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© 2009 Plant Management Network.
Accepted for publication 20 July 2009. Published 17 September 2009.

Fungicide Sensitivity of Colletotrichum cereale Isolated from Turfgrasses in the Northeastern United States

Nathaniel A. Mitkowski, Angela M. Madeiras, Arielle Chaves, Department of Plant Science, Woodward Hall, University of Rhode Island, Kingston, RI 02881; and Robert Wick, Department of Insect, Plant and Soil Sciences, University of Massachusetts, Amherst, MA 01003

Corresponding author: Nathaniel A. Mitkowski. mitkowski@mail.uri.edu

Mitkowski, N. A., Madeiras, A. M., Chaves, A., and Wick, R. 2009. Fungicide sensitivity of Colletotrichum cereale isolated from turfgrasses in the northeastern United States. Online. Applied Turfgrass Science doi:10.1094/ATS-2009-0917-01-RS.

Abstract

Twenty-seven isolates of Colletotrichum cereale collected from the northeastern United States between 1993 and 1995 and twenty-three isolates collected in 2007 were screened for resistance to fosetyl-Al, chlorothalonil, polyoxin-D, iprodione, fludioxonil, triadimefon, azoxystrobin, and thiophanate-methyl. The comparison between isolates collected from 1993 to 1995 to isolates from 2007 demonstrated significant increases in EC50 values for azoxystrobin, polyoxin-D, thiophanate-methyl, and triadimefon. Significant increases in EC90 were observed for all fungicides except chlorothalonil and fosetyl-Al. Both EC50 and EC90 values for chlorothalonil decreased significantly between the two sampling periods. Most importantly, our results indicate that the occurrence of C. cereale isolates resistant to azoxystrobin and thiophanate-methyl has increased in the Northeast over the past 12 to 14 years based on the isolates tested and that overall resistance to some commonly used turf fungicides has increased as reflected in significantly higher average EC50 and EC90 values.

Introduction

Golf course greens have been under increasing stress in the past decade, due in part to the low mowing heights required for fast ball rolling speeds. Like any other plant, turf is more susceptible to disease when stressed. Anthracnose, once considered a minor pathogen, has become a serious problem on golf courses across the United States (16). The causative organism, Colletotrichum cereale sensu lato Crouch, Clark, and Hillman (formerly Colletotrichum graminicola), has demonstrated resistance to a number of common fungicides, complicating the issue of disease control. Isolates resistant to azoxystrobin, thiophanate-methyl, and propiconazole have been observed in southern California (14,15,17). Results of fungicide resistance studies by Avila-Adame et al. (2) indicate that azoxystrobin resistant C. cereale is common in the United States and Japan.

A collection of C. cereale isolates gathered in the northeastern United States between 1993 and 1995 was kept at the University of Rhode Island (URI). These cultures were isolated from specimens submitted to the URI Turf Disease Diagnostic Lab during those years, and were used in the analyses of the pathogen by Browning et al. (3). Comparing fungicide resistance patterns in this collection with those of isolates gathered from the same region in 2007 presents a picture of resistance development in C. cereale in the Northeast over the past 12 to 14 years. Although previous research indicates that fungicide exposure over time results in the selection of resistant strains of C. cereale (2,5), it was important to assess and quantify current resistance levels to aid turfgrass managers in making appropriate fungicide selections.

The present study was conducted to determine the presence of fungicide resistance in C. cereale isolates collected in the northeastern United States between 1993 and 1995, and in 2007, and to examine differences in resistance patterns between the two sampling periods.

Isolate Collection

Between 1993 and 1995, isolates of C. cereale were collected from golf courses in Maine, New Hampshire, Massachusetts, Connecticut, Rhode Island, and New York (Table 1). With the exception of one isolate derived from Agrostis canina L., all isolates were obtained either from Poa annua L. or Agrostis palustris Huds. demonstrating symptoms of anthracnose. All isolates collected from golf courses were obtained from putting greens. Initial identification of C. cereale was based on correlation of host symptoms with conidial morphology and presence of setae. The 27 isolates obtained between 1993 and 1995 had been maintained in refrigerators at approximately 5°C on half-strength potato dextrose agar (PDA; Becton, Dickson, & Co., Sparks, MD) in 15-mm plastic Petri plates. Cultures were transferred approximately every 3 years while in storage. In 2007, isolates were obtained from 23 golf courses in Massachusetts, Rhode Island, Connecticut, and New York, including eight sites that had provided isolates in the 1990s (Table 1). One isolate from Vermont and one from Pennsylvania were included in the 2007 group. Symptomatic leaf blades and crowns from new specimens were surface disinfested with 0.6% sodium hypochlorite for 3 to 5 min and placed on acidified potato dextrose agar (1 ml lactic acid per 1000 ml liquid media). Fungal isolates were identified as C. cereale based upon colonial and conidial morphology. These colonies were subcultured to half-strength PDA. All active cultures were maintained at room temperature (~20°C). It should be noted that isolates were not derived from single spores or hyphal tip cultures and may actually be representative subpopulations that could contain a mix of genetically distinct isolates. However, 1-mm agar plugs were taken from the edges of growing cultures for at least 3 subcultures before trials began and cultures were physically uniform with no sectoring observed. Growth rate and physical appearance of the 1993 to 1995 isolates was similar to that of the 2007 isolates.

Table 1. Collection period, host species, and source of Colletotrichum cereale isolates used in this study.

 * Courses submitting specimens in both collection periods.

In addition, rDNA was amplified from all isolates collected in 2007, sequenced and compared to published data provided by GenBank to confirm the identity of the tested isolates as C. cereale. Briefly, DNA was isolated from fungal mycelium grown in 25 ml of nutrient broth (Becton, Dickson & Co.) for 5 days and extracted using the Power Plant DNA extraction kit (Mo Bio Laboratories, Carlsbad, CA). Ribosomal DNA was amplified using the ITS4 and ITS5 primers, resulting in a sequence comprising ITS1, the 5.8s rDNA gene and ITS2 (13). Among the twenty-three 2007 isolate sequences, all demonstrated very high similarity (> 99.5% in all cases) to the published C. cereale sequences and very little sequence noise was observed, suggesting that each culture as relatively homogeneous.

Fungicide Sensitivity

We chose eight chemicals to represent the major classes of fungicide used on golf course greens in the Northeast. Commercial formulations of the fungicides fosetyl-Al, chlorothalonil, polyoxin-D, iprodione, fludioxonil, triadimefon, azoxystrobin, and thiophanate-methyl were diluted to concentrations of 4, 16, 64, 256, 512, and 1024 μg/ml in half-strength PDA cooled to 55°C and poured into 100-mm Petri plates (Table 2). These higher concentrations were used because initial results suggested that many of the isolates had EC50 values substantially higher than 4.0 μg/ml of numerous active ingredients. Concentrations above 100 μg/ml sometimes exhibit solubility issues in an aqueous environment so 10 ml of ethanol was used in the highest concentrations where solubility issues were observed (> 256 μg/ml). These higher concentrations were utilized to ensure that the truly resistant isolates could be identified. Additional experiments utilizing azoxystrobin and azoxystrobin + SHAM (salicylhydroxamic acid, MP Biomedicals, Solon, OH) at 100 μg/ml were also undertaken utilizing active ingredient concentrations of 0.1, 0.25, 0.5, 1.0, 4.0 and 8.0 μg/mL. Unamended half-strength PDA was used as a negative control. Eight millimeter agar plugs containing actively growing mycelium were taken from the growing edge of twelve-day-old cultures and placed in the center of each plate. Plates were placed in a growth chamber (Percival Model 136LL, Perry, IA) in complete darkness at 21°C. Placement of plates in the growth chamber was completely randomized. Colony diameters were measured after four days of incubation. Each treatment was replicated three times and each experiment was repeated once.

Table 2. Trade name, active ingredient, labeled application rate, and labeled application rate of fungicides used in resistance testing.

 * Recommended for anthracnose control only when tank mixed with other fungicides.

Data Analysis

Data from both trials of the experiment were pooled for analysis. Effective concentration values representing reductions of mycelial growth by 50% and 90% (EC50 and EC90) were determined for each repetition by regressing probit transformed percent inhibition of growth against the log-10 concentrations of the fungicides (4). Significance of variation between sampling periods (1993 to 1995 versus 2007) for each fungicide was determined by independent sample t test after data had been log-10 transformed (Table 3). Although EC90 values are listed throughout this paper, the authors caution the reader as to the practical significant of these values. EC90 values are calculated based on the non-linear portion of the dose response curve and can have much more variability than EC50 values. While they may occasionally appear redundant, it is our contention that these values can be used to provide important insight in some situations and are therefore presented here.

Table 3. Mean and range of EC50 and EC90 (μg/ml) values in both collection periods for fungicides used in this study.

* Separation of means significant at the 0.05 level unless otherwise noted by two tailed t-test. Statistical analysis was not undertaken on fosetyl-Al (Aliette) or thiophanate-methyl because means were extremely high, indicating general ineffectiveness or putative resistance.

ns = on significant.

Fosetyl-Al

Two different formulations of fosetyl-Al were used in this study. The first formulation was Chipco Signature 80WG (Bayer, Research Triangle Park, NC). The second formulation was Aliette 80WG (Bayer). Although Chipco Signature did marginally inhibit fungal growth (the EC50 value was about 70 μg/ml for both sampling periods) Aliette provided no control at all and has been listed in Table 3 as having EC values of > 1024 μg/ml. However, no isolates of C. cereale actually showed any growth reduction at all using this formulation of fosetyl-Al. These results suggest that one of the components in Chipco Signature other than fosetyl-Al may be providing a significant amount of anti-fungal activity against C. cereale.

The data from the experiments using Chipco Signature did not indicate a significant difference in susceptibility between the two sampling periods. The ranges of EC50 and EC90 values in each sampling period were remarkably similar. These results indicate that fosetyl-Al susceptibility (or which ever ingredient is providing fungal control) of C. cereale isolates in the Northeast has remained stable for at least 12 years. Phosphonate fungicides are considered to have a low risk for resistance development because they are believed to improve innate host resistance as well as having direct (and possibly multi-site) fungicidal activity (8). Chipco Signature also contains a dye that is not present in the Aliette formulation. It was discovered in the 1990's that when fosetly-Al was combined with just the dye present in Fore, the combination gave similar results to using Aliette and Fore, despite the fact that mancozeb was no longer included in the new formulation (8). For this reason, the results of the fosetyl-Al resistance experiments may actually be examining multiple active ingredients present in the Chipco Signature formulation.

Chlorothalonil

Significant differences in EC50 and EC90 values were observed between sampling periods; however, both values were lower overall in 2007 than in 1993 to 1995. Of all the fungicides tested, chlorothalonil was the only one that elicited this type of response over time. It is unclear what mechanism may be responsible for this shift. The difference in EC50 between sampling periods, though statistically significant, is not great in practical terms (Table 3). All 50 isolates tested demonstrated EC50 values of 43 μg/ml or less. Chlorothalonil is generally not very effective against mycelial growth and this may explain the unusual results associated with this active ingredient. It may also be useful to undertake additional experiments in the greenhouse on bioassy plants to examine if these differences can be consistently observed.

Polyoxin-D

There was a statistically significant increase in both EC50 and EC90 levels between 1993 to 1995 and 2007; however, values for both parameters remained less than 63 μg/ml. This indicates that although sensitivity to polyoxin-D has decreased, it is likely that C. cereale is still adequately controlled at a concentration approximately one tenth of the labeled rate for Endorse 2.5WP (Cleary Chemical Corporation, Dayton, NJ). Descreases in sensitivity of this sort are typical of quantitative resistance development that has been best documented for the demethylation inhibitor (DMI) fungicides. It is possible that repeated polyoxin-D use has resulted in the overall decrease in the most sensitive individuals at locations where isolates were collected.

Iprodione

Although EC50 values did not differ significantly between sampling periods, the frequency of isolates demonstrating high EC90 values was markedly higher among the 2007 isolates. Among the 1993 to 1995 isolates, 11.1% demonstrated EC90 values above 1300 μg/ml and the remaining 88.9% of isolates had an EC90 distributed gradually between 8 and 714 μg/ml. Among the 2007 isolates, 34.8% demonstrated EC90 values above 1300 μg/ml and the remaining 65.2% of isolates had an EC90 distributed gradually between 92 and 795 μg/ml.

Although iprodione is not recommended for use against turf anthracnose, it is commonly used to control dollar spot (Sclerotinia homoeocarpa Bennett) and brown patch (Rhizoctonia solani Kühn) and thus it is likely that C. cereale has been repeatedly exposed to this fungicide. Other fungi are known to develop resistance to iprodione rapidly upon exposure (7,10). Selection of more resistant strains of C. cereale through iprodione use may be considered a non-target effect of this fungicide.

Fludioxonil

EC50 values did not change significantly between sampling periods. One 2007 isolate demonstrated an EC50 of 110 μg/ml, while all others remained below 10 μg/ml. EC90 values were significantly higher in 2007. Among the 1993 to 1995 isolates, 22.2% demonstrated EC90 values above 250 μg/ml and the remaining 88.9% of isolates had an EC90 distributed gradually between 3 and 114 μg/ml. Among the 2007 isolates, 43.5% demonstrated EC90 values above 250 μg/ml and the remaining 56.5% of isolates had an EC90 distributed gradually between 4 and 150 μg/ml. Although fludioxonil was not commercially available before 1997, one isolate from the 1993 to 1995 collection period demonstrated an EC90 value greater than 1024 μg/ml. There is also evidence that some iprodione-resistant fungi are cross-resistant to fludioxonil (6,9). In the current study, a total of eight isolates were found to have EC90 values greater than 1000 μg/ml for fludioxonil. Seven of these isolates also demonstrated EC90 values greater than 1600 μg/ml for iprodione.

Triadimefon

While the values for both EC50 and EC90 remained relatively low for both sampling periods (the highest EC50 for a single isolate was 56 μg/ml; the highest EC90, 153 μg/ml), significant increases were observed for both EC50 and EC90 over time. Among the 1993 to 1995 isolates, 22.7% demonstrated EC50 values greater than 20 μg/ml, and among the 2007 isolates, 78.3% demonstrated EC50 values greater than 20 μg/ml.

The current study revealed a range of EC50 values for triadimefon from > 4 to 56 μg/ml. Our results are similar to those of Wong and Midland (15), who found a range of EC50 values for triadimefon from 2.2 to 64 μg/ml in isolates of C. cereale from southern California. Unfortunately, means from the two studies cannot be compared due to substantial differences in methodology.

Our results indicate that resistance to triadimefon has increased in populations of C. cereale in the Northeast. Resistance to DMI fungicides has been observed in several other fungal pathogens, including dollar spot (4).

Triadimefon appears to be less toxic to C. cereale than some other DMI fungicides (H. T. Wilkinson, personal communication). Despite a high affinity for binding to leaf surfaces, this localized systemic has been found to have a half-life of approximately 3.3 days in the turf canopy (12). Lower toxicity, a short half-life and a long spray interval (Table 2) may account for perceived control failures.

Azoxystrobin

A marked rise in both EC50 and EC90 values was observed between sampling periods. Among the 1993 to 1995 isolates, only one isolate demonstrated an EC50 value greater than 13 μg/ml for azoxystrobin (at 92 μg/ml); conversely, only six of the 2007 isolates had EC50 values less than 120 μg/ml (between 0.7 to 6.8 μg/ml). The same six isolates also demonstrated EC90 values less than 30 μg/ml, while all other isolates from 2007 had EC90 values > 1024 μg/ml. These results indicate that widespread resistance to azoxystrobin has developed in C. cereale in the Northeast in the decade since its introduction, based on our sample. Other workers have also reported a high proportion of resistant isolates. Wong et al. (17) observed resistance in all 347 isolates taken from eight sites that had been treated with azoxystrobin, and susceptibility in all 174 isolates taken from two untreated sites. Similarly, Avila-Adame et al. (2) found azoxystrobin resistance in five isolates taken from five treated sites, and susceptibility in four isolates taken from four untreated sites. Unfortunately, differences in methodology prevent direct comparison of means. Fungicide application records were not considered in the current study.

It is interesting to note that two of the isolates collected in 1993 to 1995 demonstrated an EC90 greater than 1000 μg/ml for azoxystrobin, a fungicide that was not commercially available before 1996. A point mutation is responsible for azoxystrobin resistance (11); therefore, the probability of resistance development is high even in the absence of selective pressure, assuming that the mutation does not impart a fitness cost.

Previous studies have shown that azoxystrobin-sensitive isolates mat test falsely as "resistant" due to the presence of an alternative oxidative (AO) that is expressed by fungi in vitro (2). As a result, it is possible to overestimate the level of stobilurin resistance unless the AO pathway is disabled. To confirm the presence of azoxystrobin resistance, isolates were retested using 100 μg/ml salicylhydroxamic acid (SHAM) in the media to exclude possible false positives (Table 4). The use of SHAM had little effect on the results of trials undertaken on the 1993 to 1995 isolates. This can likely be explained by the fact that very little resistance was found among these isolates. However, the use of SHAM did have a substantial effect on the number of 2007 isolates deemed resistant. Without SHAM, 70% of the tested isolates appeared resistant. When SHAM was included in the media, only 61% of the tested isolates appeared resistant, suggesting that the OA pathway resulted in a 10% increase in the number of isolates perceived to be resistant to azoxystrobin.

Table 4. Response of isolates in the study to azoxystrobin and azoxystrobin + SHAM.

 ^x Resistant isolates are defined as those that showed no growth reduction at any of the rates tested in the study.

 ^y The number in parentheses represents the mean EC50 for this set of isolates in μg/ml. EC50 values could not be determined for those isolates that were controlled by < 0.10 μg/ml active ingredient because this was the lowest concentration of active ingredient tested. Within treatment (azoxystrobin and azoxystrobin + SHAM), differences between 1993 to 1995 and 2007 isolates are statistically significant at the 0.05 level otherwise by two tailed t-test.

Thiophanate-methyl

Several isolates from both sampling periods showed little or no response to thiophanate-methyl at the highest concentration tested. Accurate EC50 and EC90 values could not be obtained for these isolates; therefore, EC50 and EC90 values can only be reported as > 1024 μg/ml (the highest concentration of active ingredient tested in these trials). Among the 1993 to 1995 isolates, 33.3% demonstrated EC50 values > 1024 μg/ml, and 51.9% demonstrated EC90 values > 1024 μg/ml. Among the 2007 isolates, 73.9% demonstrated EC50 values > 1024 μg/ml, and 78.3% demonstrated EC90 values > 1024 μg/ml. This represents a significant increase between sampling periods; however, this does not change the overall means (Table 3). The actual means from both collection periods are well over 1024 μg/ml and statistical analysis is not presented for this data.

In order to test the reliability of our dose-response curve, media amended with 3500 and 7500 μg/ml thiophanate-methyl were made and isolates from both sampling periods that had demonstrated EC90 values > 1024 μg/ml were tested for sensitivity as described above. These isolates continued to demonstrate a similar pattern of resistance at these higher levels (data not shown), a behavior indicative of qualitative resistance. Our results indicate that thiophanate-methyl resistance is widespread in the Northeast, and may have resulted in anthracnose control failures as early as 1993.

Other researchers have noted a high proportion of isolates resistant to thiophanate-methyl. Wong et al. (14) observed that most but not all isolates from populations exposed to thiophanate-methyl were resistant. In one location, 95% (57 of 60) of the isolates were resistant. All isolates from two unexposed sites remained susceptible. All of 32 isolates from one site that had been "sparingly" exposed were also susceptible. In the current study, 74% (17 of 23) of the 2007 isolates were resistant to thiophanate-methyl. This fungicide has been used on golf course greens since its introduction in the early 1970s. Again, fungicide application records were not considered in the current study.

Conclusion

Our results demonstrate that resistance to some commonly used turf fungicides has increased in C. cereale in the northeastern United States over the past 12 to 14 years. There were significant increases in EC50 and EC90 values for four and six of the eight fungicides tested, respectively (Table 3).

A large, relatively contiguous geographical area is represented by 42 different golf courses in this study, amounting to 50 different isolates. This stands in contrast to studies by Wong et al., in which a large sample size represents a smaller area (three sites in the San Francisco Bay area and seven sites in the Los Angeles area). Nevertheless, our results for triadimefon, azoxystrobin, and thiophanate-methyl are similar to results obtained from those studies. Our results also support the findings of Dry et al. (6) and Mitkowski and Browning (9), which describe iprodione-resistant field isolates of fungi that were cross-resistant to fludioxonil.

The study and others have found that resistance to azoxystrobin is prevalent among in populations of C. cereale (2,17). Cross-resistance among the QoI fungicides is considered commonplace and the implication from this study is that turfgrass practitioners who have experienced C. cereale resistance to azoxystrobin are likely to experience resistance to trifloxystrobin, pyracloastrobin and fluoxastrobin. However, cross-resistance does not always imply complete resistance and other active ingredients within the QoI group may still provide some partial activity against the pathogen. Fungicide resistance to any active ingredient from a specific site can only be confirmed through in vitro testing in the laboratory.

Labeled application rates and recommended spray intervals for the fungicides used in this study are presented in Table 2. The application rate of a fungicide must be considered in any discussion of practical field resistance. An EC50 of 50 μg/ml may not imply practical resistance when the fungicide is applied at a rate of 7000 μg/ml, but for a fungicide applied at a rate of 375 ppm, the likelihood of practical resistance may be much greater.

The concentrations of active ingredients in different commercial fungicide formulations differ widely, as do the concentrations of solutions applied by individual golf course superintendents (ie: full rates, half rates, volumes of water carrier). The final concentration of active ingredient encountered by the pathogen in the field is likely to be much lower than the rate applied due to dilution in water on leaf surfaces and, for systemics, in plant tissue fluids. Additionally, as fungicides degrade in the environment, fungal strains with higher EC90 values may be capable of rebounding more quickly between applications, thereby creating a persistent problem.

A final caveat is that the results obtained in this study were based solely on Petri dish assays undertaken in a laboratory setting. Consequently, while these results do suggest a pattern of resistance occurring over time, these results cannot provide a complete basis for making decisions as to the reactions of the tested isolates in the field. It is entirely possible that isolates controlled easily in Petri dish experiments may not be easily controlled in field settings.

Acknowledgments

The authors wish to thank the New England Regional Turf Foundation for financial assistance.

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