The fungal pathogen Sclerotinia sclerotiorum causes substantial economic damage to agriculture in the United States and globally. This pathogen can infect an unusually broad range of plant hosts, including many economically important crop species. S. sclerotiorum produces dormant resting structures referred to as sclerotia at the end of its disease cycle which can remain viable in soil for many years, presenting a major challenge for disease management. Relatively little is currently known about genes and molecular processes governing sclerotial development, survival and longevity, and subsequent germination. Isolates of S. sclerotiorum exhibit considerable variation in morphological and developmental traits, including traits relating to production of sclerotia. In this study, growth and development traits were evaluated for a collection of 227 S. sclerotiorum isolates obtained from 26 different host plants across 27 U.S. states. Genome-wide association studies were carried out for the traits: 1) time to sclerotia production and; 2) number of sclerotia produced. These studies identified seven marker-trait associations for time to sclerotia production and three associations for number of sclerotia produced. A single gene previously described to impact sclerotial development was found near a significant association for time to sclerotia production, while several other genes with predicted functions related to processes implicated in sclerotial development were also located near associated markers. Further studies of these candidate genes may provide new information about the processes governing sclerotial development in this destructive fungal pathogen.

Exserohilum spike blight, caused by Exserohilum rostratum, is an emerging constraint in wheat production, and improving host resistance is a sustainable strategy because the disease is governed largely by quantitative, environment-responsive loci rather than single major genes. Identifying genomic regions and biological pathways underlying quantitative resistance is therefore essential for developing durable resistant varieties. In this study, the Wheat Association Mapping Initiative spring wheat panel (n = 289) was evaluated across two contrasting Indian agroclimatic zones over two seasons. Resistance was quantified using two traits: incubation period (IP) and area under the disease progress curve (AUDPC). Significant genotype, environment, and genotype × environment interaction effects were observed for both traits (p < 0.001). IP and AUDPC were weakly correlated (r = −0.18 to 0.16), indicating that these traits represent partially distinct resistance components. Genome-wide association mapping identified 25 marker–trait associations, 13 for AUDPC and 12 for IP, with most associations showing environment dependence. Putative candidate genes highlighted defense-relevant loci, including an LRR receptor-like kinase and a WRKY transcription factor for IP, and an RGA2-like resistance gene and PHLOEM UNLOADING MODULATOR-like gene for AUDPC. These findings provide the first GWAS-based framework for E. rostratum resistance in wheat, prioritizing loci for validation and marker deployment.

IntroductionThe necrotrophic fungus Sclerotinia sclerotiorum is a destructive plant pathogen that can infect a broad range of host plants, including many agriculturally important crop species. Resistance to S. sclerotiorum is partial and quantitative, controlled by many genes. The identities of genes influencing resistance and the molecular mechanisms governing defense against this pathogen are poorly understood. To improve understanding of resistance, we performed genome-wide association studies of Arabidopsis thaliana response to inoculation with two isolates of the pathogen differing in aggressiveness and sought to validate our results by identifying the causal gene at a single mapped locus.MethodsA total of 325 A. thaliana ecotypes were evaluated for resistance at two timepoints after inoculation with S. sclerotiorum isolate 1980 or BN325. Genome-wide association studies were carried out using two different models to identify loci associated with resistance. A. thaliana mutant lines were then evaluated for candidate genes at a single locus to identify the most likely candidate gene influencing resistance, and sequencing of the candidate gene and promoter region was performed to identify putative causal variants.Results and discussionGenome-wide association studies mapped 30 loci associated with resistance to S. sclerotiorum. Surprisingly, correlations for response to the two isolates among A. thaliana ecotypes were relatively weak and no overlapping loci were mapped for resistance to both isolates. A. thaliana med20a mutants impaired in a subunit of the transcriptional Mediator complex were more susceptible to S. sclerotiorum and a single variant upstream of the MED20a gene was associated with resistance. These results improve our mechanistic understanding of resistance to this important plant pathogen.

Phomopsis stem canker disease of cultivated sunflower (Helianthus annuus) is most commonly caused by the fungal pathogen Diaporthe helianthi. This disease is characterised by brown stem lesions centred around petioles accompanied by destruction of the pith, often resulting in plant death. In this study, Phomopsis stem canker disease lesions were collected from commercial sunflower fields in Minnesota, North Dakota, and South Dakota over 2 years in 2018 and 2019. The Diaporthe species associated with the disease lesion was isolated and identified for 363 total samples, and D. helianthi was isolated from 93% of lesions, with the remaining lesions primarily associated with D. gulyae or both D. helianthi and D. gulyae. Population analyses of 280 D. helianthi isolates, genotyped using genotyping-by-sequencing, revealed no significant differentiation among populations across the three states and indicated primarily outcrossing reproductive behaviour. To determine if sunflower resistance to stem lesion expansion is isolate-specific, 15 sunflower lines previously determined to exhibit resistance to stem lesioning after inoculation with D. helianthi were evaluated for resistance to 20 D. helianthi isolates. Only five sunflower lines were strongly or moderately resistant to all isolates, while the remaining lines exhibited isolate-specific resistance. These results will be useful to facilitate breeding efforts aimed at improving sunflower resistance to Phomopsis stem canker.

Basal stalk rot (BSR) of cultivated sunflower (Helianthus annuus) is caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum. This disease is economically significant and limits sunflower production in the northern Great Plains region of the United States. Resistance to BSR is quantitative, controlled by many genes exerting small effects on the level of resistance. This genetic complexity hinders efforts to develop sunflower hybrids with sufficient resistance. Field-based disease trials have successfully identified cultivated sunflower genotypes with partial BSR resistance but failed to determine the degree of resistance. Consequently, the objectives of this study were to (i) distinguish between highly and moderately resistant genotypes by reevaluating 60 cultivated sunflower genotypes exhibiting partial resistance in field trials using a newly developed greenhouse inoculation method with improved resolution; (ii) determine if selected genotypes identified in objective (i) are broadly resistant to diverse S. sclerotiorum isolates; (iii) assess potential host genotype-pathogen isolate interactions between sunflower genotypes and S. sclerotiorum isolates; and (iv) determine if resistant genotypes carry alleles of molecular markers previously associated with BSR resistance loci and assess the frequency of these alleles in resistant germplasm. The results of this study identified sunflower inbred lines HA 124 and HIR 34 exhibiting high levels of BSR resistance against all tested S. sclerotiorum isolates. Additionally, significant host genotype-pathogen isolate interactions were detected between sunflower lines and S. sclerotiorum isolates. This information will guide breeding efforts to improve BSR resistance and facilitate prioritizing highly resistant lines for genetic mapping and further characterization.

Cultivated sunflower (Helianthus annuus L.) is a globally important oilseed crop that is grown primarily in the Northern Great Plains region of the United States. In September 2018, sunflower stems exhibiting brown stem lesions centered on the leaf axils and accompanied by pith degradation, consistent with symptoms of Phomopsis stem canker (PSC) disease, were sampled from a commercial field of approximately 520 hectares in Polk County, MN (47°50'24" N, 96.34'13" W). Incidence of PSC in this field was approximately 44%. Ten diseased stem samples were collected from plants spaced at least ten meters apart. Pieces of diseased stems 15 mm in length and 10 mm in width were excised, surface sterilized in 10% sodium hypochlorite and plated onto potato dextrose agar (PDA) containing 50 µg/ml streptomycin. Plates were incubated at 22°C for 3-5 days and hyphal tips of emerging mycelia were transferred twice to new PDA plates. Genomic DNA was extracted from each isolate and polymerase chain reaction (PCR) was carried out using primers specific to either Diaporthe helianthi or D. gulyae, the most common species causing PSC on sunflower (Elverson et al., 2020). Eight isolates were identified as D. helianthi by PCR, while the remaining two isolates, designated H48 and H49, exhibited morphologies on PDA plates distinct from those of D. helianthi or D. gulyae. The ITS region, along with portions of the EF-1α and β-tubulin (TUB) genes were amplified and sequenced, and the sequences were deposited in Genbank (H48: ITS-OP429627.1, EF-1α-OP429589.1, TUB-OP429587.1; H49: ITS-OP429628.1, EF-1α-OP429590.1, TUB-OP429588.1). BLAST analyses using the H48 and H49 ITS sequences revealed 100% identity with D. caulivora isolates including the type specimen CBS 127268 (Genbank accessions AF000567.2 and type specimen MH864501.1). Additionally, 100% identity of EF-1α and TUB sequences from H48 and H49 with those of type specimen CBS 127268 (Genbank accessions KC343771.1 and KC344013.1) were also observed. A multi-locus phylogenetic tree was constructed aligning the ITS, EF-1α, and TUB sequences of H48 and H49 with those of twenty Diaporthe species. Isolates H48 and H49 formed a clade with three other D. caulivora isolates, including the type specimen. Pathogenicity of isolate H49 on sunflower was confirmed by inoculating susceptible sunflower inbred line HA 410 using a stem wound method (Mathew et al., 2018; Thompson et al., 2011; Underwood & Misar, 2024). Plants were inoculated by creating a wound approximately 5 mm deep and 8 mm long using a scalpel and affixing a PDA plug carrying mycelium of isolate H49, D. helianthi isolate H45 known to be pathogenic, or mock inoculum of PDA with no mycelium over the wound using laboratory film. Three independent experimental runs were conducted in which two 6-week-old plants grown in 1-gallon pots under greenhouse conditions at 22 ± 3°C were inoculated with each treatment, resulting in inoculation of six total HA 410 plants per treatment. Isolate H49 formed lesions with mean length of 61.27 mm on HA 410 at 14 days-post-inoculation compared to mean lesion length of 173.05 mm for D. helianthi isolate H45 and no disease symptoms were observed upon mock inoculation. D. caulivora was re-isolated from inoculated stems and confirmed by observing morphological features on PDA plates as well as sequencing the ITS region, fulfilling Koch's postulates. D. caulivora is typically associated with stem canker of soybean and has been previously reported to cause PSC on sunflower in Argentina (Zambelli et al., 2021). Additionally, this species was identified as an endophyte on asymptomatic sunflowers in the U.S., though pathogenicity was not confirmed in this study (Dangel, 2022). To our knowledge, this is the first report of D. caulivora causing Phomopsis stem canker on sunflower in the U.S. It will likely be prudent to monitor this species as a potential causal agent of PSC on sunflower and potentially to determine if sunflower lines resistant to other Diaporthe species are also resistant to D. caulivora should a need arise to deploy resistance to this species in commercial sunflower hybrids.

Phomopsis stem canker of cultivated sunflower (Helianthus annuus L.) can be caused by multiple necrotrophic fungi in the genus Diaporthe, with Diaporthe helianthi and D. gulyae being the most common causal agents in the United States. Infection begins at the leaf margins and proceeds primarily through the vasculature, progressing from the leaf through the petiole to the stem, resulting in formation of brown stem lesions centered around the petiole. Sunflower resistance to Phomopsis stem canker is quantitative and genetically complex. Due to the intricate disease process, resistance is possible at different stages of infection, and multiple forms of defense may contribute to the overall level of quantitative resistance. In this study, sunflower lines exhibiting field resistance to Phomopsis stem canker were evaluated for stem and leaf resistance to multiple isolates of D. helianthi and D. gulyae in greenhouse experiments, and responses to the two species were compared. Additionally, selected resistant and susceptible lines were evaluated for petiole transmission resistance to D. helianthi. Lines with distinct forms of resistance were identified, and results indicated that responses to stem inoculation were strongly correlated (Spearman's coefficient 0.598, P < 0.001) for the two fungal species, while leaf responses were not (Spearman's coefficient 0.396, P = 0.076). These results provide a basis for genetic dissection of distinct forms of sunflower resistance to Phomopsis stem canker and will facilitate combining different forms of resistance to potentially achieve durable control of this disease in sunflower hybrids.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

IntroductionSclerotinia sclerotiorum is a necrotrophic fungal pathogen causing disease and economic loss on numerous crop plants. This fungus has a broad host range and can infect over 400 plant species, including important oilseed crops such as soybean, canola, and sunflower. S. sclerotiorum isolates vary in aggressiveness of lesion formation on plant tissues. However, the genetic basis for this variation remains to be determined. The aims of this study were to evaluate a diverse collection of S. sclerotiorum isolates collected from numerous hosts and U.S. states for aggressiveness of stem lesion formation on sunflower, to evaluate the population characteristics, and to identify loci associated with isolate aggressiveness using genome-wide association mapping.MethodsA total of 219 S. sclerotiorum isolates were evaluated for stem lesion formation on two sunflower inbred lines and genotyped using genotyping-by-sequencing. DNA markers were used to assess population differentiation across hosts, regions, and climatic conditions and to perform a genome-wide association study of isolate aggressiveness.Results and discussionWe observed a broad range of aggressiveness for lesion formation on sunflower stems, and only a moderate correlation between aggressiveness on the two lines. Population genetic evaluations revealed differentiation between populations from warmer climate regions compared to cooler regions. Finally, a genome-wide association study of isolate aggressiveness identified three loci significantly associated with aggressiveness on sunflower. Functional characterization of candidate genes at these loci will likely improve our understanding of the virulence strategies used by this pathogen to cause disease on a wide array of agriculturally important host plants.

SUMMARYRust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R11 in sunflower line HA‐R9 shows broad‐spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long‐read sequencing to clone the R11 gene. Sequencing of a 60‐kb region spanning the R11 locus from the R11‐HA‐R9 rust‐resistant line and three EMS‐induced susceptible mutants facilitated the identification of R11 and definition of induced mutations. The R11 gene is predicted to have a single 3996‐bp open reading frame and encodes a protein of 1331 amino acids with CC‐NBS‐LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R11 rust‐susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R11‐HA‐R9 and R11‐susceptible mutants revealed substantial differences in gene expression patterns associated with R11‐mediated resistance at 7 days post‐inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

Introduction:Sclerotinia sclerotiorum is a serious pathogen causing severe basal stalk rot (BSR) disease on cultivated sunflower (Helianthus annuus L.) that leads to significant yield losses due to insufficient resistance. The wild annual sunflower species H. petiolaris, commonly known as prairie sunflower is known for its resistance against this pathogen. Sunflower resistance to BSR is quantitative and determined by many genes with small effects on the resistance phenotype. The objective of this study was to identify loci governing BSR resistance derived from H. petiolaris using a quantitative trait loci (QTL) mapping approach.Methods:BSR resistance among lines of an advanced backcross population (AB-QTL) with 174 lines developed from a cross of inbred line HA 89 with H. petiolaris PI 435843 was determined in the field during 2017-2019, and in the greenhouse in 2019. AB-QTL lines and the HA 89 parent were genotyped using genotyping-by-sequencing and a genetic linkage map was developed spanning 997.51 cM and using 1,150 SNP markers mapped on 17 sunflower chromosomes.Results and discussion:Highly significant differences (p<0.001) for BSR response among AB-QTL lines were observed disease incidence (DI) in all field seasons, as well as disease rating (DR) and area under the disease progress curve (AUDPC) in the greenhouse with a moderately high broad-sense heritability (H 2) of 0.61 for the tested resistance parameters. A total of 14 QTL associated with BSR resistance were identified on nine chromosomes, each explaining a proportion of the phenotypic variation ranging from 3.5% to 28.1%. Of the 14 QTL, eight were detected for BSR resistance in the field and six were detected under greenhouse conditions. Alleles conferring increased BSR resistance were contributed by the H. petiolaris parent at 11 of the 14 QTL.

The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter contributes to penetration resistance against nonadapted powdery mildew fungi and is targeted to papillae deposited at sites of interaction with the fungus. Timely recruitment of PEN3 and other components of penetration resistance to the host-pathogen interface is important for successful defense against this biotrophic pathogen. A forward genetic screen was previously carried out to identify Arabidopsis mutants that mistarget the PEN3 transporter or fail to accumulate PEN3 at sites of attempted powdery mildew penetration. This study focuses on PEN3 mistargeting in the aberrant localization of PEN3 4 (alp4) mutant and identification of the causal gene. In the alp4 mutant, PEN3 accumulates within the endomembrane system in an apparently abnormal endoplasmic reticulum and is not exported into papillae at powdery mildew penetration sites. This targeting defect compromises defenses at the host-pathogen interface, resulting in increased penetration success by a nonadapted powdery mildew. Genetic mapping identified alp4 as an allele of GOLGI DEFECTS 36 (GOLD36), a gene encoding a GDSL-lipase/esterase family protein that is involved in maintaining normal morphology and organization of multiple endomembrane compartments. Genetic complementation confirmed that mutation in GOLD36 is responsible for the PEN3 targeting and powdery mildew penetration resistance defects in alp4. These results reinforce the importance of endomembrane trafficking in resistance to haustorium-forming phytopathogens such as powdery mildew fungi.

The necrotrophic fungal pathogen Sclerotinia sclerotiorum can cause disease on numerous plant species, including many important crops. Most S. sclerotiorum-incited diseases of crop plants are initiated by airborne ascospores produced when fungal sclerotia germinate to form spore-bearing apothecia. However, basal stalk rot of sunflower occurs when S. sclerotiorum sclerotia germinate to form mycelia within the soil, which subsequently invade sunflower roots. To determine whether other plant species in the Asteraceae family are susceptible to root infection by S. sclerotiorum, cultivated sunflower (Helianthus annuus L.) and seven other Asteraceae species were evaluated for S. sclerotiorum root infection by inoculation with either sclerotia or mycelial inoculum. Additionally, root susceptibility of sunflower was compared with that of dry edible bean and canola, two plant species susceptible to S. sclerotiorum but not known to display root-initiated infections. Results indicated that multiple Asteraceae family plants are susceptible to S. sclerotiorum root infection after inoculation with either sclerotia or mycelium. These observations expand the range of plant hosts susceptible to S. sclerotiorum root infection, elucidate differences in root inoculation methodology, and emphasize the importance of soilborne infection to Asteraceae crop and weed species.

Sclerotinia head rot (HR), caused by Sclerotinia sclerotiorum, is an economically important disease of sunflower with known detrimental effects on yield and quality in humid climates worldwide. The objective of this study was to gain insight into the genetic architecture of HR resistance from a sunflower line HR21 harboring HR resistance introgressed from the wild perennial Helianthus maximiliani. An F2 population derived from the cross of HA 234 (susceptible-line)/HR21 (resistant-line) was evaluated for HR resistance at two locations during 2019–2020. Highly significant genetic variations (p < 0.001) were observed for HR disease incidence (DI) and disease severity (DS) in both individual and combined analyses. Broad sense heritability (H2) estimates across environments for DI and DS were 0.51 and 0.62, respectively. A high-density genetic map of 1420.287 cM was constructed with 6315 SNP/InDel markers developed using genotype-by-sequencing technology. A total of 16 genomic regions on eight sunflower chromosomes, 1, 2, 10, 12, 13, 14, 16 and 17 were associated with HR resistance, each explaining between 3.97 to 16.67% of the phenotypic variance for HR resistance. Eleven of these QTL had resistance alleles from the HR21 parent. Molecular markers flanking the QTL will facilitate marker-assisted selection breeding for HR resistance in sunflower.

Crop wild relatives of the cultivated sunflower ( Helianthus annuus L.) are a valuable resource for its sustainable production. Helianthus praecox ssp. runyonii is a wild sunflower known for its resistance against diseases caused by the fungus, Sclerotinia sclerotiorum (Lib.) de Bary, which infects over 400 broadleaf hosts including many important food crops. The objective of this research was to dissect the Sclerotinia basal stalk rot (BSR) resistance introgressed from H. praecox ssp. runyonii into cultivated sunflower. An advanced backcross quantitative trait loci (AB-QTL) mapping population was developed from the cross of a H. praecox accession with cultivated sunflower lines. The AB-QTL population was evaluated for BSR resistance in the field during the summers of 2017–2018 and in the greenhouse in the spring of 2018. Highly significant genetic variations ( p < 0.001) were observed for the BSR disease in the field and greenhouse with a moderately high broad-sense heritability ( H 2 ) ranging from 0.66 to 0.73. Genotyping-by-sequencing approach was used to genotype the parents and the progeny lines of the AB-QTL population. A genetic linkage map spanning 1,802.95 cM was constructed using 1,755 single nucleotide polymorphism (SNP) markers mapped on 17 sunflower chromosomes. A total of 19 BSR resistance QTL were detected on nine sunflower chromosomes, each explaining 2.21%–16.99% of the phenotypic variance for resistance in the AB-QTL population. Sixteen of the 19 QTL had alleles conferring increased BSR resistance derived from the H. praecox parent. SNP markers flanking the identified QTL will facilitate marker-assisted breeding to combat the disease in sunflower.