Neil Gudmestad - NDSU Publications

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Augmenting Fungicidal Activity of Tetraconazole with Chemosensitization Agents for Cercospora Leaf Spot Management
(North Dakota State University, 2017) Metz, Nicholas James
Cercospora 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.
Biology and Management of Fusarium Species on Sugar Beet
(North Dakota State University, 2017) Lai, Xiao
Minnesota 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.
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 Cytochrome B from European Field Isolates of Cercospora Beticola with Quinone Outside Inhibitor Resistance
(North Dakota State University, 2012) Birla, Keshav
Cercospora 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.
Characterization of Sensitivity of Sclerotinia Sclerotiorum Isolates from North Central U.S. to Thiophanate-Methyl and Metconazole
(North Dakota State University, 2013) Ameen, Gazala
Sclerotinia 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.
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.
Downy Mildew of Sunflowers: Establishment of Baseline Sensitivity to Azoxystrobin and Monitoring for the Development of Fungicide Resistance and Plasmopara Halstedii Virulence Phenotype Changes
(North Dakota State University, 2017) Gilley, Michelle A.
Downy mildew, caused by Plasmopara halstedii (Farl.) Berl. and de Toni, is an economically important disease in cultivated sunflowers, Helianthus annuus L. The objectives of this study were to determine disease pressure in North Dakota and South Dakota, determine the virulence phenotypes in the pathogen population, determine the baseline sensitivity to azoxystrobin and evaluate select isolates for fungicide insensitivity. While downy mildew was present in many fields, incidence was typically low. To determine virulence phenotypes, selected isolates were evaluated on an expanded set of differential lines. New virulence was found to the Pl8 resistance gene, but no virulence was observed on the PlArg, Pl15, Pl17 and Pl18 genes. Using a discriminatory dose of 10 ug ai azoxystrobin/seed, no isolate approached infection levels found in inoculated, nontreated controls; therefore, the pathogen is considered sensitive to azoxystrobin in the greenhouse and azoxystrobin should still suppress downy mildew in the field.
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.
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.
Plant-Parasitic Nematodes in Field Pea and Potato and their Effect on Plant Growth and Yield
(North Dakota State University, 2018) Upadhaya, Arjun
In this study, surveys were conducted in pea and potato fields in North Dakota and Central Minnesota to investigate the incidence and abundance of plant-parasitic nematodes in these fields. Moreover, the effect of the pin nematode, Paratylenchus nanus, on plant growth and yield of six field pea cultivars was determined under greenhouse conditions. Similarly, the influence of lesion nematode, Pratylenchus penetrans, and wilt fungi, Fusarium oxysporum alone and together on growth and yield of potato cultivar ‘Red Norland’, was evaluated in microplots under field conditions. The results indicate Paratylenchus spp. and Pratylenchus spp. are the most frequent nematodes, respectively, in pea and potato fields. Pin nematodes reproduced on field pea cultivars and caused up to 37% reduction in plant height and 40% reduction in yield. Additionally, both P. penetrans and F. oxysporum alone, and together had significant negative effect on growth and yield of potato.
Rhizoctonia Solani Damping-Off of Sugarbeet: Effect of Plant Growth Stage on Disease Severity and Management Using Penthiopyrad
(North Dakota State University, 2015) Liu, Yangxi
Rhizoctonia solani is the most damaging pathogen on sugarbeet (Beta vulgaris L.) in North Dakota and Minnesota. Research was conducted to evaluate penthiopyrad for controlling R. solani and determine when the plants are most susceptible to infection. Penthiopyrad applied in-furrow and as a soil drench resulted in significantly higher percent survival than the positive control whereas penthiopyrad applied in a band was ineffective at controlling R. solani. Penthiopyrad can also be used as a seed treatment at the 14 g rate to provide effective control of R. solani. Sugarbeet plants, irrespective of their inherent level of resistance, were easily infected by R. solani up to three weeks after planting, even longer for susceptible varieties, highlighting the need for additional protection in the form of seed treatment or fungicide application that may be required to protect vulnerable sugar beet planted in fields with a history of the disease.
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.
Sensitivity of Sclerotinia Sclerotiorum Isolates From North Central US to Azoxystrobin and Boscalid
(North Dakota State University, 2016) Munoz, Claudia Lucia
Azoxystrobin and boscalid are fungicides frequently used to manage diseases caused by Sclerotinia sclerotiorum (Lib.) de Bary in many broadleaf crops in North Central US. . Continued use of these compounds can induce development of fungicide-resistant strains and thus their sensitivity should be monitored periodically. The objective of this research was to generate baseline sensitivity information of S. sclerotiorum to azoxystrobin and boscalid. Salicylhydroxamic acid was toxic to ascospores even at concentrations of 0.1 μg/ml and therefore was not used to estimate sensitivity to azoxystrobin. Azoxystrobin EC50 for ascospores and mycelium were 3.05 and 1.01 μg/ml, respectively. Boscalid EC50 for ascospores and mycelium were 8.38 and 0.11 μg/ml, respectively. S. sclerotiorum isolates collected before 2004 had statistically (P > 0.05) similar sensitivity to azoxystrobin but where more sensitive to boscalid (P =0.05) than isolates collected in 2014. Both fungicides continue to be effective tools to manage S. sclerotiorum.

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