E Gachango

G Clark

G D Franc

J Stewart

L E Hanson

N Rosenzweig

Q W Jiang

W L Stump

William W Kirk

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ASSESSMENT OF STROBILURIN RESISTANCE IN CERCOSPORA BETICOLA ON 
SUGAR BEET IN MICHIGAN AND NEBRASKA, USA 
 
 
William W. Kirk1*, L.E. Hanson1,2, N. Rosenzweig1, G.D. Franc3, W.L. Stump3,  
Q.W. Jiang1, E. Gachango1, G. Clark4 and J. Stewart4 
1 Department of Plant, Soil and Microbial Science, Michigan State University, 612, Wilson 
Road, 35 Plant Biology Building, East Lansing, MI 48824, 2 USDA-ARS, 1066 Bogue Street, 
Room 494, East Lansing, MI,48824, 3 Department of Plant Sciences, University of Wyoming, 
Laramie, WY 82071, 4Michigan Sugar Company, Euclid Road, Bay City, MI 48706. 
 
ABSTRACT 
 
Cercospora leaf spot (CLS) caused by Cercospora beticola Sacc. is the most important 
foliar disease of sugar beet (Beta vulgaris) worldwide (Jacobsen & Franc, 2009). CLS is 
controlled mainly with fungicides, including strobilurins (FRAC group 11). Resistance to 
strobilurins in C. beticola was first confirmed in 2011   from several fields in Michigan and in 
one field in Nebraska, USA (Kirk et al. 2012) following anecdotal reports of reduced fungicide 
efficacy.  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 conidial germination 
bioassay was done on SBLEA 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 (Olaya et al., 1998). After 24 h incubation at 22°C, under 
ambient light, the percentage of germinated conidia (n = 50) was calculated from three replicates 
per treatment. Germination was recorded as positive when the germ tube was at least half the 
length of the conidium. A representative wild type isolate was unable to germinate over the 0.01 
μg/ml concentration. Effective concentration for 50% reduction in germination (EC50) values for 
each isolate were calculated by regression analysis of percentage spore germination vs. the log 
fungicide concentration using Sigmaplot Version 9.01 (Systat Software, Chicago). 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 (Förster et al., 2004) amended with the three strobilurins. 
Two isolates were obtained from Nebraska and each showed a 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) to amplify the region of the CYTB 
gene likely to contain resistance mutations (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 (Ma & Michailides, 2005).  All 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.  A high proportion of 
isolates (~90%) from the commercial growing region that were 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 CLS 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 the Michigan 
State University (MSU) Saginaw Valley Research and Extension Center. 
In addition to the strobilurin sensitivity monitoring, the program at MSU also tests for 
development of insensitivity in the CLS population to Difenoconazole (Inspire); Tetraconazole 
(Eminent); Prothioconazole (Proline), Flutriafol (Topguard), Thiophanate-methyl (Topsin) and 
TPTH (Tin). Some triazole-insensitive isolates have been recovered in MI beet fields over the 
past 10 years but the proportion is low for all these fungicides, however in 2012, nearly 100% of 
isolates tested were insensitive to Thiophanate-methyl.  
The challenges for the sugarbeet industry with respect to CLS control include a lack of 
products from diverse FRAC groups; a general increase in inoculum due to the prevalence of 
CLS susceptible cultivars grown in the Michigan and Ontario sugar beet production regions; an 
increase in proportion of isolates insensitive to strobilurins (e.g. pyraclostrobin); the industry is 
inexperienced with tank mixes and has a perceived distrust of TPTH (label restrictions). In 
addition, to the lack of available chemistries from diverse FRAC groups there is confusion on 
what to start and end a program with in order to delay the onset of insensitivity to other 
fungicides. A strobilurin is the mainstay for Rhizoctonia control in MI therefore there is a need 
for new products with alternative modes of action so that a CLS disease management program 
starts with a product not used earlier in the season for other disease management.  
 
Acknowledgements  
The authors would like to thank Tom Goodwill for his excellent technical assistance. 
 
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ASSESSMENT OF STROBILURIN RESISTANCE IN CERCOSPORA BETICOLA ON SUGAR BEET IN MICHIGAN AND NEBRASKA, USA

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ASSESSMENT OF STROBILURIN RESISTANCE IN CERCOSPORA BETICOLA ON SUGAR BEET IN MICHIGAN AND NEBRASKA, USA

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