Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance

Background: Stem rust is an economically important disease of wheat and barley. However, studies to gain insight into the molecular basis of these host-pathogen interactions have primarily focused on wheat because of its importance in human sustenance. This is the first extensive study utilizing a transcriptome-wide association mapping approach to identify candidate Puccinia graminis f. sp. tritici (Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifying Pgt elicitors that interact with the rpg4-mediated resistance locus (RMRL), the only effective source of Pgt race TTKSK resistance in barley. Results: Thirty-seven Pgt isolates showing differential responses on RMRL were genotyped using Restriction Site Associated DNA-Genotyping by Sequencing (RAD-GBS), identifying 24 diverse isolates that were used for transcript analysis during the infection process. In planta RNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to the Pgt race SCCL reference genome identifying 114 K variants in predicted genes that would result in nonsynonymous amino acid substitutions. Transcriptome wide association analysis identified 33 variants across 28 genes that were associated with dominant RMRL virulence, thus, representing candidate suppressors of resistance. Comparative transcriptomics between the 9 RMRL virulent-vs-the 15 RMRL avirulent Pgt isolates identified 44 differentially expressed genes encoding candidate secreted effector proteins (CSEPs), among which 38 were expressed at lower levels in virulent isolates suggesting that they may represent RMRL avirulence genes. Barley transcript analysis after colonization with 9 RMRL virulent and 15 RMRL avirulent isolates inoculated on the susceptible line Harrington showed significantly lower expression of host biotic stress responses specific to RMRL virulent isolates suggesting virulent isolates harbor effectors that suppress resistance responses. Conclusions: This transcriptomic study provided novel findings that help fill knowledge gaps in the understanding of stem rust virulence/avirulence and host resistance in barley. The pathogen transcriptome analysis suggested RMRL virulence might depend on the lack of avirulence genes, but evidence from pathogen association mapping analysis and host transcriptional analysis also suggested the alternate hypothesis that RMRL virulence may be due to the presence of suppressors of defense responses

Identification of Quantitative Trait Loci (QTL) Associated with Resistance to Initial Infection of Fusarium Head Blight in Spring Wheat

Video summarizing a Ph.D. dissertation for a non-specialist audience.Triticeae CAPUS Wheat and Barley Scab Initiative (USWBSI)State Board of Agricultural Research and EducationNDSU AgricultureNorth Dakota Wheat Commissio

Histology of Spot Blotch Infection in Barley, QTL Mapping of Resistance to Fusarium Head Blight, and Characterization of Root Rot Diseases in Wheat

Three independent studies were conducted for spot blotch (Bipolaris sorokiniana), Fusarium head blight (FHB) (Fusarium graminearum), and root rot diseases (Fusarium species and B. sorokiniana). Histopathology of compatible and incompatible interactions between different pathotypes of B. sorokiniana and different genotypes of barley was examined with red fluorescent protein-tagged fungal isolates. The fungus penetrated the host cell wall and developed multicellular globular infection hyphae (IH) in the lumen of epidermal cells, but infected epidermal cells appeared to be alive till 16 hours post-inoculation (HPI). In the susceptible plants, the tip of IH was found to grow ahead of the dead tissue and invade the surrounding live mesophyll cells, whereas growth of IH in the resistant plants was restricted to the dead tissue after 20 HPI. The amount of H2O2 accumulation and the fungal biomass were also significantly higher in the susceptible hosts than in the resistant hosts. To map resistance to FHB, two populations consisting 130 doubled haploid lines from the cross Grandin × PI277012 and 237 recombinant inbred lines from the cross Bobwhite × ND2710 were phenotyped and genotyped. QTL for Type I resistance were identified on chromosomes 1A, 2B, 4B, 5B and 6B in the GP population. These QTL explained 10.7-19 % of the total phenotypic variation. With the BN population, QTL for Type I resistance were identified on chromosomes 2A, 5A and 6B, explaining 6.2-13.7% of the total phenotypic variation. To assess the prevalence, incidence and severity of wheat crown rot (CR) and common root rot (CRR) in ND, wheat root samples were collected from fields across the state in 2012, 2013, and 2014. Fungal isolations indicated that B. sorokiniana was most frequently recovered in all sampled years. Seedling tests on ten spring wheat lines showed that Glenn was the least susceptible while Steele-ND was the most susceptible to one F. culmorum isolate and one B. sorokiniana isolate tested. Evaluation of 20 spring wheat genotypes for reaction to CRR at the adult plant stage showed that Freyr and RB07 were more resistant while Len and Briggs were more susceptible to CRR compared to other wheat genotypes evaluated.North Dakota Wheat Commission,Minnesota Wheat Research and Promotion CouncilND State Board of Agricultural Research and EducationTriticeae-CAP project (2011-68002-30029) of the US Department of Agriculture National Institute of Food and AgricultureU.S. Wheat and Barley Scab Initiative (USWBSI

Identification of Fungal Species Associated with Crown and Root Rots of Wheat and Evaluation of Plant Reactions to the Pathogens in North Dakota

Common root rot (CRR) and crown rot (CR), caused by Bipolaris sorokiniana and Fusarium species, respectively, can cause significant yield losses in cereal crops. To assess the prevalence, incidence, and severity of these diseases in North Dakota, wheat samples were collected from spring wheat fields across the state in 2012, 2013, and 2014. Based on subcrown internode symptoms, a greater incidence and severity of CRR was observed in 2012 (warm and dry year) than in 2013 and 2014. Also, the Northwestern Glaciated Plains and Northwestern Great Plains ecoregions showed greater CRR incidence and severity compared with the Northern Glaciated Plains and Lake Agassiz Plains ecoregions in the state. B. sorokiniana and Fusarium species, including Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium equiseti, Fusarium pseudograminearum, Fusarium oxysporum, Fusarium redolens, Fusarium sporotrichioides, and Fusarium solani were isolated and identified from the root and crown tissues of the wheat samples. B. sorokiniana was isolated more frequently than other fungal species in all sampled years and ecoregions of North Dakota. F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. pseudograminearum, and F. redolens were pathogenic causing infections on seedlings of the two wheat genotypes (ND652 and Alsen), whereas isolates of F. oxysporum and F. solani were nonpathogenic and considered as secondary invaders associated with the root and CR diseases. Evaluation of some spring wheat genotypes for reactions to one B. sorokiniana isolate at seedling and adult plant stages, and one F. culmorum isolate at the seedling stage, indicated that susceptibility to these pathogens varied among different wheat genotypes tested. This study provides useful information on fungal species associated with CRR and CR of wheat in North Dakota and on resistant/susceptible reactions of some spring wheat lines to the different fungal isolates evaluated. </jats:p

Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance

AbstractBackgroundStem rust is an economically important disease of wheat and barley. However, studies to gain insight into the molecular basis of these host-pathogen interactions have primarily focused on wheat because of its importance in human sustenance. This is the first extensive study utilizing a transcriptome-wide association mapping approach to identify candidatePuccinia graminisf. sp.tritici(Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifyingPgtelicitors that interact with therpg4-mediated resistance locus (RMRL), the only effective source ofPgtrace TTKSK resistance in barley.ResultsThirty-sevenPgtisolates showing differential responses on RMRL were genotyped using Restriction Site Associated DNA-Genotyping by Sequencing (RAD-GBS), identifying 24 diverse isolates that were used for transcript analysis during the infection process.In plantaRNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to thePgtrace SCCL reference genome identifying 114 K variants in predicted genes that would result in nonsynonymous amino acid substitutions. Transcriptome wide association analysis identified 33 variants across 28 genes that were associated with dominant RMRL virulence, thus, representing candidate suppressors of resistance. Comparative transcriptomics between the 9 RMRL virulent -vs- the 15 RMRL avirulentPgtisolates identified 44 differentially expressed genes encoding candidate secreted effector proteins (CSEPs), among which 38 were expressed at lower levels in virulent isolates suggesting that they may represent RMRL avirulence genes. Barley transcript analysis after colonization with 9 RMRL virulent and 15 RMRL avirulent isolates inoculated on the susceptible line Harrington showed significantly lower expression of host biotic stress responses specific to RMRL virulent isolates suggesting virulent isolates harbor effectors that suppress resistance responses.ConclusionsThis transcriptomic study provided novel findings that help fill knowledge gaps in the understanding of stem rust virulence/avirulence and host resistance in barley. The pathogen transcriptome analysis suggested RMRL virulence might depend on the lack of avirulence genes, but evidence from pathogen association mapping analysis and host transcriptional analysis also suggested the alternate hypothesis that RMRL virulence may be due to the presence of suppressors of defense responses.</jats:sec

MOESM2 of Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance

Additional file 2. A FASTA file containing coding sequences of the updated gene models of Pgt using the transcript sequences generated from this study in addition to the publicly available gene models of reference genome of Pgt race SCCL

MOESM3 of Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance

Additional file 3. A FASTA file containing amino acid sequences of the updated gene models of Pgt using the transcript sequences generated from this study in addition to the publicly available gene models of Pgt race SCCL

MOESM1 of Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance

Additional file 1: Contains supplementary tables Table S1-S13. Microsoft Excel Workbook containing 13 tables. Table S1. Group of Pgt isolates based on their virulence on barley lines containing stem rust resistance gene rpg4/5 and/or Rpg1. Table S2. Infection type of group1 Pgt isolates on barley differential lines. Table S3. Infection type of group2 Pgt isolates on barley differential lines. Table S4. Infection type of group3 Pgt isolates on barley differential lines. Table S5. Read and mapping statistics of RAD-GBS data of Pgt Samples. Table S6. RNAseq read mapping statistics for the Pgt inoculated Harrington samples. Table S7. RNAseq read mapping statistics for the non-inoculated Harrington samples. Table S8. Differentially expressed gene in between virulent rpg4/5 and avirulent rpg4/5 inoculated sampled and its comparison with the non-inoculated controls. Table S9. Gene enrichment analysis of differentially expressed gene in virulent vs avirulent rpg4/5 inoculated Harrington in R bioconductor package TopGO, Table S10. List of gene that are differentially regulated in Rpg1 virulent isolates compared to Rpg1 avirulent isolates. Table S11. List of gene that are differentially regulated in rpg4/5 virulent isolates compared to rpg4/5 avirulent isolates. Table S12. Identification of Pgt Candidate Secreted Effector Protein (CSEP) and their classification based on level of expression. Table S13. Variants associated with Pgt virulence on rpg4/5

Rapid Detection of <i>Cercospora beticola</i> in Sugar Beet and Mutations Associated with Fungicide Resistance Using LAMP or Probe-Based qPCR

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection “toolbox” for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in β-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates. </jats:p