Plant Physiol. (1999) 119: 795-804
Biochemical Analysis of Plant Protection Afforded by a
Nonpathogenic Endophytic Mutant of Colletotrichum
magna1
Regina S. Redman,
Stanley Freeman,
David R. Clifton,
Jed Morrel,
Gayle Brown, and
Rusty J. Rodriguez*
Western Fisheries Research Center, Biological Resources Division,
United States Geological Survey, 6505 N.E. 65th Street, Seattle,
Washington 98115 (R.S.R., D.R.C., J.M., G.B., R.J.R.); Department of
Botany, University of Washington, Seattle, Washington 98195-5325
(R.S.R., R.J.R.); and Department of Plant Pathology, The Volcani
Center, Bet Dagan 50250, Israel (S.F.)
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ABSTRACT |
A nonpathogenic mutant of
Colletotrichum magna (path-1) was previously shown to
protect watermelon (Citrullus lanatus) and cucumber
(Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was
observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) "plant-defense" response were investigated in
anthracnose-resistant and -susceptible cultivars of cucurbit seedlings
exposed to four treatments: (1) water (control), (2) path-1 conidia,
(3) wild-type conidia, and (4) challenge conditions (inoculation into
path-1 conidia for 48 h and then exposure to wild-type conidia).
Collectively, these analyses indicated that disease protection in
path-1-colonized plants was correlated with the ability of these plants
to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone.
Watermelon plants colonized with path-1 were also protected against
disease caused by Colletotrichum
orbiculare and Fusarium oxysporum. A
model based on the kinetics of plant-defense activation is presented to
explain the mechanism of path-1-conferred disease protection.
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INTRODUCTION |
The mechanism by which fungi from the genus
Colletotrichum cause disease (anthracnose) is governed by a
series of events that begins with the adhesion of fungal spores to host
surface tissue, followed by spore germination, appressoria formation,
and penetration into the first subcuticular cell (Bailey et al., 1992
).
If a compatible interaction ensues, the pathogen will exhibit
necrotrophic characteristics and quickly disseminate through host
tissues, ultimately resulting in necrotic lesions and, hence, plant
disease. In an incompatible interaction, a rapid, localized collapse of
tissue surrounding the initial infection zone occurs, resulting in
disease resistance. The resistant reaction, designated the
hypersensitive response, is believed to be genetically programmed and
confers disease resistance due to the recognition and interaction of
biochemical components from both the pathogen and host (Flor, 1971;
Keen, 1982, 1986, 1990; Klement, 1982; Kuc, 1990;
Alfano and Collmer, 1996; Hammond-Kosack and Jones, 1996; Jackson and
Taylor, 1996; Knogge, 1996). In compatible interactions, widespread
plant cell death may occur because of the delayed response of the plant
to the presence of the pathogen and the ability of the pathogen to
overcome the plant host-response system (Darvill and Albersheim, 1984;
Davis et al., 1986; Ebel, 1986; Bailey et al., 1992; Jackson and
Taylor, 1996).
Susceptibility or resistance to disease by Colletotrichum
sp. or other plant pathogens appears to follow a common theme involving the temporal and spatial expression of plant-defense components activated by a number of fungal and/or plant metabolites (Anderson, 1978; DeWit et al., 1985; Ebel, 1986; Hamdan and Dixon, 1986; Kombrink
and Hahlbrock, 1986; Anderson, 1988; Cuypers et al., 1988; Dixon and
Lamb, 1990; Bailey et al., 1992; Nicholson and Hammerschmidt, 1992;
Kamoun et al., 1993; Knogge, 1996). Kuc and Strobel (1992) implied that
susceptible cultivars may be manipulated to resist pathogen attack by
altering the timing and magnitude of the defense response.
An additional dimension to incompatible interactions involves the
extent and spatial distribution of the plant-defense response resulting
in a rapid, localized, and/or systemic form of protection. A localized
response occurs around the site of pathogen ingress and protection is
afforded to the plant cells in the surrounding area. Often, a small,
necrotic lesion will be formed as a result of the hypersensitive
response.
Key components postulated to play important roles in localized
resistance include increased activity in peroxidase, deposition of
lignin, and PAL activity. Peroxidase is involved in cross-linking extensin molecules and in the polymerization of hydroxycinnamyl alcohols to form lignin (Hammerschmidt et al., 1982; Irving and Kuc,
1990; Dalisay and Kuc, 1995a, 1995b). Increased lignin deposition is
believed to play a role in barricading the pathogen from invading the
plant through physical exclusion (Hammerschmidt and Kuc, 1982a, 1982b;
Hammerschmidt et al., 1984; Nicholson and Hammerschmidt, 1992;
Hammond-Kosack and Jones, 1996). PAL is responsible for the conversion
of Phe to trans-cinnamic acid, a key intermediate in the
pathway for production of lignin and SA. Depending on the plant species
PAL may play a role in either localized resistance or SAR
(Hammond-Kosack and Jones, 1996; Ryals, et al., 1996). It is believed
that PAL activity is correlated with the synthesis of phenols in
response to pathogen infection (Nicholson and Hammerschmidt, 1992).
Several factors are postulated to be involved in the SAR pathway,
including SA, benzoic acid, and jasmonic acid. Upon conversion of Phe
into trans-cinnamic acid via PAL, trans-cinnamic
acid is then converted through either the oxidative or nonoxidative
pathway into benzoic acid. The conversion of benzoic acid into SA is
catalyzed by benzoic acid 3-hydroxylase, which is believed to be the
rate-determining step in SA synthesis. SA has been recognized for some
time as a response to pathogen infection and, at present, is the only plant-derived substance shown to induce SAR (Ryals et al., 1996).
Recently, a nonpathogenic fungal mutant (path-1) of
Colletotrichum magna, the causal agent of anthracnose in
cucurbits, was isolated (Freeman and Rodriguez, 1992). path-1 was no
longer capable of eliciting disease symptoms but retained the ability
to adhere, infect, and disseminate through plant tissue (Freeman and
Rodriguez, 1993). Cucurbit plants colonized by the path-1 mutant showed
no disease symptoms when exposed to lethal concentrations of the wild-type (L2.5) C. magna or Fusarium oxysporum
f. sp. niveum (Freeman and Rodriguez, 1993). Data presented
in this study indicate that a hypersensitive response did not
occur in plants colonized with path-1 and that protection was localized
and tissue specific. Biochemical analyses (peroxidase, PAL, SA, and
lignin deposition) indicated that resistance to disease correlated with
the ability of path-1-colonized plants to respond more quickly to the
pathogen. We propose a working model to explain the basis of path-1
protection and how this system may be useful for biological control of
fungal disease.
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MATERIALS AND METHODS |
Fungal Isolates and Plant Cultivars
The pathogenic wild-type isolate (L2.5) of Colletotrichum
magna (Jenkins and Winstead, 1964) was obtained from S. Brown and O.C. Yoder (Cornell University, Ithaca, NY). The nonpathogenic mutant
(path-1) of C. magna was isolated following UV mutagenesis of isolate L2.5 (Freeman and Rodriguez, 1992). Colletotrichum orbiculare (isolate 254) was obtained from the
Colletotrichum Culture Collection (D. TeBeest, University of
Arkansas, Fayetteville). Fusarium oxysporum isolate f. sp.
niveum was obtained from C. Kistler (Florida State
University, Tallahassee). Fungi were cultured in either liquid or solid
modified Mather's medium (Tu, 1985) as previously described (Rodriguez
and Owen, 1992). Anthracnose-susceptible cultivars of watermelon
(Citrullus lanatus cv Sugar Baby) and cucumber
(Cucumis sativus cv Marketmore) and resistant cultivars of
watermelon (cv Jubilee) and cucumber (cv Pepino) were from Petoseed
(Woodland, CA). All plant assays were carried out in growth
chambers operated at 95% RH and 12-h light regimes at 22°C.
Experimental Treatments and Design
Plant bioassays were performed independently a minimum of three
times for each cucurbit variety, treatment, and time tested. Each
sampling consisted of 30 cucurbit seedlings. Similar results were
obtained in each replicate. One representative replicate is presented
for the bioassay and all results presented were taken from the same
replicate sample.
Plant bioassays and biochemical analysis were conducted on cucurbit
plants exposed to four treatments: treatment 1, water (control);
treatment 2, mutant (path-1) conidia; treatment 3, wild-type (L2.5)
conidia; and treatment 4, challenge conditions (inoculation into path-1
conidia for 48 h followed by exposure to lethal concentrations of
a virulent wild-type pathogen). All assays were conducted with a
minimum of 30 plant seedlings of equal size and age and were repeated a
minimum of three times.
There were three separate challenge treatments: path-1-colonized plants
exposed to 1.0 to 2.0 × 106 conidia
mL
1 C. magna isolate L2.5 (challenge
A), C. orbiculare isolate 254 (challenge B), and F. oxysporum isolate f. sp. niveum (challenge C). Although
it is not mentioned in each instance, all studies conducted contained
an additional control (designated treatment 7 in Table I). Cucurbit
plants were exposed to water for 48 h and then exposed to lethal
concentrations of C. magna isolate L2.5, C. orbiculare isolate 254, or F. oxysporum isolate f. sp. niveum as the treatment 7 control for challenge A, B, and C,
respectively. This additional control was included to ensure that
wild-type concentrations were lethal and produced 100% mortality in
all susceptible cucurbit plants tested. In addition, treatment 7 was included as a control to ensure that age effects in cucurbit seedlings was not a contributing factor to the decrease in mortality observed in
challenge treatments.
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Table I.
Mortality and colonization in
anthracnose-susceptible cucurbit cultivars
The data presented represent the mortality (assessed at d 5) and
colonization (assessed at d 3) of 30 anthracnose-susceptible watermelon
(cv Sugar Baby) and cucumber (cv Marketmore) seedlings exposed to
various treatments. The assays were repeated a minimum of three times.
The only variations between experiments occurred with challenge
conditions B and C.
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Inoculation Procedures
Conidial suspensions of C. magna, C. orbiculare, and F. oxysporum isolates were obtained
using standard procedures (Freeman and Rodriguez, 1992). Inoculation
procedures were optimized to maximize susceptible and resistant plant
reactions (data not shown). Utilizing conidial concentrations of 1.0 to
2.0 × 106 conidia
mL1, cvs Sugar Baby and Marketmore were 100%
susceptible to anthracnose elicited by L2.5. Conversely, cvs
Jubilee and Pepino were 100% resistant to anthracnose when exposed to
L2.5. Root and lower stem inoculation and incubation techniques were
identical to those previously described (Freeman and Rodriguez,
1992). The cotyledons were inoculated by briefly submerging them in
spore suspensions ranging from 1.0 to 2.0 × 106 conidia mL1 followed
by incubation in chambers with 95% RH.
Recovering Fungi from Plant Tissue
Colonization of plants by fungal isolates was determined by
surface-sterilization of plants and outgrowth of fungi on Mather's medium and was expressed as the percentage of plants colonized. Plants
were submerged in 2.0% (v/v) sodium hypochlorite for 20 to 30 min with
moderate agitation and then thoroughly rinsed with 10 to 20 volumes of
sterile distilled water. Plants were cut, using an aseptic technique,
into sections representing the lower, middle, and upper stem sections,
the roots, and the cotyledons. The sections were plated onto Mather's
medium supplemented with 100 µg mL1
ampicillin and incubated at room temperature under cool fluorescent lights for 5 to 7 d. Identification of fungi was verified after conidiation by microscopic analysis.
Fungal Inhibition Assays
In vitro inhibition assays were performed by inoculating both
mycelial plugs and spore suspensions (1.0 to 3.0 × 106 spores mL1) of path-1
and L2.5 on Mather's medium (Freeman and Rodriguez, 1992) with a
separation distance of 1.5 cm. Inoculated replicate plates were
incubated at 25°C in the dark and under cool fluorescent lights.
Growth inhibition was assessed by the occurrence of clear zones at the
interface between colonies.
To determine whether path-1 was producing growth inhibitors in vivo,
approximately 100 mg from each of the 10 path-1-colonized plant stems
was ground with a tissue homogenizer (Tekmar, Cincinnati, OH) and
resuspended in an equivalent volume of a 0.01 M Tris-HCl buffer (pH 7.0). Growth inhibition was measured by mixing ratios of
plant extract to water of 1:1 to 1:100 (v/v), spotting 50 µL on
Mather's medium, and inoculating with single mycelial plugs of L2.5
adjacent to the plant extract.
Peroxidase and PAL Activities
Peroxidase and PAL assays were performed independently a minimum
of three times for each cucurbit variety, treatment, and time tested.
Similar trends in enzyme activities were observed between replicate
samples. One representative replicate is presented for peroxidase and
PAL activities and all biochemical results presented were taken from
the same replicate samples.
Time-course studies during a 2-week span indicated that high levels of
peroxidase and PAL activities were detected within the first 4 d
(data not shown). All subsequent studies (with the exception of lignin
deposition) presented in this paper were conducted during a period of
4 d.
The stems of 30 cucurbit seedlings of similar size and age were ground
in liquid nitrogen, and 100 mg of ground tissue was placed into 1 mL of
0.01 M sodium phosphate buffer (pH 6.0). After the sample
was centrifuged (10,000g for 5 min at 4°C), 5 to 200 µL
of the supernatant was used to quantify protein and determine peroxidase and PAL activities. Protein content was quantified using BCA
protein assay reagents (Pierce). Peroxidase activity was determined
with 0.25% (v/v) guaiacol and 0.3% (v/v)
H2O2 in 1 mL of 0.01 M sodium phosphate buffer (pH 6.0) (Hammerschmidt et al., 1982). The reaction was initiated by the addition of 5 µL of
the supernatant extract to 995 µL of the reaction mixture. Activity was measured as a change in the
A470 and expressed as the change in
absorbance per minute per microgram of protein.
The reaction mixture for PAL activity consisted of 6 µM
L-Phe, 0.5 M Tris-HCl buffer (pH 8.0), and 200 µL of plant extract. After 60 min at 37°C, the reaction was
terminated by the addition of 0.05 mL of 5 N HCl. PAL
activity was assessed by measuring the amount of cinnamic acid produced
at 290 nm and is expressed as micrograms of cinnamic acid per microgram
of protein, similar to the procedure described by Beaudoin-Eagan and
Thorpe (1985).
Lignin Deposition
Lignin-deposition assays were performed independently a minimum of
three times for each cucurbit variety, treatment, and time tested. A
minimum of 30 cucurbit stems was assayed for each treatment and time
tested. Similar trends in lignin deposition were observed between
replicate samples. One representative replicate is presented for lignin
deposition and all biochemical results presented were taken from the
same replicate sample.
The pg-HCl test denoting lignin deposition was performed using a
modification of the "phloroglucinol" method described by Gurr
(1965). Watermelon seedlings were decolorized in 70% (v/v) ethanol for
24 to 48 h, washed with distilled water, and exposed to 1% (w/v)
phloroglucinol (Sigma) for 1 to 2 h. The seedlings were then
exposed to 6 M HCl until a red color developed, which denoted lignin deposition. The level of lignin deposition was qualitatively measured using water-inoculated plants as the negative background control and ranged from absent, basal, low, moderately high,
to high.
SA Accumulation
SA assays were performed independently a minimum of three times
for each cucurbit variety, treatment, and time tested. Ten cucurbit
stems were assayed for each treatment and time tested. The same results
were observed between replicate samples.
Stem sections of 10 cucurbit seedlings of similar size and age were
ground in liquid nitrogen and resuspended in 5 mL of water. After the
sample was centrifuged (5000g for 10 min at 4°C), an equal
volume of acetone and chloroform was mixed with the supernatant and
centrifuged (5000g for 10 min at 4°C), and the organic
phase was passed through anhydrous sodium sulfate to remove any
residual water. The organic phase was evaporated and resuspended in 0.5 mL of acetone, and 0.5 to 25 µL was spotted on TLC silica-gel plates
(Whatman). TLC plates were chromatographed in a mixture of
toluene:ethyl acetate:glacial acetic acid (80:10:10, v/v). The TLC
plates were dried, sprayed with 20% (w/v) aluminum chloride in 95%
(v/v) ethanol, and baked at 95°C for 10 min, and SA was visualized
under long-wave UV. SA standard (1-100 ng, Sigma) was spotted onto the
TLC plates as a positive control (Dr. N. Keller, personal
communication).
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RESULTS |
Colonization and Protection of Cucurbit Roots and Stems
Previously, we demonstrated that the nonpathogenic mutant of
C. magna (path-1) was capable of colonizing stem
tissue and protecting watermelon seedlings from disease caused by
wild-type C. magna (isolate L2.5) and by the wilt pathogen
F. oxysporum f. sp. niveum (Freeman and
Rodriguez, 1993). Although path-1 was capable of protecting plants
against these pathogens, it was not known whether protection was
afforded toward aggressive foliar pathogens. To test this,
path-1-colonized plants were challenged with lethal conidial
concentrations of C. orbiculare, an aggressive foliar
pathogen of cucurbits. The data from several plant inoculation and
challenge experiments indicated that path-1 was capable of protecting
plants against virulent C. orbiculare (Table
I).
To determine whether the protection to the various pathogens was
systemic, the roots and stems of seedlings were colonized with path-1
for 48 h, and then the cotyledons were inoculated with lethal
concentrations of C. magna L2.5 (challenge A), C. orbiculare 254 (challenge B), or F. oxysporum f. sp.
niveum (challenge C; Table I). In all of these experiments,
100% plant mortality was observed. In surface-sterilization
experiments path-1 was present in the root and stem sections but absent
in the cotyledons of all cucurbit plants tested (Table I). These data
indicated that path-1 induced a localized form of plant protection.
To determine whether path-1 protection was plant-tissue specific,
cotyledons were colonized with path-1 for 48 h, and then inoculated with lethal concentrations of C. magna L2.5
(challenge A), C. orbiculare 254 (challenge B), or F. oxysporum f. sp. niveum (challenge C). Visible signs of
necrosis and plant death were absent in cotyledons exposed to path-1.
In contrast, 100% plant mortality was observed in cotyledons exposed
to challenge A, B, and C treatments. In surface-sterilization
experiments path-1 was absent in the cotyledons of all cucurbit plants
tested (Table I). These data indicated that path-1 was able to colonize
the roots and stems but not the cotyledons of cucurbits and, therefore, affords tissue-specific plant protection from disease.
To determine whether L2.5 was able to disseminate through host tissue
colonized by path-1, genetically marked strains of L2.5 resistant to
either benlate (wtB) or chlorate (wtC) were derived (Freeman and
Rodriguez, 1993). The marked strains were not recovered from
path-1-colonized plant stems after 1 week of exposure. However, both
wtB and wtC were recovered from plants that had not been colonized with
path-1. Plants inoculated with either wtB or wtC expressed pathological
characteristics similar to plants inoculated with L2.5 (100%
mortality, data not shown).
Fungal-Inhibition Assays
We were interested in determining whether path-1 was directly
inhibiting the growth of L2.5 in vitro and/or in plant tissues. The in
vitro assays involved screening for inhibition of L2.5 by path-1 after
co-inoculation of these fungi on Mather's medium. Inhibition in plant
tissues was determined by extracting total fluids from plants colonized
with path-1 and performing liquid-inhibition assays with L2.5. Neither
of these assays revealed any inhibition of L2.5 (data not shown),
indicating that path-1 was not producing fungal inhibitors.
Lignin Deposition
The anthracnose-susceptible watermelon cv Sugar Baby and cucumber
cv Marketmore and the anthracnose-resistant watermelon cv Jubilee and
cucumber cv Pepino were assessed for lignin deposition during a 5-d
period. Plants were exposed to treatments 1 through 4 (see ``Materials and Methods''). Pg-HCl caused intense staining in plant tissue of both susceptible and resistant cultivars exposed to L2.5 (Table
II). The highest levels of lignin
deposition occurred in resistant cultivars exposed to L2.5 and in
challenge-A treatments for susceptible cultivars. When path-1-colonized
plants were challenged with L2.5 (at d 2), lignin deposition increased
rapidly within a 24-h period to levels that either nearly paralleled or
exceeded those of resistant and susceptible plants inoculated for
72 h with L2.5, respectively (Tables II and
III). No detectable pg-HCl staining was
observed in the stems of water-exposed plants and little to no staining was detected in plants exposed to path-1 in either resistant or susceptible cultivars. Although the timing of lignin deposition was
similar, the kinetics of challenge-A conditions were different in
resistant versus susceptible cultivars.
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Table II.
Lignin deposition in cucurbits
The data presented represent the average levels of lignin deposition
from 30 pooled plant stems of equal size and age (variations between
plant stems were negligible). Lignin deposition was measured on a
qualitative basis and ranged from absent ( ), basal (+), low (++),
moderate (+++), moderately high (++++), to high (+++++) levels.
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Table III.
Enzyme activities and lignin deposition after 24, 48, 72, and 96 h of exposure to C. magna wild-type L2.5 in susceptible
cucurbit plant varieties
Peroxidase activity, PAL activity, and lignin deposition on
anthracnose-susceptible cultivars after exposure to the wild-type L2.5
isolate. The data presented were obtained from 30 cucurbit seedling
stems of similar size and age and were processed as required for each
assay. Peroxidase activity was monitored as a change in
A470. PAL activity was monitored at 290 nm.
Lignin deposition was measured on a qualitative basis and ranged from
absent (), basal (+), low (++), moderate (+++), to moderately high
(++++) levels.
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Statistical analysis of the data using a two-factor ANOVA without
replication revealed statistically significant differences among the
treatments and time points in cvs Sugar Baby (treatments: F3,12 = 6.073, P = 0.009; time points:
F4,12 = 4.095, P = 0.026), Marketmore
(treatments: F3,12 = 5.776, P = 0.011;
time points: F4,12 = 4.254, P = 0.023), Jubilee (treatments: F3,12 = 11.543, P = 0.001; time points: F4,12 = 4.826, P = 0.015), and Pepino (treatments:
F3,12 = 6.738, P = 0.00; time points:
F4,12 = 4.673, P = 0.017; Zar, 1984).
Collectively, these data indicate that in challenge-A treatments lignin
deposition increased rapidly within a 24-h period post challenge to
levels that paralleled and/or exceeded those of plants inoculated with
L2.5 alone (Tables II and III).
Peroxidase Activity
Peroxidase activity was measured in cucurbit plant stems exposed
to four different treatments (see ``Materials and Methods''). A
summary of peroxidase activity in anthracnose-susceptible watermelon cv
Sugar Baby and cucumber cv Marketmore and anthracnose-resistant
watermelon cv Jubilee and cucumber cv Pepino during a 4-d period
postinoculation is presented in Figure 1.
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| Figure 1.
Peroxidase activity in plant stems of
anthracnose-susceptible (A and B) and anthracnose-resistant (D and C)
watermelon (A and C) and cucumber (B and D) cultivars were monitored
for 4 d in plants exposed to the four different treatments. The
arrows on the graphs indicate the time at which path-1-colonized plants
were exposed to the wild type. Each point represents 30 plants that
were combined for the analysis. Peroxidase activity was monitored as a
change in A470 and is reported as absorbance
per minute per microgram of protein. Statistical analysis (ANOVA)
revealed significant differences (P < 0.05) between the
treatments among time points for all varieties. Susceptible and
resistant plants in treatments 1, 2, and 4 were healthy throughout the
experiment and identical in appearance. Susceptible plants in treatment
3 expressed disease symptoms on d 3 and were dead on d 5. Resistant
plants did not show disease symptoms with any of the treatments.
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In susceptible cultivars, a higher level of peroxidase activity was
observed in plants exposed to path-1 compared with the water control
plants, and the highest peroxidase activities were observed in
challenge-A treatment plants. In addition, the kinetics of peroxidase
activity in susceptible plants exposed to challenge-A treatments was
different from plants inoculated with L2.5 alone. During the period
before challenge inoculation (d 1 and 2), plants exposed to L2.5
expressed peroxidase activities that were slightly lower (Fig. 1A) or
paralleled (Fig. 1B) the activities observed in path-1-exposed plants.
When path-1-colonized plants were challenged with L2.5 (at d 2),
peroxidase activity increased rapidly within a 24-h period to levels
that paralleled and then exceeded those of plants inoculated for
72 h with L2.5 (Fig. 1; Table III).
In resistant cultivars plant mortality was not observed in any of the
treatments, and the highest level of peroxidase activity occurred
in plants exposed to the virulent wild type (L2.5). Peroxidase activities in mutant (path-1) exposed plants were only slightly higher
than the water control plants. The activity of peroxidase in
challenge-A treatment plants was slightly lower or nearly paralleled the activities observed in L2.5-exposed plants over time (Fig. 1, C and
D). When path-1-colonized plants were challenged with L2.5 (at d 2),
peroxidase activity increased rapidly within a 24-h period to levels
that nearly paralleled but did not exceed those of plants inoculated
for 72 h with L2.5 (Fig. 1). Although the timing of peroxidase
expression was similar, the kinetics of challenge-A conditions were
different in resistant versus susceptible cultivars.
Statistical analysis of the data using a two-factor ANOVA without
replication revealed statistical differences among the treatments and
among time points sampled in cvs Sugar Baby (treatments:
F3,9 = 6.114, P = 0.015; time points:
F3,9 = 14.933, P = 0.0010), Marketmore (treatments: F3,9 = 10.181, P = 0.003;
time points: F3,9 = 26.323, P = 0.000), Jubilee (treatments: F3,9 = 3.530, P = 0.062; time points: F3,9 = 9.310, P = 0.004), and Pepino (treatments:
F3,9 = 4.474, P = 0.035;
time points: F3,9 = 14.943, P = 0.001;
Zar, 1984). Collectively, these data indicate that the rapid
accumulation of peroxidase activity within a 24-h period in
anthracnose-susceptible Sugar Baby and Marketmore varieties exposed to
challenge-A treatments was statistically significant.
PAL Activity
PAL activity was determined in anthracnose-susceptible watermelon
cv Sugar Baby and cucumber cv Marketmore and anthracnose-resistant watermelon cv Jubilee and cucumber cv Pepino exposed to treatments 1 to
4 during a 4-d period (see ``Materials and Methods''). Initial
studies indicated that the majority of PAL activity was localized in
the lower stem sections of cucurbits (data not included). Plants of
similar size and age were used, and the lower halves of stems from 30 plants were pooled and processed in the same manner as described for the peroxidase activity studies. In susceptible cultivars, a trend was
observed for PAL activity (Fig. 2; Table
III) that was similar to that observed for peroxidase activity (Fig. 1;
Table III). Prior to d 3, the highest level of PAL activity was
observed as a transient response in plants exposed to L2.5, with
significantly lower levels of PAL activity occurring in path-1- and
water-inoculated plants. Within 24 h of exposure to the wild-type
L2.5 isolate (challenge A), a rapid increase in PAL activity was noted
in the path-1-colonized plant and was found to be statistically
significant. PAL activity increased to levels that exceeded those
of plants exposed to L2.5, alone (Fig. 2; Table III). A two-factor
ANOVA without replication revealed statistically significant
differences between the treatments of watermelon cv Sugar Baby
(F3,9=4.536, P = 0.034) and cucumber cv Marketmore (F3,9=3.032, P = 0.086;
Zar, 1984).
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| Figure 2.
PAL activity in lower stem sections of
anthracnose-susceptible watermelon (A) and cucumber (B) cultivars were
monitored for 4 d in plants exposed to the four different
treatments. The arrows indicate the time at which path-1-colonized
plants were exposed to the wild type. PAL activity was monitored at 290 nm and reported as the absorbance per microgram of protein. Statistical
analysis (ANOVA) revealed significant differences in the treatments
among time points for both cultivars (P < 0.05). Plants in
treatments 1, 2, and 4 were healthy throughout the experiment and
identical in appearance. Plants in treatment 3 expressed disease
symptoms on d 3 and were dead on d 5.
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In resistant cultivars PAL activity in plants exposed to the different
treatments was not observed to be significantly different from plants
exposed to the water treatment (data not shown).
Detection of SA Accumulation
SA was not detected in anthracnose-susceptible watermelon cv Sugar
Baby or cucumber cv Marketmore or in anthracnose-resistant watermelon
cv Jubilee or cucumber cv Pepino exposed to treatments 1 to 4 during a
5-d period post inoculation (data not shown). These results
corroborated the plant colonization and protection assay results
(Table I) showing that cotyledons of path-1-colonized plants
were not protected from disease under challenge A conditions, indicating localized and not systemic protection.
| |
DISCUSSION |
The genetic and biochemical factors that allow
Colletotrichum spp. to cause plant disease are unknown. A
variety of factors (toxins, cutinases, chitinases, cellulases, and
phytoalexin demethylases) have been implicated as causal components of
fungal plant disease; however, the exact mechanism by which fungi cause
disease is largely unknown (Ciuffetti et al., 1983; Kolattukudy, 1985;
Dickman and Patil, 1986; Johal and Briggs; 1992). The path-1 mutant of
C. magna provides a useful tool for investigating the basis
of disease expression, plant host response, and protection against
fungal disease. To understand how path-1 was able to grow through host tissue without causing necrosis and protect plants against virulent pathogens, two strategies were followed: (a) inoculation and
colonization assays and (b) biochemical analysis of cellular pathways
correlated with plant defense responses. In each study plants were
tested with each of the four treatments.
Inoculation studies confirmed that plant death was not observed in
path-1-exposed plants and 100% mortality occurred in L2.5-exposed susceptible cultivars (Table I). Furthermore, 100% of the plants exposed to path-1 were colonized in the roots and stems but not in the
cotyledons. To determine whether protection from disease was systemic
or localized and/or tissue specific, the cotyledons or roots and stems
of path-1-colonized plants were exposed to lethal spore concentrations
of C. magna, C. orbiculare, or
F. oxysporum (challenge A, B, or C, respectively). The
mortality in challenged cotyledons was found to be 100%, whereas root
and stem challenge ranged from 0% to 30% (Table I). These results collectively indicated that path-1-colonized plants were afforded tissue-specific, localized plant protection. Induced systemic resistance has been reported in certain cucurbit hosts by prior inoculation with specific concentrations of pathogenic, hypovirulent, and nonpathogenic isolates (Dean and Kuc, 1987; Kuc, 1990). However, fungus-induced systemic resistance is accompanied by some degree of
necrosis in plant tissue, a phenomenon that was not observed with
path-1 (Keen, 1982, 1990). Therefore, we are designating this unique
phenomenon of path-1-conferred resistance as
"endophyte-associated resistance."
Colonization studies indicated that under challenge conditions L2.5 was
not capable of surviving or being recovered in path-1-colonized plants,
and that path-1 did not produce chemicals in vitro or in vivo that
inhibited the growth of L2.5. Since Colletotrichum and
Fusarium spp. have different mechanisms of pathogenesis, it is unlikely that path-1 confers protection by physically occluding these pathogens (Bailey et al., 1992; Alabouvette et al., 1993; Larkin
et al., 1993). Therefore, we conclude that the basis of path-1-induced
protection involves biochemical interactions between path-1 and the
host plant.
Biochemical analyses indicated that, in general, path-1-exposed plants
showed null to low levels of PAL, peroxidase, or lignin activity,
indicating little or no induction of these indicators of the
plant-defense response. In contrast, with the exception of PAL activity
in resistant cultivars, plants exposed to L2.5 displayed a significant
plant-defense response. PAL activity was not detected at significant
levels in resistant cultivars exposed to any of the treatments. These
results corroborate findings from earlier studies conducted on
anthracnose-resistant cucurbits that suggested that plant resistance
was correlated with elevated levels of lignin deposition and peroxidase
activity (Hammerschmidt and Kuc, 1982a, 1982b; Hammerschmidt et al.,
1982). Collectively, these results suggest that PAL does not play as
significant a role as peroxidase activity or lignin deposition in the
cucurbit defense response. Statistical analysis of PAL activity using a two-factor ANOVA indicated that significant differences between the
treatments occurred in anthracnose-susceptible watermelon cv Sugar Baby
(P = 0.034 (Fig. 2). No significant differences were observed in
anthracnose-resistant watermelon cv Jubilee and cucumber cv Pepino
(data not shown). These results suggest that the usefulness of PAL
activity as an indicator of the plant defense response may be cultivar
and/or species specific.
Within 24 h after exposure to virulent pathogens, both
susceptible and resistant path-1-colonized plants exhibited a rapid accumulation of peroxidase and lignin that paralleled and/or exceeded those of plants exposed only to L2.5 for 72 h (Fig. 1; Tables II
and III). These results indicated that path-1-colonized plants were
able to mount a rapid defense response that inevitably resulted in
protection from pathogen ingress and, hence, protection from disease.
The absence of detectable levels of SA (data not shown) in any of the
cultivars corroborated the results from our inoculation studies (Table
I), which indicated that path-1-induced disease protection was not
systemic; it was localized and correlated with the physical presence of
path-1 in plant tissues. In addition, the fact that path-1 did not
colonize or protect cotyledons may indicate some level of tissue
specificity.
The mechanism(s) that allows path-1 to protect plants against disease
appears to involve an interaction between the mutant and the plant
defense system. It has been suggested in several host-pathogen systems
that the most critical component of host resistance involves the timing
and activation of the defense response following recognition of the
pathogen (Madamanchi and Kuc, 1991; Kuc and Strobel, 1992). In view of
the data presented here, we propose a working model to explain the
protection of plants by path-1. This model, designated
endophyte-associated resistance, is based on the fact that path-1
either activates plant defenses to very low levels or primes the
defense system without activation. When path-1-colonized plants are
challenged with virulent fungi, a strong defense response is activated
in less than 24 h. Activation of the defense system may be the
result of a "threshold response" similar to action potentials in
nerve fibers (Stevens, 1979). For nerve fibers to "fire" and
transmit signals, they must receive a stimulus of proper strength and
duration. The stimulus induces a depolymerization of nerve cell
membranes that establishes an electrochemical gradient. If the
electrochemical gradient reaches a certain threshold potential, the
nerve fiber will fire. If the duration or strength of the stimulus is
insufficient to achieve the threshold potential, the nerve fiber will
be excited but will not fire. Perhaps plants do not activate defense
systems until the concentration of fungal metabolites reaches a
threshold to activate the system. path-1 may expose the plant to
concentrations of fungal metabolites slightly below threshold levels,
thus preventing defense system activation. Under challenge conditions,
path-1-colonized plants are exposed to concentrations of fungal
metabolites from the virulent fungus that, through an additive effect,
potentiate the system, and threshold concentrations necessary for
defense system activation are achieved. Disease-resistant cultivars may simply have lower threshold potentials to specific fungal metabolites than do susceptible cultivars.
The development of biological control agents for protecting plants
against fungal root diseases is complicated by the fact that the
organisms of interest must compete in the soil environment (Deacon,
1988). In addition, it is assumed that fungal biological control is
based on the production of antifungal chemicals, mycoparasitism, or
physical occlusion, all of which are sensitive to environmental factors
(Scher and Baker, 1982; Alabouvette et al., 1993; Deacon and Berry,
1993; Larkin et al., 1993; Nielsen and Sorensen, 1997). path-1 avoids
the potential biocontrol problems by colonizing plant tissues and
stimulating the plant to protect itself in a multigenic manner. As a
result, it may be very difficult for a virulent pathogen to overcome
this type of resistance, which allows for the development of long-term
control strategies. Finally, we have conducted in vivo experiments for
the last 6 years (data not shown) that indicate that the path-1
nonpathogenic phenotype is stable and reversion to a pathogenic
phenotype is unlikely because of the pleitrophic nature of the
path-1 mutant (Freeman and Rodriguez, 1993). Collectively, our studies
indicate that the path-1 mutant may result in the development of a
novel, long-term biocontrol strategy for plant protection.
| |
FOOTNOTES |
1
This research was supported in part by a
postdoctoral fellowship grant from the U.S.-Israel Agricultural
Research and Development Fund (BARD) awarded to S.F. Partial support
was also provided by a joint National Science Foundation, Department of
Energy-U.S. Department of Agriculture grant (to R.J.R. as co-private
investigator) and by a BARD grant awarded to R.J.R. and S.F.
*
Corresponding author; e-mail Rusty_Rodriguez{at}usgs.gov; fax
1-206-526-6654.
Received July 20, 1998;
accepted November 4, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ANOVA, analysis of variance.
PAL, Phe
ammonia-lyase.
SA, salicylic acid.
SAR, systemic acquired resistance.
| |
ACKNOWLEDGMENTS |
We would like to thank Judy Ranson, Dr. Jennifer Smith, Dr.
Richard Stange, and Dr. Ray Hammerschmidt for helpful suggestions. We
would also like to thank Dr. Jennifer Lorang for editorial assistance.
| |
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Top
Abstract
Introduction