Plant Pathology
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Research from the Department of Plant Pathology. The department website may be found at https://www.ag.ndsu.edu/plantpath
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Item Assessing Cereal Aphid Diversity and Barley Yellow Dwarf Risk In Hard Red Spring Wheat and Durum(North Dakota State University, 2018) Haugen, Samuel Arthur McGrathBarley yellow dwarf (BYD), caused by Barley yellow dwarf virus and Cereal yellow dwarf virus, and is a yield limiting disease of small grains. A research study was initiated in 2015 to identify the implications of BYD on small grain crops of North Dakota. A survey of 187 small grain fields was conducted in 2015 and 2016 to assess cereal aphid diversity; cereal aphids identified included, Rhopalosiphum padi, Schizaphis graminum, and Sitobion avenae. A second survey observed and documented field absence or occurrence of cereal aphids and their incidence. Results indicated prevalence and incidence differed among respective growth stages and a higher presence of cereal aphids throughout the Northwest part of North Dakota than previously thought. Field and greenhouse screenings were conducted to identify hard red spring wheat and durum responses to BYD. Infested treatments in the greenhouse had significantly lower number of spikes, dry shoot mass and yield.Item Association Mapping of Resistance to Common Root Rot and Spot Blotch in Barley, and Population Genetics of Coch/iobolus sativus(North Dakota State University, 2010) Gyawali, SanjayaCochliobolus sativus (Ito & Kurib.) Drechsl. ex Dast. [anamorph, Bipolaris sorokiniana (Sacc. in Sorok.) Shoem.] is an important fungal pathogen, which causes common root rot (CRR), spot blotch (SB) and black point/kernel blight in barley in North America. Use of genetic resistance has been effective against SB presumably due to presence of durable resistance in North America. However, recently emerged virulence groups have overcome durable resistance in barley. Additionally, the genetics of resistance to CRR is poorly understood. Therefore, the objectives of current studies are multifaceted. To identify the sources of resistance to CRR, 824 contemporary barley lines from the USDA-CSREES Barley Coordinated Agricultural Project (CAP) were evaluated for resistance to CRR under natural inoculum pressure in the field during 2006 - 2008. Additionally, resistance to CRR (n = 384 lines) and SB (n = 386 lines) was also evaluated in the greenhouse. The results indicate that only 0.9% of breeding lines showed resistance to CRR in field experiments during 2006-2008. None of the genotypes showed resistance to virulent isolates in greenhouse experiments. Hordeum jubatum sp. jubatum (Accession # CGN 13044) showed 12% CRR severity against highly virulent isolates. Therefore, this accession can be used as a potential source of resistance to CRR in the future. In the SB experiment, only 0.5% of the 386 genotypes showed resistant responses to isolate 4008. The barley line NDB112 with durable resistance to SB also showed a highly susceptible reaction to this isolate. To map QTL for CRR and SB resistance, association mapping was employed using the CAP06 population (n = 384), CAP07 population (n = 384) for CRR resistance and the CAP06 population (n = 384) for SB resistance. In all association analyses, 3072 single nucleotide polymorphism (SNP) markers were used. The results suggest five QTL resistance to CRR, CRR-3H-28-51, CRR-SH- 180-195, CRR-6H-30-64, CRR-6H-91-97, and CRR-lH-50-86 were detected in chromosomes 3H, 5H, 6H, and 7H. Two QTL resistance to spot blotch, Rcs-1H- 84.6 and Rcs-2H-106-122 were identified in chromosomes 1 Hand 2H, respectively. These QTL didn't coincide with any of the QTL reported earlier and confer resistance to virulence group 7.7.3.6. To investigate the virulence spectrum of C. sativus isolates collected from North Dakota (ND), 12 barley genotypes were inoculated with 12 virulent C. sativus isolates on both root and leaf. The results suggest that different virulence groups for CRR and SB diseases exist in the pathogen population. To understand the population structure of C. sativus populations collected from Australia and the USA (ND), 289 single-spore isolates were analyzed for amplified fragment length polymorphism (AFLP) using three AFLP primer combinations. Moderate to high gene diversity (H = 0.27 - 0.35) and high genotypic diversity (GD= 1) within C. sativus populations indicate occurrence of genetic recombination other than sexual in C. sativus populations. The moderate to high population differentiation (Gst = 0.196), moderate multilocus linkage disequilibrium (rd = 0.046 - 0.118), and low gene flow (Nm= 2.0) suggest the occurrence of different populations of C. sativus in the field.Item Augmenting Fungicidal Activity of Tetraconazole with Chemosensitization Agents for Cercospora Leaf Spot Management(North Dakota State University, 2017) Metz, Nicholas JamesCercospora beticola (Sacc.) is the causal agent of Cercospora leaf spot (CLS). CLS is considered to be one of the most destructive foliar diseases of sugar beet in the world. CLS is managed in part through resistant cultivars, crop rotation, and cultural practices, but timely fungicide applications are necessary to manage disease effectively. Heavy reliance on fungicides to manage CLS has led to the development of resistance to multiple classes of fungicides. The most widely used class of fungicides is the demethylation inhibitors (DMIs). DMI-resistant C. beticola isolates have been increasing in incidence over the past decade. Chemosensitization agents (CAs) are compounds that have little to no antifungal activity, but may increase efficacy of commercial fungicides when co-applied. CAs could lead to better management of CLS and reduced production costs.Item Biology and Development of Two Wilt Fungi of Potato: Verticilliam Dahliae and Colletotrichum Coccodes(North Dakota State University, 2012) Pasche, Julie ShermanThe demand for high quality tubers for the frozen processing industry has exerted increased pressure on producers to control diseases that can compromise tuber quality, including, C. coccodes and V. dahliae. Infection of potato plants by C. coccodes, the causal agent of black dot, can result in foliar necrosis, plant wilting, yield loss, tuber vascular discoloration and skin blemishes. Black dot of potato can originate from foliar-, seed- of soil-borne infections and, all plant tissues can be affected. The development of colonization in above- and below-ground plant tissues, from each inoculation/infestation source, was evaluated at two sites across two growing seasons. Colonization of potato tissue was detected at the first sampling date, even prior to plant emergence. While disease resulted from natural inoculum only, as inoculation/infestation sources increased, so did host colonization. Disease development also was greater in treatments with more than one site of inoculation/infestation. Overall, it was determined that stem tissue was colonized at greater frequency than root or stolon tissue. Infection of potato plants with V. dahliae, the causal agent of Verticillium wilt, occurs mainly through root contact with infested soil and can result in premature senescence as well as losses from decreased tuber yield and quality. Control of this pathogen is difficult and expensive and, therefore, efforts recently have increased towards the development of resistance. Resistance to V. dahliae in potato is thought to be multigenic, and therefore, quantification of the host:pathogen interaction is required to accurately define the level of resistance in a particular cultivar. Unfortunately, current methods used for quantification of V. dahliae in potato stems are time and labor intensive. A real-time duplex quantitative PCR assay was developed to simplify pathogen quantification to help breeding programs and researchers identify resistance in germplasm and cultivars. QPCR assays were validated using plant material from greenhouse and field trials, demonstrating specificity for V. dahliae, as well as sensitivity and accuracy when compared to traditional plating assays. Results from greenhouse and field evaluations also indicated that resistance is present among the eight russet-skinned cultivars evaluated in these studies.Item Biology and Management of Fusarium Species on Sugar Beet(North Dakota State University, 2017) Lai, XiaoMinnesota and North Dakota together produce about 51% of the beet sugar in the United States of America. Fusarium diseases caused by Fusarium oxysporum f. sp. betae and F. secorum on sugar beet cause significant reduction in both root yield and sucrose concentration. This research was conducted to determine the best inoculation methods to induce Fusarium diseases on sugar beet seeds and plants and to evaluate fungicides for their efficacy at controlling Fusarium diseases in greenhouse conditions. The use of Fusarium colonized barley seeds in close proximity to sugar beet seeds and seedlings caused similar level of disease severity as the standard root-dipping method, and reduced the time for evaluation by directly inoculating seeds and 4-leaf stage plants rather than using older plants which have to be transplanted into new pots. Pydiflumetofen and metconazole fungicides used in-furrow have the potential to provide effective control of Fusarium diseases on sugar beet.Item Characteristics and Host Range of a Novel Fusarium Species Causing Yellowing Decline of Sugarbeet(North Dakota State University, 2013) Villamizar-Ruiz, Johanna PatriciaIn 2008, a novel and distinct Fusarium species was reported in west central Minnesota causing early-season yellowing and severe decline of sugarbeet. This study was conducted to (i) establish optimum conditions for fungal growth and (ii) determine the host range of the novel Fusarium. The optimum temperature for fungal growth is 24°C and root injury is not needed to penetrate, infect, and cause disease of sugarbeet plants. Of the fifteen common crops and weeds tested for susceptibility to the new Fusarium sp. in field and greenhouse trials, disease symptoms were only observed in sugarbeet. Host range plants were tested for the presence of latent infection by root isolations and PCR. The pathogen was only present in canola and sugarbeet. The results suggest that canola has implications in the sugarbeet production system and management strategies for the novel Fusarium species. The name and description of the new Fusarium sp. is pending.Item Characterization and Detection of Bacterial Pathogens of Common Bean(North Dakota State University, 2022) Nelson, KatieCommon bacterial blight (CBB) (Xanthomonas axonopodis pv. phaseoli (Xap) and X. fuscans pv. fuscans (Xff), halo blight (Pseudomonas savastanoi pv. phaseolicola (Psp)), and bacterial brown spot (P. syringae pv. syringae (Pss)), are yield-limiting diseases of common bean in North Dakota and Minnesota. The objectives of this research were to optimize a multiplex quantitative PCR (qPCR) assay for rapid detection and quantification of four bacterial pathovars in common bean seed, leaf, and pod tissue; determine the aggressiveness of Xap and Xff isolates; and determine the race types of Psp isolates in North Dakota and Minnesota. A fourplex qPCR assay was optimized, and novel primers and probe were designed for pathovar-specific detection of Pss. Significant differences were observed in Xap/Xff aggressiveness across isolates evaluated and in susceptibility of the differential lines (P <.0001). The 60 Psp isolates were identified as Race 6, the predominant race in the region.Item 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 DanielWheat 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.Item Characterization of Cytochrome B from European Field Isolates of Cercospora Beticola with Quinone Outside Inhibitor Resistance(North Dakota State University, 2012) Birla, KeshavCercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugar beet worldwide. Control strategies for CLS rely heavily on fungicides including quinone outside inhibitor (QOI) fungicides. We collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed their sensitivity to the QOI fungicide pyraclostrobin. To gain an understanding of the molecular basis of QOI resistance, we cloned the full-length coding region of Cbcytb. All tested QOI-resistant isolates harbored a point mutation in Cbcytb at nucleotide position 428 that conferred an exchange from glycine to alanine at amino acid position 143 (G143A). A PCR assay was developed to discriminate QOI-sensitive and QOI-resistant isolates based on the G143A mutation. Our results indicate that QOI resistance has developed in some European C. beticola populations in Italy and monitoring the G143A mutation is an essential fungicide resistance management strategy.Item Characterization of Effector Encoding Genes from the Novel Sugar Beet Pathogen Fusarium Secorum(North Dakota State University, 2015) Bian, ZhuyunA new disease of sugar beet, named Fusarium yellowing decline, was recently found in in the Red River Valley of MN and ND. This disease is caused by a novel pathogen named Fusarium secorum. Pathogens such as F. secorum secrete proteins during infection called ‘effectors’ that help establish disease. Since pathogenicity and disease development may depend on effector proteins produced by F. secorum during infection, effector protein identification furthers our understanding of the biology of this important pathogen. A list of 11 candidate effectors was generated previously. In this study, to characterize putative effectors, we developed a transformation system using polyethylene glycol–mediated transformation. Several mutant lines were created with an effector deleted from the genome using a split-marker knock-out strategy. To explore their role in pathogenicity, mutant strains have been inoculated to sugarbeet and compared to WT F. secorum.Item Characterization of Fusarium Head Blight Resistance in Wheat(North Dakota State University, 2017) Zhao, MingxiaFusarium 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.Item Characterization of North Dakota Hard Red Spring Wheat for Stem and Stripe Rust Resistance(North Dakota State University, 2022) Kim, YongJaePuccinia graminis f. sp. tritici (Pgt) and Puccinia striiformis f. sp. tritici (Pst) are causal agents of devastating wheat stem and stripe rust diseases, respectively. Both diseases sporadically occur in North Dakota (ND), and stripe rust incidence has been increasing in the Midwest of United States over the last decade. Complete information of rust resistance in ND hard red spring wheat (HRSW) germplasm is not well-established. This study focused on the phenotypic characterization of ND HRSW germplasm for stripe and stem rust resistance and the identification of existing and novel rust resistance genomic loci in this population through genome-wide association study (GWAS). The GWAS has identified several marker-trait associations (MTAs) for both all-stage and adult plant resistance for each rust disease. This information will support the deployment of these resistant loci in wheat varieties by the NDSU HRSW breeding program.Item 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 LouisePSbMV 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.Item Characterization of Pectobacterium carotovorum subsp. brasiliense as a Causal Agent of Sugarbeet Soft Rot(North Dakota State University, 2018) Metzger, Michael StewartA 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.Item Characterization of Programmed Cell Death Responses Involved in Disease Resistance/Susceptibility Responses in Barley(North Dakota State University, 2015) Sager-Bittara, Lauren PaigeThe plant innate immune system relies on regulated programmed cell death (PCD) responses which provide resistance against biotrophic fungal pathogens and are utilized by necrotrophic pathogens. The gene-for-gene interactions leading to resistance against biotrophs has been co-evolving over millions of years and provides many targets for necrotrophic pathogens to subvert the plants PCD for their benefit. Two PCD pathways involved in plant immunity were characterized. The first, nec3, appears to control runaway PCD in response to infection by many pathogens who mainly elicit pathogen or damage associated molecular pattern (PAMP or DAMP) triggered immunity. The nec3 mutant was mapped to chromosome 6H and a pool of candidate genes were identified by RNAseq. The second, Rcs5, is a dominant susceptibility gene or a putative necrotrophic effector triggered susceptibility (NETS) target that confers susceptibility to Cochliobolus sativus. The candidate Rcs5 genes are three Wall Associated Kinases (WAKs) found on chromosome 7H (1).Item Characterization of Sensitivity of Sclerotinia Sclerotiorum Isolates from North Central U.S. to Thiophanate-Methyl and Metconazole(North Dakota State University, 2013) Ameen, GazalaSclerotinia sclerotiorum (Lib.) de Bary causes Sclerotinia stem rot on canola and many other crops of economic importance in the U.S. SSR is primarily controlled with fungicides applied at flowering time. Most fungicides currently used to control SSR can promote resistance buildup in their target populations making monitoring of sensitivity important. In this study the reaction of S. sclerotiorum to thiophanate-methyl (TM) and metconazole (MTZ) was characterized. Samples collected in several states of north central U.S. were used. Three and ten isolates were considered to be moderately insensitive to TM and MTZ, respectively. Greenhouse trials indicated, however, that diseases caused by these isolates could be effectively controlled using currently recommended doses of each compound. In vitro sensitivity to TM was temperature dependent. A previously unreported mutation at codon E111D in the β-tubulin gene of a TM-moderately insensitive isolate was identified.Item 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 MariePyrenophora 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.Item 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, GazalaUpon 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.Item Comparative Population Genetics of Fusarium Graminearum and Novel Sources of Resistance to Fusarium Head Blight in Spring Wheat(North Dakota State University, 2013) Puri, Krishna DattaFusarium graminearum is the major causal agent of Fusarium head blight (FHB) in cereal crops. The fungus produces several types of trichothecenes [Deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV)]. Characterization of 123 isolates collected during 1980 to 2000 (old collection), and 43 isolates collected in 2008 (new collection) from North Dakota revealed that 15ADON producing isolates were predominant (98%) in the old collection while the 3ADON producing isolates accounted for 43% in the new collection. Further, evaluation showed that the 3ADON isolates caused a higher disease severity and accumulated more DON in spring wheat than the 15ADON isolates. The 3ADON also exhibited higher DON in rice culture, and produced more spores on agar media. Population genetic analyses revealed a significant genetic differentiation between the two populations. To elucidate the transcriptomic differences between the two populations in vitro and in planta, RNA-sequencing was used. The in vitro gene expression comparison identified 479 up- and 801 down- regulated genes in the 3ADON population compared to 15ADON population. The in planta pair-wise comparisons between the two populations revealed 185, 89, and 62 unique genes to 3ADON at 48, 96 and 144 hours after inoculation (HAI), respectively. In a different study, population genetic analysis was conducted on 160 isolates collected in 2008 and 2009 from a FHB disease nursery located in China. All isolates analyzed were F. asiaticum except one (F. avenaceum). Of the 159 isolates, 79% were NIV producing, 18% were 15ADON and 3% were 3ADON. The two populations grouped based on year of collection exhibited low genetic differentiation (Fst = 0.032). To identify new sources of FHB resistance, 71 wheat accessions of diverse origins were re-evaluated for FHB severity and haplotyped using seventeen DNA markers associated with known resistance quantitative trait loci (QTL). Twenty two accessions had a haplotype different from all known sources used, suggesting that they may carry novel loci for FHB resistance. In conclusion, the information obtained in this study could have an impact on development of effective disease management measures and on improvement of FHB resistance in wheat.Item Detection of Qol Fungicide Resistance and Genomic Analysis for Identification of Polyketide Synthases and Methyltransferases in Ascochyta rabiei(North Dakota State University, 2011) Delgado, Javier AndresAscochyta blight is the most important disease of chickpeas in North America since the fungal pathogen Ascochyta rabiei was introduced to this region in 1986. Ascochyta blight epidemics can cause yield and economic losses of up to 100%. Currently available chickpea cultivars are partially resistant to A. rabiei, and the disease is primarily managed with the aid of seed and foliar fungicide treatment throughout the growing season. Development of resistance to highly effective fungicides such as the quinone outside inhibitor (Qol) fungicides has therefore significantly threatened chickpea production in the US and Canada. In this dissertation, the mutation associated with resistance to Qol fungicides was identified in A. rabiei in North Dakota from which a quick and accurate diagnostic assay was developed for the detection of Qol resistant isolates of A. rabiei. The detection of Qol resistant isolates is important for designing management strategies aimed to controlling A. rabiei in the field. Polyketide synthases (PKSs) and methyltransferases have been associated with virulence in several fungi but these genes have not been identified or characterized previously in A. rabiei. Therefore, a bioinformatic resource was developed for the identification of PKS proteins from the genome of A. rabiei. This was developed using the 13-ketoacyl synthase and acyltransferase domains from PKS proteins exclusively belonging to the fungal species and was used to identify PKS genes from the A. rabiei genome. Several methyltransferase genes were also identified using a similar strategy. The characterization of the identified A. rabiei PKSs and methyltransferases was conducted to study the effect of the divalent cations present in chickpea seed extracts on the accumulation of phytotoxic compounds, vegetative growth, and conidial production. This study provides an insight into the effect of these divalent cations on the expression of genes that modulate biological processes that may be directly associated with infection and colonization of the host.