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United States Department of Agriculture

Agricultural Research Service

Research Project: BREEDING SELECTION AND MOLECULAR CHARACTERIZATION FOR IMPROVED SUGAR BEET GERMPLASM

Location: Sugarbeet and Bean Research

Title: First report of strobilurin resistance in Cercospora beticola in sugar beet (Beta vulgaris) in Michigan and Nebraska, USA

Authors
item Kirk, W -
item Hanson, Linda
item Franc, G -
item Stump, W -
item Gachango, E -
item Clark, G -
item Stewart, J -

Submitted to: New Disease Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 17, 2012
Publication Date: July 31, 2012
Citation: Kirk, W.W., Hanson, L.E., Franc, G.D., Stump, W.L., Gachango, E.N., Clark, G., Stewart, J. 2012. First report of strobilurin resistance in Cercospora beticola in sugar beet (Beta vulgaris) in Michigan and Nebraska, USA. New Disease Reports. Available: http://dx.doi.org/10.5197/j.2044-0588.2012.026.003.

Interpretive Summary: Cercospora leaf spot (CLS) caused by Cercospora beticola Sacc. is the most important foliar disease of sugar beet (Beta vulgaris) worldwide. CLS is controlled mainly with fungicides, including strobilurins. In 2011, fields from several areas in Michigan that were treated with strobilurins had severe CLS and diminished control was found in small plot trials. Individual leaf spot lesions were sampled and tested on three strobilurins at concentrations between 0 and 100 µg/ml supplemented with salicylhydroxamic acid to block the alternate oxidation pathway. A representative wild type isolate was unable to germinate at 0.01 µg/ml concentration and the effective concentration for 50% reduction in germination (EC50) for the sensitive isolate was <0.01 µg/ml. Isolates from several counties in Michigan had uninhibited germination and EC50 values exceeded the highest concentration tested. Two isolates from Nebraska showed similar response to strobilurin fungicides in amended plate assays. DNA was extracted from four representative resistance isolates and a fragment of the cytochrome b gene likely to contain resistance mutations was amplified by PCR. This fragment showed 99% identity with both the C. beticola and the C. kikuchii cytochrome b partial sequences. Sequence results revealed that each resistant isolate contained a change in codon 143 that predicts to lead to a substitution which was demonstrated to confer QoI resistance in several other fungi. All tested isolates with the mutation germinated at 100 µg/ml pyraclostrobin, while sensitive isolates that lacked the mutation failed to grow. Isolates that contained the mutation included representatives from Nebraska and different counties in Michigan. These findings indicate that reduced Cercospora leaf spot control in some commercial sugar beet fields may be due to the development of resistance to strobilurins.

Technical Abstract: Cercospora leaf spot (CLS) caused by Cercospora beticola Sacc. is the most important foliar disease of sugar beet (Beta vulgaris) worldwide. CLS is controlled mainly with fungicides, including strobilurins (FRAC group 11). Resistance to strobilurins in C. beticola has not been reported in the field but insensitive mutations have been artificially developed. In 2011, fields from several areas in Michigan, USA treated with strobilurins had severe CLS and diminished control was also noted in small plot trials. Individual leaf spot lesions were sampled and grown on sugar beet leaf extract agar (SBLEA). In a conidium germination bioassay with pyraclostrobin, azoxystrobin or trifloxystrobin at 0, 0.001, 0.01, 0.1, 1, 10, or 100 µg/ml supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway a representative wild type isolate was unable to germinate over the 0.01 µg/ml concentration and the EC50 for the sensitive isolate was <0.01 µg/ml. Isolates from several counties in Michigan had uninhibited germination and EC50 values exceeded the highest concentration tested. Isolates also grew on spiral gradient dilution plates amended with the three strobilurins. Two isolates from Nebraska showed similar response to strobilurin fungicides in amended plate assays. DNA was extracted from four representative resistance isolates and a fragment of the cytochrome b (CYTB) gene likely to contain resistance mutations was amplified by PCR using the C. beticola primers of Malandrakis et al. (2011). This fragment showed 99% identity with both the C. beticola cytochrome b mRNA, partial sequence and the C. kikuchii mitochondrial gene for cytochrome b partial sequence. Sequence results revealed that each resistant isolate contained a change in codon 143 that predicts to lead to a substitution of G143A, which was demonstrated to confer QoI resistance in several other fungi. All four isolates with the G143A mutation germinated at 100 µg/ml pyraclostrobin (50% of conidia), while sensitive isolates that lacked the mutation failed to grow. Isolates that contained the G143A mutation included representatives from two different counties in Michigan as well as Nebraska, USA. These findings reveal that reduced Cercospora leaf spot control in some commercial sugar beet fields may be due to the development of resistance to strobilurins.

Last Modified: 4/18/2014
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