Genome-Wide Association Analysis Uncovers Genes Associated with Resistance to Head Smut Pathotype 5 in Senegalese Sorghum Accessions.

A newly documented pathotype 5 of the soil-borne fungus Sporisorium reilianum, causing head smut in sorghum, was tested against 153 unexplored Senegalese sorghum accessions. Among the 153 sorghum accessions tested, 63 (41%) exhibited complete resistance, showing no signs of infection by the fungus. The remaining 90 accessions (59%) displayed varying degrees of susceptibility. Sorghum responses against S. reilianum were explored to analyze the potential link with previously known seed morphology-related traits and new phenotype data from 59 lines for seed weight. A genome-wide association study (GWAS) screened 297,876 SNPs and identified highly significant associations (p < 1 × 10-5) with head smut resistance in sorghum. By mapping these significant SNPs to the reference genome, this study revealed 35 novel candidate defense genes potentially involved in disease resistance.

Genetic and Pathogenic Variability among Isolates of Sporisorium reilianum Causing Sorghum Head Smut.

Sporisorium reilianum, the causal agent of sorghum (Sorghum bicolor (L.) Moench) head smut, is present in most sorghum-producing regions. This seed replacement fungal disease can reduce yield by up to 80% in severely infected fields. Management of this disease can be challenging due to the appearance of different pathotypes within the pathogenic population. In this research, the genetic variability and pathogenicity of isolates collected from five Texas Counties was conducted. Due to the lack of available space, 21 out of 32 sequenced isolates were selected and evaluated for virulence patterns on the six sorghum differentials, Tx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), Tx414, and BTx643. The results reveal the occurrence of a new pathotype, 1A, and four previously documented US pathotypes when the 21 isolates were evaluated for virulence patterns on the differentials. The most prevalent was pathotype 5, which was recovered from Brazos, Hidalgo, Nueces, and Willacy Counties, Texas. This pathotype was followed by 1A and 6 in frequency of recovery. Pathotype 4 was identified only from isolates collected from Hidalgo County, while pathotype 1 was from Burleson County, Texas. It appeared that the previous US head smut pathotypes (2 and 3) are no longer common, and the new pathotypes, 1A, 5, and 6, are now predominant. The phylogenetic tree constructed from the single-nucleotide polymorphism (SNP) data through the neighbor-joining method showed high genetic diversity among the tested isolates. Some of the diverse clades among the tested isolates were independent of their sampled locations. Notably, HS37, HS49, and HS65 formed a clade and were classified as 1A in the virulence study, while HS 61 and HS 66, which were collected from Nueces County, were grouped and identified as pathotype 5.

A Genome-Wide Association Study of Nigerien and Senegalese Sorghum Germplasm of Exserohilum turcicum, the Causal Agent of Leaf Blight.

In Senegal, sorghum ranks third after millet and maize among dryland cereal production and plays a critical role in the daily lives of millions of inhabitants. Yet, the crop's productivity and profitability are hampered by biotic stresses, including Exserohilum turcicum, causing leaf blight. A total of 101 sorghum accessions collected from Niger and Senegal, SC748-5 and BTx623, were evaluated in three different environments (Kaymor, Kolda, and Ndiaganiao) in Senegal for their reactions against the leaf blight pathogen. The results showed that 11 out of the 101 accessions evaluated exhibited 100% incidence, and the overall mean incidence was 88.4%. Accession N15 had the lowest incidence of 50%. The overall mean severity was 31.6%, while accessions N15, N43, N38, N46, N30, N28, and N23 from Niger recorded the lowest severity levels, ranging from 15.5% to 25.5%. Accession N15 exhibited both low leaf blight incidence and severity, indicating that it may possess genes for resistance to E. turcicum. Also, the accessions evaluated in this study were sequenced. A GWAS identified six novel single-nucleotide polymorphisms (SNPs) associated with an average leaf blight incidence rate. The candidate genes were found in chromosomes 2, 3, 5, 8, and 9. Except for SNP locus S05_48064154, all five SNPs associated with the leaf blight incidence rate were associated with the plant defense and stress responses. In conclusion, the candidate genes identified could offer additional options for enhancing plant resistance against E. turcicum through plant breeding or gene editing.

Multi-Trait Genome-Wide Association Studies of Sorghum bicolor Regarding Resistance to Anthracnose, Downy Mildew, Grain Mold and Head Smut.

Multivariate linear mixed models (mvLMMs) are widely applied for genome-wide association studies (GWAS) to detect genetic variants affecting multiple traits with correlations and/or different plant growth stages. Subsets of multiple sorghum populations, including the Sorghum Association Panel (SAP), the Sorghum Mini Core Collection and the Senegalese sorghum population, have been screened against various sorghum diseases such as anthracnose, downy mildew, grain mold and head smut. Still, these studies were generally performed in a univariate framework. In this study, we performed GWAS based on the principal components of defense-related multi-traits against the fungal diseases, identifying new potential SNPs (S04_51771351, S02_66200847, S09_47938177, S08_7370058, S03_72625166, S07_17951013, S04_66666642 and S08_51886715) associated with sorghum's defense against these diseases.

Genome-Wide Association Study of Seed Morphology Traits in Senegalese Sorghum Cultivars.

Sorghum is considered the fifth most important crop in the world. Despite the potential value of Senegalese germplasm for various traits, such as resistance to fungal diseases, there is limited information on the study of sorghum seed morphology. In this study, 162 Senegalese germplasms were evaluated for seed area size, length, width, length-to-width ratio, perimeter, circularity, the distance between the intersection of length & width (IS) and center of gravity (CG), and seed darkness and brightness by scanning and analyzing morphology-related traits with SmartGrain software at the USDA-ARS Plant Science Research Unit. Correlations between seed morphology-related traits and traits associated with anthracnose and head smut resistance were analyzed. Lastly, genome-wide association studies were performed on phenotypic data collected from over 16,000 seeds and 193,727 publicly available single nucleotide polymorphisms (SNPs). Several significant SNPs were found and mapped to the reference sorghum genome to uncover multiple candidate genes potentially associated with seed morphology. The results indicate clear correlations among seed morphology-related traits and potential associations between seed morphology and the defense response of sorghum. GWAS analysis listed candidate genes associated with seed morphologies that can be used for sorghum breeding in the future.

Inoculation and Screening Methods for Major Sorghum Diseases Caused by Fungal Pathogens: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina.

Sorghum is the fifth most important crop globally. Researching interactions between sorghum and fungal pathogens is essential to further elucidate plant defense mechanisms to biotic stress, which allows breeders to employ genetic resistance to disease. A variety of creative and useful inoculation and screening methods have been developed by sorghum pathologists to study major fungal diseases. As inoculation and screening methods can be keys for successfully conducting experiments, it is necessary to summarize the techniques developed by this research community. Among many fungal pathogens of sorghum, here we summarize inoculation and screening methods for five important fungal pathogens of sorghum: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina. The methods described within will be useful for researchers who are interested in exploring sorghum-fungal pathogen interactions. Finally, we discuss the latest biotechnologies and methods for studying plant-fungal pathogen interactions and their applicability to sorghum pathology.

The genome of the oomycete Peronosclerospora sorghi, a cosmopolitan pathogen of maize and sorghum, is inflated with dispersed pseudogenes.

Several species in the oomycete genus Peronosclerospora cause downy mildew on maize and can result in significant yield losses in Asia. Bio-surveillance of these pathogens is a high priority to prevent epidemics on maize in the United States and consequent damage to the US economy. The unresolved taxonomy and dearth of molecular resources for Peronosclerospora spp. hinder these efforts. P. sorghi is a pathogen of sorghum and maize with a global distribution, for which limited diversity has been detected in the southern USA. We characterized the genome, transcriptome, and mitogenome of an isolate, representing the US pathotype 6. The highly homozygous genome was assembled using 10× Genomics linked reads and scaffolded using Hi-C into 13 chromosomes. The total assembled length was 303.2 Mb, larger than any other oomycete previously assembled. The mitogenome was 38 kb, similar in size to other oomycetes, although it had a unique gene order. Nearly 20,000 genes were annotated in the nuclear genome, more than described for other downy mildew causing oomycetes. The 13 chromosomes of P. sorghi were highly syntenic with the 17 chromosomes of Peronospora effusa with conserved centromeric regions and distinct chromosomal fusions. The increased assembly size and gene count of P. sorghi is due to extensive retrotransposition, resulting in putative pseudogenization. Ancestral genes had higher transcript abundance and were enriched for differential expression. This study provides foundational resources for analysis of Peronosclerospora and comparisons to other oomycete genera. Further genomic studies of global Peronosclerospora spp. will determine the suitability of the mitogenome, ancestral genes, and putative pseudogenes for marker development and taxonomic relationships.

Genetic Diversity and Classification of Colletotrichum sublineola Pathotypes Using a Standard Set of Sorghum Differentials.

Anthracnose, incited by Colletotrichum sublineola, is the most destructive foliar disease of sorghum and, under severe conditions, yield losses can exceed 80% on susceptible cultivars. The hyper-variable nature of the pathogen makes its management challenging despite the occurrence of several resistant sources. In this study, the genetic variability and pathogenicity of 140 isolates of C. sublineola, which were sequenced using restriction site-associated sequencing (RAD-Seq), resulted in 1244 quality SNPs. The genetic relationship based on the SNP data showed low to high genetic diversity based on isolates' origin. Isolates from Georgia and North Carolina were grouped into multiple clusters with some level of genetic relationships to each other. Even though some isolates from Texas formed a cluster, others clustered with isolates from Puerto Rico. The isolates from Puerto Rico showed scattered distribution, indicating the diverse nature of these isolates. A population structure and cluster analysis revealed that the genetic variation was stratified into eight populations and one admixture group. The virulence pattern of 30 sequenced isolates on 18 sorghum differential lines revealed 27 new pathotypes. SC748-5, SC112-14, and Brandes were resistant to all the tested isolates, while BTx623 was susceptible to all. Line TAM428 was susceptible to all the pathotypes, except for pathotype 26. Future use of the 18 differentials employed in this study, which contains cultivars/lines which have been used in the Americas, Asia, and Africa, could allow for better characterization of C. sublineola pathotypes at a global level, thus accelerating the development of sorghum lines with stable resistance to the anthracnose pathogen.

A Genome-Wide Association Study of Senegalese Sorghum Seedlings Responding to Pathotype 5 of Sporisorium reilianum.

Sporisorium reilianum is a fungal pathogen that causes head smut in sorghum. In addition to pathotypes (P) 1-4, P5 and P6 were identified recently. In this study, seedlings of Senegalese sorghum, comprising 163 accessions, were evaluated for response to Sporisorium reilianum. Teliospores of pathotype P5 of the pathogen in dilute agar were pipetted onto seedling shoots while still in soil, and inoculated seedlings were submerged under water at 4 days post-inoculation. Signs of infection (noticeable spots) on the first leaf were checked daily up to 6 days post submergence. A genome-wide association study (GWAS) was conducted using 193,727 single-nucleotide polymorphisms (SNPs) throughout the genome based on two types of phenotypic data: whether noticeable spots were shown or not and the average time for an observation of the spots across 163 accessions. When mapped back to the reference sorghum genome, most of the top candidate SNP loci were associated with plant defense or plant stress response-related genes. The identified SNP loci were associated with spot appearance in sorghum seedlings under flooding following inoculation with P5 of Sporisorium reilianum.

Genome-wide association study of Senegalese sorghum seedlings responding to a Texas isolate of Colletotrichum sublineola.

Colletotrichum sublineola is a destructive fungal pathogen that causes anthracnose in sorghum. Senegalese sorghum germplasm is currently being considered as an option of sources for genetic resistance. In a recent study, Senegalese sorghum accessions were evaluated for response to a mixture of Texas isolates of C. sublineola at the 8-leaf stage in the greenhouse. As a comparison, 159 Senegalese sorghum accessions at the 1-leaf developmental stage were evaluated against a single Texas isolate of C. sublineola (FSP53) using an excised-leaf assay. A genome-wide association study (GWAS) was conducted based on the phenotypic data acquired to discover genetic variation associated with response to C. sublineola using 193,727 single nucleotide polymorphisms (SNPs) throughout the genome. Sorghum seedlings tended to be more resistant when compared with sorghum plants inoculated at the 8-leaf stage in the greenhouse in previous experiments. Based on the highest score evaluated in the 1-leaf developmental stage excised leaf assay for each accession, 16 accessions were labeled as susceptible. GWAS identified the SNP locus S01_72868925 that is associated with protein kinase domain // Leucine rich repeat N-terminal domain at a level of confidence that surpassed Bonferroni correction. Along with the SNP locus S01_72868925, other top SNP loci were also associated with genes that are known to play critical roles in plant defense or plant stress responses.

Interaction of Fusarium Wilt Race 4 with Root-Knot Nematode Increases Disease Severity in Cotton.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum, is a severe disease of cotton (Gossypium spp.). Strains of the wilt pathogen in the United States, such as race 1, require the presence of nematodes such as southern root-knot nematode (Meloidogyne incognita) to cause appreciable disease. The exception is the race 4 strain of the wilt pathogen, which can attack cotton without concomitant infection by plant-parasitic nematodes and was first identified in California in 2001 and in Texas and New Mexico since 2017. The effects of the interaction between M. incognita and race 1 or race 4 on wilt severity and nematode reproduction on two Gossypium hirsutum cultivars, Acala 44 and FM 966, and a G. barbadense cultivar, Pima S-4, were directly compared in growth chamber assays. All three cultivars were susceptible to M. incognita. Suppression of nematode reproduction by the wilt pathogen was detected only for race 4 on all three cultivars on a per plant basis but not on a per gram root tissue basis. The control, M. incognita alone, and race 1 alone treatments caused no symptoms. Inoculation with race 1 and M. incognita caused moderate wilt symptoms in 'Acala 44' and 'FM 966' and mild symptoms in 'Pima S-4'. However, race 4 treatment caused severe wilt in 'Pima S-4' and moderate wilt severity in 'Acala 44' and 'FM 966'. The symptom severity of 'Acala 44' and 'FM 966' further increased in the presence of M. incognita. Thus, race 4 is not only capable of causing wilt in the absence of M. incognita but can also interact with the nematode to further increase disease severity. Though control of wilt caused by race 1 can be achieved mainly through breeding for nematode resistance, it will be imperative to incorporate both southern root-knot nematode and race 4 resistance to effectively control the disease should race 4 expand into southern root-knot nematode-infested fields.

RNA-Sequencing in Resistant (QL3) and Susceptible (Theis) Sorghum Cultivars Inoculated With Johnsongrass Isolates of Colletotrichum sublineola.

Gene expression was analyzed at 0- and 24-h post-inoculation of two inbred sorghum cultivars known to differ in response to inoculation with Colletotrichum sublineola, the fungal pathogen that causes anthracnose. QL3 is reported to have quantitative resistance, while Theis is susceptible to most pathotypes of the pathogen; RNASeq identified over 3,000 specific genes in both cultivars as showing significant changes in expression following inoculation; in all but one gene, the changes in QL3 and Thies were in the same direction. Many other genes showed significant changes in only one of the two cultivars. Overall, more genes were downregulated than upregulated. Differences in changes in expression levels of a few genes suggested potential roles for the difference in disease response between QL3 and Theis, but did not identify known resistance genes. Gene ontology (GO) and pathway enrichment analysis identified upregulation of 23 transcription factor encoding genes as well as genes involved in the production of secondary metabolites, which are part of a typical host defense reaction.

Genome-wide association analysis for response of Senegalese sorghum accessions to Texas isolates of anthracnose.

Anthracnose disease of sorghum is caused by Colletotrichum sublineola, a filamentous fungus. The genetic basis of resistance to anthracnose in sorghum is largely unclear, especially in Senegalese sorghum germplasm. In this study, 163 Senegalese sorghum accessions were evaluated for response to C. sublineola, and a genome-wide association study (GWAS) was performed to identify genetic variation associated with response to C. sublineola using 193,727 single nucleotide polymorphisms (SNPs) throughout the genome. Germplasm diversity analysis showed low genetic diversity and slow linkage disequilibrium (LD) decay among the Senegalese accessions. Phenotypic analysis resulted in relatively low differences to C. sublineola among the tested population. Genome-wide association study did not identify any significant association based on a strict threshold for the number of SNPs available. However, individual analysis of the top eight SNPs associated with relative susceptibility and resistance identified candidate genes that have been shown to play important roles in plant stress tolerance in previous studies. This study identifies sorghum genes whose annotated properties have known roles in host defense and thus identify them as candidates for use in breeding for resistance to anthracnose.

Genetic Diversity and Pathogenicity of Verticillium dahliae Isolates and Their Co-occurrence with Fusarium oxysporum f. sp. vasinfectum Causing Cotton Wilt in Xinjiang, China.

Cotton production in Xinjiang, the largest cotton-producing area in China, has an increasingly serious disease threat from Verticillium dahliae. Eighty-five V. dahliae isolates were obtained from wilted cotton plants collected from eight counties in Xinjiang. The isolates were assessed for genotypic diversity by DNA sequence analysis and PCR molecular genotyping with specific markers for race 1, race 2, defoliating (D) pathotype, nondefoliating (ND) pathotype, and mating type idiomorph Mat1-2. Isolates belonged to lineages 1A or 2B, with three subgenotypes found in each lineage. All isolates tested positive for race 2 and Mat1-2 markers. All isolates in lineage 2B tested positive for the ND pathotype marker but only isolates in the major subgenotype in lineage 1A tested positive for the D pathotype marker. Pathogenicity assays on Gossypium hirsutum 'Acala 44' demonstrated no significant difference among subgenotypes within each lineage. Isolates in lineage 1A caused greater shoot weight reductions, percent leaf drop, and percent diseased leaves than isolates in lineage 2B. One isolate in each lineage for 1A and 2B was avirulent. Isolates in lineage 1A caused greater than 50% leaf drop and a 17-g shoot weight reduction compared with a 9% leaf drop and a 6-g shoot weight reduction by isolates in lineage 2B. Overall, 42% of the V. dahliae isolates from Xinjiang were D pathotype but the percentage varied widely among locations. Two plants had both pathotypes. Nineteen isolates of Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) also were recovered from wilted plants in Xinjiang. Two plants had both Verticillium wilt and Fusarium wilt pathogens. Both pathogens should be considered when using or developing wilt resistant or tolerant materials for Xinjiang.

Leaf angle distribution in Johnsongrass, leaf thickness in sorghum and Johnsongrass, and association with response to Colletotrichum sublineola.

Basal leaf angle distribution was surveyed in twenty-one Johnsongrass cultivars near the end of the vegetative stage. The angles increased from the top to the bottom leaves, and compared to cultivated grain sorghums, the average angle was larger in Johnsongrass. When basal leaf angle distribution data were correlated with pathogenicity test data from excised-leaf assays for three isolates of Colletotrichum sublineola, the results showed a weak positive correlation between basal leaf angle and pathogenicity level in Johnsongrass. In order to investigate a protective role of leaf thickness to C. sublineola, leaf thickness was measured in three sorghum cultivars and one Johnsongrass cultivar at the 8-leaf-stage. Leaf thickness near the apex, near the base, and half-way between the two points were measured in the top four leaves of each plant. Thickness of leaf blade and midrib were recorded separately. Using an excised-leaf-assay, the three points were inoculated with C. sublineola, and pathogenicity level was recorded 4-days-post-inoculation. Results showed strong negative correlations between leaf midrib thickness and pathogenicity level in sorghum and Johnsongrass but not in leaf blades.

Genome wide association analysis of sorghum mini core lines regarding anthracnose, downy mildew, and head smut.

In previous studies, a sorghum mini core collection was scored over several years for response to Colletotrichum sublineola, Peronosclerospora sorghi, and Sporisorium reilianum, the causal agents of the disease anthracnose, downy mildew, and head smut, respectively. The screening results were combined with over 290,000 Single nucleotide polymorphic (SNP) loci from an updated version of a publicly available genotype by sequencing (GBS) dataset available for the mini core collection. GAPIT (Genome Association and Prediction Integrated Tool) R package was used to identify chromosomal locations that differ in disease response. When the top scoring SNPs were mapped to the most recent version of the published sorghum genome, in each case, a nearby and most often the closest annotated gene has precedence for a role in host defense.

GWAS analysis of sorghum association panel lines identifies SNPs associated with disease response to Texas isolates of Colletotrichum sublineola.

KEY MESSAGE: SNPs identify prospective genes related to response to Colletotrichum sublineola (anthracnose) in the sorghum association panel lines. Sorghum association panel (SAP) lines were scored over several years for response to Colletotrichum sublineola, the causal agent of the disease anthracnose. Known resistant and susceptible lines were included each year to verify successful inoculation. Over 79,000 single-nucleotide polymorphic (SNP) loci from a publicly available genotype by sequencing dataset available for the SAP lines were used with TASSEL association mapping software to identify chromosomal locations associated with differences in disease response. When the top-scoring SNPs were mapped to the published sorghum genome, in each case, the nearest annotated gene has precedence for a role in host defense.

Specific PCR Detection of Fusarium oxysporum f. sp. vasinfectum California Race 4 Based on a Unique Tfo1 Insertion Event in the PHO Gene.

A highly virulent race 4 (Cal race 4) of Fusarium oxysporum f. sp. vasinfectum was identified in California cotton fields in 2001, and has since been found in increasing numbers of fields. Cal race 4 isolates contain a unique Tfo1 transposon insertion in the PHO gene that was not found in other F. oxysporum f. sp. vasinfectum genotypes. Based on this insertion, a multiplex polymerase chain reaction method was developed to detect the Cal race 4 pathogen. A panel of F. oxysporum f. sp. vasinfectum isolates representing different vegetative compatibility groups (VCG) and DNA sequence types was assembled to test the specificity of the detection method. In all, 16 of 17 Cal race 4 isolates produced a 583-bp amplicon; the other isolate produced a 396-bp amplicon reflecting the absence of the Tfo1 insertion. This isolate was a moderately virulent pathogen among Cal race 4 isolates. In total, 80 other F. oxysporum isolates associated with cotton and 11 other formae speciales of F. oxysporum produced only the 396-bp amplicon. The method also distinguished Cal race 4 isolates from India race 4 isolates and China race 7 isolates, which did not possess the unique Tfo1 insertion but otherwise had identical DNA sequences, and all belong to VCG0114. The method is capable of detecting the pathogen directly from infected stem tissues even before external symptom appears and, thus, provides an effective tool for timely identification of infested fields and seed lots, and should help reduce dissemination of Cal race 4 in the U.S. Cotton Belt.

Virulence and Molecular Genotyping Studies of Sporisorium reilianum Isolates in Sorghum.

Head smut, caused by the fungal pathogen Sporisorium reilianum, has been reported with increasing frequency in the grain sorghum growing areas of Texas. To facilitate analysis of changes in pathogen virulence, four inoculation techniques were examined: soil and teliospore mixture, seed coating, media placement, and syringe injection. Of the four, syringe injection was determined to be the most effective. Inoculations of sorghum host differentials BTx643, BTx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), and Tx414 showed 23 of 32 Texas isolates were race 4. Two isolates from College Station, TX, were classified as race 1, but no race 2 or 3 isolates were found. New, virulent races 5 and 6 were identified among isolates from south Texas. Using 16 amplified fragment length polymorphism (AFLP) primer combinations, genetic diversity was assessed in DNA samples from 49 S. reilianum isolates, including 44 sorghum isolates from Texas, two from Uganda, and one from Mali; and two maize isolates from Mexico. Single-base extensions with EcoRI and MseI primers in the selective amplification increased the number of informative polymorphic bands. High genetic dissimilarity (50%) was observed between isolates originating from maize and those originating from sorghum. The resultant dendrogram, made using cluster analysis, grouped the Texas S. reilianum isolates into four small clusters with ≥82% similarity. Other than for two race 6 isolates from Weslaco, TX, no evidence for geographical or other restrictions on gene flow was evident.