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Applied and Environmental Microbiology, May 1999, p. 2179-2183, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Localized, Positive Charge Mediates Adhesion of
Rhodosporidium toruloides to Barley Leaves and
Polystyrene
James W.
Buck and
John H.
Andrews*
Plant Pathology Department, University of
Wisconsin, Madison, Wisconsin 53706
Received 23 October 1998/Accepted 3 March 1999
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ABSTRACT |
The physicochemical forces that mediate attachment of yeasts to the
phylloplane are unknown. Cell surface charge and hydrophobicity and
adhesion to polystyrene, glass, and barley were assessed for wild-type
Rhodosporidium toruloides and attachment-minus
(Att
) mutants. Cells were grown under conditions
promoting (excess carbon) or not promoting (excess nitrogen) capsule
production. Hydrophobicity was measured by adhesion to xylenes, and
surface charge characteristics were assessed by attachment to either
DEAE (positive)- or carboxymethyl (CM) (negative)-Sephadex ion-exchange beads. Hydrophobicity and adhesiveness of nonencapsulated, wild-type R. toruloides decreased from mid-log to late stationary
phase. Encapsulated wild-type R. toruloides cells were more
hydrophobic and more adhesive than nonencapsulated cells. However, two
encapsulated Att mutants were more hydrophobic than the
wild type and levels of adhesion of R. toruloides were
similar on polystyrene and less hydrophobic glass surfaces. Adhesion of
wild-type yeast to barley and polystyrene was correlated with
attachment to CM-Sephadex beads, indicating a positive cell surface
charge. Sixteen Att mutants did not exhibit a positive
cell surface charge, and wild-type yeast cells that did not attach to
CM-Sephadex did not adhere to either polystyrene or barley. Wild-type
R. toruloides attached to CM-Sephadex beads by the poles of
the cells, indicating a localization of positive charge which was also
visualized with India ink. We conclude that localized, positive charge,
and not hydrophobic interactions, mediates attachment of R. toruloides to barley leaves.
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INTRODUCTION |
Leaf surfaces of temperate zone
plants are populated by epiphytic yeasts commonly classified as pink or
red (predominantly Rhodotorula spp. and
Sporobolomyces spp.) and white (Cryptococcus spp.) yeasts. These yeasts provide a natural buffer against plant pathogens (12), yet essentially nothing is known about their biology, including how they attach to the phylloplane.
The initial events leading to microbial adhesion are primarily due to
nonspecific, physicochemical forces between microbe and test surfaces.
Since most microbial cells and both natural and test surfaces are
negatively charged, these events can be partially explained by what is
now known as the Derjagiun, Landau, Verwey, and Overbeek (DLVO) theory
of colloid stability (9, 44). In short, a balance between
attractive London-van der Waals forces and electrostatic forces, which
are usually repulsive, results in weak attachment at a distance called
the secondary minimum (>10 nm). The DLVO theory has been used to
describe the initial steps of adhesion for five isolates of bacteria to
polystyrene (42) and for the human pathogenic yeast
Candida albicans to various surfaces (for a review, see
reference 20). Additional physicochemical forces,
including hydrophobic interactions or chemical bonds (e.g.,
electrostatic, ion-dipole interactions), can result in stronger
adhesion at the primary minimum (less than 1 nm) (13, 20,
43). Similar interactions are speculated to mediate the
attachment of yeasts to the phylloplane (10), yet no
experimental evidence is available.
Leaves are covered, to various degrees, with surface waxes which
function to repel water due to their hydrophobic nature (15, 28). Not surprisingly, many plant-pathogenic fungi, including Colletotrichum spp. (31, 38, 46),
Magnaporthe grisea (14), Uromyces
appendiculatus (41), and Botrytis cinerea
(11), adhere more tenaciously to hydrophobic than to
hydrophilic surfaces. Hydrophobic interactions are involved in the
attachment of Uromyces viciae-fabae to various synthetic
surfaces (8). Cell surface hydrophobicity of microbes is
also positively correlated with increased adhesion (e.g., references
11 and 21). However, not all
plant-pathogenic fungi attach better to more hydrophobic surfaces;
there is no correlation between adhesion of Cochliobolus heterostrophus conidia and germlings and substratum hydrophobicity (5).
The role of hydrophobic interactions in adhesion has been investigated
with artificial test substrata, including glass or chemically treated
glass (e.g., references 5, 31, and
41), Teflon (e.g., reference 5),
and polystyrene (e.g., references 31 and
41), which exhibit various degrees of
hydrophobicity. Removal of leaf surface waxes with chloroform,
resulting in a more wettable or less hydrophobic surface, results in
decreased adhesion of Colletotrichum lindemuthianum to bean
hypocotyls (46).
Microbial cell surface charge can also influence adhesion. For example,
chemical modification of C. albicans producing positively charged cell surfaces results in increased adhesion to plastic (23) and intestinal epithelium (24). Ion bridging
by divalent cations, including Ca2+, between negatively
charged acrylic and the negatively charged surface groups (e.g., acidic
polysaccharides) of C. albicans is hypothesized to be a
mechanism of adhesion (29). While most microbes are
naturally negatively charged, there are exceptions: the
bacterium Stenotrophomonas (Xanthomonas)
maltophilia is positively charged, which promotes adhesion
to glass and Teflon (19). Adhesion of the fungal plant
pathogen Bipolaris sorokiniana sporelings to glass is
mediated by positively charged polymers of galactosamine (36), and the adhesive material of the fungus
Buergenerula spartinae contains positively charged basic
proteins (33).
Previously, we have shown (6) that the basidiomycete yeast
Rhodosporidium toruloides Banno (anamorph, Rhodotorula
glutinis) adheres to barley leaves and polystyrene by a region of
material, including mannose residues, localized at sites of bud
development. The object here was to determine the physicochemical
forces responsible for this attachment. This problem was approached by
analyzing certain cell surface characteristics of wild-type and
attachment-minus (Att) mutants of R. toruloides. Specifically, our hypothesis was that adhesion of
R. toruloides to polystyrene and barley leaves depends on
hydrophobic interactions. We provide evidence that hydrophobic interactions are not responsible for the observed adhesion of R. toruloides, either to barley leaves or to polystyrene. Unlike most
other fungal or yeast systems studied, attachment of R. toruloides is mediated by a localized region of positively charged material.
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MATERIALS AND METHODS |
Yeast strains and inoculum.
The parental strain R. toruloides NRRL Y-1588 was obtained from the Agricultural Research
Service Culture Collection, Peoria, Ill. Att mutants were
described previously (6). For all experiments, cells were
grown in 50-ml volumes of liquid medium (see below), harvested by
centrifugation (3,000 × g, 10 min), and washed twice in 10 mM sodium phosphate buffer (pH 7.0).
Plant growth conditions.
Barley (Hordeum vulgare
cv. Hazen) was provided by the Wisconsin Foundation Seed Program,
Madison, Wis. H. vulgare cv. Bonus and the waxless mutant
cer-j59 were provided by P. von Wettstein-Knowles,
Copenhagen, Denmark. Barley seeds were sown in Redi-Earth Peat Lite Mix
(Scotts-Sierra Horticultural Products Co., Marysville, Ohio) and
incubated at 24°C with a light regimen consisting of 12 h of
light (approximately 180 microeinsteins s1
m2 at pot level) and 12 h of darkness. The first
fully expanded leaf of individual 9- to 11-day-old plants was used for
the assays.
Adhesion assay.
Adhesion of R. toruloides was
assessed as described previously (6). Briefly, yeast cells
were suspended in 10 mM sodium phosphate buffer (pH 7.0) at 3.5 × 106 cells ml1 and were incubated on the test
surfaces for 90 min. Yeast inoculum and surfaces were placed into 2-ml
silicon-coated microcentrifuge tubes (Sigma, St. Louis, Mo.) with 1 ml
of sodium phosphate buffer and vortexed for 10 s (setting 3, Vortex Genie; Scientific Products, McGaw Park, Ill.), and nonadherent
cells were counted with an electronic particle counter (Elzone model 80 XY; Particle Data Inc., Elmhurst, Ill.). The initial amount of inoculum
applied to the surfaces was determined by enumerating cells in similar yeast suspensions without incubation of the test surfaces. Adhesion was
determined with the formula (number of cells recovered after incubation
on test surface/initial number of cells in inoculum) × 100%.
Determination of relative surface charge.
Relative surface
charges of wild-type R. toruloides and Att
isolates were assessed by attachment to Sephadex ion-exchange beads
(16). Sephadex beads were washed six times (DEAE-Sephadex A-25 anion exchanger and carboxymethyl [CM]-Sephadex C25 cation exchanger; Sigma) in 10 mM sodium phosphate buffer (pH 7.0). Yeast cells (107 cells ml1) in sodium phosphate
buffer were mixed with either CM- or DEAE-Sephadex beads in 2-ml
silicon-coated microcentrifuge tubes (Sigma) for a final bead-to-buffer
ratio of 1:4. Cell-bead suspensions were incubated for 1 h with
agitation (75 rpm) at 22°C. Beads and attaching cells were allowed to
sediment, 500 µl of the remaining suspension was collected from each
tube, and the nonadherent cells were enumerated with a particle counter
(6). The initial amount of inoculum was determined by
enumerating cells in similar samples without addition of beads.
Adhesion was determined with the formula 1 
(number of cells
recovered after incubation with beads/initial number in inoculum) × 100%. Positive surface charge was determined by attachment to
negatively charged CM-Sephadex, and negative surface charge was
assessed by adhesion to positively charged DEAE-Sephadex. Beads were
viewed microscopically to determine orientations of attaching yeast cells.
Cell surface hydrophobicity.
Cell surface hydrophobicity was
determined by the ability of yeast isolates to adhere to xylenes
(40). Yeast cells (1 ml, 107 cells
ml1) suspended in 10 mM sodium phosphate buffer (pH 7.0)
were overlaid with 250 µl of xylenes (Fisher Scientific, Pittsburgh,
Pa.) in 2-ml silicon-coated microcentrifuge tubes. Tubes were vortexed for 30 s (setting 10) and then were allowed to equilibrate for 30 min at 22°C. A volume of 500 µl of the lower, aqueous phase was
removed from each tube, and the yeast cells were enumerated as
described above. Cells in control tubes without xylenes were quantified
to determine the total initial number of yeast cells. Hydrophobicity
was determined with the formula 1 (number of cells recovered
from the aqueous phase after incubation with xylenes/initial number in
inoculum) × 100%. Higher values represented greater percentages of
cells adhering to the xylenes and therefore higher cell surface
hydrophobicity. Xylenes were examined microscopically to ensure that
yeast cells were intact.
Effect of culture age or capsule on adhesion, cell surface
hydrophobicity, and charge.
To determine the effect of culture age
on adhesion, cell surface hydrophobicity, and surface charge, wild-type
and Att yeast cultures were grown in 50-ml yeast nitrogen
base medium (YNB; Difco Laboratories, Detroit, Mich.) supplemented with
2% glucose to either mid-log phase (16 to 18 h) or late
stationary phase (72 h). Adhesion to polystyrene and H. vulgare cv. Hazen and cell surface measurements were done as
described above. Wild-type cells were positively stained with India ink
(25) to detect polar adhesive material (6). Cells
grown under these conditions lacked visible capsules as determined by
negative staining with India ink.
To assess the effect of the yeast capsule on adhesion, charge, and
hydrophobicity, cells were grown in yeast carbon base medium (YCB;
Difco) with 3% glucose for 72 h. Growth of R. toruloides in YCB induces capsule formation, which was assessed by
negative staining with India ink (6). Cells were harvested,
and adhesion and surface measurements were assessed as described above.
Substratum hydrophobicity and adhesion of R. toruloides.
The effect of substratum hydrophobicity on attachment
was determined by assessing adhesion of R. toruloides to
glass, polystyrene, and barley surfaces. Polystyrene is more
hydrophobic than glass (41). Glass coverslips (13 mm
diameter; Ernest F. Fullam, Inc., Latham, N.Y.) and polystyrene pieces
(1.5 by 1.5 cm; Ward's Plastics, Rochester, N.Y.) were washed in
distilled water and air dried. The effect of leaf surface wettability
was determined by assessing attachment of R. toruloides to
wild-type H. vulgare cv. Bonus and the waxless mutant
cer-j59. Water droplets were observed to spread and cover a
larger surface area on the waxless mutant than on wild-type barley,
indicating that the surfaces were less hydrophobic (data not shown). In
addition, surface wettability of H. vulgare cv. Hazen was
modified by gently rubbing the leaf segments once between thumb and
forefinger. Wild-type R. toruloides Y-1588 was grown to
mid-log phase in YNB and harvested, and adhesion to the above-mentioned
surfaces was determined.
Effect of chelators or surfactants on adhesion.
To determine
the possible role of cations in adhesion, R. toruloides
Y-1588 was grown in YNB to mid-log phase, harvested, and incubated with
either 50 mM EDTA or 50 mM EGTA in 100 mM Tris HCl (pH 8.0). Cells were
incubated for 4 h at 22°C with gentle agitation (100 rpm), and
then adhesion to polystyrene was determined. Cationic (cetylpyridinum
chloride), anionic (sodium dodecyl sulfate), and nonionic (Tween 20)
surfactants (final concentration, 0.75%; Sigma) were added to
mid-log-phase yeast cells in sodium phosphate buffer, and adhesion to
polystyrene was determined.
Microscopy and image analysis.
Photomicrographs were taken
on Kodak Technical Pan film with Nomarski differential interference
contrast optics on a Zeiss Universal microscope with a 40× lens
objective. The film was processed with HC-110 developer, negatives were
scanned with a Polaroid SprintScan 35-mm slide scanner, and images were
adjusted for contrast with Adobe Photoshop, version 5.0.
Replication and statistics.
Unless noted otherwise, all
experiments were done at least twice. Data were analyzed by one-way
analysis of variance (Minitab, version 10.0; Minitab Inc., State
College, Pa.).
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RESULTS |
Effect of culture age on adhesion, hydrophobicity, surface charge,
and polar staining pattern.
Wild-type cultures of R. toruloides Y-1588 grown in YNB were adhesive during mid-log growth
phase but became significantly less adhesive in late stationary phase
(Fig. 1A). Att mutants in
mid-log or late stationary phase did not adhere to polystyrene (Fig.
1A). Similarly, cell surface hydrophobicity of wild-type and two
Att isolates significantly (P = 0.05)
decreased with culture age (Fig. 1B). All 16 Att mutants
were less hydrophobic than the wild type when they were grown in YNB to
mid-log phase (data not shown).
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FIG. 1.
(A) Adhesion to polystyrene of wild-type R. toruloides Y-1588 and three Att mutants (3-3, IID2,
and 2-5) grown without detectable capsules to mid-log ( ) or late
stationary ( ) phase in YNB. Relative surface measurements of
hydrophobicity (xylenes) (B), positive charge (CM-Sephadex) (C), and
negative charge (DEAE-Sephadex) (D) are shown. Data are the means and
standard deviations of results of five replicate experiments.
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None of the Att
mutants attached to negatively charged,
CM-Sephadex beads, and wild-type adhesion to CM-Sephadex decreased
significantly (
P = 0.05) in late stationary phase (Fig.
1C). All
isolates of
R. toruloides adhered to positively
charged beads
regardless of culture age (Fig.
1D). These data suggest
that a
positively charged material(s) on the cell surface mediates
adhesion.
A decrease in positively charged, polar material on wild-type
R. toruloides from mid-log to late stationary phases in YNB
was
monitored by staining with India ink (Fig.
2). Mid-log-phase
cultures typically had
a high proportion (greater than 90%) of
cells exhibiting polar
staining patterns, but the affinity of
the cells for the India ink
decreased significantly in late stationary
phase (to less than 50%)
(Fig.
2).
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FIG. 2.
Cell concentrations ( ) of wild-type R. toruloides Y-1588 from mid-log to stationary phases and
percentages of cells with polar areas that stained positively with
India ink ( ). Cells were grown in 50-ml YNB cultures. Data are the
means and standard deviations of four counts of at least 200 cells for
polar staining and cell counts from four replicate cultures for cell
concentration (culture age).
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Effect of capsule on adhesion, surface charge, and hydrophobicity
of R. toruloides.
Wild-type R. toruloides Y-1588
cells with capsules adhered to polystyrene and barley leaf segments
significantly better (P = 0.05) and were more
hydrophobic than cells without capsules (Fig.
3). Cell surface charge characteristics
differed between the encapsulated and nonencapsulated cells.
Significantly more encapsulated cells adhered to negatively charged
(CM) beads, but fewer encapsulated cells attached to positively charged
(DEAE) beads (Fig. 3).
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FIG. 3.
Adhesion of late-stationary-phase wild-type R. toruloides Y-1588 with () or without () capsules to plastic
(polystyrene), barley (H. vulgare cv. Hazen), xylenes
(hydrophobicity), CM-Sephadex beads (CM; positive cell charge), and
DEAE-Sephadex beads (DEAE; negative cell charge). Data are the means
and standard deviations of results from nine replicates for barley and
five replicates for all others.
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Encapsulated Att
mutants did not adhere to polystyrene or
negatively charged beads (four representative mutants are shown
in Fig.
4). Two of the Att
mutants
were significantly more hydrophobic than wild-type
R. toruloides (Fig.
4B), and all isolates attached to DEAE beads.
The
common phenotype among all 16 Att
mutants was the lack of
positive charge on the cell surfaces,
indicated by the inability to
attach to CM-Sephadex (four representative
mutants are shown in Fig.
4C). Encapsulated wild-type cells, but
not encapsulated
Att
mutants, displayed polar staining patterns with India
ink.
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FIG. 4.
Adhesion of wild-type R. toruloides Y-1588
and four Att mutants (3-3, 3K, IID2, and 1n) grown with
capsules to polystyrene (A), xylenes (hydrophobicity) (B), CM-Sephadex
beads (positive cell charge) (C), and DEAE-Sephadex beads (negative
cell charge) (D). Data are the means and standard deviations of results
from five replicates.
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Localization of positively charged areas on the surface of R. toruloides.
Wild-type R. toruloides adhered to
negatively charged beads by the polar regions and were oriented
perpendicularly to the bead surface (Fig.
5A), suggesting that the positively
charged material is localized at the poles of the cells. In addition, wild-type cells stained with India ink did not attach to negatively charged beads (data not shown). India ink localizes at the poles of the
cells (6), further suggesting that the polar regions are
sites of attachment. Cells attached to positively charged beads along
the long axes of the cells (Fig. 5B).
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FIG. 5.
Wild-type R. toruloides cells adhering to a
negatively charged CM-Sephadex bead (A) or a positively charged
DEAE-Sephadex bead (B). Note the difference in orientation of attaching
cells at the edges of the beads. Bar, 5 µm.
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Effect of substratum on adhesion of R. toruloides.
R. toruloides adhered significantly (P = 0.05) better to the waxless barley mutant cer-j59
(71.7%) and polystyrene (66.9%) than to wild-type H. vulgare cv. Bonus (29.6%). Attachment of the yeast to H. vulgare cv. Hazen increased (54.9 versus 28.7% based on the
results of one experiment) if the leaf segments were rubbed gently
prior to application of the yeast. Inoculum droplets were observed to
lie flatter and cover a larger surface area on the leaves that were
rubbed (i.e., were more wettable). Adhesion of R. toruloides
to glass was equal to or greater than adhesion to the more hydrophobic
polystyrene surfaces (data not shown).
Effect of surfactants and chelators on adhesion.
Surfactants
significantly reduced adhesion to polystyrene (Fig.
6), suggesting the possible role of
hydrophobic interactions in adhesion. Chelators had no effect on
adhesion, which indicated that cations were not required for adhesion
(data not shown).
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FIG. 6.
Effects of surfactants on adhesion of R. toruloides to polystyrene. Data are the means of results from five
replicates. Data bars with different letters are significantly
different (P = 0.05). CPC, cetylpyridinum chloride;
SDS, sodium dodecyl sulfate.
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DISCUSSION |
The main significance of this work is the finding that R. toruloides adheres to surfaces by a region of localized material that is positively charged. Unlike with adhesion of many
plant-pathogenic fungi to plant surfaces (e.g., references 31,
38, and 46), hydrophobic interactions were
not important in the attachment of this epiphytic yeast to barley
leaves or polystyrene.
Several observations support the conclusion that a positive
charge mediates the adhesion of R. toruloides to leaves and
polystyrene. First, wild-type R. toruloides cells, but not
16 Att mutants, displayed a transient ability to attach
to negatively charged Sephadex beads (CM beads). Second, wild-type
cells unable to adhere to CM beads did not attach to leaves or
polystyrene (this work and reference 6). These
results are in contrast to those of other reports on the interaction of
various yeast genera, including Candida spp. and
Saccharomyces cerevisiae (16, 22), and the fungus
Nomuraea rileyi (35) with cation-exchange resins
at physiological pH. Similarly, most bacteria do not adhere to
negatively charged resins (45); however, isolates of
Aeromonas salmonicida are retained in cation-exchange resin
(4).
Localization of the positive charge to polar regions of the wild-type
cells was visualized by microscopic examination of cells attaching to
CM beads and by India ink staining. Both India ink and concanavalin
A-fluorescein staining patterns localized to the polar regions of
attachment-competent, wild-type R. toruloides cells, and
these patterns were absent in all Att mutants
(6). Kuo and Hoch (25) hypothesize that India ink binds to proteins with positive charges and/or hydrophobic regions. Our
data suggest that the ink binds to positively charged, polar areas of
R. toruloides. This suggestion is further supported by the
inability of wild-type cells stained with India ink to attach to
negatively charged beads. The bacterium S. maltophilia
(19) and avirulent isolates of A. salmonicida
(37) also possess a net positive surface charge, but there
is no information on possible localization of the charge. Anionic
sites, thought to be involved in adhesion of C. albicans,
are dispersed evenly over the surfaces of blastoconidia bearing bud
scars but not on the bud scars (17). Horisberger and Clerc
(17) do not mention if the bud scars are positively charged
or lacking charge. Jones et al. (18), using the fluorescent
probe 9-aminoacridine, have studied the electrostatic properties of
C. albicans and conclude that the yeast is electronegative. However, they state that while no information on the spatial
distribution of charge is obtained with 9-aminoacridine, the
electrostatic behavior of the population is consistent with an even
distribution over the cell surface (18). Charge distribution
has also been observed on the surfaces of fungal cells: basic amino
acids are detected with anionic colloidal gold on hyphae but not on
conidia of Colletotrichum lindemuthianum (34).
We demonstrated previously that attachment of R. toruloides
depends on the physiological condition of the cells and is correlated with sites of bud development (6). We have shown here that the adhesive phenotype of cells is lost as cultures reach late stationary or death phase. This finding is in contrast to what occurs
with another yeast, Cryptococcus neoformans, in which
maximum adhesion to glial cells is observed in late stationary phase
(32). The onset of late stationary phase corresponds to a
decrease in the cellular mannose content of R. toruloides
(39) and the disappearance of a thick layer of localized
mucilage deposited over developing buds (27), which we
proposed may be involved in adhesion (6).
The polar region of adhesive material of R. toruloides
contains mannose residues, possibly as mannoproteins (6);
however, the positive charge is probably due to basic proteins. With
few exceptions (e.g., reference 36), positively
charged regions on most organisms have been hypothesized to originate
from proteins. For example, the presence of proteins rich in basic
amino acids results in positive charge being associated with the
extracellular matrices of germ tubes and appressoria of
Colletotrichum lindemuthianum (34). The
extracellular matrix of Phyllosticta ampelicida has positively charged regions due to proteins or simply nonspecific charge
(25). While the surface charge of C. albicans is
primarily negative, resulting from phosphate groups of mannoproteins
(1), positive charge can be present in the form of
N-terminal amino groups and arginine, histidine, and lysine residues
(16).
Hydrophobic interactions did not mediate adhesion of R. toruloides to barley or polystyrene. Three lines of evidence
support this conclusion. First, adhesion of R. toruloides to
glass was equal to or greater than its adhesion to the more hydrophobic polystyrene. Second, attachment to the more wettable (i.e., less hydrophobic) barley mutant increased compared to attachment to wild-type barley and increased if barley was rubbed to alter its wax
rodlet structure. The eceriferum mutant cer-j59 has
significantly fewer wax rodlets on the surfaces of its leaves (26), which results in a more hydrophilic surface
(15). Third, when grown with a capsule, some
Att mutants were more hydrophobic than the wild type.
Other researchers (11) have shown that adhesion is disrupted
by surfactants, suggesting that hydrophobic interactions are involved.
However, in our study the surfactants may have bound nonspecifically
with lipids either in the cell wall or associated with capsular
material to disrupt adhesion.
What do our findings imply? The observation that R. toruloides adheres in high numbers to a waxless barley mutant
suggests that leaf surface characteristics may dramatically affect,
both qualitatively and quantitatively, immigration of yeasts to the phylloplane. Old leaves have much rougher surfaces than young leaves
(30) and typically become less hydrophobic (2).
Yeast populations generally become established on leaves towards the middle of the growing season, when the atmospheric inoculum and a
suitable nutrient environment become available (3). In
addition to reflecting nutrient status and inoculum availability, these patterns may also reflect the changing surface structure of the leaves.
We are currently investigating the effect of leaf surface characteristics on long-term (days) adhesion and colonization of
R. toruloides (7).
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ACKNOWLEDGMENTS |
This research was supported by U.S. Department of Agriculture
(USDA) Hatch grant 142-3995.
We thank Russ Spear for discussions and help with the photomicrograph
and Jo Handelsman, Eric Johnson, and Gary Roberts for their suggestions
for improving the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: Plant Pathology
Department, 1630 Linden Dr., University of Wisconsin, Madison, WI
53706. Phone: (608) 262-9642. Fax: (608) 263-2626. E-mail:
jha{at}plantpath.wisc.edu.
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Applied and Environmental Microbiology, May 1999, p. 2179-2183, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
