Sojina Isolates Research Articles

Determining the Distribution of QoI Fungicide-Resistant Cercospora sojina on Soybean from Indiana.

Frogeye leaf spot (FLS) is a foliar disease of soybean (Glycine max) caused by Cercospora sojina. Application of fungicide products that contain quinone outside inhibitor (QoI) active ingredients has been one of the major tools used in the management of this disease, but, since 2010, QoI-resistant C. sojina isolates have been confirmed in over 20 states in the United States, including Indiana. In summer 2019 and 2020, 406 isolates of C. sojina were collected from 32 counties across Indiana and screened for QoI resistance using a PCR-restriction fragment length polymorphism (RFLP) method. An in vitro fungicide sensitivity test was also performed on a subset of isolates to evaluate their sensitivity to three QoI fungicides: azoxystrobin, pyraclostrobin, and picoxystrobin. A discriminatory dose of picoxystrobin was established as 1 µg/ml by testing five concentrations (0.001, 0.01, 0.1, 1, and 10 µg/ml). QoI-resistant isolates were found in 29 counties, and 251 of the 406 isolates (61.8%) were confirmed to be resistant to QoI fungicides based on PCR-RFLP results. Partial nucleotide sequences of the cytochrome b gene from four resistant and four sensitive isolates corroborated the presence and absence, respectively, of the G143A mutation. Results from the sensitivity assays with discriminatory doses of azoxystrobin (1 µg/ml) and pyraclostrobin (0.1 µg/ml) also supported the findings from the PCR-RFLP assay, because all QoI-resistant isolates were inhibited less than 50% relative to a no-fungicide control when exposed to these doses. Resistant isolates harboring the G143A mutation also exhibited resistance to picoxystrobin. The effective concentrations to inhibit mycelial growth by 50% relative to the nonamended control (EC50) in QoI-sensitive isolates ranged from 0.087 to 0.243 µg/ml, with an overall mean of 0.152 µg/ml, while EC50 values in QoI-resistant isolates were established as >10 µg/ml for picoxystrobin. Results from this study indicated that QoI-resistant C. sojina isolates are spread throughout Indiana and exhibit cross-resistance to QoI fungicides.

First Detection of Frogeye Leaf Spot in Soybean Fields in North Dakota and the G143A Mutation in the Cytochrome b Gene of Cercospora sojina

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from North Dakota soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in North Dakota. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in North Dakota will need to use an integrated approach of cultural practices, genetic resistance, and fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

The G143A Mutation in the Cytochrome b Gene Is Associated with Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina from Soybean Fields in Wisconsin

Frogeye leaf spot, caused by Cercospora sojina, is an important foliar disease of soybean (Glycine max) in the United States. Application of quinone outside inhibitor (QoI) fungicides has been an important management tool available to farmers to help manage this disease, but in 2010, C. sojina isolates with resistance to QoI fungicides were first discovered in Tennessee and then additional states in the years to follow. During the 2020 growing season, C. sojina isolates collected from Wisconsin soybean fields were tested for QoI resistance using laboratory and molecular assays. The results of these assays showed that QoI fungicide-resistant C. sojina isolates are present in Wisconsin. Similar to previous findings in other states, these QoI-resistant C. sojina isolates contain the G143A mutation. Soybean farmers in Wisconsin will need to use an integrated approach of cultural practices, genetic resistance, and using fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Sensitivity of Cercospora sojina to demethylation inhibitor and methyl benzimidazole carbamate fungicides

Cercospora sojina, the causal agent of frogeye leaf spot (FLS) of soybean (Glycine max), can cause significant yield losses. Foliar fungicide application is a common strategy used to control FLS; however, C. sojina strains with resistance to quinone outside inhibitor (QoI) fungicides have become widespread throughout U.S. soybean production regions. Because of QoI-resistance, demethylation inhibitor (DMI) and methyl benzimidazole carbamate (MBC) fungicides have become more important in managing FLS. Baseline sensitivity assays are important to monitor any shifts in fungicide sensitivity. Sensitivity of the DMI fungicides flutriafol and tetraconazole and the MBC fungicide thiophanate methyl were established for 145 C. sojina isolates collected prior to 2001(baseline isolates) and C. sojina isolates collected from 2007 to 2012 representing 12 states. Bioassays were conducted to assess effective fungicide concentration at which 50% of mycelium growth was inhibited relative to a non-fungicide control (EC50) for each isolate-fungicide combination. Baseline EC50 value means for flutriafol, tetraconazole, and thiophanate methyl were 0.268, 0.153, and 0.521 μg/ml. When means of EC50 values for isolates collected from 2007 to 2012 were compared to baseline sensitivity EC50 means, no significant difference (P < 0.05) was observed for sensitivities to flutriafol and tetraconazole. When this same comparison was done for sensitivity to thiophanate methyl, isolates collected in 2008 and 2012 were significantly more sensitive than the baseline isolates. Overall, these findings indicate no shift towards reduced sensitivity to flutriafol, tetraconazole, and thiophanate methyl. Based on these results, continued monitoring of C. sojina population sensitivity to DMI and MBC fungicides should occur.

Resistance to Quinone Outside Inhibitor Fungicides Conferred by the G143A Mutation in Cercospora sojina (Causal Agent of Frogeye Leaf Spot) Isolates from Michigan, Minnesota, and Nebraska Soybean Fields

Frogeye leaf spot, caused by Cercospora sojina, is an important disease of soybean (Glycine max) in the United States. An important tactic to manage frogeye leaf spot is to apply foliar fungicides. Isolates of C. sojina were collected from soybean fields in one county in Michigan, three counties in Minnesota, and 10 counties in Nebraska in 2019, and they were tested for resistance to quinone outside inhibitor (QoI) fungicides using a discriminatory dose assay, a PCR assay, and DNA sequencing. Results of the testing indicated that QoI fungicide-resistant isolates were detected in isolates from all counties. Testing results also indicated that the G143A mutation was responsible for the QoI fungicide resistance. This is the first report of QoI fungicide-resistant C. sojina isolates in Michigan, Minnesota, and Nebraska and expands the geographical distribution of QoI fungicide-resistant C. sojina isolates to 18 states in total.

Pathotype Grouping of Cercospora sojina Isolates on Soybean and Sensitivity to QoI Fungicides.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is a common disease of soybean in the southern and northern United States and causes significant yield loss. The use of the current race scheme for classification for C. sojina does not take into account the range of disease severity reactions within each differential. The objective of this research was to better understand the diversity among C. sojina isolates through the development and use of pathogenicity groups. In this study, 83 isolates acquired from 2006 to 2009 were screened using 12 soybean (Glycine max) differentials. Disease severity on the 12 differentials ranged from 0 to 9, where 0 is immune and 9 is very susceptible. The average severity for each isolate across differentials ranged from 1 to 7. The 83 isolates were grouped into five pathogenicity groups (PG): PG1, PG2, PG3, PG4, and PG5, reflecting the severity grouping. Using the 12 differentials, PG1 isolates were differentiated by the lack of infection on Davis, Peking, Kent, Palmetto, Hood, CNS, Tracy, and Richland. PG2 had a range of infections on a scale of 1 to 2 on all differentials except on Davis; PG3 isolates had severity ranging from 3 to 4 except on Davis. PG4 isolates caused no infection on Davis, a maximum disease severity of 5 on Peking, while the rest of differentials had severities from 5 to 6. PG5 isolates caused no infection on Davis, severity of 7 on CNS, and severity of 8 on Kent, Hood, and Palmetto. The remaining seven differentials had severities of 9. Across the geographical locations, the predominant pathotypes were PG3 and PG4 and represented 84% of the tested isolates. Azoxystrobin fungicide sensitivity tests showed that 88% of the isolates were sensitive and dominated the population, while only 6% had a high level of fungicide resistance, suggesting that FLS resistance to the QoI fungicide group was not yet completely developed and had not spread to other areas at the time when these isolates were acquired. The overall virulence profile of the isolates indicated that there was variation in disease severity, suggesting that selection of resistance for each PG may produce lines with more precisely defined interactions to specific pathotypes of C. sojina. This may improve the screening and selection of useful resistance genes that could be pyramided for resistance to each pathogenicity group.

Resistance to Quinone Outside Inhibitor Fungicides Conferred by the G143A Mutation in Cercospora sojina (Causal Agent of Frogeye Leaf Spot) Isolates from South Dakota Soybean Fields

Resistance to quinone outside inhibitor (QoI) fungicides was detected in Cercospora sojina (causal agent of frogeye leaf spot) isolates collected from soybean (Glycine max) fields in four South Dakota counties during the 2018 growing season. A discriminatory dose assay was used to detect QoI-resistant isolates, and a follow-up polymerase chain reaction assay was used to determine the presence of the G143A mutation in QoI-resistant isolates. This is the first report of resistance to QoI fungicides in C. sojina isolates from South Dakota.

Widespread Occurrence of Quinone Outside Inhibitor Fungicide-Resistant Isolates of Cercospora sojina, Causal Agent of Frogeye Leaf Spot of Soybean, in the United States

Isolates of Cercospora sojina, causal agent of frogeye leaf spot of soybean (Glycine max), were collected across Alabama, Arkansas, Delaware, Illinois, Indiana, Iowa, Kentucky, Louisiana, Mississippi, Missouri, North Carolina, Ohio, Tennessee, and Virginia and were evaluated for quinone outside inhibitor (QoI) fungicide resistance. Collection of these isolates from these 14 states occurred between 2010 and 2017. QoI fungicide-resistant C. sojina isolates were detected in all 14 states surveyed and represent a total of 240 counties or parishes. In 2017, these 240 counties and parishes represented approximately 13% of the harvested soybean hectares in the United States. In light of this widespread occurrence of QoI fungicide-resistant C. sojina isolates, management of frogeye leaf spot should focus on integrated management practices such as planting resistant soybean cultivars, rotating with nonhost crops, and tilling to speed up decomposition of infested soybean residue. When foliar fungicide application is warranted, fungicide products that contain active ingredients from chemistry classes other than the QoI class should be applied for frogeye leaf spot management.

Genetic diversity, QoI fungicide resistance, and mating type distribution of Cercospora sojina-Implications for the disease dynamics of frogeye leaf spot on soybean.

Frogeye leaf spot (FLS), caused by Cercospora sojina, causes significant damage to soybean in the U.S. One control strategy is the use of quinone outside inhibitor (QoI) fungicides. QoI resistant isolates were first reported in Tennessee (TN) in 2010. To investigate the disease dynamics of C. sojina, we collected 437 C. sojina isolates in 2015 from Jackson and Milan, TN and used 40 historical isolates collected from 2006–2009 from TN and ten additional states for comparison. A subset of 186 isolates, including historical isolates, were genotyped for 49 single nucleotide polymorphism (SNP) markers and the QoI resistance locus, revealing 35 unique genotypes. The genotypes clustered into three groups with two groups containing only sensitive isolates and the remaining group containing all resistant isolates and a dominant clonal lineage of 130 isolates. All 477 C. sojina isolates were genotyped for the QoI locus revealing 344 resistant and 133 sensitive isolates. All isolates collected prior to 2015 were QoI sensitive. Both mating type alleles (MAT1-1-1 and MAT1-2) were found in Jackson and Milan, TN and recovered from single lesions suggesting sexual recombination may play a role in the epidemiology of field populations. Analysis of C. sojina isolates using SNP markers proved useful to investigate population diversity and to elaborate on diversity as it relates to QoI resistance and mating type.

Comparison of quinone outside inhibitor fungicide-resistant and -sensitive isolates of Cercospora sojina

Isolates of Cercospora sojina, causal agent of frogeye leaf spot of soybean (Glycine max), that are resistant to quinone outside inhibitor (QoI) fungicides have been reported in the United States. Radial mycelial growth and sporulation of QoI-resistant and -sensitive C. sojina isolates were compared in the laboratory, and virulence of QoI-resistant and -sensitive isolates on a susceptible soybean cultivar (Blackhawk) and on a cultivar (Davis) with the Rcs3 resistance gene that confers resistance to frogeye leaf spot were compared. No differences in amount of sporulation between QoI-resistant and -sensitive isolates were observed. On cultivar (cv.) Blackhawk, frogeye leaf spot caused by QoI-resistant isolates was more severe than disease caused by QoI-sensitive isolates 7–8 days after inoculation, but no differences in disease between QoI-resistant and -sensitive isolates were observed 9 days after inoculation. On cv. Davis, QoI-resistant isolates caused significantly greater disease severity than sensitive isolates 8–14 days after inoculation. Although cv. Davis was affected more severely by QoI-resistant isolates, disease severity was less on cv. Davis than cv. Blackhawk, indicating that the Rcs3 gene was still effective against the C. sojina isolates tested. Our findings indicate that no fitness costs associated with QoI resistance in C. sojina were observed in the characteristics measured in our research.

Occurrence of QoI Fungicide Resistance in Cercospora sojina from Mississippi Soybean.

Frogeye leaf spot, caused by Cercospora sojina Hara, is a foliar disease affecting soybean (Glycine max (L.) Merr.), often managed by applications of quinone outside inhibitor (QoI) fungicides. In 2013 and 2014, 634 C. sojina monoconidial isolates were collected from soybean fields throughout Mississippi. Initially, in vitro bioassays were performed to evaluate the sensitivity of 14 of 634 isolates plus a baseline. Resistant and sensitive isolates were characterized by determining the effective fungicide concentrations at which 50% of conidial germination was inhibited (EC50). The molecular mechanism of resistance was determined for all 634 isolates, using a PCR-RFLP method and comparing nucleotide sequences of the cytochrome b gene. The state of Mississippi was divided into five distinct geographical regions (the Hills, Delta, Pines, Capital, and Coast) based on estimates of total soybean hectares. The greatest proportion (16.7%) of QoI-sensitive isolates was collected in the Hills while the Coast had no QoI-sensitive isolates. QoI-sensitive isolates from the Pines, Capital, and Delta ranged from 1.6 to 7.0%. Results of this study determined that more than 93% of C. sojina isolates collected in Mississippi carried the G143A amino acid substitution, indicating a shift to a QoI-resistant population throughout Mississippi soybean fields.

Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Mating-Type Distribution and Genetic Diversity ofCercospora sojinaPopulations on Soybean from Arkansas: Evidence for Potential Sexual Reproduction

Cercospora sojina causes frogeye leaf spot of soybean, which can cause serious economic losses in the United States. In this study, 132 C. sojina isolates were collected from six fields (from two counties, Cross and Crawford) in Arkansas. To determine mating type, a multiplex polymerase chain reaction assay was developed with primers specific for C. sojina. Of the 132 isolates, 68 isolates had the MAT1-1-1 idiomorph and 64 isolates had the MAT1-2 idiomorph; no isolates possessed both idiomorphs. Both mating types were present in a variety of spatial scales, including separate lesions on individual leaves. Clone-corrected data from eight microsatellites indicated that mating-type loci were present in approximately equal proportions in all populations analyzed, which suggests that Arkansas populations of C. sojina are undergoing cryptic sexual reproduction. All six populations evaluated had high genotypic diversity of 26 to 79%. In addition, among strains isolated from a single leaf, multiple and distinct haplotypes were associated with both mating types, supporting the hypothesis that sexual reproduction occurs within the populations. Most populations showed significant gametic disequilibrium but levels of disequilibrium were relatively low, particularly in populations from Crawford County. A low differentiation index (GST) was observed for all simple-sequence repeat markers across all populations. Furthermore, the value of G statistics between populations suggests that significant genetic exchange exists among the populations. Taken together, these results demonstrate that C. sojina populations from Arkansas are genetically diverse and most likely undergoing sexual reproduction.

Sensitivity of Cercospora sojina isolates to quinone outside inhibitor fungicides

Frogeye leaf spot, caused by Cercospora sojina, causes yield reductions to soybean (Glycine max) grown worldwide. Quinone outside inhibitor (QoI) fungicides have been effective in managing frogeye leaf spot, but the risk of selecting C. sojina strains with resistance to this class of fungicides is considered high. A QoI fungicide resistance monitoring program was initiated, in which sensitivities to azoxystrobin, pyraclostrobin, and trifloxystrobin were determined in C. sojina isolates collected prior to QoI fungicide use on soybean (baseline group) and C. sojina isolates collected from soybean fields in 2007, 2008 and 2009. For the baseline group, the mean effective fungicide concentration at which 50% of the conidial germination was inhibited (EC50) was determined to be 0.01287, 0.00028 and 0.00116 μg ml−1 for azoxystrobin, pyraclostrobin and trifloxystrobin, respectively. When mean EC50 values of 2007, 2008 and 2009 C. sojina isolates were compared to baseline C. sojina EC50 values, a small but statistically significant (P ≤ 0.05) shift towards less sensitivity was observed for trifloxystrobin in 2009. Although small (<1.5-fold), this shift in sensitivity indicates a risk of selecting for C. sojina strains with reduced sensitivity to QoI fungicides, and fungicide sensitivities should continue to be monitored in the future.

Genetic diversity of Cercospora sojina revealed by amplified fragment length polymorphism markers

Cercospora sojina Hara, the causal agent of frogeye leaf spot of soybean (Glycine max (L.) Merr.), causes yield reductions worldwide. Although the phenotypic diversity (physiological races) of this pathogen has been assessed through its ability to affect soybean lines with different resistant genes (Rcs genes), little is known about the pathogen's genetic diversity. In order to better understand the genetic diversity that exists with C. sojina, a historical collection of 62 C. sojina isolates from Brazil (10 isolates), China (7 isolates), Nigeria (1 isolate), and United States (44 isolates) was used for genetic diversity analysis with amplified fragment length polymorphism (AFLP) markers. The average genetic similarity of the isolates was 0.56 on a scale between 0 and 1, indicating a high degree of genetic diversity within the species. Cluster analysis resulted in two major clusters and seven sub-clusters. Two isolates collected from Georgia were clustered together, and two isolates from China were clustered together. Besides these four isolates, no clear separation of isolates based on origin was found. Our results provide evidence that substantial genetic diversity exists within the species of C. sojina and that selection for broad-spectrum host-resistance should be targeted in soybean breeding programmes.

First Report of the Soybean Frogeye Leaf Spot Fungus (Cercospora sojina) Resistant to Quinone Outside Inhibitor Fungicides in North America.

Quinone outside inhibitor (QoI; also known as strobilurin) fungicides sometimes are applied to soybean (Glycine max) fields to help manage frogeye leaf spot of soybean (caused by Cercospora sojina) in the United States. In August 2010, soybean leaflets exhibiting severe frogeye leaf spot symptoms were collected from a field in Lauderdale County, TN that had been treated twice with pyraclostrobin during that growing season. The field had been planted into soybean annually since at least 2008, and a QoI fungicide had been applied to the field in each of those years. Fifteen single-spore isolates of C. sojina were recovered from the affected soybean leaflets. These isolates were identified as C. sojina based on the observed symptoms on the soybean leaflets and the morphology and size of conidiophores and conidia (3). In addition, DNA was extracted from the cultures, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region was conducted (2), and the resulting PCR product was sequenced at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequences were compared with sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of C. sojina. The isolates were tested for their sensitivity to technical-grade formulations of the QoI fungicides azoxystrobin, pyraclostrobin, and trifloxystrobin with an in vitro conidial germination assay with fungicide + salicylhydroxamic acid (SHAM)-amended potato dextrose agar as described by Bradley and Pedersen (1). The effective concentration at which 50% conidial germination was inhibited (EC50) was determined for all 15 C. sojina isolates, with mean values of 3.1644 (2.7826 to 4.5409), 0.3297 (0.2818 to 0.6404), and 0.8573 (0.3665 to 2.5119) μg/ml for azoxystrobin, pyraclostrobin, and trifloxystrobin, respectively. When compared with previously established mean EC50 values of C. sojina baseline isolates (4), EC50 values of the C. sojina isolates collected from the Lauderdale County, TN soybean field were approximately 249- to 7,144-fold greater than the EC50 values of the baseline isolates. These results indicate that all isolates recovered from the Lauderdale County, TN soybean field were highly resistant to QoI fungicides. To our knowledge, this is the first report of QoI fungicide resistance occurring in C. sojina, and surveys for additional QoI fungicide-resistant C. sojina isolates are needed to determine their prevalence and geographic distribution. In light of these findings, soybean growers in Tennessee and adjacent states should consider utilizing alternative frogeye leaf spot management practices such as planting resistant cultivars, rotating to nonhost crops, and tilling affected soybean residue (3).

Differential Responses of Soybean Cultivars to Cercospora sojina Isolates, the Causal Agent of Frogeye Leaf Spot in Korea

During the summer of 2005, specimens of soybean cultivars (Daepung, Daewon, Hwanggeum and Taegwang) showing frogeye leaf spot (FLS) were obtained from various areas in Korea. Eight isolates identified as Cercospora sojina were inoculated on the adaxial leaf surfaces of 63 Korean soybean cultivars; the disease responses to each isolate were evaluated 14 days and 21 days after inoculation. Based on the disease responses (resistant or susceptible) of the cultivars by the isolates, a set of cultivars (Anpeong, Bogwang, Cheongdu No. 1, Cheongja No. 3, Dachae, Daemang, Jangwon, Namhae, Sowon, Taegwang) were selected and inoculated with seven isolates for further testing pathogenic variance. Interestingly, 6 out of 7 tested C. sojina isolates revealed differential ability in infecting different soybean cultivars. This result may indicate the possibility of new race occurrence or pathogenic variation; this also presents evidence for prevalent FLS occurrence during humid and hot weather in Korea.

Frogeye Leaf Spot of Soybean: A Review and Proposed Race Designations for Isolates of Cercospora sojina Hara

Frogeye leaf spot (FLS), caused by Cercospora sojina K. Hara, is a common disease of soybean [Glycine max (L.) Merr.] in most soybean‐growing countries of the world. Significant yield losses of soybean (10–60%) have been attributed to FLS under hot and humid growing conditions. The FLS in the southern United States has been kept under control by planting resistant cultivars. Cercospora sojina is a dynamic pathogen with extensive virulence or race diversity. The current number of C. sojina isolates at the University of Georgia collection is 93. Assigning race designations to this large number of isolates has been difficult because of the lack of a well‐defined set of differentials. To facilitate future studies of the genetics of soybean resistance to FLS and the identification and comparison of existing and future races of C. sojina, we have reassessed both the soybean differentials and the C. sojina races. Based on the reactions of 93 isolates of C. sojina on 38 putative soybean differentials, we propose a core set of 12 differentials and 11 races that represent the major diversity among the 93 C. sojina isolates. The definition of these 12 differentials and 11 races should provide the foundation for the identification and comparison of additional soybean resistance genes and new races of C. sojina