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Item Managing Economically Important Diseases of Sunflower and Oilseed Rape in North Dakota, California, and Schleswig-Holstein(North Dakota State University, 2021) Berghuis, Brandt GregoryRust, caused by Puccinia helianthi, and blackleg, caused by Leptosphaeria maculans, are economically important diseases on sunflower and oilseed rape, respectively. In order to recommend the most effective disease management practices to growers, management strategies need to be evaluated as new tools are developed and pathogen populations change. The objectives of these studies were to; 1) evaluate the efficacy of foliar fungicides on rust in confection and oilseed sunflowers in the U.S. Northern Great Plains, 2) determine virulence phenotypes of Puccinia helianthi in the sunflower seed production region of Northern California, and 3) evaluate the seed applied fungicide adepidyn for efficacy of blackleg on oilseed rape in Northern Germany. Efficacy of 11 foliar fungicides against sunflower rust were demonstrated on both confection and oilseed sunflowers, however, yield differences were only observed in confection experiments. In total, 21 races of Puccinia helianthi were identified in the sunflower seed production region of California, of which 18 were reported for the first time. To the best of our knowledge, this work presents the only data generated from wild Helianthus annuus populations in the region, and results demonstrate the threat that rust on wild species presents to the commercial seed production industry. Results of experiments conducted to evaluate the suitability of the novel seed treatment adepidyn for management of blackleg demonstrated efficacy under climate-controlled conditions, but efficacy was not observed in field studies. Results of these studies detail relevant and timely information that will help agriculture professionals better manage these economically important diseases of oilseed crops.Item Molecular Genetic Characterization of Ptr ToxC-Tsc1 Interaction and Comparative Genomics of Pyrenophora tritici-repentis(North Dakota State University, 2018) Kariyawasam, Gayan KanishkaTan 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.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 Parasitic Fitness of SDHI-Sensitive and -Resistant Isolates of Alternaria Solani(North Dakota State University, 2017) Bauske, Mitchell JamesEarly 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.Item Genetic and Molecular Characterization of Genes Involved in Barley-Cochliobolus Sativus Interaction(North Dakota State University, 2015) Wang, RuiSpot 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.Item 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, SubidhyaVideo summarizing a Ph.D. dissertation for a non-specialist audience.Item Towards Identifying the Physical and Molecular Components Involved in Resistance to Leaf Rust Pathogen Puccinia Triticina in Wheat and Barley(North Dakota State University, 2016) Dugyala, SheshankaGenetic resistance is the preferred method to reduce yield losses caused by many diseases including wheat leaf rust. However, in many cases host resistance seems to be short-lived. For decades, scientists have tried and continue trying to understand genetics and physical mechanisms involved in durable host resistance. Incompatible host-pathogen interactions in wheat- Puccinia triticina (Pt) pathosystem can be classified into pre-and post-haustorial. Post-haustorial resistance tends to be involved in race specific resistance and is commonly characterized by presence of hypersensitive reaction (HR), while pre-haustorial resistance do not generally involve HR. The objective of this work was to understand the physical and molecular components associated with resistance mechanisms to Pt. A set of Thatcher near isogenic lines (NIL) carrying different leaf rust (Lr) resistance genes and barley (non-host) genotypes were evaluated in time course experiments for histological analysis and relative fungal DNA quantification. Histological evaluation and q-PCR assay showed differences in time of fungal structures formation and amount of fungal DNA among genotypes. The q-PCR assay could differentiate between resistant and susceptible genotypes at 24 hpi. Additionally, Cq ratio of q-PCR assay allowed for classification of resistant genotypes carrying pre-haustorial (Lr9, Q21860) and those carrying post-haustorial resistance (Lr21, Lr34 and Harrington). To our surprise, despite carrying pre-haustorial resistance, Tc-Lr9 presented HR as early as 6 hpi. Tc-Lr21 presented HR, before (5 hpi) and after haustoria formation (24 hpi). To better characterize the HR and determine if the HR observed was the product of H2O2 accumulation, histochemical and gene expression studies were used. Both methods confirmed that the HR observed in Tc-Lr9 and Tc-Lr21 indeed involved H2O2 accumulation. In addition, up-regulation of hypersensitive induced resistance genes TaHIR1, TaHIR2 was observed in Tc-Lr9. Up-regulation of Tc-Lr21 gene at 5 hpi was observed in Tc-Lr21. The protocols developed and data obtained from this study provide opportunities for quantitatively assess components of resistance and suggest that some previous assumptions about plant-pathogen interaction in host and non-host systems should be revisited.Item Development of Management Tools for Sunflower Downy Mildew (Plasmopara Halstedii) and Rust (Puccinia Helianthi)(North Dakota State University, 2016) Humann, RyanDowny 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.Item Genomics and Management of Fusarium Root Rot of Field Peas(North Dakota State University, 2012) Chittem, KishoreDry Pea or field pea (Pisum sativum L.) is an important cool season legume crop grown in the United States. Field peas are vulnerable to many diseases of which, soil borne diseases including wilt and root rot are of major economic importance and can cause significant reduction in yield. There is a dearth of satisfactory methods for control of root rot and no varieties with complete resistance to Fusarium root rot are currently available. Root rot disease was found to be prevalent in all the major pea growing counties of North Dakota surveyed in 2004, 2005, 2010 and 2011. Fusarium species were the most frequently isolated fungal species from the infected pea roots of which, F. oxysporum and F. avenaceum were the most common. 21 Field pea varieties were screened for resistance against F. avenaceum and F. solani f. sp. pisi, the Fusarium species traditionally associated with root rots of field pea in growth chamber experiments and field trials. Low levels of resistance were detected in a few cultivars but no variety was found to be completely resistant to any of the pathogens tested. Efficiency of precipitated calcium carbonate (PCC) in controlling Fusarium species most commonly associated with root rots was evaluated under in vitro and field conditions. Significant reduction in spore production, spore germination, and dry mycelial weight of Fusarium spp. were detected on PCC amended media in laboratory studies. In greenhouse and field experiments significant reduction in root rot disease severity was observed with PCC application compared to control. Fungal gene expression in artificially infected field pea roots and F. graminearum grown in culture was assessed using the Illumina mRNA-Seq technology. A total of 613 F. graminearum genes were found to be differentially expressed in planta on pea. Functional classes associated with amino acid metabolism, nitrogen metabolism, extracellular polysaccharide degradation, detoxification by degradation and defense related proteins were found to be significantly enriched in the up-regulated gene set as determined using FunCatDB. Expression of four up-regulated genes was confirmed by RT-PCR to validate the inferences from the sequencing results.Item Identification and Characterization of Pythium Spp. on Glycine Max (Soybean) in North Dakota(North Dakota State University, 2013) Zitnick-Anderson, Kimberly KorthauerThe Oomycete Pythium comprises one of the most important groups of seedling pathogens affecting soybean, causing both pre- and post-emergence damping off. Numerous species of Pythium have been identified and found to be pathogenic on a wide range of hosts. Recent research on Pythium sp. infecting soybean has been limited to regions other than the Northern Great Plains and has not included North Dakota. In addition, little research has been conducted on the pathogenicity of various Pythium species on soybean or associations between Pythium communities and soil properties. Therefore, the objectives of this research were to isolate and identify the Pythium sp. infecting soybean in North Dakota, test their pathogenicity and assess if any associations between Pythium sp. and soil properties exist. Identification of the Pythium sp. was achieved using molecular techniques and morphological features. A total of 26 described Pythium sp. and several unknown species were recovered from soybean roots collected from 138 fields between 2011 and 2012. The majority of Pythium species (P. attrantheridium, P. debaryanum, P. diclinum, P. dissotocum, P. heterothallicum, P. hypogynum, P. inflatum, P. intermedium, P. irregulare, P. kashmirense, P. lutarium, P. minus, P. oopapillum, P. perplexum, P. terrestris, P. viniferum, P. violae,and an unknown Pythium sp.) caused pre-emergence damping off on soybean seedlings with less than 50% emergence and survival. In contrast, P. orthogonon, P. nunn, and P. rostratifingens had approximately 80% or greater emergence and survival of soybean seedlings. The negative and positive controls had 100% and 0% emergence and survival of soybean seedlings. Associations between soil properties and three Pythium groups were performed using logistic regression analysis. Logistic regression analysis determined that the presence of group one characterized by P. ultimum was correlated with zinc levels. Group two was characterized by P. kashmirense and an unknown Pythium sp. and was correlated with cation exchange capacity (CEC) values. Group three was characterized by P. irregulare and P. heterothallicum and was correlated with calcium carbonate exchange and CEC.