Genome-wide association of single nucleotide polymorphism loci and candidate genes for frogeye leaf spot (Cercospora sojina) resistance in soybean

Background Frogeye leaf spot (FLS) is a destructive fungal disease that affects soybean production. The most economical and effective strategy to control FLS is the use of resistant cultivars. However, the use of a limited number of resistant loci in FLS management will be countered by the emergence of new high-virulence Cercospora sojina races. Therefore, we identified quantitative trait loci (QTL) that control resistance to FLS and identified novel resistant genes using a genome-wide association study (GWAS) on 234 Chinese soybean cultivars. Results A total of 30,890 single nucleotide polymorphism (SNP) markers were used to estimate linkage disequilibrium (LD) and population structure. The GWAS results showed four loci (p < 0.0001) distributed over chromosomes (Chr.) 5 and 20, that are significantly associated with FLS resistance. No previous studies have reported resistance loci in these regions. Subsequently, 45 genes in the two resistance-related haplotype blocks were annotated. Among them, Glyma20g31630 encoding pyruvate dehydrogenase (PDH), Glyma05g28980, which encodes mitogen-activated protein kinase 7 (MPK7), and Glyma20g31510, Glyma20g31520 encoding calcium-dependent protein kinase 4 (CDPK4) in the haplotype blocks deserves special attention. Conclusions This study showed that GWAS can be employed as an effective strategy for identifying disease resistance traits in soybean and narrowing SNPs and candidate genes. The prediction of candidate genes in the haplotype blocks identified by disease resistance loci can provide a useful reference to study systemic disease resistance.

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 use fungicides with multiple modes of action to manage this disease in light of QoI-resistant C. sojina isolates being present in the state.

Assessing Missouri soybean fields for azoxystrobin resistant cercospora sojina

Successful management of Cercospora sojina, the causal agent of frogeye leaf spot, can be achieved through utilizing resistant varieties and fungicide applications. Fungicides in the quinone outside inhibitor (QoI) class are most effective in controlling C. sojina in the field. Unfortunately, azoxystrobin fungicide-resistant isolates of C. sojina have been recovered in many soybean growing areas of the U.S. Fungicide-resistant isolates of C. sojina were first detected in 2011 and 2012 in two counties in southeast Missouri, but no further assessment was conducted. In this study, 121 isolates were collected from 15 surveyed counties between 2019 and 2020 in an effort to understand the geographical distribution of fungicide-resistant C. sojina. Isolates were collected from fields based on the presence of frogeye leaf spot symptoms. Samples were brought to the laboratory and isolates were recovered from individual lesions. A fungicide sensitivity bioassay was conducted to determine which isolates were resistant to the QoI class of fungicides. The fungicide sensitivity bioassay consisted of full-strength PDA amended with technical grade azoxystrobin at five different concentrations and a no fungicide control. Out of 121 isolates, 81 were fungicide-resistant representing 13 of the 15 Missouri counties included in the survey. Notably, the northwest corner of Missouri had the highest concentration of fungicide-resistant isolates, consistent with the recent recovery of fungicide-resistant C. sojina isolates in Iowa and Nebraska. The widespread recovery of fungicide-resistant C. sojina in multiple counties throughout Missouri provides new insight into disease management in the state.

Genome-wide Association of Single Nucleotide Polymorphism Loci and Candidate Genes for Frogeye Leaf Spot (Cercospora Sojina) Resistance in Soybean

Background: Frogeye leaf spot (FLS) is a destructive fungal disease in soybean production. The most economical and effective strategy to control FLS is to use resistant cultivars. However, the use of a limited number of resistant loci in FLS management will be countered by the emergence of new, and more virulent, Cercospora sojina races. Thus, we identified quantitative trait loci (QTL) that control resistance to FLS and identified novel resistant genes using a genome-wide association study (GWAS) on 234 Chinese soybean cultivars.Results: A total of 30,890 single nucleotide polymorphism (SNP) markers were used to estimate linkage disequilibrium (LD) and population structure. The GWAS results identified six loci (p<0.001) distributed over chromosomes (Chr.) 2, 5, 20 comprising 69 SNPs significantly associated with FLS resistance. No previous studies have reported resistance loci in these regions. Subsequently, 55 genes in three resistance-related haplotype blocks were annotated. Glyma20g31630 encoding pyruvate dehydrogenase (PDH), Glyma05g28980 encoding mitogen-activated protein kinase 7 (MPK7), and Glyma20g31510, Glyma20g31520 encoding the calcium-dependent protein kinase 4(CDPK4) in the haplotype blocks were found to be associated with FLS resistance.Conclusions: The proteins encoded by these four genes directly, or indirectly participate in the biological pathway of salicylic acid (SA) and jasmonic acid (JA). These two plant hormones can induce the expression of disease resistance related genes and are essential for plant systemic acquired resistance (SAR). Therefore, the novel loci and candidate genes provide genomic location information for further advances in FLS resistance gene cloning and molecular breeding.

Assessment of quinone outside inhibitor sensitivity and frogeye leaf spot race of Cercospora sojina in Georgia soybean

Frogeye leaf spot (FLS), caused by the fungal pathogen Cercospora sojina K. Hara, is a foliar disease of soybean (Glycine max L. (Merr.)) responsible for yield reductions throughout the major soybean producing regions in the world. In the United States, management of FLS relies heavily on the use of resistant cultivars and in-season fungicide applications, specifically within the class of quinone outside inhibitors (QoIs), which has resulted in the development of fungicide resistance in many states. In 2018 and 2019, 80 isolates of C. sojina were collected from six counties in Georgia and screened for QoI fungicide resistance using molecular and in vitro assays, with resistant isolates being confirmed from three counties. Additionally, 50 isolates, including a “baseline isolate” with no prior fungicide exposure, were used to determine the percent reduction of mycelial growth to two fungicides, azoxystrobin and pyraclostrobin, at six concentrations: 0.0001, 0.001, 0.01, 0.1, 1, and 10 g ml-1. Mycelial growth observed for resistant isolates varied significantly from both the sensitive isolates and the baseline isolate for azoxystrobin concentrations of 10, 1, 0.1, and 0.01 g ml-1 and for pyraclostrobin concentrations of 10, 1, 0.1, 0.01 and 0.001 g ml-1. Moreover, 40 isolates were used to evaluate pathogen race on six soybean differential cultivars by assessing susceptible or resistant reactions. Isolate reactions suggested 12 races of C. sojina present in Georgia, four of which have not been previously described. Species richness indicators (rarefaction and abundance-based coverage estimator - ACE) indicated that within-county C. sojina race numbers were undersampled in the present study, suggesting the potential for the presence of either additional undescribed races or known but unaccounted for races in Georgia. However, no isolates were pathogenic on differential cultivar ‘Davis’, carrying the Rcs3 resistance allele, suggesting the gene is still an effective source of resistance in Georgia.