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United States Department of Agriculture

Agricultural Research Service

Research Project: BREEDING SELECTION AND MOLECULAR CHARACTERIZATION FOR IMPROVED SUGAR BEET GERMPLASM Title: First report of QoI insensitive Cercospora beticola on sugarbeet in Ontario, Canada

Authors
item Trueman, Cheryl -
item Hanson, Linda
item Rosenweig, N -
item Jiang, Q -
item Kirk, W -

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 22, 2013
Publication Date: September 1, 2013
Citation: Trueman, C.L., Hanson, L.E., Rosenweig, N., Jiang, Q.W., Kirk, W.W. 2013. First report of QoI insensitive Cercospora beticola on sugarbeet in Ontario, Canada. Plant Disease. 97:1255.

Interpretive Summary: Cercospora beticola causes Cercospora leaf spot (CLS) of sugarbeet and is the most destructive foliar disease of sugarbeet worldwide. The quinone outside inhibitor (QoI) fungicide pyraclostrobin has been an important management tool for CLS in Canada since 2003. Beginning in 2010 some growers reported unsatisfactory disease control after applying pyraclostrobin. Tests were conducted on CLS from 16 commercial fields in Kent and Lambton Counties in Ontario, Canada. These counties encompass the entire commercial sugarbeet production area in Ontario. Leaf spot of up to 5 mm in diameter with tan, light brown, or sometimes gray centers were collected. PCR was used to amplify a fragment of the gene for the fungicide target (CYTB) using C. beticola primers. DNA was sequenced for 32 samples and 25 were confirmed to be identical to a QoI-resistant C. beticola from Michigan. The other seven had identical sequence to a QoI-sensitive isolate. Each resistant isolate contained the same change in the gene. This mutation (known as G143A) has been associated with QoI resistance in a number of fungi. To confirm the result, a spore germination assay was carried out using fungal isolates on sugar beet leaf agar covered with water agar amended with the fungicide at concentrations ranging from 0 to 54.0 µg/mL distributed on a spiral gradient. The medium was supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway. Following incubation at 25°C for 2 d, the calculated effective concentration for 50% reduction in germination (EC50) values were determined for each isolate. The estimated EC50 for a representative sensitive isolate was 0.03 µg/mL, while the value for the resistant isolates could not be calculated because it was greater than the highest concentration tested (54 µg/mL). This indicates a change of over a thousand fold in fungicide sensitivity. The spore germination was reduced for the resistant isolates compared to the sensitive isolate when no fungicide was present. Confirmation of fungicide resistance will require a re-evaluation of current management practices in Ontario to minimize economic losses from CLS.

Technical Abstract: Cercospora beticola causes Cercospora leaf spot (CLS) of sugarbeet (Beta vulgaris) and is the most destructive foliar disease of sugarbeet worldwide. The QoI fungicide pyraclostrobin has been an important management tool for CLS in Canada since 2003. Beginning in 2010 some growers reported unsatisfactory disease control after applying pyraclostrobin. Leaf disk samples with CLS lesions were collected in September 2012. Samples were collected from 16 commercial fields located in the southwestern region of the province in Kent and Lambton Counties, Ontario, Canada. Kent and Lambton Counties encompass the entire commercial sugarbeet production area in Ontario (ca. 300,000 ha). CLS symptoms ranged from light to severe among the sampling sites. Leaf discs with a single leaf spot were cut from leaves using a hole-punch. Spots were up to 5 mm in diameter with tan, light brown, or sometimes gray centers. DNA was extracted from leaf discs. PCR was used to amplify a fragment of the cytochrome b (CYTB) using C. beticola primers. DNA was sequenced for 32 samples and 25 were confirmed to have 100% identity with the sequence of QoI-resistant C. beticola from Michigan and showed 99% identity with the C. beticola cytochrome b mRNA, partial sequence from a QoI-sensitive strain. The remaining seven had 100% identity with the sensitive isolate. Each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A in the cytochrome b gene. This G143A mutation has been associated with QoI resistance in a number of fungi. To confirm the result, a conidium germination bioassay was carried out on sugar beet leaf agar covered with water agar amended with pyraclostrobin at concentrations ranging from 0 to 54.3 µg/mL, distributed on a spiral gradient. The medium was supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway. Following incubation at 25°C for 2 d, the distance between the center of the plate at which conidial germination was 50% of the maximum observed growth (EC) and the point at which conidial germination terminated were measured (TEC). The EC50 values were determined for each isolate from the EC and TEC values. The estimated EC50 for a representative wild type (sensitive) isolate was 0.03 µg/mL, while the value for the resistant isolate could not be calculated because it was greater than the highest concentration tested (54 µg/mL). Additionally, in the controls with no SHAM or fungicide, the resistant isolate showed a consistent reduced germination rate compared to the sensitive isolate (30% and 93.5% germination, respectively). Confirmation of fungicide insensitivity will require a re-evaluation of current management practices in Ontario to minimize economic losses due to CLS.

Last Modified: 10/31/2014
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