Assessment of strobilurin resistance in Cercospora beticola in on sugar beet in Michigan and Nebraska, USA

Authors: KIRK, WILLIAM - Michigan State University; Hanson, Linda; ROSENZWEIG, N - Michigan State University; FRANC, G - University Of Wyoming; STUMP, W - University Of Wyoming; JIANG, Q - Michigan State University; GACHANGO, E - Michigan State University; CLARK, G - Michigan Sugar Company; STEWARD, J - Michigan Sugar Company

Submitted to: American Society of Beet Sugar Technologists Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 3/3/2013
Publication Date: 6/14/2013
Citation: Kirk, W.W., Hanson, L.E., Rosenzweig, N., Franc, G.D., Stump, W.L., Jiang, Q.W., Gachango, E.N., Clark, G., Steward, J. 2013. Assessment of strobilurin resistance in Cercospora beticola in on sugar beet in Michigan and Nebraska, USA. IN: Proceedings of the American Society of Beet Sugar Technologists. 37th Biennial Meeting, February 27 - March 3, 2013, Anaheim, California. 2013 CD-ROM.

Interpretive Summary:

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 was first identified in 2011 from several fields in Michigan and in one field in Nebraska, USA. In these fields, sugar beet treated with strobilurins had severe CLS and diminished control was also noted in small plot trials in Michigan. Individual leaf spot lesions were sampled from leaves and grown on sugar beet leaf extract agar (SBLEA). A conidium germination bioassay was done on SBLEA covered with water agar amended 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. 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. Isolates also grew on spiral gradient dilution plates amended with the three strobilurins. Two isolates were obtained from Nebraska and each showed similar response to strobilurin fungicides in amended plate assays. In 2012, widespread strobilurin resistance was recorded in isolates of C. beticola collected in Michigan although a few isolates submitted to the program were sensitive. Pure cultures of a subset of resistant isolates were grown in potato dextrose broth at 125 rpm, and DNA extracted. A fragment of the cytochrome b (CYTB) gene was amplified by PCR using the C. beticola primers of Malandrakis et al. (2011). This fragment was sequenced at the Genomics Technology Support Facility (MSU, East Lansing, MI) and showed 99% identity with both the C. beticola cytochrome b mRNA, partial sequence (GenBank Accession No. EF176921.1) and the C. kikuchii mitochondrial gene for cytochrome b partial sequence (AB231863.1). Sequence results revealed that each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A, which was demonstrated to confer QoI resistance in several other fungi. All four Michigan isolates with the G143A mutation germinated at 100 µg/ml pyraclostrobin (50% of conidia), while sensitive isolates that lacked the mutation failed to grow. Additional isolates that contained the G143A mutation included representatives from Michigan and Nebraska, USA. A high proportion of isolates (90%) screened in 2012 have been found to contain the G143A mutation by PCR-RFLP screening using digestion of the above PCR products. 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. In 2012, three consecutive applications of pyraclostrobin treatments failed to adequately control C. beticola at MSU bean and Beet Research Farm.