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Plant Physiol. (1998) 117: 1171-1178
Chemotropic and Contact Responses of Phytophthora
sojae Hyphae to Soybean Isoflavonoids and Artificial
Substrates1
Paul F. Morris*,
Elizabeth Bone, and
Brett M. Tyler
Department of Biological Sciences, Bowling Green State University,
Bowling Green, Ohio 43403 (P.F.M., E.B.); and Department of Plant
Pathology, University of California, Davis, California 95616 (B.M.T.)
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ABSTRACT |
We have investigated the role of the
isoflavones daidzein and genistein on the chemotropic behavior of
germinating cysts of Phytophthora sojae. Hyphal
germlings were shown to respond chemotropically to daidzein and
genistein, suggesting that hyphal tips from zoospores that have
encysted adjacent to the root may use specific host isoflavones to
locate their host. Observations of the contact response of hyphal
germlings were made on several different substrates in the presence and
absence of isoflavones. Hyphal tips of germlings detected and
penetrated pores in membranes and produced multiple appressoria on
smooth, impenetrable surfaces. Hyphae that successfully penetrated the
synthetic membrane were observed to grow away from the membrane
surface. The presence of isoflavones in the medium surrounding the
hyphal germlings did not appear to alter any of those habits. Daidzein
and genistein did not inhibit germination or initial hyphal growth at
concentrations up to 20 µM.
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INTRODUCTION |
For host-specific pathogens or symbionts, the ability to recognize
and move in the direction of a plant signal may be critical to the
survival of the organism. Germination or chemotropism in response to a
host-specific signal has now been described in several plant-fungus
associations (Coley-Smith, 1990 ; Podila et al., 1993; Ruan et
al., 1995). In bacteria, expression of nodulation genes in
Rhizobium and Bradyrhizobium species is induced
by flavones or isoflavones (Fisher and Long, 1992), and some species
such as Rhizobium meliloti also respond chemotactically to
the nodulation signals (Dharmatilake and Bauer, 1992). In addition,
chemotaxis to plant exudates appears to be an important factor
contributing to the virulence of Agrobacterium species in
heterogenous soil mixtures (Hawes and Smith, 1989).
Zoospores of oömycetes also exhibit positive chemotaxis to
plant-derived compounds (Zentmyer, 1961; Carlile, 1983; Horio et al.,
1992; Morris and Ward, 1992; Sekizaki et al., 1993). Zoospores of the
soybean pathogen Phytophthora sojae are highly sensitive to
the isoflavones daidzein and genistein, which are exuded from the roots
of soybeans into the rhizosphere (Morris and Ward, 1992; Tyler et al.,
1996). Because six other species of Phytophthora and one
species of Pythium displayed no sensitivity to these
isoflavones, Morris and Ward (1992) suggested that the specific
attraction to soybean isoflavones might be part of the mechanism that
determines host range.
Although oömycetes have a predominantly filamentous hyphal growth
pattern, the relationship between soil water and disease severity
suggests that zoospores are the predominant means by which pathogenic
oömycetes spread throughout the soil and infect plant roots,
especially in flooded soils (Duniway, 1983; Erwin and Ribeiro, 1996).
Despite the complex structure and small pore size of most soils,
zoospores are capable of traveling substantial distances (25-35 mm) to
initiate infection within a 24-h period (Duniway, 1976). However, the
majority of zoospores released from the zoosporangium on the hyphae do
not reach their host and eventually form cysts. Hardham and Gubler
(1990) established that both adhesion and the initial direction from
which the germ tube emerges are determined at the time of encystment.
Factors that influence the direction of hyphal growth after germination
are less clearly defined. Autoaggregation of zoospores in the absence
of an available host is characteristic of some but not all
oömycetes (Reid et al., 1995). Zentmyer (1961) demonstrated that
Phytophthora cinnamoni cysts that were adjacent to the
root of their host germinated rapidly and grew in the direction of the
root, but the chemical signal was not identified. A chemotropic
response of hyphae to nutrient sources has also been demonstrated in
several saprophytic and parasitic oömycetes (Musgrave et al.,
1977; Manavathu and Thomas, 1985; Jones et al., 1991). Thus,
chemotropic responses of oömycetes hyphae might also contribute
to their effectiveness as plant pathogens.
In this study we report on an in vitro system that we
have developed to study the encystment and subsequent germination of zoospores in response to soybean isoflavones. We show that both thigmotropic (contact) and specific chemotropic determinants control the direction of growth in germinating hyphae.
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MATERIALS AND METHODS |
Preparation of Zoospores and Chemotropism Assays
Phytophthora sojae Kauf. & Gerd., strains P6497 and
P7063 (Förster et al., 1994), were grown on vegetable juice agar,
and zoospores were produced by repeated washing of plates with
distilled water as described previously (Morris and Ward, 1992). These
strains were used because their differential responses to daidzein and genistein have been well characterized (Tyler et al., 1996). In capillary assays, genistein is 10 and 100 times more potent than daidzein in P6497 and P7063, respectively. Chemotropism assays were
performed in an assay chamber that was created by supporting a
coverslip with two glass pieces on a glass slide. The weight of the
coverslip was sufficient to hold a 250-µL drop of zoospores in place,
and small soybean roots or 1-µL capillary tubes (Drummond microcaps)
containing the isoflavones daidzein or genistein could be inserted
directly into the chamber. Daidzein and genistein were obtained from
Indofine Chemical (Somerville, NJ). Stock solutions of 40 µM in water were stored at  20°C.
To demonstrate the chemotropic properties of hyphal germlings, a 1-µL
capillary tube containing 20 µM daidzein or genistein was
introduced into a chemotaxis chamber and left undisturbed for 3 h.
Hyphal germlings surrounding the mouth of the capillary tube were
photographed under phase-contrast optics using a BH-2 microscope
(Olympus). To test the ability of uniformly dispersed cysts to
germinate and reorient in response to host roots or an isoflavone
gradient, P6497 zoospores were induced to encyst by adding 10 mM CaCl2 combined with vortexing for
15 s. The zoospores were then pipetted into the chemotaxis chamber
and left undisturbed for 1 h. A soybean root or a capillary tube
containing 20 µM genistein was then inserted into the
chemotaxis chamber. A 50-µL drop of genistein was also applied to the
open end of the capillary tube, which was then left undisturbed for
3 h.
Quantitative Analysis of Hyphal Chemotropism
The angle of hyphal growth relative to the root surfaces was
determined by defining a plane parallel to the surface of the root, and
measuring the angle of the hyphal tip as a deviation (in degrees) from
a direction directly toward this plane. Thus, hyphal tips growing
directly toward the "source" were given a value of 0°, and hyphal
tips growing directly away from the source were assigned a value of
180°. The angle of hyphal growth relative to the capillary tube was
determined by drawing a line from the mouth of the capillary tube to
the hyphal tip. The angle was measured as a deviation from that line,
so that hyphal tips that pointed directly at the tube were given a
value of 0°. The distance of each hyphal tip from the source was
determined by measuring the distance in the photographs and converting
this value to actual distances using a photograph of a stage micrometer
taken at the same magnification. A total of 231 and 279 data points
were calculated from 13 and 15 photographs of capillary tubes and
roots, respectively. For each data set, all points were ordered by
distance, and the mean of the cosine was calculated for a 200-µm
window sliding from 0 µm to the end at 20-µm intervals. A 95%
confidence interval was calculated for the points in each data window
using a t distribution. The number of points in each window
ranged from 30 to 74.
Assessment of Chemotropic and Contact Responses of Hyphal Germlings
Cell-culture plates and inserts (Falcon, Fisher Scientific) were
used to test the chemotropic and thixotropic (contact) responses of
hyphae on a solid surface. In a typical experiment, 1 mL of swimming
zoospores was placed in one well of the cell-culture plate. The
isoflavone attractant was dissolved in a 0.2% agarose solution, which
was added to the cell-culture insert cup. The bottom layer of the cup
consisted of a porous PET membrane with 3-µm pores. When the insert
cup was placed in the well containing the zoospores, the isoflavones
diffused through the pores, producing a concentration gradient that
caused zoospores to swim toward the membrane. After 40 min the insert
was removed from the chamber and the membrane surface carefully rinsed
to remove any zoospores that had not encysted on the membrane surface.
The insert was then placed in a second well containing only water or
water plus isoflavonoids. To compare how gravity might influence the
chemotropic response of hyphae, the zoospores were also placed in the
cell-culture insert, and the agarose containing the isoflavone
attractant was added directly to the cell-culture well.
To test the response of hyphae to a soft, smooth surface, a drop of
Vitrogen 100 (Collagen Corp., Palo Alto, CA) was applied to one side of
the PET membrane on the cell-insert cup and cross-linked by incubating
in the presence of NH4OH vapor in a desiccation chamber for 12 h. The proteinaceous surface was rinsed briefly in
distilled water and a 200-µL drop of 1 µM genistein was
applied to the inner chamber of the cup. The cell-culture insert was
then placed in a sample well containing swimming zoospores. After
4 h the cell-culture insert was removed and germinating hyphae on the membrane were fixed and prepared for scanning electron microscopy as described below.
To test the response of hyphae to a smooth, impermeable surface, a
piece of clear plastic wrap (Dow Chemical, Indianapolis, IN) was glued
to the lower end of the cell-culture insert. The cell-culture insert
was inverted and a 30-µL drop of 20 µM daidzein or
genistein was placed on the membrane surface. The cell-culture insert
was quickly turned right-side up again and placed in a well containing
1 mL of swimming zoospores (1 × 106).
Encysted zoospores were left undisturbed for 40 min because initial
experiments confirmed the earlier observations by Donaldson and Deacon
(1992) that further mechanical perturbation of newly formed cysts
delayed germination of hyphae. The insert was then removed, rinsed free
of swimming zoospores, and transferred to a new well containing water
or water plus isoflavones. After 4 h cells were fixed and prepared
for electron microscopy.
Effect of Isoflavones on Cyst Germination
A cell-culture insert with affixed clear plastic wrap membrane was
placed in a sample well containing swimming zoospores. Stock solutions
of daidzein and genistein were added to the sample well to adjust the
final isoflavone concentrations to 1 or 20 µM.
CaCl2 (10 mM final concentration) was
used to induce encystment of all zoospores (Griffith et al., 1988).
After 2 h the cells on the plastic membrane were fixed with 2%
glutaraldehyde in 0.05 M
Na2HPO4, pH 7.2. Germination, hyphal germling length, and the percentage of hyphae with
appressoria were counted by surveying several microscopic fields. Each
experiment was repeated at least four times with both strains.
Fixation and Microscopy
For scanning electron microscopy, germinating cysts were fixed on
membranes for 1 h in 2% glutaraldehyde in 0.05 M
Na2HPO4, pH 7.2, and then
rinsed in buffer. Some samples were postfixed for 2 h in 1%
OsO4 in 0.05 M
Na2HPO4 buffer, pH.7.2, and
rinsed twice with buffer. After dehydration in a graded ethanol series, the zoospores were critical-point dried, mounted on aluminum stubs, and
coated with 10 nm of gold-palladium using a sputter coater (Polaron,
Milton Keynes, UK). The zoospores were viewed with a S-2700
scanning electron microscope (Hitachi, Tokyo, Japan).
Hyphae that had penetrated the PET membrane were photographed by light
microscopy 16 h after encystment by adjusting the plane of focus
to distances progressively closer to the membrane surface. Visualization of the hyphal threads was improved by prior addition of
0.1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazollium bromide
in 1 mM KH2PO4,
pH 7.0.
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RESULTS |
When a capillary tube containing 20 µM daidzein or
genistein was introduced to the chemotaxis chamber and left
undisturbed, zoospores rapidly plugged the capillary tube and others
encysted around the mouth of the tube. Some zoospores that had encysted farthest from the capillary tube opening germinated and grew away from
the capillary tube before turning in the direction of the isoflavone
source (Fig. 1). Similarly, when a
soybean root was introduced to swimming zoospores, encystment occurred
both on and adjacent to the root tip. Cysts adjacent to the root
germinated and grew toward the root surface (not shown). To confirm
that the chemotropic signal was an isoflavone and was not derived from adjacent germinating cysts, zoospores were induced to encyst randomly, then a soybean root tip or a capillary tube containing genistein was
introduced into the chemotaxis chamber after cyst germination had
occurred. Visual inspection of hyphae indicated that both encystment
and the initial direction of hyphal growth were random. The tropic
response of the hyphae toward the root was significant at distances up
to about 300 µM from the root surface (Fig.
2). The tropic response of hyphae toward
genistein was significant at distances up to 750 µM from
the mouth of the capillary tube (Fig. 3).
Hyphae behaved in a similar manner in reorientation experiments using
daidzein (not shown).
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| Figure 1.
Chemotropism of hyphae toward a capillary tube
containing genistein (magnification ×165). Zoospores encysted
around the capillary tube and the majority of cysts produced hyphae
that grew in the direction of the source. Hyphae farthest from the
source changed direction to grow toward the capillary tube.
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| Figure 2.
Chemotropic growth of hyphae toward soybean root
tips. Zoospores were induced to encyst in a random orientation.
Approximately 3 h after the introduction of the root tip into the
chemotaxis chamber, the angles of hyphal tips were measured relative to
a line drawn perpendicular to the root surface.  , Cosine of
growth angle; thick line, cosines averaged over a 200-µm window at
20-µm increments; thin lines, upper and lower 95% significance
limits for cosine means calculated using a t test. The
number of data points in each window ranged from 38 to 74. Chemotropism
was considered significant when the lower confidence limit was greater
than 0.
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| Figure 3.
Chemotropic growth of hyphae toward capillary
tubes containing 20 µM genistein. Zoospores were induced
to encyst in a random orientation. Approximately 3 h after the
introduction of the capillary into the chemotaxis chamber, the angles
of hyphal tips were measured relative to a line drawn from the
counterpoint of the mouth of the capillary.  , Cosine of
growth angle; thick line, cosines averaged over a 200-µm window at
20-µm increments; thin line, upper and lower 95% significance limits
for cosine means calculated using a t test. The number
of data points in each window ranged from 30 to 66. Chemotropism was
considered significant when the lower confidence limit was greater than
0.
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To determine the effect of isoflavones on newly formed cysts, zoospores
were induced to encyst on clear plastic wrap membranes glued to
cell-culture inserts in the presence of varying levels of isoflavones.
After 2 h, germination in both strains was 100% in the presence
of 0, 1, or 20 µM levels of daidzein and genistein for
both strains there were no significant differences in the length of the
germinating hyphae or the percentage of hyphae that had formed
appressoria (Table I). The wide variation
in the length of the germlings appeared to be dependent on whether the
germinating cyst immediately produced an appressorium or continued to
grow along the membrane surface.
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Table I.
Effect of isoflavones on initial growth of
germinating cysts
Data from a typical experiment are shown. Germination in all treatment
groups at 2 h was 100%. Differences in length of the hyphae and
cyst or in the percentage of hyphae forming appressoria are not
significant.
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To determine how germinating hyphae responded to tactile and chemical
signals, zoospores were induced to encyst on the porous PET membranes
of cell-culture inserts by adding isoflavones to the adjacent or
opposite side of the PET membrane. In the presence of isoflavones,
hyphae germinated and grew along the membrane surface until they came
in contact with a pore (Fig. 4). The
hyphae grew through the pores and emerged on the other side of the
membrane. Hyphae emerging from the pores grew away from the membrane
rather than remaining in contact with the membrane surface (Fig.
5).
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| Figure 4.
Hyphal germlings growing along and penetrating
pores of a PET membrane. Zoospores were induced to encyst on the
membrane by application of an agar plug containing isoflavones to the
opposite side of the membrane. Germinating hyphae penetrated pores
detected by the hyphal tip. The distance indicated is the distance
between two hatch marks.
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| Figure 5.
Hyphae growing away from a membrane after having
penetrated a pore (magnification ×27). Zoospores were induced
to encyst on a PET membrane. Hyphae that had successfully penetrated
through pores were visualized by staining with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazollium bromide. The
plane of focus in each picture (A-D) is progressively closer to the
membrane surface and is slightly above the membrane in D. Arrows point
to hyphae that are more visible in frames A, B, or C, indicating that
they must be growing away from the membrane surface (D).
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Several attempts were made to maintain an unequal distribution of
isoflavones across the porous membrane to try to change the initial
tactile responses of the hyphae. For these experiments we placed an
agar plug containing isoflavones on the same or the opposite side of
the membrane as the encysted zoospores. In no case were we able to
induce the hyphae to emerge from the cyst and grow away from the
membrane surface (not shown); nor were we able to inhibit the hyphae
from growing through the membrane pores.
Zoospores that were induced to encyst on a smooth surface consisting of
cross-linked collagen overlying the PET membrane germinated and
immediately penetrated the collagen layer. The hyphae grew along the
PET membrane a short distance before forming an appressorial structure,
possibly over a membrane pore (Fig. 6).
Zoospores that were induced to encyst on a smooth template of clear
plastic wrap germinated and produced multiple appressoria (Fig.
7) in what appeared to be successive
attempts to penetrate the membrane surface. None of the hyphae were
observed to grow away from the template surface. In chemotropism assays
the majority of the zoospores encysted on the coverslip and also tended
to grow along the surface rather than away from it.
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| Figure 6.
Germination of cysts on a surface of cross-linked
collagen overlying a PET membrane. Zoospores were induced to encyst on
the collagen by application of an agar plug containing isoflavones to
the opposite side of the membrane. Germinating hyphae penetrated the
collagen layer and subsequently formed appressorial structures
that are believed to be located over membrane pores. The distance
indicated is the distance between two hatch marks.
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| Figure 7.
Multiple appressoria were produced by a
germinating hyphae on clear plastic wrap (magnification ×1000).
Zoospores were induced to encyst on a smooth surface. The hyphal
germlings produced multiple appressoria as they grew along the
surface.
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DISCUSSION |
Zoospores that encyst adjacent to the root tend to germinate and
grow toward the root surface (Zentmyer, 1961; Musgrave et al., 1977;
Manavathu and Thomas, 1985; Jones et al., 1991). Here we have shown
that when P. sojae zoospores have been induced to encyst and
germinate before the introduction of a soybean root, the hyphal tips
closest to the root grew chemotropically toward the root surface.
Because the hyphal tips from germinating zoospores are also
chemotropically attracted to a capillary tube containing genistein, the
release of isoflavones from the root tips (Graham, 1991) may function
as a chemotropic signal for P. sojae hyphae. Thus, both
P. sojae zoospores (Morris and Ward, 1992) and hyphal germlings respond to host signals. The chemotropic response of hyphae
to daidzein and genistein is significant, because in our laboratory
experiments, many zoospores encysted before reaching the root. In the
soil zoospores that encyst adjacent to the root can produce hyphae that
can use an isoflavone gradient to reorient in the direction of a
growing root tip. Although genistein is more potent than daidzein in
chemotaxis assays (Tyler et al., 1996) for these isolates, daidzein is
the most abundant isoflavone excreted into the rhizosphere by root tips
and may be the most important isoflavone used by P. sojae
zoospores and germlings to locate soybean roots.
These experiments do not rule out the possibility that hyphal tips of
P. sojae also respond to an attractant from adjacent masses
of zoospores, such as those clustered around a root (Reid et al.,
1995). However, because the zoospores encysted without clustering in
the chemotaxis chamber, and the direction of initial hyphal growth was
fixed before introduction of the root or capillary tube, we observed
that an autoaggregation signal could not account for the tropic
response of hyphae to roots or isoflavones. The isoflavones daidzein
and genistein function as chemical signals directing several key steps
in the early stages of the infection response: (a) They mediate
chemotaxis of swimming zoospores toward the root tips (Morris and Ward,
1992), where most of the isoflavones are exuded by the root (Graham,
1991). (b) Sudden exposure of zoospores to elevated levels of
isoflavones was effective in inducing the encystment of zoospores on
artificial surfaces such as a capillary tube or plastic membrane
(Morris and Ward, 1992; this paper). (c) Isoflavones induce
chemotropic growth of germlings toward the roots (this paper).
At elevated concentrations, genistein (concentration that
results in 50% inhibition of growth = 150 µM) but not
daidzein appears to function as a phytoalexin (Rivera-Vargas et al.,
1993), and Vedenyalpina et al. (1993) reported that at lower
levels (11 µM), genistein altered the pattern of mycelial
branching. Genistein acts to inhibit Tyr kinase activity (inhibitor
concentration for 50% displacement = 3-50 µM;
Akiyama et al., 1987), so inhibition at very high levels of genistein
might be expected unless this pathogen has also evolved to become more
tolerant. Isoflavones are stored in the endosperm tissue as malonylated
and glucosylated compounds (Graham, 1991; Morris et al., 1991),
but there is no evidence that the localized concentrations of these
conjugates contribute to either cultivar-specific resistance of
soybeans to P. sojae or nonspecific age-related resistance
in mature tissues such as leaves (Morris et al., 1991). In
chemotactic assays, zoospores are strongly attracted to the rhizosphere
surrounding the root cap and the area of the root immediately behind
the meristem. Graham (1991) estimated that in this region of the
rhizosphere, concentrations of genistein and its conjugates would not
exceed 2 µM. However, in results reported here,
germination of cysts and initial hyphal elongation were not inhibited
by 20 µM daidzein or genistein. Thus, it seems unlikely
that in vivo levels of these isoflavones in the roots are sufficient to
restrict the initial growth of the pathogen.
The ability of hyphal germlings to respond to surface topography has
been described for both plant and animal pathogens (Allen et al., 1991;
Read et al., 1992; Gow et al., 1994). Surface cues such as pores,
grooves, and ridges are primary determinants of hyphal growth on both
their hosts and on artificial templates. In experiments reported here,
hyphae penetrated pores on the membrane surface or, in the absence of
pores, formed appressoria to attempt the penetration of plastic
membranes or glass surfaces. Germinating hyphae typically infect
soybean hypocotyl tissue by penetrating between the anticlinal walls of
epidermal cells (Stössel et al., 1980; Ward et al., 1989).
The preference for this site may be attributable to the hyphae
detecting a concentration gradient of plant metabolites at this site or
its ability to recognize grooves and ridges on the host surface.
Isoflavones appear not to be necessary to induce appressorial
formation, because zoospores that encysted on clear plastic wrap
germinated and produced appressoria in the presence and absence of
isoflavones. Although the isoflavones stimulated hyphal germination and
chemotropic growth, they would not induce the hyphal tip to grow away
from the membrane surface. Thus, the thigmotropic response appears to
be a more important determinant of the direction of hyphal growth in
germinating cysts. Nevertheless, chemotropic orientation of hyphae on
the coverslip of the chemotaxis chamber was still observed in response
to an isoflavone gradient. The initial thigmotropic response of
P. sojae hyphae to surfaces is consistent with previous
accounts of the importance of surface features for both plant and
animal fungal pathogens. In contrast to the growth habit of
Candida albicans, in which the hyphae continued to be
appressed to the membrane surface after emergence on the opposite side
of the membrane (Gow et al., 1994), the hyphae of P. sojae
grew away from the membrane surface into the aqueous medium. Because
this behavior was not observed on smooth, impermeable templates despite
the production of multiple appressoria, the process of successful
penetration could trigger a new developmental response that might be
similar to what happens during the normal course of infection (Ward et al., 1989).
In summary, we have shown that the chemotropic response of germinating
hyphae toward roots may be explained by their ability to respond to
soybean isoflavones. Isoflavones in the range of concentrations likely
to be present in the rhizosphere are not toxic to zoospores or
germinating hyphae. However, the tactile response of hyphae appears to
be a more important determinant of early hyphal growth than the
isoflavones, since germinating hyphae could not be induced to grow away
from the substrate on which they had encysted.
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FOOTNOTES |
1
This work was supported by U.S. Department of
Agriculture grant no. 94-37303-0700 to P.F.M. and B.M.T., and by
National Science Foundation equipment grants BIR-9009697 and
BIR-9249275.
*
Corresponding author; e-mail pmorris{at}bgnet.bgsu.edu; fax
1-419-372-2024.
Received February 2, 1998;
accepted May 12, 1998.
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ABBREVIATIONS |
Abbreviation:
PET, polyethylene terephthalate.
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ACKNOWLEDGMENTS |
We thank Dan Schwab and Carol Heckman for their assistance with
scanning microscopy.
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Top
Abstract
Introduction