2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify and characterize virulence factors of Cercospora beticola.
Sub-objective 1.A: Characterize the genome sequence of Cercospora beticola with emphasis on effector prediction.
Sub-objective 1.B: Identify Cercospora beticola effectors induced during colonization of sugarbeet using next generation sequencing.
Sub-objective 1.C: Identify effector proteins secreted by Cercospora beticola using proteomic techniques.
Objective 2: Identify molecular markers in sugarbeet that are associated with the resistance response to Cercospora beticola.
Sub-objective 2.A: Identify sugarbeet genes differentially expressed during the compatible and incompatible interaction using next generation sequencing.
Sub-objective 2.B: Quantify defense-related hormone levels in sugarbeet during the compatible and incompatible interaction.
Objective 3: Determine feasibility and validate new high-throughput PCR-based methods to detect fungicide resistance to aid in fungicide management programs.
Sub-objective 3.A: Clone and sequence genes encoding the targets for demethylation inhibitor (DMI) or Qo inhibitor (QoI) fungicides in Cercospora beticola.
Sub-objective 3.B: Analyze sequences of genes encoding fungicide targets to determine if mutations correlate with fungicide resistance and assess whether such mutations can be identified using PCR-based technology to identify resistant isolates in field populations.
Objective 1 addresses current knowledge gaps in the pathology of Cercospora beticola by identifying effectors using genomic, transcriptomic, and proteomic approaches. This knowledge is particularly useful in conjunction with Objective 2, which seeks to identify sugarbeet resistance mechanisms using transcript and hormone profiling strategies that will aid in the development of improved molecular markers for Cercospora leaf spot resistance and/or susceptibility. Objective 3 will focus on the molecular basis of fungicide resistance, which may lead to novel means of identifying resistance in pathogen populations to improve fungicide efficacy.
1b.Approach (from AD-416):
The $2.1 billion U.S. sugarbeet industry is the primary provider of domestic sucrose. Sugarbeet provides an important source of stable, environmentally safe, and low-cost dietary carbohydrate to the public. In addition, beet sugar production and allied industries are an important source of employment regionally and nationally. Yield and quality losses are primarily a result of disease. These losses, in conjunction with expenses associated with pesticide application, cost the U.S. sugarbeet industry up to $150 million annually. The most important foliar disease of sugarbeet in the U.S. is Cercospora leaf spot, caused by the fungus Cercospora beticola. Despite the agricultural importance of this pathogen, stable genetic resistance does not exist and disease is often controlled through application of pesticides. Critical information is lacking on the basic biology of the sugarbeet-C. beticola interaction and the molecular basis of fungicide resistance. The goals of this project are to identify virulence factors of C. beticola, identify molecular markers in sugarbeet that are involved in the regulation of the response to Cercospora leaf spot, and determine if molecular methods can be utilized to detect fungicide resistance in C. beticola populations to aid in fungicide resistance management. This project utilizes state-of-the-art technology with results that will be utilized by all sectors of the sugarbeet industry from growers to processors. The concepts and discoveries generated by this research will lead to a more thorough understanding of sugarbeet pathology as well as host-pathogen interactions that negatively affect worldwide production agriculture.
Under Objective 1, significant progress was made in the identification of Cercospora beticola virulence factors using multifaceted approaches. A high-quality genome sequence from an aggressive C. beticola strain was obtained. The genome was assembled and refined by sequencing three DNA libraries and three RNA libraries, which will provide a robust framework for downstream bioinformatic analyses. Using culture growth conditions that were validated last year, we have identified ion exchange chromatography fractions that induce necrosis when infiltrated into sugarbeet leaves. Under Objective 2, significant progress was made in identifying sugarbeet markers associated with susceptibility to C. beticola. After C. beticola inoculation of susceptible plant genotypes, fungal growth was monitored using quantitative PCR to identify timepoints of interest for subsequent RNA-Seq and hormone analyses. Under Objective 3, significant progress towards validating molecular approaches to identify fungicide-resistant strains was achieved. The cytochrome b gene, which encodes the target of the quinone outside inhibitor (QoI) class of fungicides, was cloned. This led to the identification of a single mutation associated with QoI-resistance in C. beticola strains harvested from several major sugarbeet growing areas of the world. The Cyp51 gene, which encodes the target of the sterol demethylation inhibitor (DMI) class of fungicides, was cloned last year. Using primers designed from this sequence, we sequenced Cyp51 from C. beticola isolates harvested throughout sugarbeet growing areas of MN and ND. Although no Cyp51 mutation was associated with DMI resistance, we found that Cyp51 was over-expressed in DMI-resistant isolates. Therefore, DMI-resistant isolates can be identified by monitoring Cyp51 expression level or potentially through the identification of the transcription factor that promotes increased Cyp51 expression in resistant isolates.
Cercospora leaf spot, caused by the fungus Cercospora beticola, is managed by timely application of fungicides. Although DMI fungicides have been used for over a decade to control this disease, recent outbreaks have suggested that this fungicide class is exhibiting decreased efficacy in some populations. In collaboration with plant pathologists at North Dakota State University, ARS scientists in Fargo, ND have characterized DMI-resistant isolates to show that they are as aggressive as DMI-sensitive isolates in the absence of fungicide application, suggesting that DMI resistance will be maintained in growers’ fields even if they do not apply DMI fungicides. Moreover, a multi-year analysis of nearly 2,700 strains of the fungus has shown that DMI-resistant strains exhibit cross-resistance to all tested DMI fungicides. This information is useful for growers because it has significant implications for fungicide resistance management and will help guide decisions on choosing the optimum fungicide for effective disease control, which will decrease production costs and increase yields.
Cercospora leaf spot, caused by the fungus Cercospora beticola, is the most important foliar disease of sugarbeet and is managed by the application of fungicides. Quinone outside inhibitor (QoI) fungicides are one of the most common classes of fungicides used to control this disease worldwide. However, outbreaks of the disease were noted in major sugarbeet growing regions in Italy and Michigan despite QoI application. In collaboration with plant pathologists at North Dakota State University, ARS scientists in Fargo, ND identified a specific gene mutation in all QoI-resistant strains of the fungus. This mutation was exploited to develop a high throughput molecular technique to identify resistant strains. Since this technology allows for the identification of resistant strains within 24 to 48 hours of receiving a sample, this technique has replaced traditional fungicide sensitivity methods that are time-consuming, laborious, and require several weeks to carry out.
Birla, K., Rivera-Varas, V., Secor, G.A., Khan, M.F.R., Bolton, M.D. 2012. Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance. European Journal of Plant Pathology. 134(3):475-488.
Bolton, M.D., Rivera-Varas, V., del Rio Mendoza, L.E., Khan, M., Secor, G.A. 2012. Efficacy of variable tetraconazole rates against Cercospora beticola isolates with differing in vitro sensitivities to DMI fungicides. Plant Disease. 96(12):1749-1756.
Bolton, M.D., Rivera-Varas, V., Secor, G. 2013. Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan, United States. Pest Management Science. 69:35-39.
Bolton, M.D., Thomma, B.P. 2012. Plant Fungal Pathogens: Methods and Protocols. New York, NY: Humana Press. p 648.