Neil Gudmestad - NDSU Publications

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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.
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.
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.
Identification and Genomic Analysis of Stagonospora Nodorum Blotch Susceptibility Genes in Wheat
(North Dakota State University, 2014) Shi, Gongjun
Parastagonospora nodorum is a necrotrophic fungal pathogen that causes the disease Stagonospora nodorum blotch (SNB) on wheat. The fungus produces necrotrophic effectors (NEs), that when recognized by corresponding host genes, cause cell death leading to disease. A novel NE, designated SnTox7, was identified from culture filtrates of isolate Sn6 of P. nodorum. SnTox7 is a small protein with estimated size less than 30 kDa. The interaction between SnTox7 and its corresponding host sensitivity gene, Snn7, explained 33% of the disease variation among a segregating F2 population. The Snn7 gene governs sensitivity to SnTox7 and was delineated to a 2.7 cM interval on the long arm of wheat chromosome 2D. Another host sensitivity gene Snn3- B1, conferring sensitivity to SnTox3, was previously mapped on the short arm of wheat chromosome 5B. Forty-four molecular markers were added to the genetic map to saturate the Snn3-B1 gene region. High-resolution mapping of the Snn3-B1 locus in 5,600 gametes delineated the gene to a 1.5 cM interval. The closely linked markers should be very useful for marker-assisted selection against Snn3-B1. A third host gene, Snn1, confers sensitivity to the NE Tox1. Snn1 was isolated through map-based cloning, and its structure, expression and allelic diversity were further characterized. A bacterial artificial chromosome (BAC) contig of about 2.5 Mb in size was identified to span the Snn1 locus through screening of Chinese Spring chromosome arm 1BS minimum tiling path (MTP) pools. Additional markers developed from BAC end sequences (BESs) delineated the Snn1 gene to a physical segment consisting of four BAC clones. Sequencing and bioinformatic analysis of these clones led to the identification of seven candidate genes. Six of the seven candidates were excluded through critical recombinants. The seventh gene, a cell wall-associated kinase (WAK), was verified as Snn1 through comparative sequence analysis with ethylmethane sulfonate (EMS)-induced mutants. The Snn1 transcription profile showed that it was regulated by light and possibly circadian rhythms. These results demonstrate that P. nodorum can hijack multiple host pathways driven by different classes of genes that typically confer resistance to biotrophic pathogens, thus demonstrating the surprisingly intricate nature of plant-necrotrophic pathogen interactions.
Inoculation Techniques, Development of Brassica Napus Breeding Lines and Identification of Markers Associated with Resistance to Sclerotinia Sclerotiorum (Lib.) De Bary
(North Dakota State University, 2012) Burlakoti, Pragyan
Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economic disease affecting canola (Brassica napus L). Since expression of sclerotinia stem rot symptoms shows much variability and the trait is quantitative in nature, reliable phenotypic evaluation methods for characterization are needed. The three major objectives of this dissertation were to: i) evaluate eight different inoculation methods to discriminate between S. sclerotiorum-resistant and susceptible B. napus germplasm; ii) develop breeding lines with resistance to multiple diseases, and; iii) to identify QTL associated with resistance to sclerotinia stem rot using association mapping (AM). The eight methods evaluated were the detached leaves, detached stems, petiole inoculation (PIT), straw-inoculation, stem-piercing with toothpick, mycelial spray (MSI), petal inoculation and oxalic acid assay. MSI and PIT can better discriminate between the isolates and germplasm. Breeding lines resistance to S. sclerotiorum, Leptosphaeria maculans, and Rhizoctonia solani were developed from a cross between two moderately sclerotinia stem rot resistant plant introductions (PI). F2 seedlings were screened for sclerotinia stem rot using PIT. Surviving plants were self pollinated and their progeny screened again. This process was repeated until the F6 generation. In addition, F5 seedlings were evaluated for their reaction to R. solani and F5 and F6 seedlings for their reaction to L. maculans. Eight lines were identified as moderately resistance to these three pathogens. The genomes of a group of 278 B. napus plant introductions were screened using Diversity Array Technology to detect QTL associated with resistance to sclerotinia stem rot. The population was classified into nine sub-populations and 32 significant markers each explaining between 1.5 and 4.6% of the variation were identified. Blastn search indicates that similar nucleotide sequences are distributed throughout the genomes of B. oleracea, B. rapa, and A. thaliana. Results of these studies suggest the PIT and MSI are reliable screening tools to evaluate materials for resistance to sclerotinia stem rot; materials identified as resistant to S. sclerotiorum were also moderately resistant against R. solani and L. maculans and could be valuable sources for canola improvement programs; and AM allowed us to identify QTL associated with resistance to sclerotinia stem rot.
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 Characterization and Pathogenicity of Sunflower Stem Pathogens
(North Dakota State University, 2014) Mathew, Febina
Sunflower (Helianthus annuus L.) production can be limited by several stem diseases. Among these, Phomopsis stem canker causes frequent yield reductions in Australia, Europe and North America. In the U.S., while Diaporthe helianthi was assumed to be the sole causal agent, Diaporthe gulyae was found to cause Phomopsis stem canker in Australia. In order to determine the causal agent in the U.S., 234 isolates were cultured from 275 infected sunflower stems collected from the Northern Great Plains. Phylogenetic analyses of the internal transcribed spacer region, elongation factor subunit 1-α, and actin gene sequences confirmed two species, D. helianthi and D. gulyae. Four methods were tested to assess the Phomopsis stem canker response using four D. helianthi isolates on sunflowers. Stem-wound method was adopted for subsequent experiments based on the recovery of D. helianthi and its correlation with disease severity at 14- d after inoculation. Aggressiveness of two Diaporthe species was determined in greenhouse and results suggest they did not vary significantly (p=0.0012) in their aggressiveness, except at 3-d after inoculation. Among the nine genotypes screened for resistance, USDA ‘PI 162784’ and ‘PI 219649’ were less susceptible to the two Diaporthe spp. Fusarium is commonly regarded a minor pathogen on sunflowers in most production regions of the world. A total of 110 Fusarium isolates were recovered from 1,637 stalks randomly sampled for stem diseases in the Northern Great Plains and identified to species level. Phylogenetic analyses of repetitive-sequence-based polymerase chain reaction fingerprints and the translation elongation factor 1-alpha gene revealed that Fusarium isolates from sunflowers represented clades of eight species; namely, F. graminearum, F. proliferatum, F. culmorum, F. avenaceum, F. oxysporum, F. acuminatum, F. sporotrichioides and F. equiseti. Pathogenicity studies of eight Fusarium spp. in the greenhouse suggests F. sporotrichioides and F. equiseti were most aggressive. The study comparing the aggressiveness of three Fusarium spp. and V. dahliae isolates representing six VCGs showed V. dahliae VCG4B and VCG2A were significantly more aggressive (p ≤ 0.05) than F. sporotrichioides, F. oxysporum and F. equiseti. The identification of Diaporthe spp. and Fusarium spp. on sunflowers has implications for breeding for resistance and disease management.
Sensitivity of Rhizoctonia Solani and Aphanomyces Cochlioides to Fungicides, and Fitness of Tetraconazole-Resistant Isolates of Cercospora Beticola after Exposure to Different Temperature Regimes
(North Dakota State University, 2015) Arabiat, Sahar Ibrahim
North Dakota and Minnesota produce 55% of USA sugarbeet production. Diseases caused by Rhizoctonia solani, Aphanomyces cochlioides, and Cercospora beticola are the major diseases affecting sugarbeet production in North Dakota and Minnesota. Growers mainly use partial resistant varieties and fungicides to manage diseases of sugarbeet. Sensitivity of R. solani and A. cochlioides to fungicides were evaluated in vitro using mycelium radial growth assay and by evaluating disease severity on inoculated plants treated with fungicides in the greenhouse. Phenotypic stability of tetraconazole-resistant isolates of C. beticola after exposure to different temperature regimes was evaluated. For R. solani, mean EC50 values for baseline isolates were 49.7, 97.1, 0.3, 0.2, and 0.9 μg ml-1 and for non-baseline isolates were 296.1, 341.7, 0.9, 0.2, and 0.6 μg ml-1 for azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole, respectively. The mean EC50 values of azoxystrobin, trifloxystrobin, and pyraclostrobin increased with a change factor of 6.0, 3.5, and 2.7, respectively. All fungicides at labeled rates effectively controlled R. solani in vivo. For A. cochlioides, tetraconazole, prothioconazole, and pyraclostrobin reduced mycelium radial growth in vitro with mean EC50 values of 3.5, 2.4, and 0.8 μg ml-1, respectively. However, these fungicides were not effective at controlling A. cochlioides in vivo. Sugarbeet plants up to three weeks old were found susceptible to A. cochlioides. Resistant isolates of C. beticola had no fitness penalty as measured by spore production, spore germination, mycelium radial growth, and disease severity after exposure to different temperature regimes. However, isolate 09-347, resistant to tetraconazole, reverted to a moderate resistance level after exposure to -20ºC, and -20ºC to 4ºC to -20ºC to 4ºC with a factor of change of 38.6 and 32.8, respectively. This research indicated that R. solani sensitivity to the evaluated QoIs had decreased, but they were still effective at labeled rates under greenhouse conditions, and rotation of different fungicide classes could be a useful strategy to manage fungicide resistance. No fitness penalty was found after exposure of C. beticola isolates to cold treatments. However, C. beticola isolates resistant to tetraconazole became more sensitive to this fungicide after exposure to cold treatments.

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