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Resistance to Azoxystrobin in the Gummy Stem Blight Pathogen Documented in Georgia
Katherine L. Stevenson, David B. Langston, Jr., and Kenneth W. Seebold, Department of Plant Pathology, University of Georgia, Tifton 31793
Corresponding author: Katherine L. Stevenson. ks@uga.edu
Abstract
Gummy stem blight, caused by the fungus Didymella bryoniae, is the
most destructive disease of watermelon in Georgia and in many other watermelon
producing areas of the U.S. The QoI
fungicide azoxystrobin has been
used for gummy stem blight control in Georgia since 1998. As early as 1999,
reduced control of gummy stem blight with azoxystrobin was noted in several
research sites and commercial fields of cucumber and watermelon in Georgia.
Isolates from several of these fields were later confirmed to be resistant to
azoxystrobin. To determine how widespread the resistance problem was in Georgia,
extensive surveys of watermelon and muskmelon fields and transplant houses were
conducted in 2001 and 2002 to determine the frequency of azoxystrobin-resistant
isolates in populations of D. bryoniae. Of the 272 isolates collected in
2001, 247 (91%) were resistant to azoxystrobin. In 2002, 82% of the 170 isolates
collected were resistant to azoxystrobin, and of the 40 isolates collected from
watermelon transplants, all but one were resistant to azoxystrobin, suggesting
that resistant isolates in the field may have originated from seed or
transplants. Georgia melon growers are now advised to use alternative fungicides
that are chemically unrelated to the QoIs for gummy stem blight
control.
Introduction
Fig. 1. A field of watermelons in Georgia severely damaged by gummy stem blight, caused by Didymella bryoniae. |
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Gummy stem blight, caused by the fungus Didymella bryoniae (Auersw.)
Rehm, is the most widespread and destructive disease of watermelon in Georgia
and in many other watermelon-producing areas of the U.S. (Fig. 1). Although
watermelon suffers the greatest losses from gummy stem blight, severe epidemics
are observed in cucumber and muskmelon each year. Management options for this
disease are crop rotation, deep turning to bury diseased tissue, avoiding
irrigation that prolongs leaf wetness, and preventive fungicide applications. Of
these management options, application of preventive fungicides is the most
effective. Fungicides labeled for control of gummy stem blight include: ethylenebisdithiocarbamates (EBDCs such as Dithane, Maneb, Manzate, and
Penncozeb); chlorothalonil (Bravo, Echo, Equus); thiophanate-methyl (Topsin M); azoxystrobin (Quadris), a fungicide in the QoI class of chemistry;
and a new carboximide fungicide, boscalid, which was labeled for cucurbits in
July of 2003. Benomyl, or thiophanate-methyl tank-mixed with EBDCs and alternated
with chlorothalonil products, provided good control of gummy stem blight until
resistance to the benzimidazoles was observed in the early 1990s (10).
Chlorothalonil products have shown good efficacy on gummy stem blight but are
not used because they have been implicated in causing phytotoxicity to mature
watermelon rinds, as indicated by a warning on the product label. Azoxystrobin
provided excellent control of gummy stem blight in the early 1990s
(1,9,15,16,22) and was granted Section 18 emergency exemption status in Georgia
in 1997 and 1998 specifically for gummy stem blight control. A full Section 3
national label was granted for azoxystrobin use on cucurbit crops in March of
1999, which led to the widespread and routine use of the fungicide to control a
broad spectrum of foliar cucurbit diseases. However, compared to previous
reports of disease control with azoxystrobin (22), reduced efficacy of
azoxystrobin on gummy stem blight was first observed in Georgia as early as 1999
in watermelon field trials (13) and commercial watermelon fields treated with
Quadris. Isolates of the pathogen collected in 2000 from watermelon fields in
Delaware, Maryland, and Georgia, where disease control was unsatisfactory, were
confirmed to be resistant to azoxystrobin in in vitro laboratory assays (17). In
2001 and 2002, extensive surveys were conducted to determine the frequency of
azoxystrobin-resistant isolates in commercial watermelon fields in Georgia.
Preliminary reports of these results have been published (20,21).
Sample Collection and Fungal Isolations
Isolates of the fungus were obtained from samples of infected leaves, stems,
or seedlings of watermelon and muskmelon showing typical symptoms of gummy stem
blight (Figs. 2 and 3). In 2001, samples were collected from 26 commercial
watermelon fields and research sites in 13 counties in Georgia, as the disease
appeared during June through October. In 2002, isolates of the fungus were
obtained from infected watermelon and muskmelon during April through October
from transplant houses, commercial fields, and research sites from 15 different
locations in Georgia, representing at least six different counties. Counties in
Georgia where samples were collected in 2001 and 2002 are shown in Figure 4.
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Fig. 2. A watermelon leaf showing typical symptoms of gummy stem blight, caused by Didymella bryoniae. |
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Fig. 3. Symptoms of gummy stem blight, caused by Didymella bryoniae, on watermelon seedlings. |
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Fig. 4. Counties in Georgia where samples of watermelon and muskmelon infected with gummy stem blight were collected for sensitivity assays to azoxystrobin in 2001 and 2002. |
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Portions of infected tissue were surface disinfested in 10% household bleach,
rinsed in sterile distilled water and placed on ¼-strength potato dextrose
agar (QPDA). Cultures were incubated at room temperature (23 to 25°C) for up to 1
week until visible characteristic growth of D. bryoniae was observed.
Plugs of agar containing mycelium of the fungus were transferred to fresh QPDA
and incubated at room temperature under continuous fluorescent light for 2 weeks
to encourage sporulation. Three milliliters of sterile distilled water was added
to each culture and the surface of the mycelium was gently scraped to release
conidia. One drop of the conidial suspension was spread across the surface of
QPDA in a petri dish and incubated at room temperature overnight. A single
germinated conidium from each culture was transferred to fresh QPDA and
incubated at room temperature for 2 weeks to obtain monoconidial isolates for
fungicide sensitivity assays.
Fungicide Sensitivity Assays
Sensitivity of each isolate to azoxystrobin was determined using a conidial
germination assay on 4% water agar medium amended with azoxystrobin at
concentrations of 0.0001, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, or 10 µg a.i./ml, or nonamended (0 µg a.i./ml). Technical grade azoxystrobin (Syngenta
Crop Protection, Greensboro, NC) was dissolved in acetone, serially diluted to
the appropriate concentration, and added to autoclaved water agar cooled to 60°C, such
that the concentration of acetone was 0.1% (v/v) in all treatments. The medium
was also amended with 100 µg/ml salicylhydroxamic acid (SHAM) to inhibit an
alternative respiratory pathway in the fungus that can interfere with the
activity of the fungicide.
Each monoconidial isolate was transferred to four petri dishes of QPDA and
incubated at room temperature under continuous fluorescent light to encourage
sporulation. Conidial suspensions of each isolate were prepared by flooding each
culture with 3 ml sterile distilled water and gently scraping the surface of the
mycelium to release conidia. The suspensions from all 4 cultures were combined
and filtered through 2 layers of sterile cheesecloth to remove mycelial
fragments. The suspensions were centrifuged at 5,000 rpm for 10 min to
concentrate the conidia and then re-suspended in 5 ml of sterile water. The
final concentration of conidia ranged from 1.0 × 105 to 1.5 × 106.
Twenty-five microliters of each conidial suspension were transferred to
fungicide-amended or nonamended medium in small petri dishes (60 × 15 mm). Two
replicate dishes of each fungicide concentration and isolate combination were
prepared. After 24 h (in 2001) or 48 h (in 2002) of incubation at room
temperature, 50 conidia per dish were examined microscopically and the
percentage of germinated conidia was recorded. A conidium was considered
germinated if the length of the germ tube was equal to or greater than half the
length of the conidium. Relative germination (RG) was calculated as the
percentage germination on fungicide-amended medium divided by the percentage
germination of the same isolate on medium without fungicide. Percent inhibition
(100 minus RG) was converted to a proportion, probit-transformed, and linearly
regressed on log10-transformed fungicide concentration. Fungicide
sensitivity for each isolate was expressed as the EC50 value (the
fungicide concentration that inhibits spore germination by 50% relative to the
control), estimated from linear regressions. As reported in the previous study
(17), an isolate was considered resistant to azoxystrobin if the EC50
value was greater than 10 µg
/ml.
In both years, three different types of dose-response relationships were
observed (Fig. 5). A relatively small proportion of the isolates in both years
showed a typical S-shaped dose-response relationship between germination
inhibition and fungicide concentration, with estimated EC50 values
less than 10 µg/ml; these isolates were considered sensitive to the fungicide.
Other isolates showed some dose response, but EC50 values could not
be accurately estimated because germination was not inhibited by more than 50%,
even on the highest concentration tested (10 µg/ml). The EC50 values
of isolates in this group clearly exceeded 10 µg/ml and were considered to be
resistant to azoxystrobin. A third group of isolates showed no dose-response to
the fungicide; germination was not significantly inhibited, even at the highest
fungicide concentration tested. This group of isolates was considered to be
highly resistant to azoxystrobin.
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Fig. 5. Examples of 3 different categories of dose-response curves observed in in vitro spore germination assays to determine sensitivity of Didymella bryoniae to azoxystrobin. The red curve shows a typical S-shaped dose-response relationship, with an EC50 value less than 10 µg/ml; isolates with this type of dose response were considered sensitive (S) to the fungicide. The blue curve shows some dose response, and the EC50 value is greater than 10 µg/ml, but could not be accurately estimated because germination was not inhibited by more than 50%, even on the highest fungicide concentration tested (10 µg/ml); isolates with this type of dose-response were considered resistant (R). The green curve shows no dose response to the fungicide and germination was not significantly inhibited by the fungicide, even at the highest concentration tested; isolates with this type of dose response were considered highly resistant (HR). |
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A total of 272 isolates from 26 fields in 13 counties in Georgia was assayed
in 2001 for sensitivity to azoxystrobin. The frequencies of resistant isolates
in each field sampled in 2001 are shown in Table 1. Of the 272 isolates tested,
247 isolates (91%) were found to be resistant to azoxystrobin based on the spore
germination assay. In contrast to the 2001 season, gummy stem blight was not as
severe in 2002. As a result of lower disease incidence throughout the state,
only 170 isolates were collected and assayed for sensitivity to azoxystrobin in
2002. The frequencies of resistant isolates in each field sampled in 2002 are
shown in Table 2. Of the 170 isolates collected in 2002, 82% were resistant to
azoxystrobin. Of the 40 isolates collected from watermelon transplants, all but
one isolate were resistant to azoxystrobin. Interestingly, the frequency of
resistant isolates ranged from 57 to 64% in the samples from border rows at
research sites where fungicide trials had been conducted for many years and
included tests of many different fungicides and application schedules. The
frequency of resistant isolates was considerably higher (93 to 100%) in all but one
of the samples from commercial fields in 2002. A relatively small number of
sensitive isolates was detected in both years (25 in 2001 and 31 in 2002). The
minimum EC50 values of these sensitive isolates were 0.021 µg/ml and
0.010 µg/ml and the geometric mean EC50 values were 1.476 µg/ml and
0.301 µg/ml, in 2001 and 2002, respectively. Based on results of Fisher's Exact
Test (2-tailed, = 0.05), there was no significant association between the number
of resistant or sensitive isolates and the use of DMI fungicides during the
current season, for isolates collected in both 2001 and 2002.
Table 1. Frequencies of azoxystrobin-resistant isolates of the gummy stem
blight pathogen, Didymella bryoniae, in watermelon fields in Georgia
sampled in 2001.
Georgia
County |
Field |
Azoxystrobin
use in 2001 |
Total #
isolates |
# Resistant
isolatesy |
% Resistant
isolatesy |
Berrien |
1 |
Yes |
19 |
19 |
100 |
2 |
Yes |
12 |
10 |
83 |
Colquitt |
1 |
naz |
6 |
6 |
100 |
Cook |
1 |
Yes |
6 |
6 |
100 |
2 |
Yes |
15 |
15 |
100 |
Crisp |
1 |
No |
6 |
4 |
67 |
2 |
na |
13 |
10 |
77 |
Decatur |
1 |
Yes |
8 |
8 |
100 |
2 |
No |
17 |
15 |
88 |
3 |
Yes |
14 |
14 |
100 |
Dodge |
1 |
No |
8 |
8 |
100 |
Dooly |
1 |
No |
7 |
7 |
100 |
2 |
na |
6 |
4 |
67 |
Early |
1 |
Yes |
6 |
6 |
100 |
Mitchell |
1 |
Yes |
14 |
14 |
100 |
Stephens |
1 |
Yes |
14 |
13 |
93 |
Telfair |
1 |
Yes |
11 |
11 |
100 |
Tift |
1 |
Yes |
6 |
5 |
83 |
2 |
na |
8 |
8 |
100 |
3 |
na |
6 |
5 |
83 |
4 |
na |
9 |
7 |
78 |
5 |
No |
12 |
12 |
100 |
Wilcox |
1 |
na |
14 |
14 |
100 |
2 |
na |
10 |
10 |
100 |
3 |
na |
9 |
7 |
78 |
4 |
na |
16 |
9 |
56 |
All isolates |
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272 |
247 |
91 |
Table 2. Frequencies of azoxystrobin-resistant isolates of the gummy stem
blight pathogen, Didymella bryoniae, in samples of watermelon transplants
and field-grown watermelon (W) and muskmelon (M) plants in Georgia in 2002.
Georgia
County |
Sample |
Azoxy-
strobin
use in 2002 |
Total #
isolates |
#
Resistant
isolatesy |
%
Resistant
isolatesy |
NAz |
Transplants-W |
NA |
9 |
9 |
100 |
NA |
Transplants-W |
NA |
9 |
8 |
89 |
Cook |
Field-W |
Yes |
7 |
6 |
86 |
Crisp |
Field-W |
No |
7 |
7 |
100 |
Decatur |
Field-W |
No |
22 |
14 |
64 |
Field-W |
No |
3 |
2 |
67 |
Field-W |
NA |
6 |
5 |
83 |
Field-W |
NA |
7 |
4 |
57 |
Telfair |
Transplants-W |
NA |
9 |
9 |
100 |
Tift |
Transplants-W |
NA |
13 |
13 |
100 |
Field-M |
NA |
15 |
14 |
93 |
Field-M |
NA |
18 |
17 |
94 |
Field-W |
No |
14 |
14 |
100 |
Field-W |
No |
14 |
8 |
57 |
Worth |
Field-W |
Yes |
6 |
0 |
0 |
All isolates |
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170 |
139 |
82 |
Conclusions and Implications for Disease Management
Results of in vitro sensitivity assays conducted in 2001 and 2002
provided evidence of widespread resistance to azoxystrobin in the gummy stem
blight pathogen in Georgia. Of the 272 isolates collected in 2001 from 26 fields
in 13 counties, 247 (91%) were found to be resistant to azoxystrobin, and of the 170 isolates collected in 2002,
82% were found to be resistant, based on the spore germination assay. It
is very difficult to determine with certainty the origin of the azoxystrobin-resistant
isolates. However, based on high frequencies of resistant isolates detected in
greenhouse transplants and transplanted and direct-seeded field-grown plants,
overuse of the product both in the greenhouse and in the field is the suspected
cause. Commercial transplant producers in Georgia have indicated that they have
used azoxystrobin for years in the greenhouse to suppress diseases before
transplants are sold (Langston and Gay, personal communication). Some
watermelon growers have also relied too heavily on azoxystrobin to suppress
gummy stem blight and other diseases and have made three or more sequential
applications in one crop (Langston, personal communication). Another
possible source of resistant isolates is inoculum from previous greenhouse crops
of infected transplants treated with azoxystrobin. And because D. bryoniae
has been isolated from seeds of cucumber, pumpkin, and other cucurbit hosts
(3,14), contaminated seed cannot be ruled out as a potential source of resistant
pathogen isolates.
The risk of resistance development in pathogen populations treated with QoI
fungicides can range from low to high depending on several factors associated
with pathogen biology and epidemiology (6). Pathogens with a short generation
time, which produce abundant spores that are readily and widely dispersed, are
generally associated with greater risk of resistance (2). Indeed, rapid
selection of resistance to QoI fungicides in field populations has
been documented in several such pathogens, including Magnaporthe grisea
(23), Pseudoperonospora cubensis (8), Podosphaera xanthii (=Podosphaera
fusca) (8), Blumeria graminis (4), and Mycosphaerella fijiensis
(5). In most cases, rapid development of resistance is associated with a
single point mutation (G143A) in the cytochrome b gene of mitochondrial DNA in
the fungus, which essentially confers immunity to the fungicide (7,11). However,
based on recent work with the apple scab pathogen V. inaequalis,
mutations other than G143A may cause a more gradual (quantitative) development
of resistance to QoIs, and that both types of resistance may develop
consecutively in a pathogen population (12). Although the genetic basis for
resistance in D. bryoniae was not determined in this study, the rapid
development and high level of resistance observed in the isolates we collected
are consistent with the G143A mutation documented in other plant pathogens.
Research on QoI resistance in the apple scab pathogen suggests
that if initial shifts toward reduced sensitivity develop gradually, in a
quantitative fashion, then rotation of QoIs with unrelated fungicides
may restore satisfactory levels of disease control by reducing resistant
populations before they can reproduce and spread (12). Unfortunately, however,
rotation with unrelated fungicides is not likely to restore a satisfactory level
of disease control if resistance is the result of the G143A mutation (12).
Therefore, to protect their crops, melon growers in Georgia are advised to rely
exclusively on alternative fungicides that are chemically unrelated to the QoIs,
rather than alternations or tank mixtures, and eliminate any potential sources
of inoculum to reduce severity of gummy stem blight epidemics. Mancozeb and
chlorothalonil products both suppress gummy stem blight to some degree. Mancozeb
products alone are usually marginally effective and chlorothalonil products have
been associated with a rind burn when applied within 2 weeks of harvest. A
pre-packaged mixture of the new carboximide fungicide boscalid and the QoI,
pyraclostrobin was labeled for cucurbits in July of 2003 and has shown very
good efficacy against gummy stem blight in field trials (18,19). Like the QoIs,
carboximides also inhibit fungal respiration and are site-specific, but the
target site is different from that of the QoIs, so cross-resistance
between members of these two groups is unlikely, but the risk of multiple
resistance remains a concern. Seed companies and
producers have been advised to limit the use of boscalid and practice
recommended fungicide rotations on seed production melons and production fields,
respectively, to reduce the exposure of the fungus to this new product.
Transplant growers have been and will continue to be warned of the dangers of
using site-specific fungicides on greenhouse transplants. Hopefully, these
strategies will help us preserve the efficacy of boscalid for gummy stem blight
control.
Acknowledgment
The authors thank the Georgia Fruit and Vegetable Growers Foundation for
partial financial support of this research, and Jason Brock, Doug Denney, and
Sydney Goddard for technical assistance.
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