Plant Pathology

Permanent URI for this communityhdl:10365/32426

Research from the Department of Plant Pathology. The department website may be found at https://www.ag.ndsu.edu/plantpath

Browse

Now showing 1 - 20 of 27

Characterization and Identification of Genetic Resistance to Puccinia Graminis F. Sp. Tritici in Triticum Aestivum and Hordeum Vulgare
(North Dakota State University, 2015) Zurn, Jason Daniel
Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a major threat to wheat (Triticum aestivum) and barley (Hordeum vulgare) production. The emergence of the highly virulent Ug99-lineage stem rust races has stimulated research toward the identification and characterization of rust resistance genes in wheat and barley. Populations were developed to elucidate the inheritance and location of Pgt resistance genes in the common wheat landraces PI 626573 and PI 362698. The resistance present in PI 626573 was shown to be conferred by a single dominant gene (SrWLR) and was mapped to a 1.9 cM region on the long arm of chromosome 2B. This region is known to contain Sr9h which is effective against Ug99. SrWLR provides resistance to Pgt race RKQQC and Sr9h does not, suggesting SrWLR may be a new gene or allele of Sr9. Subsequent work has delimited the SrWLR region to 0.36 cM using a synteny-based approach. QTL analysis of the PI 362698 population using Pgt races identified significant (P < 0.1) resistance QTLs on multiple chromosomes. QTLs identified on chromosome 3B map to a similar location as Sr12 which does not provide resistance to Ug99-lineage races, suggesting a new allele or novel resistance gene. The QTLs identified on chromosomes 2B and 6A are thought to be Sr16 or an allele of Sr28 and Sr8a. Sr57 is known to be present in PI 362698 and is thought to be associated with Pgt QTLs detected on chromosome 7D. QTLs on chromosomes 5A and 5B are in regions where Pgt resistance genes have not been previously identified. Relative qPCR, fluorescence microscopy, and infection type approaches were utilized to phenotype barley for seedling resistance to Pgt race MCCFC at multiple time points. Statistical differences (P < 0.05) were found between accessions at 24 hours post inoculation using qPCR and displayed similar hierarchical ordering to microscopy observations. At early stages, the susceptible cultivar Steptoe had less fungal DNA than barley accessions containing resistance genes suggesting potential pre-haustorial resistance contributions. Temporal variation in resistance ranking suggests the qPCR assay may be valuable for dissecting pre- and post-haustorial resistance mechanisms.
Characterization of Fusarium Head Blight Resistance in Wheat
(North Dakota State University, 2017) Zhao, Mingxia
Fusarium head blight (FHB) is a devastating fungal disease threatening wheat production worldwide. Utilization of resistant wheat cultivars is generally considered as the most effective, economic and environmental friendly approach for management of the disease. This research aimed to identify and map quantitative trait loci (QTL) for FHB resistance in two spring wheat lines (ND2710 and PI 277012) and two durum wheat lines (10Ae564 and Joppa). Using a mapping population consisting of 233 recombinant inbred lines (RILs) from a cross between ND2710 (with FHB resistance derived from Sumai 3) and the spring wheat cultivar ‘Bobwhite’ (susceptible to FHB), four QTL (Qfhb.ndwp-3B, Qfhb.ndwp-6B, Qfhb.ndwp-2A, and Qfhb.ndwp-6A) were mapped on chromosomes 3B, 6B, 2A, and 6A, respectively, in ND2710. Qfhb.ndwp-3B and Qfhb.ndwp-6B were mapped to the same genomic regions as Fhb1 and Fhb2, confirming that they originated from Sumai 3. Two FHB resistance QTL, Qfhb.rwg-5A.1 and Qfhb.rwg-5A.2, were previously identified on chromosome 5AS and 5AL, respectively, in PI 277012. In this study, Qfhb.rwg-5A.2 was delimited in a 1.09-Mbp genomic region, and DNA markers tightly linked to Qfhb.rwg-5A.2 were developed using 947 RILs from the cross between PI 277012 and Grandin (susceptible to FHB). Using a mapping population consisting of 205 RILs from the cross between the durum cultivar Joppa and the durum wheat line 10Ae564 (with FHB resistance derived from PI 277012), one QTL (Qfhb.ndwp-2A) on chromosome 2A from Joppa and two QTL (Qfhb.ndwp-5A and Qfhb.ndwp-7A) each on 5A and 7A from 10Ae564 were detected. Qfhb.ndwp-5A was mapped to the same genomic region as Qfhb.rwg-5A.2 on 5AL, and thus confirming that this QTL was derived from PI 277012. The DNA markers closely linked to the FHB resistance QTL identified in two spring wheat lines and two durum wheat lines will be useful for marker-assisted selection of FHB resistance in wheat breeding programs.
Characterization of Pea Seed-Borne Mosaic Virus, Efficacy of Foliar Applications for Common Bacterial Blight Management in Dry Beans and Impact of Common Bacterial Blight on Prostrate and Upright Beans
(North Dakota State University, 2018) Beck, Amanda Louise
PSbMV in field pea has resulted in substantial yield and seed quality losses world-wide and has recently been reported in North Dakota. Traditional management of this virus includes preventative measures such as removal of alternate hosts, planting virus free seed and the use of cultivar resistance. The objectives of this research were to screen field pea cultivars commonly grown in North Dakota for a response to North Dakota PSbMV isolate ND14-1 and ascertain the effect on plant symptoms, seed size and weight, the number of pods and seeds and seed transmission. Two cultivars were identified as highly resistant and one as partially resistant. The results from this study were combined into a risk assessment. Cultivars were categorized based on inherent risk of PSbMV infection, transmission and reduction in total seed weight. Common bacterial blight (CBB) in dry bean is capable of causing substantial yield losses and has been reported in up to 75% of fields in the Northarvest region in the last five years. Current management practices include the use of planting clean seed, crop rotation, partial host resistance and the application of cupric bactericides, although inconsistent for the management of CBB. Growers in this Northarvest region have recently shifted to growing upright (Type II) dry beans rather than prostrate (Type III) dry beans for ease of harvest. The objectives of this research were to evaluate copper products, surface sanitizers and growth promoters for the management of CBB and to discern if Type II dry beans experienced greater yield losses under CBB disease pressure than Type III dry beans. Numerous products were identified that significantly reduced CBB disease severity and spread; however, no significant yield benefit was observed. Across a wide range of disease severity (0-46%), no significant yield losses were observed between high and low disease severity any of the cultivars screened.
Characterization of Pectobacterium carotovorum subsp. brasiliense as a Causal Agent of Sugarbeet Soft Rot
(North Dakota State University, 2018) Metzger, Michael Stewart
A soft rot decay of sugarbeet was observed in commercial fields in North Dakota and Minnesota from 2012 to 2016. Symptoms reported are similar to those for bacterial vascular necrosis and rot caused by Pectobacterium betavasculorum including soft decay of internal root tissues, reddening of affected tissue after cutting, blackening of petiole vascular bundles, half-leaf yellowing, and root frothing. The disease can cause serious yield losses in the field, and additional economic losses in storage and during processing due to accumulation of invert sugars that reduce sugarbeet quality. Sap from the margin of diseased root tissue was streaked on pectate agar medium and incubated. Single pectolytic colonies were selected and transferred to nutrient broth for bacterial identification and completion of Koch’s postulates. Pathogenicity of isolates was assessed by inoculating greenhouse-grown sugarbeet roots. Symptoms characteristic of the disease were observed at 30 days after inoculation included all of the aforementioned, previously stated symptomology. Bacterial DNA was extracted from 46 pathogenic isolates and analyzed by restriction-associated DNA genotype-by-sequencing (RAD-GBS). Ion-torrent sequencing reads (n = 8.54 million) were assembled de novo producing ∼6,000 sequence tags representing approximately 21% of each bacterial genome analyzed. Partial sequences of five of the seven genes previously used in Pectobacterium subspecies phylogenetic analysis were represented in the RAD-GBS isolate sequences. Gene sequences were aligned using Workbench 8.0.3 software to the corresponding reference gene sequences of P. carotovorum subsp. carotovorum, P. atrosepticum, P. betavasculorum, P. carotovorum subsp. odoriferum, and P. wasabiae. The alignments showed 99.76% nucleotide sequence identity on average across all five genes to the P. carotovorum subsp. brasiliense reference sequences. The alignments to P. cartovoroum subsp. carotovorum, P. atrosepticum, P. betavasculorum, P. carotovorum subsp. odoriferum and P. wasabiae reference sequences showed 96, 95.4, 94.3, 97 and 94.4% identity, respectively, on average across the five genes. The nearly 100% identity across all five genes previously utilized in multi-locus sequencing and divergence from the closely related subspecies strongly suggests that the isolates are P. carotovorum subsp. brasiliense. To our knowledge, this is the first report of this pathogen causing field decay of sugarbeet in North America.
Characterizing Pyrenophora Teres F. Maculata in the Northern United States and Impact of Spot Form Net Blotch on Yield of Barley
(North Dakota State University, 2015) Kinzer, Kasia Marie
Pyrenophora teres f. maculata causes spot form net blotch (SFNB) on barley and was recently documented in North Dakota. The impact of SFNB on barley, the genetic diversity of the pathogen, and virulence structure are unknown for the state. Yield and quality loss in North Dakota due to SFNB was investigated over eleven year-sites, and simple linear regression of percent yield loss on adjusted percent disease using year-site means of treatments predicted a 0.77% increase in yield loss for every 1% increase in disease. When virulence of isolates of P. teres f. maculata collected from geographically diverse regions in the northern United States was evaluated on differential barley genotypes, few isolates were identical in terms of virulence patterns, and the virulence profile of a population from Idaho differed from other populations. To understand population structure and genetic diversity, SNPs of 140 isolates were generated using genotyping-by-sequencing for analysis of population genetics and structure. Evidence for sexual recombination in each population includes the ratio of mating-type idiomorphs that do not significantly differ from a 1:1 ratio; low index of association values for most populations; and high variation within and low variation among populations. Association mapping detected forty-five significant marker-trait associations of SNPs associated with virulence or avirulence across 19 P. teres f. maculata scaffolds using 82 isolates of P. teres f. maculata from diverse areas in the northern United States. The most significant marker, 01700_198, was found on P. teres f. maculata-scaffold 8 when the population was challenged with four different barley lines. This research demonstrates that SFNB causes significant yield loss; that high diversity exists in the pathogen, with respect to virulence and population genetics; and that association mapping can be used to identify virulence/avirulence marker-trait associations to fill gaps in our understanding of host-parasite genetic interactions in this pathosystem.
Cloning and Characterization of rcs5, Spot Blotch Resistance Gene and Pathogen Induced Nec3 Gene Involved in Programmed Cell Death in Barley
(North Dakota State University, 2019) Ameen, Gazala
Upon sensing pathogens, plants initiating defense responses typically resulting in programmed cell death (PCD). PCD effectively subdues biotrophic pathogens but is hijacked by necrotrophs that colonize the resulting dead tissues. We showed that barley wall associated kinase (WAK) genes, underlying the rcs5 QTL, are manipulated by the necrotrophic fungal pathogen Bipolaris sorokiniana to cause spot blotch disease. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four WAK genes, designated HvWak2, Sbs1, Sbs2, and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in the susceptible barley cultivars Steptoe and Harrington resulted in resistance, suggesting a dominant susceptibility function. Sbs1&2 expression is undetectable in barley prior to pathogen challenge; however, specific upregulation of Sbs1&2 occurred in the susceptible lines post inoculation. Promotor sequence polymorphisms were identified in the allele analysis of Sbs1&2 from eight resistant and two susceptible barley lines, which supported the possible role of promotor regulation by virulent isolates contributing to susceptibility. Apoplastic wash fluids from virulent isolates induced Sbs1expression, suggesting regulation by an apoplastic-secreted effector. Thus, the Sbs1&2 genes are the first susceptibility/resistance genes that confer resistance against spot blotch, a disease that threatens barley and wheat production worldwide. The nec3 mutants of barley are hyper-susceptible to many necrotrophs and show distinctive cream to orange necrotic lesions that are induced by infection, representing aberrant PCD. The γ- irradiation induced necrotic mutant, nec3-γ1 (Bowman) was confirmed as a nec3 mutant by allelism tests. The F2 progeny of a cross of nec3 x Quest inoculated with B. sorokiniana segregated as a single recessive gene fitting a 3 WT: 1 mutant ratio. The homozygous F2 mutant progeny were genotyped with four SSR and 25 SNP markers at nec3 locus on chromosome 6H, a physical region spanning ~ 16.96 Mb containing 91 high and low confidence annotated genes. Exome capture sequencing of nec3 mutants failed to identify a candidate gene, however, RNAseq analysis identified two candidates in the nec3 region with >three-fold downregulation. We hypothesize that the underlying aberrant PCD mechanism in the nec3 barley mutant facilitates extreme susceptibility to multiple adapted fungal pathogens of barley.
Development of Management Tools for Sunflower Downy Mildew (Plasmopara Halstedii) and Rust (Puccinia Helianthi)
(North Dakota State University, 2016) Humann, Ryan
Downy mildew (Plasmopara halstedii) and rust (Puccinia helianthi) are two economically important diseases of sunflower (Helianthus annuus) in North Dakota. Both diseases are capable of causing significant reductions in yield and quality. Effective disease management tools for both diseases are limited. Genetic resistance to both pathogens is frequently overcome by new pathogen races and only one efficacious fungicide is currently available to manage downy mildew. In order to identify additional management tools for downy mildew and rust, three research studies were done. The objective of the first study was to evaluate the efficacy of a novel fungicide, oxathiapiprolin, for the management of sunflower downy mildew. Seventeen inoculated field trials were conducted from 2011-2015 to test the efficacy of oxathiapiprolin. Results indicate that oxathiapiprolin significantly and consistently reduced downy mildew incidence and determined the optimal effective rate, which ranged from 9.37 – 18.75 µg active ingredient per seed. The second and third objectives focused on identifying accessions with novel sources of genetic resistance to P. halstedii and P. helianthi isolates collected in North Dakota. In the past, a disproportionate amount of resistance genes have been identified in wild Helianthus germplasm originating from Texas. For both studies, 182 wild H. annuus and 33 wild H. argophyllus accessions originating from Texas were obtained from the USDA North Central Regional Plant Introduction Station and screened to both pathogens in a greenhouse environment. Results from these individual studies identified numerous accessions with high levels of resistance to P. halstedii and P. helianthi, some accessions had high levels of resistance to both. Overall, results from these three studies will provide information and tools that will be useful for the long-term management of both diseases.
Dissecting the Mystery Behind the Rpg5 Mediated Puccinia graminis Resistance in Barley Using Genetics, Molecular and Bioinformatics Approaches
(North Dakota State University, 2017) Solanki, Shyam
Barley rpg4/Rpg5 locus harbors three tightly linked genes, two NLRs Rpg5 and HvRga1, and HvAdf3, togather providing resistance against Puccinia graminis f. sp. tritici, causal agent of wheat stem rust including race TTKSK, considered a threat to global food security. The integrated decoy hypothesis proposes role for head-to-head genome architecture present in the dual plant NLR immunity receptors, where one NLR partner contains an integrated sensory domain (ISD). The ISDs represent mimics of virulence effector targets translocated to the immunity receptors and act as baits to recognize virulent effectors to initiate defense responses. Alleles of Rpg5 contain two diverse C-terminal, the Rpg5 resistance allele has a serine threonine protein kinase (STPK) ISD whereas the major class of rpg5 susceptible alleles contain a protein phosphatase 2C (PP2C) ISD. Genetic and functional analysis shows that in the heterozygous state rpg5-PP2C allele acts as a dominant susceptibility factor suppressing Rpg5-STPK mediated Pgt resistance. This is the first integrated decoy NLR gene identified that contains two distinct ISDs. Liabrary scale Y2H screeing using Rpg5-STPK as bait identified HvVoz1. HvVoz1 was also interacts with the HvRga1, Rpg5-LRR, and rpg5-PP2C domains suggesting that it may act as a scaffold to hold the R-protein complex together until effector manipulation. We identified Rpg5-STPK ISD progenitor HvGak1, ortholog of the Arabidopsis guard cell AT5G15080 and AtAPK1b, shown to function in stomatal aperture opening in response to light. We hypothesize that several forma specialis of P. graminis contain virulence effector/s, that manipulate HvGak1, mimicking the presence of light to open the stomates, allowing the pathogen to gain entry in to the plant during dark period that P. graminis spores evolved to germinate. We identified dark period pathogen penteration through stomata by deveoping a novel staining method and using confocal microscopy. To further characterize the Rpg5 immunity pathway fast-neutron irradiation was utilized to generate rpr9 mutant, compromised for rpg4/Rpg5-mediated resistance. Utilizing genetic mapping and exom capture we identified candidate genes for rpr9 mutants. Based on our understanding of this resistance mechanism it would be a good candidate system for generating synthetic resistances utilizing different ISD baits fused to the Rpg5 NLR.
Fitness and Management of Site-specific Fungicide Resistant Cercospora beticola Isolates from Sugar Beet
(North Dakota State University, 2020) Liu, Yangxi
Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is one of the most destructive foliar diseases on sugar beet. It severely affects productivity and profitability of the sugar beet industry. CLS is managed by using resistant cultivars, rotating with non-host crops, and applying effective fungicides in a timely manner. In North Dakota and Minnesota, site-specific fungicides, such as quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs), had been widely and extensively applied to control C. beticola which has developed resistances to these fungicides. The mycelial growth, spore production, spore germination, and aggressiveness of QoI and/or DMI resistant isolates were compared to sensitive isolates in a laboratory and greenhouse study. Results indicated that the QoI and/or DMI resistant isolates had a relatively slower disease development on sugar beet leaves due to their fitness penalty in sporulation and mycelial growth but still caused high disease severities as sensitive isolates. Fungicides were evaluated to manage the QoI and/or DMI resistant C. beticola isolates in a sensitivity study and a greenhouse and field study. Copper-based multisite activity fungicides were evaluated for controlling fungicide resistant C. beticola. The mean EC50 values for nine copper-based chemicals ranged from 1 to 10 ug/ml using a spore germination assay. In a greenhouse study, Fertileader (a copper-based fertilizer) caused leaf injury and was not evaluated, but the other tested chemicals provided significantly better control of CLS compared to the control check with Badge X2, Champion, Cuprofix, COCS and Ridomil having significantly small AUDPC. Newer site specific fungicides with different modes of action that have never been used for CLS management in North Dakota and Minnesota were also tested. The mean EC50 values were 4.9, 33.1, 99.4, and 481.6 ug/ml using mycelial growth assay and 5.7, 4.1, 9.2, and 4.2 ug/ml using spore germination assay for cyprodinil, fluazinam, pydiflumetofen, and Chlorothalonil, respectively. In a two-year field study, all the fungicides resulted in significantly better disease control, significantly higher beet tonnage, and recovered sucrose compared to the nontreated check. However, none of tested fungicide treatments performed better than the industry’s standard fungicide, triphenyltin hydroxide.
From Bioinformatics to Identifying R-genes, Enhancers, Signaling Pathways and Pathogen Elicitors in the Barley-Stem Rust Pathosystem
(North Dakota State University, 2018) Sharma Poudel, Roshan
Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt) is a threat to wheat and barley. Rpg1 is the only deployed stem rust resistance gene in barley that provides resistance to the predominant races in North America, except to the local race QCCJB and the widely virulent race TTKSK (aka Ug99) and its lineages. The barley rpg4-mediated resistance locus (RMRL) confers resistance against the majority of Pgt races, including TTKSK and its lineage. With the goal of identifying Pgt effectors/suppressors that elicit/suppress RMRL resistance responses twenty-four Pgt isolates showing differential infection types were genotyped utilizing in planta RNAseq. The RNAseq experiment identified 114K SNPs within genes that resulted in predicted nonsynonymous amino acid changes and were utilized to identify genes associated with virulence/avirulence. Twenty-two genes were identified that were associated with RMRL virulence that represent candidate suppressors of resistance. Host differential gene expression analysis comparing virulent vs avirulent isolates identified virulent isolate specific down regulation of stress response genes, genes involved in chloroplastic ROS, and non-host resistance responses, suggesting that Pgt isolates may contain a conserved virulence factor that elicits RMRL responses and virulent isolates contain suppressors of virulence rather than dominant avirulence genes. The second chapter focused on the observations that introgression of RMRL into the elite malting variety Pinnacle (Rpg1+) resulted in susceptibility to Pgt race QCCJB (RMRL) and HKHJC (Rpg1) suggesting the presence of a gene required for rpg4/Rpg5 and Rpg1 resistance. Utilizing a Pinnacle RMRL-NIL X Q21861 derived RIL population and PCR-GBS genotyping, the required for rpg4- and Rpg1-mediated resistance 1, Rrr1 gene was mapped ~5cM proximal to RMRL on barley chromosome 5H. A second gene required for Rpg1- mediated resistance 2, Rrr2, complimentary to Rrr1 was mapped to the telomeric region of the short arm of barley chromosome 7H. A novel Pgt race TTKSK resistance gene designated RpgHv645 was identified in an unimproved swiss landrace Hv645. Utilizing a RIL population developed from a Hv645 X Harrington cross and Pgt race TTKSK phenotyping data generated at the adult plant stage in Njoro, Kenya, RpgHv645 was mapped distal of RMRL and delimited to an ~11cM region.
Genetic and Molecular Characterization of Genes Involved in Barley-Cochliobolus Sativus Interaction
(North Dakota State University, 2015) Wang, Rui
Spot blotch caused by ascomycete fungus Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dastur. [anamorph: Bipolaris sorokiniana (Sacc.) Shoem.] is one of the most common and economically important diseases on barley. To better understand the molecular interaction between the different pathotypes of this pathogen and barley differential lines, fungal genes involved in virulence and barley genes for spot blotch resistance were characterized in this study. Previous studies have revealed that the virulence factor in the pathotype 2 isolate ND90Pr making cv. Bowman susceptible is a secondary metabolite peptide synthesized by two non-ribosomal peptide synthesdases (NRPSs). However, the global regulation of biosynthesis of this secondary metabolite is not well understood in this fungus. Recently, the velvet-complex proteins containing LaeA and velvet proteins (VeA, VelB, VelC and VosA) have been shown to be involved in global regulation of secondary metabolism and fungal development in many fungal pathogens. To characterize the functions of the orthologous of velvet-complex proteins in C. sativus, single and double gene knockout mutants were generated. The results indicated that the velvet-complex factors affect virulence of the fungus by regulating expression of the NRPSs involved in virulence. In addition, velvet-complex proteins were found to coordinately and distinctly regulate fungal development, such as conidiogenesis and conidia germination. To identify and characterize genes for resistance to the new pathotype, 2,062 barley accessions from a USDA barley core collection were screened for spot blotch resistance to this pathotype and 24.5% of them showed resistance or moderate resistance at the seedling stage. Genome-wide association analysis identified four QTLs associated with the seedling resistance, which were located on chromosome 1H, 2H, 3H, and 6H, respectively. A genetic analysis of the cross between a highly resistant line (PI 235186) and a highly susceptible accession (PI 356741) suggested a single dominant gene confers the resistance in PI 235186. The resistance gene was further mapped to the short arm of chromosome 6H based on bulk segregant analysis using 194 SSR markers and genotyping-by-sequencing using 20 SNP markers in a F2 population. Additional markers were developed to fine map the resistance gene to a ~6.5 cM genomic region.
Genetic and Molecular Characterization of Host Resistance and Susceptibility to Pyrenophora Teres F. Teres in Hordeum vulgare
(North Dakota State University, 2016) Richards, Jonathan
Pyrenophora teres f. teres, a necrotrophic fungal pathogen and causal agent of net form net blotch (NFNB), is an economically important pathogen of barley (Hordeum vulgare) and has potential to cause significant yield losses in barley production regions of the world. Host resistance is the most desirable means of disease management, yet the genetic nature of this pathosystem is exceedingly complex. With the goal of identifying novel sources of resistance to NFNB, a diverse population of barley accessions was utilized to conduct a genome wide association study which identified a total of 78 significant markers associated with disease reaction to three North American P. teres f. teres isolates, corresponding to 16 genomic loci. Five novel loci were detected and will be of importance for barley breeders for the improvement of elite barley lines. Dominant susceptibility harbored by barley cultivars Rika and Kombar to P. teres f. teres isolates 6A and 15A, respectively, were previously identified to exist in repulsion and mapped at low-resolution. Using 2976 recombinant gametes derived from a cross of Rika x Kombar and markers developed through mining of syntenous genes in Brachypodium distachyon, we mapped the Spt1 locus to ~0.24 cM near the centromere of chromosome 6H. Within the delimited Spt1 region, a receptor-like protein was identified as the primary candidate Spt1 gene designated Spt1.cg. Allele analysis of diverse barley lines exhibited a strong correlation with the presence of a Rika, Kombar, or Morex allele of Spt1.cg and susceptibility to P. teres f. teres isolates 6A, 15A, or Tra-A5/Tra-D10, respectively. Alleles of Spt1.cg appear highly diverged, stemming from selection pressures in wild barley populations and may be targeted by several unique necrotrophic effectors. The barley cultivar Morex rpr2 mutant, previously characterized to have lost Rpg1-mediated resistance to Puccinia graminis f. sp. tritici, also has compromised resistance to P. teres f. teres. Exome capture revealed a 12 base-pair deletion in a gene containing fibronectin and plant homeodomain domains with homology to Arabidopsis VIN3-like proteins. This gene may function in the perception of pathogen effector proteins, that disrupt cell wall integrity, eliciting early damage associated molecular pattern immunity responses.
Genetic Mapping and Characterization of Net Blotch Dominant Resistance and Dominant Susceptibility Loci in Barley
(North Dakota State University, 2017) Tamang, Prabin
Net blotch is an economically important foliar disease of barley that occurs in two distinct forms: Spot Form Net blotch (SFNB) and Net Form Net Blotch (NFNB) caused by the necrotrophic fungal pathogens Pyrenophora teres f. maculata (Ptm) and Pyrenophora teres f. teres (Ptt), respectively. The recent emergence and the identification of both Ptm and Ptt isolates virulent on popular ND malting barley varieties have warranted the identification of new resistance sources. Association mapping was conducted on 2,062 diverse barley accessions phenotyped at the seedling stage with four diverse P. teres f. maculata isolates and genotyped with the 9k Illumina barley iSelect chip. A total of 138 significant marker-trait associations (MTA; −log10P value > 3.0) corresponding to 27 resistance loci were identified of which 21 loci were novel and six corresponded to previously characterized SFNB resistance QTL. Further, two higly resistant lines PI67381 and PI84314 were crossed with the two susceptible cultivars Tradition and Pinnacle grown in the Upper Midwestern US to develop three bi-parental recombinant inbred line (RIL) mapping populations of Tradition x PI67381, Pinnacle x PI67381 and Pinnacle x PI84314. These RIL populations were phenotyped with six diverse Ptm isolates and genotyped using PCR-GBS. MapDisto and Qgene were used to analyze the data and a total of twelve QTL were identified on chromosome 2H, 3H, 4H, 6H and 7H, of which nine were previously reported and the remaining three are considered novel. These resistances and the markers delimiting the QTL are being utilized to develop prebreeding lines by introgressing SFNB resistance into the cultivars Pinnacle and Tradition utilizing marker assisted selection. The barley line CI5791 exhibits a high level of resistance to diverse Ptt isolates collected from around the world. A forward genetics approach and an exome capture-mediated mapping-by-sequencing identified a candidate HvWRKY6 transcription factor gene required for NFNB resistance on chr 3H. We hypothesize that the HvWRKY6 gene function as a component of a conserved basal defense mechanism, which regulates the expression of other defense response genes that restrict lesion growth. The resistance/susceptibility loci identified in this study will facilitate the development of net blotch resistant cultivars.
Genomic Mapping of Leaf Rust and Stem Rust Resistance Loci in Durum Wheat and Use of RAD-Genotype by Sequencing for the Study of Population Genetics in Puccinia Titicina
(North Dakota State University, 2016) Aoun, Meriem
Leaf rust, caused by Puccinia triticina Erikss. (Pt), and stem rust, caused by Puccinia graminis f. sp. tritici Erikss. and E. Henn (Pgt), are among the most devastating diseases of durum wheat (Triticum turgidum L. var. durum). This study focused on the identification of Lr and Sr loci using association mapping (AM) and bi-parental population mapping. From the AM conducted on the USDA-National Small Grain Collection (NSGC), 37 loci associated with leaf rust response were identified, of which 14 were previously uncharacterized. Inheritance study and bulked segregant analysis on bi-parental populations developed from eight leaf rust resistance accessions from the USDA-NSGC showed that five of these accessions carry single dominant Lr genes on chromosomes 2B, 4A, 6BS, and 6BL. The other three accessions have Lr genes with more complex inheritance. All eight accessions carry different genes than those already mapped in durum cultivars except one accession with Lr61. Linkage mapping in two of the bi-parental populations showed that the gene in PI 209274 (LrCA) was mapped to 6BS between SNPs IWA3298 and IWB39456, while the gene in PI 192051 (LrPort) was mapped to 4AL, flanked by IWA4254 and IWA8341. Resistance to Pgt-race TTKSK was also observed in PI 534304 and PI 192051. PI 534304 was found to carry Sr13 on chromosome 6AL, while PI 192051 carries a novel Sr gene (SrPort) mapped to 7AS flanked by IW8390 and IWA1805. The genotype PI 192051 has an additional QTL (QSr.ndsu-5B) to Pgt races in a field trial in Ethiopia in 2016. The QSr.ndsu-5B was mapped to 5BL and delimited by IWA6992 and IWA2181. The study of virulence diversity in Pt isolates collected from several countries identified seven races among 51 isolates collected from durum wheat and 21 races among 40 isolates collected from common wheat. The phylogeny study on 30 Pt isolates based on the Restriction-Associated DNA (RAD)-Genotype by Sequencing (GBS), clustered the isolates into eight clades, with higher diversity in the SNP genotypes in common wheat isolates compared to that in durum wheat isolates. RAD-GBS is identified as a suitable and informative genotyping technique to study the population genetics of Pt.
Identification and Functional Characterization of Necrotrophic Effectors in Parastagonospora Nodorum
(North Dakota State University, 2015) Gao, Yuanyuan
The necrotrophic fungus Parastagonospora nodorum (teleomorph; Phaeosphaeria nodorum), is the causal agent of Septoria nodorum blotch (SNB) on common wheat (Triticum aestivum L.) and durum wheat (Triticum turgidum L.). SNB is a serious foliar and glume disease which causes significant yield losses in major wheat growing areas and has serious impact on grain quality. P. nodorum produces necrotrophic effectors (NEs) that are recognized by and interact with dominant host sensitivity genes in an inverse gene-for-gene manner. The NE-host interaction is critical to induce necrotrophic effector-triggered susceptibility (NETS), resulting in SNB disease. To date, nine NE-host sensitivity gene interactions, following a NETS model, have been identified in the P. nodorum-wheat pathosystem. One of the NE-host sensitivity gene interactions, SnTox6-Snn6 interaction was characterized in this study. The SnTox6-Snn6 interaction was shown to be light dependent and Snn6 was located to a major disease susceptibility QTL on wheat chromosome 6A. SnTox1, another NE first identified in our lab, interacts with the corresponding wheat sensitivity gene Snn1. SnTox1 was further characterized in this study. The SnTox1 protein harbors C-terminal domains with a high degree of structural homology to plant chitin binding proteins and was subsequently shown to bind chitin, a main component of the fungal cell wall. Therefore, SnTox1 was hypothesized to compete with wheat chitinases to bind chitin, preventing fungal cell wall degradation. To investigate this hypothesis, the SnTox1 binding affinity with chitin was tested, as well as its potential function in the protection against chitinases during fungal mycelial growth. To identify additional NE regions, genome wide association study (GWAS) technology was used. A global collection of 191 P. nodorum isolates were genotyped using a restriction-site associated DNA genotyping by sequencing (RAD-GBS) protocol to identify SNP markers. Phenotypic data including fungal inoculations and culture filtrate infiltrations were collected using 191 P. nodorum isolates across several wheat lines. GWAS analyses were performed by regressing the phenotypic data and genotypic data by running multiple GWAS models.
Identification of Brassica Napus L. Sources of Resistance Against Blackleg (Leptosphaeria Maculans)
(North Dakota State University, 2017) Mansouripour, Seyedmojtaba
Blackleg, caused by the fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [anamorph = Phoma lingam (Tode: Fr.) Desm.] has become the most important disease affecting canola around the world. A study was conducted to identify sources of resistance to L. maculans in a collection of B. napus plant introduction accessions. Approximately, 5% of accessions showed highly resistance (median severity <3) reaction to the L. maculans PG-4 under the greenhouse conditions and three of them performed better than commercial hybrids in the field conditions. At the same time, DNA extracted and genome-wide association study (GWAS) was done for 213 and 78 accessions for PG-4 and PG-3. The 0.1 and 0.01 percentile tails of an empirical distribution, obtained from 5,000 bootstraps, was used to determine the cut off P-value to identify significant markers. Finally, 10 and 26 significant single nucleotide polymorphism (SNP) markers associated with resistance to PG-4 and PG-3, respectively. These markers were located across 14 chromosomes (A01, A02, A03, A04, A05, A07, A08, A10, C03, C04, C05, C07, C08 and C09) out of B. napus 19 chromosomes. These markers were validated under field conditions. With exploring flanking region of each significant marker eight candidate genes were identified which involved in plant defense family such as defensing and leucine-rich repeat and serine-threonine protein kinase protein. To infer the presence of R genes in commercial canola hybrids, elite B. napus plant introduction materials, and elite canola breeding lines, they were inoculated with different L. maculans races. The results showed that, resistance gene Rlm9 was present in 18% of the genotypes evaluated; Rlm2 and Rlm3 were each present in 16% of them, while LepR1, Rlm4 and Rlm5 with present in 11, 5, and 5% each, respectively. However, we were not able to infer R gene(s) on 29% of the genotypes evaluated. Approximately 18% of genotypes were susceptible to all the races used. The hybrids with different R genes could use for hybrid rotation.
Identification of Quantitative Trait Loci (QTL) Associated with Resistance to Initial Infection of Fusarium Head Blight in Spring Wheat
(North Dakota State University, 2017) Shrestha, Subidhya
Video summarizing a Ph.D. dissertation for a non-specialist audience.
Molecular and Histological Study of Sphaerulina musiva-Populus spp. Interaction
(North Dakota State University, 2017) Abraham, Nivi Deena
Sphaerulina musiva, the causal agent of leaf spot and stem canker, is responsible for critical yield loss of hybrid poplar in agroforestry. This research examined quantification of S. musiva in host tissue, and infection of leaf tissue, plus gene expression between resistant and susceptible poplar genotypes. This study reports the first use of a multiplexed hydrolysis probe qPCR assay for faster and accurate quantification of S. musiva in inoculated stems of resistant, moderately resistant and susceptible genotypes of hybrid poplar at three different time points -1 wpi (weeks post-inoculation ), 3 wpi and 7 wpi. This assay detected significant differences in the level of resistance among the different clones at 3 wpi (p < 0.001) and significant differences among isolates at 1 wpi (p < 0.001), that were not detected by visual phenotyping. Histological and biochemical comparisons were made between resistant and susceptible genotypes inoculated with conidia of S. musiva in order to study the mode of leaf infection and defense response of hybrid poplar. Leaf infection was examined at 48 h, 96 h, 1 wpi, 2 wpi and 3 wpi using scanning electron microscopy (SEM) and fluorescent and laser scanning confocal microscopy. Infection process of S. musiva on Populus spp. was further characterized by transforming S. musiva with red fluorescent protein through Agrobacterium tumefaciens. Results indicated that there was no difference in pre-penetration processes, however, differences were observed in post-penetration between resistant and susceptible genotypes. The host response was also studied by examining the accumulation of hydrogen peroxide (H2O2) using fluorescent microscopy after DAB staining, and a significant difference (p < 0.0001) was observed by 2 wpi. The molecular mechanism underlying host-pathogen interaction was elucidated by studying temporal differentially expressed genes of both the interacting organisms, simultaneously, using RNA-seq. Genes involved in cell wall modification, antioxidants, antimicrobial compounds, signaling pathways, ROS production and necrosis were differentially expressed in the host. In the pathogen, genes involved in CWDE, nutrient limitation, antioxidants, secretory proteins and other pathogenicity genes were differentially expressed. The results from this research provide an improved understanding of poplar resistance/susceptibility to S. musiva.
Molecular Genetic Characterization of Ptr ToxC-Tsc1 Interaction and Comparative Genomics of Pyrenophora tritici-repentis
(North Dakota State University, 2018) Kariyawasam, Gayan Kanishka
Tan spot of wheat, caused by Pyrenophora tritici-repentis, is an economically important disease worldwide. The disease system is known to involve three pairs of interactions between fungal-produced necrotrophic effectors (NEs) and the wheat sensitivity genes, namely Ptr ToxA-Tsn1, Ptr ToxB-Tsc2 and Ptr ToxC-Tsc1, all of which result in susceptibility. Many lines of evidence also suggested the involvement of additional fungal virulence and host resistance factors. Due to the non-proteinaceous nature, Ptr ToxC, has not been purified and the fungal gene (s) controlling Ptr ToxC production is unknown. The objective for the first part of research is to map the fungal gene (s) controlling Ptr ToxC production. Therefore, A bi-parental fungal population segregating for Ptr ToxC production was first developed from genetically modified heterothallic strains of AR CrossB10 (Ptr ToxC producer) and 86-124 (Ptr ToxC non-producer), and then was genotyped and phenotyped. Using the data, the gene (s) was mapped to the distal end of chromosome 2 in the reference genome of Pt-1c-BFP. The objective for the second part of my research is to develop genomic and genetic resources for the fungal pathogen. A high quality of genome sequence for AR CrossB10 and the first P. tritici-repentis genetic linkage map was generated. The AR CrossB10 genome and genetic linkage map is highly comparable to newly published reference genome except some noticeable chromosomal structural variations (SVs). Comparison of the genome sequences between parental isolates and twenty progeny isolates also revealed some SVs including deletion, insertion and inversion were detected that likely occurred during the fungal sexual reproduction. The objective for the third of my research is to characterize genetic resistance in Nebraskan winter wheat cultivar ‘Wesley’ using QTL mapping in a recombinant inbred line population. The results showed that resistance in Wesley is largely due to the lack of susceptibility genes Tsc1 and Tsn1. My Ph.D. research provides a further understanding of the genetics of host-pathogen interaction in wheat tan spot and contributes knowledge and tools for breeding tan spot resistant cultivars.
Parasitic Fitness of SDHI-Sensitive and -Resistant Isolates of Alternaria Solani
(North Dakota State University, 2017) Bauske, Mitchell James
Early blight of potato (Solanum tuberosum L.) is caused by Alternaria solani Sorauer. The single-site mode of action chemistries of the succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs) have been widely used for early blight control but resistance has developed rapidly to a number of fungicide chemistries. QoI resistance in A. solani has been attributed to the F129L mutation, or the substitution of phenylalanine with leucine at position 129. Resistance to SDHI fungicides in A. solani is conferred by five known point mutations on three AsSdh genes. Over 1,300 A. solani isolates collected from 11 states in 2013 through 2015 were characterized for the presence of mutations associated with QoI and SDHI resistance through real-time, SDH multiplex, and mismatch amplification analysis (MAMA) polymerase chain reaction (PCR). Approximately 95% of isolates collected from 2013 to 2015 possessed the F129L mutation. Additionally, 95% of the A. solani population was determined to be SDHI-resistant, with the most prevalent mutation being on the AsSdhC gene. All A. solani isolates collected that were characterized as possessing the D123E mutation, or the substitution of aspartic acid for glutamic acid at position 123 in the AsSdhD gene, were evaluated for boscalid and fluopyram sensitivity in vitro. Furthermore, 15 isolates characterized as being SDHI-sensitive or -resistant, including five D123E-mutant isolates, were evaluated in vivo for percentage disease control to boscalid and fluopyram. Sensitivity of D123E-mutant isolates to fluopyram ranged from 0.8 to 3 ug/ml, and were found to be sensitive or only slightly higher than those of baseline isolates, ranging from 0.1 to 0.6 ug/ml. Disease control of all five D123E isolates evaluated in vivo was reduced significantly with the application of fluopyram compared to SDHI-sensitive isolates. Fitness was compared among 120 SDHI-sensitive and -resistant A. solani isolates using the parameters of spore germination and mycelial growth in vitro and aggressiveness in vivo. Spore germination and mycelial growth between SDHI-sensitive and -resistant isolates was not significantly different. However, D123E-mutant isolates were significantly more aggressive in in vivo assays compared to other SDHI-resistant and SDHI-sensitive isolates. These results illustrate the importance of implementing fungicide resistance management strategies.

Browse

Browsing Plant Pathology by browse.metadata.type "Video"