Genetic mapping of sorghum resistance to an Illinois isolate of Colletotrichum sublineola.

Anthracnose leaf blight (ALB) is an economically important disease of sorghum [Sorghum bicolor (L.) Moench] caused by the fungal pathogen Colletotrichum sublineola Henn. ex Sacc. & Trotter. Although qualitative and quantitative resistance have been identified for ALB, the usefulness of resistance loci differs depending on the pathogen pathotype. Identifying resistance effective against unique pathogen pathotypes is critical to managing ALB, as the disease is managed primarily through the deployment of host resistance. We isolated C. sublineola from ALB-infected leaves collected in Illinois and found that the strain was a novel pathotype, as it produced a unique combination of virulence against a set of differential lines. Using this isolate, we inoculated 579 temperate-adapted sorghum conversion lines in 2019 and 2020. We then conducted a genome-wide association study (GWAS) and a metabolic pathway analysis using the Pathway Associated Study Tool (PAST). We identified 47 significant markers distributed across all chromosomes except chromosome 8. We identified 32 candidate genes based on physical proximity with significant markers, some of which have a known role in host defense. We identified 47 pathways associated with ALB resistance, indicating a role for secondary metabolism in defense to ALB. Our results are important to improve the understanding of the genetic basis of ALB resistance in sorghum and highlight the importance of developing durable resistance to ALB.

Genomic regions associated with virulence in Setosphaeria turcica identified by linkage mapping in a biparental population.

Northern corn leaf blight (NCLB) and sorghum leaf blight (SLB) are significant diseases of maize and sorghum, respectively, caused by the filamentous fungus Setosphaeria turcica. Strains of S. turcica are typically host-specific and infect either maize or sorghum. Host specificity in this pathogen is attributed to a single locus for maize and a second distinct locus for sorghum. To identify the genetic basis of host specificity in S. turcica, we generated a biparental population of S. turcica by crossing strains specific to maize and sorghum, phenotyped the population for leaf blight on sorghum and maize, genotyped the population to create a linkage map of S. turcica, and located candidate virulence regions. A total of 190 ascospores from 35 pseudothecia were isolated from the cross of maize and sorghum-specific strains. Greenhouse phenotyping of the biparental population (n = 144) showed independent inheritance of virulence, as indicated by a 1:1:1:1 segregation for virulence to maize, sorghum, both maize and sorghum, and avirulence to both crops. The population and host-specific parent strains were genotyped using genome skim sequencing on an Illumina NovaSeq 6000 platform resulting in over 780 million reads. A total of 32,635 variants including single nucleotide polymorphisms and indels were scored. There was evidence for a large deletion in the sorghum-specific strain of S. turcica. A genetic map consisting of 17 linkage groups spanning 3,069 centimorgans was constructed. Virulence to sorghum and maize mapped on distinct linkage groups with a significant QTL detected for virulence to maize. Furthermore, a single locus each for the in vitro traits hyphal growth rate and conidiation were identified and mapped onto two other linkage groups. In vitro traits did not correlate with in planta virulence complexity, suggesting that virulence on both hosts does not incur a fitness cost. Hyphal growth rate and conidiation were negatively correlated, indicating differences in hyphal growth versus dispersal ability for this pathogen. Identification of genetic regions underlying virulence specificity and saprotrophic growth traits in S. turcica provides a better understanding of the S. turcica- Andropogoneae pathosystem.

Differential Regulation of Maize and Sorghum Orthologs in Response to the Fungal Pathogen Exserohilum turcicum.

Pathogens that infect more than one host offer an opportunity to study how resistance mechanisms have evolved across different species. Exserohilum turcicum infects both maize and sorghum and the isolates are host-specific, offering a unique system to examine both compatible and incompatible interactions. We conducted transcriptional analysis of maize and sorghum in response to maize-specific and sorghum-specific E. turcicum isolates and identified functionally related co-expressed modules. Maize had a more robust transcriptional response than sorghum. E. turcicum responsive genes were enriched in core orthologs in both crops, but only up to 16% of core orthologs showed conserved expression patterns. Most changes in gene expression for the core orthologs, including hub genes, were lineage specific, suggesting a role for regulatory divergent evolution. We identified several defense-related shared differentially expressed (DE) orthologs with conserved expression patterns between the two crops, suggesting a role for parallel evolution of those genes in both crops. Many of the differentially expressed genes (DEGs) during the incompatible interaction were related to quantitative disease resistance (QDR). This work offers insights into how different hosts with relatively recent divergence interact with a common pathogen. Our results are important for developing resistance to this critical pathogen and understanding the evolution of host-pathogen interactions.

Colonization and Movement of Green Fluorescent Protein-Labeled Clavibacter nebraskensis in Maize.

Clavibacter nebraskensis causes Goss's bacterial wilt and leaf blight, a major disease of maize. Infected crop residue is the primary inoculum source and infection can occur via wounds or natural openings, such as stomata or hydathodes. The use of resistant hybrids is the primary control method for Goss's wilt. In this study, colonization and movement patterns of C. nebraskensis during infection were examined using green fluorescent protein (GFP)-labeled bacterial strains. We successfully introduced a plasmid to C. nebraskensis via electroporation, which resulted in GFP accumulation. Fluorescence microscopy revealed that in the absence of wounding, bacteria colonize leaf tissue via entry through the hydathodes when guttation droplets are present. Stomatal penetration was not observed under natural conditions. Bacteria initially colonize the xylem and subsequently the mesophyll, which creates the freckles that are characteristic of the disease. Bacteria infiltrated into the mesophyll did not cause disease symptoms, could not enter the vasculature, and did not spread from the initial inoculation point. Bacteria were observed exuding through stomata onto the leaf surface, resulting in the characteristic sheen of diseased leaves. Resistant maize lines exhibited decreased bacterial spread in the vasculature and the mesophyll. These tools to examine C. nebraskensis movement offer opportunities and new insights into the pathogenesis process and can form the basis for improved Goss's wilt management through host resistance.

Genome-wide association analysis of the strength of the MAMP-elicited defense response and resistance to target leaf spot in sorghum.

Plants have the capacity to respond to conserved molecular features known as microbe-associated molecular patterns (MAMPs). The goal of this work was to assess variation in the MAMP response in sorghum, to map loci associated with this variation, and to investigate possible connections with variation in quantitative disease resistance. Using an assay that measures the production of reactive oxygen species, we assessed variation in the MAMP response in a sorghum association mapping population known as the sorghum conversion population (SCP). We identified consistent variation for the response to chitin and flg22-an epitope of flagellin. We identified two SNP loci associated with variation in the flg22 response and one with the chitin response. We also assessed resistance to Target Leaf Spot (TLS) disease caused by the necrotrophic fungus Bipolaris cookei in the SCP. We identified one strong association on chromosome 5 near a previously characterized disease resistance gene. A moderately significant correlation was observed between stronger flg22 response and lower TLS resistance. Possible reasons for this are discussed.

Genetic variation associated with PPO-inhibiting herbicide tolerance in sorghum.

Herbicide application is crucial for weed management in most crop production systems, but for sorghum herbicide options are limited. Sorghum is sensitive to residual protoporphyrinogen oxidase (PPO)-inhibiting herbicides, such as fomesafen, and a long re-entry period is required before sorghum can be planted after its application. Improving sorghum for tolerance to such residual herbicides would allow for increased sorghum production and the expansion of herbicide options for growers. In this study, we observed sorghum tolerance to residual fomesafen. To investigate the underlying tolerance mechanism a genome-wide association mapping study was conducted using field-collected sorghum biomass panel (SBP) data, and a greenhouse assay was developed to confirm the field phenotypes. A total of 26 significant SNPs (FDR<0.05), spanning a 215.3 kb region on chromosome 3, were detected. The ten most significant SNPs included two in genic regions (Sobic.003G136800, and Sobic.003G136900) and eight SNPs in the intergenic region encompassing the genes Sobic.003G136700, Sobic.003G136800, Sobic.003G137000, Sobic.003G136900, and Sobic.003G137100. The gene Sobic.003G137100 (PPXI), which encodes the PPO1 enzyme, one of the targets of PPO-inhibiting herbicides, was located 12kb downstream of the significant SNP S03_13152838. We found that PPXI is highly conserved in sorghum and expression does not significantly differ between tolerant and sensitive sorghum lines. Our results suggest that PPXI most likely does not underlie the observed herbicide tolerance. Instead, the mechanism underlying herbicide tolerance in the SBP is likely metabolism-based resistance, possibly regulated by the action of multiple genes. Further research is necessary to confirm candidate genes and their functions.

Identification of Loci That Confer Resistance to Bacterial and Fungal Diseases of Maize.

Crops are hosts to numerous plant pathogenic microorganisms. Maize has several major disease issues; thus, breeding multiple disease resistant (MDR) varieties is critical. While the genetic basis of resistance to multiple fungal pathogens has been studied in maize, less is known about the relationship between fungal and bacterial resistance. In this study, we evaluated a disease resistance introgression line (DRIL) population for the foliar disease Goss's bacterial wilt and blight (GW) and conducted quantitative trait locus (QTL) mapping. We identified a total of ten QTL across multiple environments. We then combined our GW data with data on four additional foliar diseases (northern corn leaf blight, southern corn leaf blight, gray leaf spot, and bacterial leaf streak) and conducted multivariate analysis to identify regions conferring resistance to multiple diseases. We identified 20 chromosomal bins with putative multiple disease effects. We examined the five chromosomal regions (bins 1.05, 3.04, 4.06, 8.03, and 9.02) with the strongest statistical support. By examining how each haplotype effected each disease, we identified several regions associated with increased resistance to multiple diseases and three regions associated with opposite effects for bacterial and fungal diseases. In summary, we identified several promising candidate regions for multiple disease resistance in maize and specific DRILs to expedite interrogation.

Conserved defense responses between maize and sorghum to Exserohilum turcicum.

BACKGROUND: Exserohilum turcicum is an important pathogen of both sorghum and maize, causing sorghum leaf blight and northern corn leaf blight. Because the same pathogen can infect and cause major losses for two of the most important grain crops, it is an ideal pathosystem to study plant-pathogen evolution and investigate shared resistance mechanisms between the two plant species. To identify sorghum genes involved in the E. turcicum response, we conducted a genome-wide association study (GWAS). RESULTS: Using the sorghum conversion panel evaluated across three environments, we identified a total of 216 significant markers. Based on physical linkage with the significant markers, we detected a total of 113 unique candidate genes, some with known roles in plant defense. Also, we compared maize genes known to play a role in resistance to E. turcicum with the association mapping results and found evidence of genes conferring resistance in both crops, providing evidence of shared resistance between maize and sorghum. CONCLUSIONS: Using a genetics approach, we identified shared genetic regions conferring resistance to E. turcicum in both maize and sorghum. We identified several promising candidate genes for resistance to leaf blight in sorghum, including genes related to R-gene mediated resistance. We present significant advancements in the understanding of host resistance to E. turcicum, which is crucial to reduce losses due to this important pathogen.

How Well Can Multivariate and Univariate GWAS Distinguish Between True and Spurious Pleiotropy?

Quantification of the simultaneous contributions of loci to multiple traits, a phenomenon called pleiotropy, is facilitated by the increased availability of high-throughput genotypic and phenotypic data. To understand the prevalence and nature of pleiotropy, the ability of multivariate and univariate genome-wide association study (GWAS) models to distinguish between pleiotropic and non-pleiotropic loci in linkage disequilibrium (LD) first needs to be evaluated. Therefore, we used publicly available maize and soybean genotypic data to simulate multiple pairs of traits that were either (i) controlled by quantitative trait nucleotides (QTNs) on separate chromosomes, (ii) controlled by QTNs in various degrees of LD with each other, or (iii) controlled by a single pleiotropic QTN. We showed that multivariate GWAS could not distinguish between QTNs in LD and a single pleiotropic QTN. In contrast, a unique QTN detection rate pattern was observed for univariate GWAS whenever the simulated QTNs were in high LD or pleiotropic. Collectively, these results suggest that multivariate and univariate GWAS should both be used to infer whether or not causal mutations underlying peak GWAS associations are pleiotropic. Therefore, we recommend that future studies use a combination of multivariate and univariate GWAS models, as both models could be useful for identifying and narrowing down candidate loci with potential pleiotropic effects for downstream biological experiments.

Dominant, Heritable Resistance to Stewart's Wilt in Maize Is Associated with an Enhanced Vascular Defense Response to Infection with Pantoea stewartii.

Vascular wilt bacteria such as Pantoea stewartii, the causal agent of Stewart's bacterial wilt of maize (SW), are destructive pathogens that are difficult to control. These bacteria colonize the xylem, where they form biofilms that block sap flow leading to characteristic wilting symptoms. Heritable forms of SW resistance exist and are used in maize breeding programs but the underlying genes and mechanisms are mostly unknown. Here, we show that seedlings of maize inbred lines with pan1 mutations are highly resistant to SW. However, current evidence suggests that other genes introgressed along with pan1 are responsible for resistance. Genomic analyses of pan1 lines were used to identify candidate resistance genes. In-depth comparison of P. stewartii interaction with susceptible and resistant maize lines revealed an enhanced vascular defense response in pan1 lines characterized by accumulation of electron-dense materials in xylem conduits visible by electron microscopy. We propose that this vascular defense response restricts P. stewartii spread through the vasculature, reducing both systemic bacterial colonization of the xylem network and consequent wilting. Though apparently unrelated to the resistance phenotype of pan1 lines, we also demonstrate that the effector WtsE is essential for P. stewartii xylem dissemination, show evidence for a nutritional immunity response to P. stewartii that alters xylem sap composition, and present the first analysis of maize transcriptional responses to P. stewartii infection.