2013. Plant Management Network. This article is in the public domain. Accepted for publication 31 May 2013. Published. Identification of the G143A Mutation in Cytochrome b Associated with QoI Resistance in Cercospora beticola Isolates from the Red River Valley Melvin D. Bolton, Northern Crop Science Laboratory, USDA-ARS, Fargo, ND 58102; Viviana Rivera-Varas, and Gary A. Secor, Department of Plant Pathology, North Dakota State University, Fargo, ND 58108; Allan W. Cattanach, American Crystal Sugar Company, Moorhead, MN 56560; and Michael S. Metzger, Minn-Dak Farmers Cooperative, Wahpeton, ND 58075 Corresponding author: M. D. Bolton. Melvin.Bolton@ars.usda.gov Bolton, M. D., Rivera-Varas, V., Secor, G. A., Cattanach, A. W., and Metzger, M. S. 2013. Identification of the G143A mutation in cytochrome b associated with QoI resistance in Cercospora beticola isolates from the Red River Valley. Online. Plant Health Progress doi:10.1094/php-2013-0812-02-rs. Abstract Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugarbeet. The disease is managed in part by timely applications of quinone outside inhibitor (QOI) fungicides. However, pathogen resistance to QOI fungicides is associated with the exchange of glycine to alanine at amino acid position 143 (G143A) in the C. beticola cytochrome b gene. To assess whether QOI resistance has developed in C. beticola in the Red River Valley (RRV) of Minnesota and North Dakota, a real-time PCR procedure was used to determine whether the G143A mutation could be identified in samples harvested from 922 fields across the RRV. In total, 12 fields located in diverse locations within the RRV contained the G143A mutation, suggesting that QOI resistance arose independently at each location and in several genetic backgrounds. This is the first report of QOI resistance in the RRV. Careful monitoring of the G143A mutation will be necessary to ensure that QOI fungicides remain efficacious for CLS management in the RRV region. Introduction The Red River Valley (RRV) of North Dakota and Minnesota produces ~50% of the total US sugarbeet (Beta vulgaris L.) production (13), which contributes ~$3 billion in economic activity annually from sugarbeet and allied industries in the region (1). Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola (Sacc.), is the most important foliar disease of sugarbeet (12). Management of CLS includes the use of tolerant sugarbeet varieties and crop rotation, but the disease is managed most effectively when these measures are combined with timely fungicide applications (12). Resistance to fungicides used for CLS control has been a persistent problem for sugarbeet growers in the RRV, most often due to wide-spread and repeated use of fungicides with the same or similar modes-of-action (12). In the 1970s, three benzimidazole fungicides were commonly used for CLS management, but fungicide resistance developed that facilitated an epidemic of CLS in 1981 (14). As such, growers then relied predominantly on triphenyltin hydroxide, but extensive resistance in the C. beticola population was established by 1996 in the RRV (8). Favorable environmental conditions for disease development and wide-spread resistance to triphenyltin hydroxide and other fungicide chemistries led to another major CLS epidemic in 1998, costing the local industry in excess of $100 million (12). Currently, sterol demethylation inhibitor (DMI) fungicides are widely used for CLS management, but resistance to DMIs has been reported in C. beticola in some RRV populations (4,5). Strobilurin, or
quinone outside inhibitor (QoI), fungicides are widely used in the RRV for CLS management as well as for non-disease physiological purposes on sugarbeet and other rotational crops. Resistance to QoIs in C. beticola field isolates was first reported in Italy in 2010 (3) and subsequently in Michigan in 2011 (6). As part of our on-going research on fungicide resistance management in this pathosystem, we are interested in understanding the molecular basis of fungicide resistance in order to develop high throughput methodologies for the identification of fungicide-resistant isolates and to monitor changes in resistance within the population on an annual basis. We have previously shown that resistance to QoI fungicides in C. beticola is associated with an exchange of glycine to alanine at amino acid position 143 (G143A) in cytochrome b, the fungal protein targeted by QoI fungicides (3,6). Since fungicide chemistries labeled for CLS control are limited, identification of fungicide resistance isolates is the major focus for fungicide resistance management to ensure continued efficacy of all available chemistries. Therefore, the objectives of this research were to determine if C. beticola isolates from RRV harbor the mutation in cytochrome b encoding the G143A mutation associated with QoI resistance. Sample Collection, Fungal Isolation, and DNA Extraction At least one leaf with CLS was collected from each of five separate plants from 922 commercial fields of the American Crystal Sugar Company and Minn- Dak Farmers Cooperative growing region of North Dakota and Minnesota between August and September 2012. Each sample consisted of conidia harvested from a minimum of five CLS lesions from each of five leaves per field. Briefly, ~20 µl of T-water (0.06% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO), 0.02% (w/v) filter-sterilized ampicillin (Sigma-Aldrich) added after the solution was autoclaved) was added to the surface of a CLS lesion. The lesion was gently scraped with a pipette tip to liberate conidia into the T-water, which was transferred to a 1.5-ml Eppendorf tube. This was repeated for each lesion, which resulted in a composite sample with an approximately 300-µl volume. 300 µl of potato dextrose broth (BD Diagnostics, Sparks, MD) was added to the spore suspension and incubated at 25 C for 48 hr under white light. The mycelium/spore mixture was then centrifuged (~21,000 g for 2 min) and the resulting pellet was transferred to a well of a 96-well PCR plate. To extract DNA, 50 µl of 250 mm NaOH was added to each sample and the PCR plate was incubated at 96 C for 2 min using a standard PCR machine. 100 µl of 500 mm Tris-HCL [ph 9.0; 0.1% (v/v) Tween-20] was added to each well and incubated again at 96 C for 2 min. Plates were centrifuged briefly and stored at 4 C until used for analysis. Detection of G143A Mutation by Real-Time PCR To detect the G143A mutation, a real-time PCR procedure (6) was used to identify samples harboring the mutation in the C. beticola cytochrome b gene. Using our previous studies with individual CLS lesions as a guideline (7), we estimated that each lesion consists of a minimum of 100 spores, for a total of approximately 2,500 spores per field in this study. Since traditional methods of identifying QoI-resistant isolates utilized a single C. beticola spore to represent a field (11,12), the use of molecular technology vastly improved CLS representation per sample while decreasing the amount of time necessary to identify QoIresistant isolates. Most samples (98.7%) in this study contained the wild-type cytochrome b sequence (Table 1). In total, three composite samples were identified that only contained C. beticola isolates harboring the G143A mutation in cytb (Table 1). Since samples in this study were derived from at least 25 lesions from several plants harvested throughout the field, this result suggests that QoI resistance was wide-spread within these fields. Nine other field samples contained varying percentages of isolates with the G143A mutation in the composite sample (Table 1). Samples from these fields were broken down in two broad categories in which the G143A mutation was either found to be more abundant (51 to 99% G143A) or less abundant (<50% G143A) than the wild-type cytochrome b sequence
(Table 1). Fields containing G143A C. beticola mutants were not localized at any specific location, but were widespread in the RRV (Fig. 1). This pathogen is not known for long-distance inoculum movement, making it likely that that QoI resistance originated independently at several locations in the RRV. QoIresistant C. beticola isolates in Michigan and Italy were also shown to be of varied genetic backgrounds (3,6). Table 1. Red River Valley Cercospora leaf spot samples analyzed for the G143A mutation in cytochrome b. Factory district v n 0% <50% 51 to 99% 100% G143A w G143A x G143A y G143A z Crookston 88 86 0 2 0 Drayton 225 225 0 0 0 East Grand Forks 169 166 1 1 1 Hillsboro 158 155 1 1 1 Moorhead 69 68 0 1 0 Wahpeton 213 210 1 1 1 Total 922 910 3 6 3 v American Crystal Sugar Company is represented by the Crookston, MN, Drayton, ND, East Grand Forks, MN, Hillsboro, ND, and Moorhead, MN factory districts. Minn-Dak Farmers Cooperative is represented by a single district at Wahpeton, ND. Except for the Crookston factory district, growers from both ND and MN supply sugarbeet at each factory district. w Number of composite samples that contained only the wild-type cytochrome b sequence. x Number of composite samples that contained < 50% G143A sequence compared to wild-type cytochrome b. y Number of composite samples that contained 51 to 99% G143A sequence compared to wild-type cytochrome b. z Number of composite samples that contained only G143A cytochrome b sequence.
Fig. 1. Locations of Red River Valley Cercospora leaf spot samples analyzed for the G143A mutation in cytochrome b. Percentage of G143A refers to the estimate of Cercospora beticola isolates harboring the G143A in each composite sample.
Conclusions and Management Implications There is typically no fitness penalty associated with the G143A mutation in other pathosystems (2,10). If the same is true in C. beticola, it is likely that the mutation will persist even in the absence of the selection pressure imposed by QoI application. Moreover, isolates with G143A express complete resistance, which is always associated with QoI disease control failures (9). Therefore, any field harboring the G143A mutation is of great concern. In fields where the C. beticola population has become QoI resistant, selection of fungicides based on mode-of-action (single or in combination) is necessary and QoI chemistries should be used judiciously and with caution. That being said, QoI fungicides may still serve as a valuable management tool in fields where resistance to other modes-of-action has been identified. However, QoI application for physiological reasons other than disease control may exacerbate QoI resistance development and spread and thus are ill-advised. Taken together, these approaches will help to ensure that QoI fungicides remain efficacious for CLS management. Acknowledgments The authors thank Xiaoyun Wang (USDA-ARS) for excellent technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. This research was supported by USDA-ARS CRIS project 5442-22000-047-00D and grants from the Beet Sugar Development Foundation and the Sugarbeet Research and Education Board of Minnesota and North Dakota. Literature Cited 1. Bangsund, D. A., and Leistritz, F. L. 2004. Economic contribution of the sugarbeet industry in Minnesota, North Dakota, and eastern Montana. Agribus. Appl. Econ. Rept. 532. 2. Banno, S., Yamashita, K., Fukumori, F., Okada, K., Uekusa, H., Takagaki, M., Kimura, M., and Fujimura, M. 2009. Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene. Plant Pathol. 58:120-129. 3. Birla, K., Rivera-Varas, V., Secor, G. A., Khan, M. F. R., and Bolton, M. D. 2012. Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance. Eur. J. Plant Pathol. 134:475-488. 4. Bolton, M. D., Birla, K., Rivera-Varas, V., Rudolph, K., and Secor, G. A. 2012. Characterization of CbCyp51 from field isolates of Cercospora beticola. Phytopathol. 102:298-305. 5. Bolton, M. D., Rivera-Varas, V., del Río Mendoza, L. E., Khan, M. F. R., and Secor, G. A. 2012. Efficacy of variable tetraconazole rates against Cercospora beticola isolates with differing in vitro sensitivities to DMI fungicides. Plant Dis. 96:1749-1756. 6. Bolton, M. D., Rivera, V., and Secor, G. 2013. Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan, United States. Pest Manag. Sci. 69:35-39. 7. Bolton, M. D., Secor, G. A., Rivera, V., Weiland, J. J., Rudolph, K., Birla, K., Rengifo, J., and Campbell, L. G. 2012. Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA. Fungal Biol. 116:511-521. 8. Campbell, L. G., Smith, G. A., Lamey, H. A., and Cattanach, A. W. 1998. Cercospora beticola tolerant to triphenyltin hydroxide and resistant to thiophanate methyl in North Dakota and Minnesota. J. Sugar Beet Res. 35:29-41. 9. Fernández-Ortuño, D., Torés, J. A., de Vicente, A., and Pérez-García, A. 2010. Mechanisms of resistance to QoI fungicides in phytopathogenic fungi. Int. Microbiol. 11:1-9. 10. Karaoglanidis, G. S., Luo, Y., and Michailides, T. J. 2010. Competitive ability and fitness of Alternaria alternata isolates resistant to QoI fungicides. Plant Dis. 95:178-182. 11. Secor, G. A., and Rivera, V. V. 2012. Fungicide resistance assays for fungal plant pathogens. Pages 385-392 in: Plant Fungal Pathogens: Methods and Protocols, Vol. 835. M. D. Bolton and B. P. H. J. Thomma, eds. Humana Press, New York, NY.
12. Secor, G. A., Rivera, V. V., Khan, M. F. R., and Gudmestad, N. C. 2010. Monitoring fungicide sensitivity of Cercospora beticola of sugar beet for disease management decisions. Plant Dis. 94:1272-1282. 13. National Agricultural Statistics Service. 2013. Crop Production 2012 Summary. USDA-NASS, Washington, DC. 14. Smith, L. J., and Cattanach, A. W. 1982. Response of sugarbeet varieties to various fungicides and fungicide combinations. Sugarbeet Res. Ext. Rept. 13:184-194.