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Peer Reviewed

2012 Plant Management Network.
Accepted for publication 16 April 2012. Published 21 May 2012.

Resistance to Frogeye Leaf Spot in Selected Soybean Accessions in MG I through MG VI

Alemu Mengistu, USDA-ARS, Jackson, TN 38301; Jason Bond, Southern Illinois University, Carbondale, IL 62901; Rouf Mian, USDA-ARS and The Ohio State University, Wooster, OH 44691; Randall Nelson, Soybean/Maize Germplasm, Pathology, and Genetics Research Unit, USDA-ARS, and Department of Crop Sciences, University of Illinois, Urbana, IL 61801; and Grover Shannon and Allen Wrather, University of Missouri, Portageville, MO 63873

Corresponding author: Alemu Mengistu.

Mengistu, A., Bond, J., Mian, M. A. R., Nelson, R., Shannon, G., and Wrather, A. 2012. Resistance to frogeye leaf spot in selected soybean accessions in MG I through MG VI. Online. Plant Health Progress doi:10.1094/PHP-2012-0521-02-RS.


Frogeye leaf spot (FLS) caused by Cercospora sojina Hara is a disease of soybean [Glycine max (L.) Merr.] that causes significant seed yield loss in warm, humid environments worldwide. The Rcs3 gene in soybean has been reported to condition resistance to all known races of C. sojina. The objectives of this study were to: (i) identify maturity group (MG) I to VI accessions resistant to C. sojina race 11 by field screening at two locations; and (ii) determine if the FLS resistance of the symptomless soybean accessions is likely to be conditioned by the Rcs3 allele. A total of 260 accessions including 12 differentials were evaluated for reaction to race 11 in field trials in Missouri and Illinois during 2009, and 20 accessions that did not develop symptoms were retested in 2010 to validate their resistance. The 20 accessions remained resistant and were tested for the potential presence of Rcs3 allele using molecular markers; and none was predicted to carry the Rcs3 allele. These accessions may contain novel loci for FLS resistance and may be used to broaden the base for developing soybean cultivars with frogeye leaf spot resistance.


Frogeye leaf spot (FLS) caused by Cercospora sojina Hara is primarily a foliar disease of soybean [Glycine max (L.) Merr.], even though seeds, pods, and stems can also be infected (Fig. 1). The disease is favored by warm, humid environments (14) and the incidence of FLS in soybean is dependent on the growing conditions (1). The yield loss from FLS is mainly the result of reduced photosynthetic area and premature defoliation (1). Soybean yield reductions of 10 to 60% due to FLS have been reported (10).


Fig. 1. Severe symptoms on leaves (A and B), petioles (C), and pods (D) infected with Cercospora sojina (courtesy of Melvin Newman).

Symptoms of FLS develop primarily on foliage of soybean even though seeds, pods, and stems can also become infected (14). The seedlings from infected seeds may have lesions on the cotyledons (16) but the lesions on leaves do not appear for nearly 7 to 14 days after invasion of the host tissue, so they are not visually observed on young expanding leaves (14).

Recently, FLS has been reported in Iowa (20), Wisconsin (8), and Ohio (5). The reason for its spread in these states is unknown but it may be due to changes in the environment, increased usage of no-till cultivation (19), or may be due to changes in the pathogen.

Frogeye leaf spot may be partially managed by planting disease-free seeds, treatment of seeds with a fungicide before planting, crop rotation, treatment of R2-R5 growth stage soybean foliage with fungicides, and planting resistant cultivars if available. Three single genes conditioning resistance to C. sojina are currently recognized by the Soybean Genetics Committee. Rcs1 in ‘Lincoln’ was the first gene identified, and it conditioned resistance to race 1 of C. sojina (2). Rcs2 in ‘Kent’ conditioned resistance to race 2 (3), and Rcs3 in ‘Davis’ conditioned resistance to race 5 and to all other known races of C. sojina in the USA (15) as well as to all isolates from Brazil (21). Other dominant genes for resistance to race 5 were found in ‘Ransom,’ ‘Stonewall,’ and ‘Lee’ in 1993, and each of these genes was non-allelic to Rcs3 and to each other (13). Another single dominant gene reported as non-allelic to Rcs3 from the cultivar Peking was found later and provided resistance against many isolates of C. sojina (4).

Some MG V to VIII FLS resistant cultivars have been developed and are being used in the southern USA, and the resistance in those cultivars is conditioned by the Rcs3 gene initially discovered in cultivar Davis (15). In contrast, very few C. sojina resistant cultivars and breeding lines adapted to the north central production region have been identified (11). The objectives of this study were (1) to identify maturity group (MG) I to VI accessions resistant to C. sojina race 11 by field screening, and (2) to determine by molecular marker assay if FLS resistance in symptomless accessions is likely to be conditioned by the Rcs3 allele.

Identifying FLS Resistance in MG I to VI Soybean Accessions

Field screening. Field plots were established in 2009 and 2010 at the University of Missouri-Delta Center near Portageville, MO, and at the Southern Illinois University near Tamms, IL. The soil in Missouri is classified as a Tiptonville silt loam (fine-silty, mixed, superactive, thermic, oxyaquic Argiudoll) and the soil in Illinois is classified as a Meadowbank silt loam (fine-silty, mixed, superactive, mesic Typic Argiudolls). The accessions tested ranged from maturity group (MG) I to VI. A total of 260 soybean accessions (including 12 differentials) were tested for their reaction to C. sojina race 11. The standard FLS differentials, Kent, Hood, Lincoln, Lee, Blackhawk, Peking, Davis, S-100, CNS, Tracy, Palmetto, and Richland, were used as controls (Table 1).

Table 1. Soybean accessions reaction to Cercospora sojina race 11 in field studies at Illinois and Missouri during 2009.

Prior to planting, the field was disked twice, and 75- to 96-cm-wide row beds were formed. The top 10 cm of the beds were pushed off just prior to planting to form a flat-top ridge. The herbicides imazaquin (0.02 kg a.i./ha) and alaclor (0.4 kg a.i./ha) were preplant incorporated into the flat-top ridges. The test sites at both locations were fields that had been rotated with corn (Zea mays L.) to minimize the presence of natural inoculums of C. sojina. Ten seeds of each genotype were planted in hills, spaced 0.6 m apart, in the center of the ridge with a hand held "jab" planter. The planting dates at both sites were May 11 and May 15 in 2009 and May 13 and May 10 at Tamms, IL, and Portageville, MO, respectively. Accessions and differentials were planted in a randomized complete block design with two replications at each test site each year. Each test site was treated with a post-emergence directed application of bentazon (0.56 kg a.i./ha) and clethodim (0.14 kg a.i./ha) at the V6 stage of soybean growth (6). Plots were cultivated once and hand weeded as needed. Overhead irrigation was applied at about 8:00 pm for 15 min just prior to inoculating plants. Plots were irrigated by overhead sprinkler up to the R7 stage.

Evaluation of genotype reaction to Cercospora sojina. Accessions were inoculated with the same race 11 isolate of C. sojina (12). Plants with only Rcs1 are susceptible to race 11, but plants with Rcs2, Rcs3, and the undefined single dominant gene in Peking are resistance to race 11 (12). This isolate was cultured on Emerson medium (18) at 25°C on a laboratory bench for 7 days. The culture was then flooded with sterile deionized water and the culture surface rubbed manually to dislodge conidia. The conidia suspension was filtered through cheesecloth, the concentration adjusted to 1 × 105 conidia/ml and Tween 20 mixed with the suspension (0.003% v/v). Forty two-day plants were sprinkle irrigated for 30 min at 8:00 pm, and the plants in each hill were then sprayed with approximately 30 ml of inoculum. A susceptible soybean cultivar was planted around the whole experiment for use as an indicator for incoming natural inoculum. One half of the border row planted around the experimental area was also inoculated, while the other half was left uninoculated. Genotype reaction to C. sojina was evaluated 14 days after inoculation using a 0 to 9 scale, developed by the Southern Soybean Disease Workers, where 0 = no observable disease symptom and 9 = 90% diseased leaf tissue (17) (Fig. 2). All accessions without symptoms 14 days after inoculation were further evaluated for reaction to C. sojina at 21 and 28 days after inoculation. Accessions without symptoms in both replications at both locations were retested for validation of resistance in 2010. The ratings are always in the top one third of the canopy. The rating was also in the top one third, 2 to 4 weeks later.

Twenty accessions (Table 1) without symptoms at both locations over the two-year period were tested for the presence of the Rcs3 allele (resistant to all known races of C. sojina) at the Rcs3 locus with the molecular marker and screening protocol reported by Ha and Boerma (7). This high-throughput single nucleotide polymorphism (SNP) genotyping assay for melting curve analysis for marker assisted selection of the Rcs3 allele uses the LightCycler 480 of Roche Applied Science. Ha and Boerma reported that the melting curve assay for the SNP marker AZ573AG159 (tightly linked to Rcs3 locus) was a highly accurate predictor for the presence of the Rcs3 allele at the locus. The presence of the Rcs3 allele from cultivar Davis is indicated by the melting peak at 66°C while the presence of the Rcs3 allele (susceptible to all known races of C. sojina) from cultivar Blackhawk is indicated by the presence of the melting peak at 60°C. Along with 20 FLS resistant accessions, Davis was used as the check for the presence of Rcs3 allele (for FLS resistance) and cultivar Blackhawk was used as the check for the presence of the Rcs3 allele (for FLS susceptibility) in the molecular assay.

Field screening analysis. Genotype reactions to FLS were subjected to analysis of variance (ANOVA) using the SAS general linear mixed model procedure (SAS Procedures Guide, Version 8, 1999; SAS Institute, Cary, NC). Genotypes within each MG were analyzed separately. Fisher’s least significant difference test was used to generate means separation groups and those genotypes without symptoms were of interest and tested for the presence of Rcs3.

260 Accessions Evaluated

Field screening. There were significant differences in reaction of accessions between locations (Pr < 0.0001) to C. sojina race 11, so data for accessions for each location was treated separately for analysis. The reaction to C. sojina race 11 ranged from 0 to 7 and varied among accessions (Table 1). Even after 28 days after inoculation, some accessions remained without symptoms. Twenty five accessions did not develop symptoms of FLS and 20 of these plant introductions (not previously characterized for any FLS resistant gene) were tested for the presence of Rcs3 using SNP marker. The other five accessions that did not develop symptoms were part of the 12 differentials used in the test. The non-inoculated border plants did not show any frogeye leaf spot symptoms but the inoculated section of the border rows had a FLS score of 7.

Many of the accessions with a score of 0 were from MG IV. Accessions with no infection were also found in MG II and III but not in MG I but very few MG 1 accessions were evaluated. Sixty nine accessions in Illinois and 37 in Missouri did not produce FLS symptoms but only 20 accession resistant in both locations were selected. Of the total 260 accessions, 154 accessions (59%) had a score > 5 in at least one of the locations.

Marker assisted selection. Of the 20 soybean accessions subjected to SNP marker melting curve analysis, none had the melting peak at 66°C for the presence of the Rcs3 allele (Table 2). However, they all had the melting peak at 60°C, same with the susceptible check Blackhawk, indicating the presence of Rcs3 allele. Thus, these results indicate that the FLS resistance of the 20 accessions tested was probably not conditioned by the Rcs3 locus and may be conditioned by other locus. Similar data was also reported by Mengistu, et al. (9) where a total of 521 accessions from maturity group (MGs) 00 to VII were assessed for reaction to race 11 in field trials in Missouri, Illinois and Tennessee during 2006, 2007 and 2008 found 72 that these lines did not develop symptoms of FLS but 67 of the accessions were not conditioned by the Rcs3 locus and may be conditioned by other locus similar to this study.

Table 2. Results of the melting curve analyses of the 20 frogeye leaf
spot resistant (symptomless) accessions for prediction of the Rcs3 allele
with the SNP marker AZ573AG159 on the LightCycler 480 according to
Ha and Boerma (7).

Melting peak
FLS resistance
Davis (Resistant) 66 Rcs3/Rcs3
Blackhawk (susceptible) 60 Rcs3/Rcs3
PI532465 60 Unknown locus
PI567302 60 Unknown locus
PI567481 60 Unknown locus
PI490766 60 Unknown locus
PI567330 60 Unknown locus
PI567333A 60 Unknown locus
PI567432A 60 Unknown locus
PI567432B 60 Unknown locus
PI567438 60 Unknown locus
PI532463A 60 Unknown locus
PI567477 60 Unknown locus
PI567478 60 Unknown locus
PI567490 60 Unknown locus
PI63945 60 Unknown locus
PI538383 60 Unknown locus
PI538386B 60 Unknown locus
PI567291 60 Unknown locus
PI567319B 60 Unknown locus
PI567316A 60 Unknown locus
PI567333B 60 Unknown locus

 * A melting peak at 66°C indicates the potential presence of the Rcs3
allele for resistance to frogeye leaf spot while the presence of a melting
peak at 60°C indicates the potential presence of Rcs3 allele for
susceptibility to frogeye leaf spot at the Rcs3 locus.

The background genetics for some of the commercial soybeans grown in the southern US is from the cultivar ‘Davis,’ the source of Rcs3 resistance. Since nearly two thirds of the commercially available varieties are susceptible to FLS, growers resort to using fungicides as a means of control. Even though fungicides are effective in managing frogeye leaf spot, there are recent reports of resistance developing to commonly used QoI fungicides (Carl A. Bradley, personal communication). Resorting to the use of host resistance will be the most economical and environmentally sound effective way to slow the progression of QoI fungicide resistance and manage frogeye leaf spot. The resistant accessions identified in this study will be further characterized in the future. It is possible that it may carry novel resistant genes and could be utilized to broaden the base of host resistance to combat the FLS disease.

Acknowledgments and Disclaimer

These studies were supported in part by the University of Missouri Agriculture Experiment Station, The Ohio State University Agriculture Experiment Station, and Southern Illinois University, Carbondale. The authors thank the North Central Soybean Research Program and the Tennessee Soybean Promotion Council for the financial support from farmer soybean check-off dollars through funding of the project. In addition the authors thank Joyce Elrod, Jason Deffenbaugh, Jamie Jordan, and Chris Vick for their efforts in this project.

The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

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