown. Clearly, this is an
area that warrants further study.
Other factors that also affected bacterial attachment included temperature (optimum at 18 to 30°C) and water
content of sands (near 100%). The fact that germination
events are optimum under these conditions further implicates germination as the influencing factor on bacterial attachment.
In the sand systems used in these studies, E. cloacae was
able to attach far more rapidly to the seed surface than the
fungi, P. ultimum. Although other studies have shown colonization by P. ultimum between 6 to 10 h after seed exposure
[25, 36], and, in one study, as early as 4 h [26], here, the
fungi could not be detected on the seed surface until 6 h after
exposure. The bacteria, in contrast, were observed on the
seed surface within 1 h of exposure. Comparison of bacterial
and fungal movement (both hyphal growth in CSE and germ
tube grown in CSE and lineolic acid) revealed that the bacteria could initially move faster than the fungi. These temporal results further support the notion that bacteria are able
to get to the seed coat much more quickly than the fungi.
The finding that bacterized cotton seeds showed far less
attachment of fungi corroborate those from earlier studies
[20, 25] which indicate that seed colonization and hyphal
growth by P. ultimum was indeed inhibited in the presence
of E. cloacae on seed surfaces. This inhibition may be a direct
result of competition on the surface of the seed. It has been
shown that Pythium spp. are highly dependent on exudate
molecules in order to germinate and colonize seeds [21].
Thus, competition between the fungi and the bacteria for
specific exudate molecules could explain the mechanism of
biological control. A limited amount of experimental evidence supports a mechanism of biological control with Pseudomonas species [2, 8, 10, 21, 27, 28], and more recently with
E. cloacae [17, 38]. In the studies with E. cloacae, it was
shown that metabolism and inactivation of linoleic acid, an
important stimulatory component in cotton seed exudates
[31] was correlated with reduction in seed infection by P.
ultimum.
It has been proposed that protecting seeds for Pythium
infections by bacterial seed treatments involves competition
and antibiosis between the bacterium and the fungal pathogen [12]. The success of any bacteria acting as a biocontrol
agent depends on the ability of the bacteria to: 1) rapidly
move to the seed surface; 2) establish high populations; and
3) actively metabolize stimulatory fungal compounds or to
produce compounds that inhibit the fungi. From the results
Attachment by Enterobacter cloacae
reported here, E. cloacae EcCT-501 seems capable of getting
to the surface of the seed and establishing high populations
before the fungal pathogen, P. ultimum. The bacterium
probably does this by taking advantage of seed germination
events. The bacterium may utilize certain fatty acids which
stimulate fungal germination [38] as well as other fungal
growth nutrients, and, as a result, block fungal attachment.
Although direct attachment of E. cloacae to cotton seeds
was shown, the question of whether such intimate attachment of the bacterium to the seed coat is essential for biological control remains unclear. Perhaps with the use of
motility, adherence, and detachment mutants of E. cloacae,
as well as fatty-acid mutants, the role of these characteristics
in biological control may be established.
Acknowledgments
We acknowledge Dr. William Ghiorse for his generous time
and expertise with laser scanning confocal microscopy. Also,
the staff at the Cornell Integrated Microscopy Center is acknowledged for its assistance.
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