Plant Pathology Doctoral Work

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Studies on the Population Biology of Colletotrichum coccodes Using AFLP and SCAR Markers
(North Dakota State University, 2010) Alananbeh, Kholoud Mohammad
Black dot is a blemish disease of potato caused by the fungus Colletotrichum coccodes (Wallr.) Hughes. with worldwide distribution. It occurs in Africa, Asia, Australia, Europe, North America, South America, and Central America. C. coccodes has no known sexual cycle, but genetic exchange is possible through vegetative conjugation. The determination of VCG among fungal isolates is usually based on the complementation tests with nitrate nonutilizing (nit) mutants which enables researchers to compare strains of pathogens. Eight VCGs have been identified for the Europe/Israel population, seven for the North American population, and six for the Australian population. Variation in aggressiveness, morphological traits, and physiological traits have been detected among the different C. coccodes VCGs. AFLP markers have been used also to study relationships within and among North American VCGs. With this method, five VCGs were separated from C. coccodes isolates, coinciding almost completely with the six VCGs of nit mutants. Additionally, a relationship of specific AFLP bands to corresponding VCGs was reported using AFLP analysis. The objectives of this study were: to develop VCG-specific sequence characterized amplified regions (SCAR) markers, to study the population biology of C. coccodes of North America using the AFLP method, and to study genetic diversity of the global populations of C. coccodes. A total of 88 isolates representing the different C. coccodes VCGs were analyzed via the AFLP method to generate SCAR markers. A total of 47 primers were designed and evaluated in PCR reactions. Only one primer, AGb6F/R, with 156 bp amplification PCR product was found to be specific for NA-VCG6 and NA V CG 7 of C. coccodes. For the second objective, 210 loci were generated and used to cluster the isolates into their NA-VCGs and to test the genetic structure of the North American population of C. coccodes. C. coccodes isolates recovered from potato plants were assigned to fourNA-VCGs: NA-VCGl, NA-VCG2, NA-VCG4/5, and NA-VCG6/7. No isolates tested belonged to NA-VCG3. NA-VCG2 was the dominant group in the population (n=238) and was the most frequently detected NA-VCG among states, fields, farms, and plants. However, in several instances there was more than one NA-VCG recovered from the same plant, field, farm, and state, indicating variability within the C. coccodes population in United States. Genetic differentiation among the nine states was 0.331, and overall gene flow for the 366 isolates was (Nm =1.01). For the genetic diversity of the global C. coccodes population, 855 isolates were studied. Three primer pairs were used and generated 210 loci. Based on this study, the population of C. coccodes exists as one large population with four main groups (NA-VCGl/3; NA-VCG2; NA-VCG4/5; and NA-VCG6/7). NA-VCG5 was the most common VCG globally followed by NA-VCG2. Among the five regions studied, there was relatively low gene diversity (0.222). The overall gene flow (Nm) was 1.25, meaning that one or more individuals are exchanged among the five regions each generation and that the populations will gradually become similar. Most of the variation among the five geographic regions originated from within population differentiation. Among VCGs of C. coccodes, there was high VCG differentiation (GsT=0.463), meaning there is a differentiation among the different C. coccodes VCGs and a population structure exists. AFLP analysis proved to be valuable in differentiating and studying the global population of C. coccodes.
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, Sanjaya
Cochliobolus 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.
Understanding Programmed Cell Death Pathways by Characterizing Barley Disease Lesion Mimic Mutants
(North Dakota State University, 2021) Alsolami, Wail
Programmed cell death (PCD) in plants refers to rapid and localized cell death that occurs as a result of regular biological processes, or in response to infection or injury. Understanding the molecular mechanisms of PCD will fill key knowledge gaps in regard to plant development or resistance mechanisms. Upon pathogen invasion salicylic acid and reactive oxygen intermediate (ROI) signaling responses are triggered that contribute to the PCD outcome. Substantial research efforts have been conducted to identify genes involved in these PCD pathways including the generation and characterization of disease lesion mimic mutants (DLMMs) that spontaneously express PCD and necrotic lesions that resemble necrotrophic diseases in the absence of infection by a pathogen. The constitutive expression of PCD may be a result of the deletion of gene/s that directly or indirectly suppress PCD when it is not needed. Five fast neutron generated DLMMs in the cultivar (cv) Steptoe background (FN360, FN361, FN365, FN370 and FN396) were crossed with the cv Morex and 400 F2 individuals of each population were screened for the mutant phenotype. Allele frequency mapping was used to map the mutation in each population to different chromosomal locations showing that each was caused by a different gene. Thus, each phenotype is the result of different mutated gene(s) in the PCD pathway that needs to be functionally validated.
Molecular Mapping of QTL for Genetic Transformation-Related Trait and CRISPR/Cas9-Mediated Gene Editing in Wheat
(North Dakota State University, 2022) Karmacharya, Anil
Plant transformation is a valuable tool for gene characterization and crop improvement. However, wheat transformation is greatly impacted by genotype dependency for successful regeneration of the transgenic plants. For improving disease resistance in wheat, targeting host genes for disease susceptibility has become one of the most attractive strategies. This study aimed to 1) identify the QTL associated with a genetic transformation-related trait (plant regeneration capability from calli derived from immature embryos); 2) targeting the wheat Tsn1 gene for susceptibility to tan spot disease, using CRISPR/Cas9 vector cassette delivered by Agrobacterium and particle bombardment mediated transformation; 3) targeting wheat TaHRC gene (encoding for reticulum histidine-rich calcium binding protein) at the Fhb1 locus involved in the resistance/susceptibility to Fusarium head blight (FHB) resistance, using haploid induction coupled with the CRISPR/Cas9 genome editing technology. Using phenotypic and genotypic data from a mapping population of 186 recombinant inbred lines derived from the cross between wheat cultivar Bobwhite and wheat line PI 277012, two QTL controlling plant regeneration capability in Bobwhite were detected on chromosome 1A and 6D. To target the Tsn1 gene in wheat, two and 30 T0 transgenic plants were generated from Fielder through the Agrobacterium-mediated transformation and Bobwhite by the biolistic transformation, respectively. However, no Tsn1 mutant was identified by ToxA infiltration on 1176 T1 transgenic plants. Previous studies revealed two allelic forms of the TaHRC gene with TaHRC-S controlling susceptibility and TaHRC-R resistance to FHB, and deletion of TaHRC-S renders wheat FHB resistance. To target TaHRC, pollens of the transgenic hybrid corn variety Hi-II with high expression of Cas9 and gRNA were used to pollinate emasculated spikes of wheat variety Dayn with TaHRC-S and TaHRC-R. After embryo rescue, of the 82 haploid plants screened, 12 plants were identified having mutations at the target sites of TaHRC-S allele and 2 plants at the target site of TaHRC-R allele. Doubled haploid plants are being generated from these gene-edited haploid plants and will be evaluated for FHB resistance. This study may facilitate our understanding of genetics of transformation-related traits and provide a novel approach for improving disease resistance in wheat.
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 Andres
Ascochyta 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.
Genetic Characterization and Engineering of Disease Resistance to Spot Form Net Blotch and Fusarium Head Blight in Barley
(North Dakota State University, 2022) Alhashel, Abdullah
Barley spot form net blotch (SFNB) caused by Pyrenophora teres f. maculata (Ptm) and Fusarium head blight (FHB) caused by Fusarium graminearum are devastating diseases of barley requiring advanced molecular breeding tools for disease management. I used genetic mapping and host-induced gene silencing (HIGS) to identify novel host genes and target existing pathogen genes to improve disease resistance in barley. Barley resistance to SFNB is often isolate specific and the Idaho isolate 13MI8.3 has a unique virulence profile. Two recombinant inbred mapping populations were utilized to characterize and map 13IM8.3 resistance. Quantitative trait loci (QTL) analysis revealed 10 significant resistance/susceptibility loci, including a previously unidentified QTL on chromosome 5H and the Rpt4 locus on chromosome 7H containing a dominant susceptibility gene (Sptm1) for broad-spectrum susceptibility to SFNB. Fine mapping of the Rpt4 locus in a F2:3 population derived from the cross Tradition (S) × PI 67381 (R) anchored the Sptm1 gene to a 400 kb region on chromosome 7H, and a putative cold-responsive protein kinase gene (HORVU.MOREX.r3.7HG0735560) was identified as a strong candidate and potential target for gene editing. As for the FHB management, HIGS was employed to silence the fungal gene FgGCN5 using the barley line Golden Promise. The FgGCN5 gene encodes a histone acetyltransferase which is essential for the F. graminearum growth. Despite demonstrated production of FgGCN5 small-interfering RNAs in the transgenic barley; the disease severity, DON accumulation, and fungal biomass showed no difference from wild-type. This research allows for more in depth analysis for the use of HIGS against FHB. Use of genetic maps, QTL, molecular markers, and transgenic technology in this research will benefit barley breeders, growers, and the industry in developing resistance to these important diseases.
Improving Management and Understanding of Major Diseases of Sugar Beet
(North Dakota State University, 2022) Bhuiyan, Md Ziaur Rahman
Sugar beet is a sugar-yielding crop, that contributes 25% of the global sucrose production. Economic production of sugar beet is hampered by Cercospora leaf spot (CLS) (Cercospora beticola), Rhizoctonia crown and root rot (RCRR) (Rhizoctonia solani), Sclerotinia root rot (SRR) (Sclerotinia sclerotiorum), and Rhizopus root rot (Rhizopus arrhizus) diseases. These diseases can reduce yield by 15 to 40%. On CLS, buildup of fungicide-resistance strains is a major issue due to poor implementation of fungicides and understanding of disease development at early stages. The identification of germplasm resistant to RCRR disease is hindered by the lack of effective inoculation methods. Identification of SRR and RRR pathogens is crucial for their proper management. The objectives of this research were 1. to evaluate the role of adjuvants in improving the efficacy of fungicides on CLS, 2. to characterize the infection process during early stages of infection by C. beticola, 3. to identify an effective inoculation method for RCRR, and 4. to identify and characterize the causal organisms of SSR and RRR. The value of adjuvants was evaluated in greenhouse and field conditions. Application of fungicides with or without adjuvants before disease onset reduced disease severity of CLS in greenhouse condition. In field conditions, additions of adjuvants did not improve the effectiveness of fungicides and few of them negatively impacted root yield. The initial stage of infection on CLS susceptible and resistant sugar beet variety were compared using confocal microscopy. C. beticola biomass accumulation, percent leaf cell death and disease severity were all significantly greater in the susceptible variety compared to the resistant variety (P<0.05). R. solani inoculated on the crown and roots were compared in a replicated trial in greenhouse conditions. The root inoculation method provided a more consistent disease rating of the sugar beet variety in the greenhouse for screening of RCRR cultivars in a resistance breeding program. Based on morphological and molecular techniques, causal organisms of SRR and RRR were characterized and was found to be pathogenic to sugar beet varieties tested in-vitro and in the greenhouse conditions.
Managing Economically Important Diseases of Sunflower and Oilseed Rape in North Dakota, California, and Schleswig-Holstein
(North Dakota State University, 2021) Berghuis, Brandt Gregory
Rust, 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.
Fitness and Management of Site-specific Fungicide Resistant Cercospora beticola Isolates from Sugar Beet
(North Dakota State University, 2020) Liu, Yangxi
Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is one of the most destructive foliar diseases on sugar beet. It severely affects productivity and profitability of the sugar beet industry. CLS is managed by using resistant cultivars, rotating with non-host crops, and applying effective fungicides in a timely manner. In North Dakota and Minnesota, site-specific fungicides, such as quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs), had been widely and extensively applied to control C. beticola which has developed resistances to these fungicides. The mycelial growth, spore production, spore germination, and aggressiveness of QoI and/or DMI resistant isolates were compared to sensitive isolates in a laboratory and greenhouse study. Results indicated that the QoI and/or DMI resistant isolates had a relatively slower disease development on sugar beet leaves due to their fitness penalty in sporulation and mycelial growth but still caused high disease severities as sensitive isolates. Fungicides were evaluated to manage the QoI and/or DMI resistant C. beticola isolates in a sensitivity study and a greenhouse and field study. Copper-based multisite activity fungicides were evaluated for controlling fungicide resistant C. beticola. The mean EC50 values for nine copper-based chemicals ranged from 1 to 10 ug/ml using a spore germination assay. In a greenhouse study, Fertileader (a copper-based fertilizer) caused leaf injury and was not evaluated, but the other tested chemicals provided significantly better control of CLS compared to the control check with Badge X2, Champion, Cuprofix, COCS and Ridomil having significantly small AUDPC. Newer site specific fungicides with different modes of action that have never been used for CLS management in North Dakota and Minnesota were also tested. The mean EC50 values were 4.9, 33.1, 99.4, and 481.6 ug/ml using mycelial growth assay and 5.7, 4.1, 9.2, and 4.2 ug/ml using spore germination assay for cyprodinil, fluazinam, pydiflumetofen, and Chlorothalonil, respectively. In a two-year field study, all the fungicides resulted in significantly better disease control, significantly higher beet tonnage, and recovered sucrose compared to the nontreated check. However, none of tested fungicide treatments performed better than the industry’s standard fungicide, triphenyltin hydroxide.
Plant-Parasitic Nematodes on Corn (Zea mays L.) and Soybean (Glycine max L.) in North Dakota
(North Dakota State University, 2020) Chowdhury, Intiaz Amin
Four studies were conducted to investigate plant-parasitic nematodes (PPN) of corn and soybean in ND. The first study investigated the incidence and abundance of vermiform PPN in ND corn fields in 2015 and 2016. Samples were collected from 300 corn fields across 20 counties. Seventy-two percent of the fields were positive for PPN. The major genera of PPN identified were Helicotylenchus, Tylenchorhynchus, Paratylenchus, Pratylenchus, Heterodera, Xiphinema, Hoplolaimus, and Paratrichodorus. The second study characterized SCN (SCN; Heterodera glycines) virulence phenotypes in ND. A total of 419 soybean fields across 22 counties were sampled during 2015, 2016, and 2017. Among these samples, 73 SCN field populations were successfully virulence phenotyped using the HG type tests. The HG types that were detected and confirmed in ND were HG type 0, 7, 2.5.7, 5.7, 1.2.5.7, and 2.7. The third study developed a new molecular method for detecting and identifying a new Pratylenchus sp. discovered in a soybean field in ND. A species-specific primer set, that can be used in both conventional and real-time polymerase chain reaction (PCR) assays, was designed from the internal transcribed spacer (ITS) region of ribosomal DNA. Laboratory experiments confirmed that the primers only amplified DNA of the target nematode species but not the non-target species used in the specificity tests. Practically, DNA from as little as a single nematode could be used to specifically identify the new Pratylenchus sp. using the molecular diagnostic methods developed in this study. The fourth study was conducted to ascertain resistance levels of 20 soybean cultivars to the new Pratylenchus sp. Combined results of four trials indicated that seven of the cultivars were moderately resistant, ten were moderately susceptible, four were susceptible, and none of the cultivars tested were resistant. Analysis of the habitat preference of the new Pratylenchus sp., revealed that above or close to 50% of the nematode population resided in roots at nine weeks after planting for a majority of the cultivars evaluated. Results from these studies will be helpful in improving nematode detection and developing management strategies to control plant-parasitic nematodes in ND corn and soybean fields.
Cloning and Characterization of rcs5, Spot Blotch Resistance Gene and Pathogen Induced Nec3 Gene Involved in Programmed Cell Death in Barley
(North Dakota State University, 2019) Ameen, Gazala
Upon sensing pathogens, plants initiating defense responses typically resulting in programmed cell death (PCD). PCD effectively subdues biotrophic pathogens but is hijacked by necrotrophs that colonize the resulting dead tissues. We showed that barley wall associated kinase (WAK) genes, underlying the rcs5 QTL, are manipulated by the necrotrophic fungal pathogen Bipolaris sorokiniana to cause spot blotch disease. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four WAK genes, designated HvWak2, Sbs1, Sbs2, and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in the susceptible barley cultivars Steptoe and Harrington resulted in resistance, suggesting a dominant susceptibility function. Sbs1&2 expression is undetectable in barley prior to pathogen challenge; however, specific upregulation of Sbs1&2 occurred in the susceptible lines post inoculation. Promotor sequence polymorphisms were identified in the allele analysis of Sbs1&2 from eight resistant and two susceptible barley lines, which supported the possible role of promotor regulation by virulent isolates contributing to susceptibility. Apoplastic wash fluids from virulent isolates induced Sbs1expression, suggesting regulation by an apoplastic-secreted effector. Thus, the Sbs1&2 genes are the first susceptibility/resistance genes that confer resistance against spot blotch, a disease that threatens barley and wheat production worldwide. The nec3 mutants of barley are hyper-susceptible to many necrotrophs and show distinctive cream to orange necrotic lesions that are induced by infection, representing aberrant PCD. The γ- irradiation induced necrotic mutant, nec3-γ1 (Bowman) was confirmed as a nec3 mutant by allelism tests. The F2 progeny of a cross of nec3 x Quest inoculated with B. sorokiniana segregated as a single recessive gene fitting a 3 WT: 1 mutant ratio. The homozygous F2 mutant progeny were genotyped with four SSR and 25 SNP markers at nec3 locus on chromosome 6H, a physical region spanning ~ 16.96 Mb containing 91 high and low confidence annotated genes. Exome capture sequencing of nec3 mutants failed to identify a candidate gene, however, RNAseq analysis identified two candidates in the nec3 region with >three-fold downregulation. We hypothesize that the underlying aberrant PCD mechanism in the nec3 barley mutant facilitates extreme susceptibility to multiple adapted fungal pathogens of barley.
Population Structure of Uromyces appendiculatus in North Dakota
(North Dakota State University, 2019) Monclova-Santana, Cecilia
North Dakota is the lead producing state of common bean in the USA, accounting for 43% of national production. Bean rust is caused by the autoecious, macrocyclic fungus Uromyces appendiculatus (Pers.:Pers.). All pathogen spores stages have been observed in North Dakota, but it is unclear how frequently sexual reproduction occurs. Traditionally, genetic resistance is the preferred management method. Sixty-seven percent of the 119 U. appendiculatus single pustule isolates collected in 2015 and 2016 were classified phenotypically as race 20-3. Virulence phenotypes of race 20-3 isolates ranged from hypersensitive to small pustules (0.2-0.3mm) on Early Gallatin, PC-50, Mexico 235, and Mexico 325 rust differential lines. This variation suggests more pathogen diversity is present than is discernable via traditional race classifications. The remaining 33% of U. appendiculatus isolates belonged to 18 additional races. Genome Wide Association Studies were conducted with the advanced breeding lines from the NDSU bean breeding program, the Middle American diversity panel, and a subset of Andean diversity panel reaction to races 20-3, 29-3 and 27-7. Significant SNP markers on chromosomes Pv01, Pv04, Pv06, Pv08, Pv10, and Pv11 were identified using genome wide association mapping. RAD-GBS was performed on 84 single pustule U. appendiculatus isolates using the Ion-Torrent S5 sequencing platform. A de novo assembly was performed on a single isolate of race 20-3 to generate reference sequence tags for variant calling. The relatedness measure using an identity by state (IBS) matrix suggested the presence of diversity within and among the isolates belonging to the same race, providing further evidence that the U. appendiculatus population in North Dakota is undergoing sexual reproduction and is more diverse than virulence phenotypes suggest. Results from this research increase our understanding of population dynamics and diversity in phenotype and genotype of U. appendiculatus and will assist common bean breeding for rust resistance.
Characterization of Pectobacterium carotovorum subsp. brasiliense as a Causal Agent of Sugarbeet Soft Rot
(North Dakota State University, 2018) Metzger, Michael Stewart
A soft rot decay of sugarbeet was observed in commercial fields in North Dakota and Minnesota from 2012 to 2016. Symptoms reported are similar to those for bacterial vascular necrosis and rot caused by Pectobacterium betavasculorum including soft decay of internal root tissues, reddening of affected tissue after cutting, blackening of petiole vascular bundles, half-leaf yellowing, and root frothing. The disease can cause serious yield losses in the field, and additional economic losses in storage and during processing due to accumulation of invert sugars that reduce sugarbeet quality. Sap from the margin of diseased root tissue was streaked on pectate agar medium and incubated. Single pectolytic colonies were selected and transferred to nutrient broth for bacterial identification and completion of Koch’s postulates. Pathogenicity of isolates was assessed by inoculating greenhouse-grown sugarbeet roots. Symptoms characteristic of the disease were observed at 30 days after inoculation included all of the aforementioned, previously stated symptomology. Bacterial DNA was extracted from 46 pathogenic isolates and analyzed by restriction-associated DNA genotype-by-sequencing (RAD-GBS). Ion-torrent sequencing reads (n = 8.54 million) were assembled de novo producing ∼6,000 sequence tags representing approximately 21% of each bacterial genome analyzed. Partial sequences of five of the seven genes previously used in Pectobacterium subspecies phylogenetic analysis were represented in the RAD-GBS isolate sequences. Gene sequences were aligned using Workbench 8.0.3 software to the corresponding reference gene sequences of P. carotovorum subsp. carotovorum, P. atrosepticum, P. betavasculorum, P. carotovorum subsp. odoriferum, and P. wasabiae. The alignments showed 99.76% nucleotide sequence identity on average across all five genes to the P. carotovorum subsp. brasiliense reference sequences. The alignments to P. cartovoroum subsp. carotovorum, P. atrosepticum, P. betavasculorum, P. carotovorum subsp. odoriferum and P. wasabiae reference sequences showed 96, 95.4, 94.3, 97 and 94.4% identity, respectively, on average across the five genes. The nearly 100% identity across all five genes previously utilized in multi-locus sequencing and divergence from the closely related subspecies strongly suggests that the isolates are P. carotovorum subsp. brasiliense. To our knowledge, this is the first report of this pathogen causing field decay of sugarbeet in North America.
Molecular Genetic Characterization of Ptr ToxC-Tsc1 Interaction and Comparative Genomics of Pyrenophora tritici-repentis
(North Dakota State University, 2018) Kariyawasam, Gayan Kanishka
Tan spot of wheat, caused by Pyrenophora tritici-repentis, is an economically important disease worldwide. The disease system is known to involve three pairs of interactions between fungal-produced necrotrophic effectors (NEs) and the wheat sensitivity genes, namely Ptr ToxA-Tsn1, Ptr ToxB-Tsc2 and Ptr ToxC-Tsc1, all of which result in susceptibility. Many lines of evidence also suggested the involvement of additional fungal virulence and host resistance factors. Due to the non-proteinaceous nature, Ptr ToxC, has not been purified and the fungal gene (s) controlling Ptr ToxC production is unknown. The objective for the first part of research is to map the fungal gene (s) controlling Ptr ToxC production. Therefore, A bi-parental fungal population segregating for Ptr ToxC production was first developed from genetically modified heterothallic strains of AR CrossB10 (Ptr ToxC producer) and 86-124 (Ptr ToxC non-producer), and then was genotyped and phenotyped. Using the data, the gene (s) was mapped to the distal end of chromosome 2 in the reference genome of Pt-1c-BFP. The objective for the second part of my research is to develop genomic and genetic resources for the fungal pathogen. A high quality of genome sequence for AR CrossB10 and the first P. tritici-repentis genetic linkage map was generated. The AR CrossB10 genome and genetic linkage map is highly comparable to newly published reference genome except some noticeable chromosomal structural variations (SVs). Comparison of the genome sequences between parental isolates and twenty progeny isolates also revealed some SVs including deletion, insertion and inversion were detected that likely occurred during the fungal sexual reproduction. The objective for the third of my research is to characterize genetic resistance in Nebraskan winter wheat cultivar ‘Wesley’ using QTL mapping in a recombinant inbred line population. The results showed that resistance in Wesley is largely due to the lack of susceptibility genes Tsc1 and Tsn1. My Ph.D. research provides a further understanding of the genetics of host-pathogen interaction in wheat tan spot and contributes knowledge and tools for breeding tan spot resistant cultivars.
Prevalence, Diversity, and Management of Goss’s Leaf Blight (Clavibacter nebraskensis) in North Dakota
(North Dakota State University, 2018) Bauske, Elizabeth Crane
Goss’s wilt and leaf blight (GWLB) (Clavibacter nebraskensis) is a yield-limiting disease of corn (Zea mays L.). Research conducted in other corn growing states have indicated yield losses as severe as 60% on susceptible hybrids. In 2011, the disease was first reported in southcentral North Dakota (ND). Concurrently, corn production was increasing in ND due to favorable grain prices. With increased production, there was concern about disease prevalence and the impact of GWLB on corn yields in the state. In order to determine which corn diseases are present in ND, and to gain a better understanding of the C. nebraskensis population in ND and its impact on corn yield, three studies were conducted. Prior to 2014, no formal corn foliar disease survey had been documented in ND, thus the first objective was to document the prevalence of foliar diseases of corn in ND. Results indicate that four diseases are common in ND; common rust, common smut, northern corn leaf blight, and GWLB. One corn disease is of economic concern in ND; GWLB. The objectives of the second study were to evaluate phenotypic and genotypic differences among isolates of the ND C. nebraskensis population. Results indicate significant differences in the amount of disease caused and the rate of disease progression by the isolates. Genetic differences among isolates also exist, but both phenotypic and genotypic differences appear to be random with no association to isolate origin. The objective of the third study was to evaluate yield loss due to GWLB based on infection timing and hybrid resistance. Results indicated that infection by C. nebraskensis at vegetative growth stages caused yield loss in excess of 40% on the susceptible hybrid, while infection at silking resulted in losses around 2%. Regardless of infection timing, yield loss on the resistant hybrid did not exceed 11%. The impacts of these studies will be to direct research efforts to economically important diseases (GWLB) and to strengthen GWLB management recommendations for ND corn growers.
Characterization of Pea Seed-Borne Mosaic Virus, Efficacy of Foliar Applications for Common Bacterial Blight Management in Dry Beans and Impact of Common Bacterial Blight on Prostrate and Upright Beans
(North Dakota State University, 2018) Beck, Amanda Louise
PSbMV in field pea has resulted in substantial yield and seed quality losses world-wide and has recently been reported in North Dakota. Traditional management of this virus includes preventative measures such as removal of alternate hosts, planting virus free seed and the use of cultivar resistance. The objectives of this research were to screen field pea cultivars commonly grown in North Dakota for a response to North Dakota PSbMV isolate ND14-1 and ascertain the effect on plant symptoms, seed size and weight, the number of pods and seeds and seed transmission. Two cultivars were identified as highly resistant and one as partially resistant. The results from this study were combined into a risk assessment. Cultivars were categorized based on inherent risk of PSbMV infection, transmission and reduction in total seed weight. Common bacterial blight (CBB) in dry bean is capable of causing substantial yield losses and has been reported in up to 75% of fields in the Northarvest region in the last five years. Current management practices include the use of planting clean seed, crop rotation, partial host resistance and the application of cupric bactericides, although inconsistent for the management of CBB. Growers in this Northarvest region have recently shifted to growing upright (Type II) dry beans rather than prostrate (Type III) dry beans for ease of harvest. The objectives of this research were to evaluate copper products, surface sanitizers and growth promoters for the management of CBB and to discern if Type II dry beans experienced greater yield losses under CBB disease pressure than Type III dry beans. Numerous products were identified that significantly reduced CBB disease severity and spread; however, no significant yield benefit was observed. Across a wide range of disease severity (0-46%), no significant yield losses were observed between high and low disease severity any of the cultivars screened.
Parasitic Fitness of SDHI-Sensitive and -Resistant Isolates of Alternaria Solani
(North Dakota State University, 2017) Bauske, Mitchell James
Early blight of potato (Solanum tuberosum L.) is caused by Alternaria solani Sorauer. The single-site mode of action chemistries of the succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs) have been widely used for early blight control but resistance has developed rapidly to a number of fungicide chemistries. QoI resistance in A. solani has been attributed to the F129L mutation, or the substitution of phenylalanine with leucine at position 129. Resistance to SDHI fungicides in A. solani is conferred by five known point mutations on three AsSdh genes. Over 1,300 A. solani isolates collected from 11 states in 2013 through 2015 were characterized for the presence of mutations associated with QoI and SDHI resistance through real-time, SDH multiplex, and mismatch amplification analysis (MAMA) polymerase chain reaction (PCR). Approximately 95% of isolates collected from 2013 to 2015 possessed the F129L mutation. Additionally, 95% of the A. solani population was determined to be SDHI-resistant, with the most prevalent mutation being on the AsSdhC gene. All A. solani isolates collected that were characterized as possessing the D123E mutation, or the substitution of aspartic acid for glutamic acid at position 123 in the AsSdhD gene, were evaluated for boscalid and fluopyram sensitivity in vitro. Furthermore, 15 isolates characterized as being SDHI-sensitive or -resistant, including five D123E-mutant isolates, were evaluated in vivo for percentage disease control to boscalid and fluopyram. Sensitivity of D123E-mutant isolates to fluopyram ranged from 0.8 to 3 ug/ml, and were found to be sensitive or only slightly higher than those of baseline isolates, ranging from 0.1 to 0.6 ug/ml. Disease control of all five D123E isolates evaluated in vivo was reduced significantly with the application of fluopyram compared to SDHI-sensitive isolates. Fitness was compared among 120 SDHI-sensitive and -resistant A. solani isolates using the parameters of spore germination and mycelial growth in vitro and aggressiveness in vivo. Spore germination and mycelial growth between SDHI-sensitive and -resistant isolates was not significantly different. However, D123E-mutant isolates were significantly more aggressive in in vivo assays compared to other SDHI-resistant and SDHI-sensitive isolates. These results illustrate the importance of implementing fungicide resistance management strategies.
Histology of Spot Blotch Infection in Barley, QTL Mapping of Resistance to Fusarium Head Blight, and Characterization of Root Rot Diseases in Wheat
(North Dakota State University, 2017) Shrestha, Subidhya
Three independent studies were conducted for spot blotch (Bipolaris sorokiniana), Fusarium head blight (FHB) (Fusarium graminearum), and root rot diseases (Fusarium species and B. sorokiniana). Histopathology of compatible and incompatible interactions between different pathotypes of B. sorokiniana and different genotypes of barley was examined with red fluorescent protein-tagged fungal isolates. The fungus penetrated the host cell wall and developed multicellular globular infection hyphae (IH) in the lumen of epidermal cells, but infected epidermal cells appeared to be alive till 16 hours post-inoculation (HPI). In the susceptible plants, the tip of IH was found to grow ahead of the dead tissue and invade the surrounding live mesophyll cells, whereas growth of IH in the resistant plants was restricted to the dead tissue after 20 HPI. The amount of H2O2 accumulation and the fungal biomass were also significantly higher in the susceptible hosts than in the resistant hosts. To map resistance to FHB, two populations consisting 130 doubled haploid lines from the cross Grandin × PI277012 and 237 recombinant inbred lines from the cross Bobwhite × ND2710 were phenotyped and genotyped. QTL for Type I resistance were identified on chromosomes 1A, 2B, 4B, 5B and 6B in the GP population. These QTL explained 10.7-19 % of the total phenotypic variation. With the BN population, QTL for Type I resistance were identified on chromosomes 2A, 5A and 6B, explaining 6.2-13.7% of the total phenotypic variation. To assess the prevalence, incidence and severity of wheat crown rot (CR) and common root rot (CRR) in ND, wheat root samples were collected from fields across the state in 2012, 2013, and 2014. Fungal isolations indicated that B. sorokiniana was most frequently recovered in all sampled years. Seedling tests on ten spring wheat lines showed that Glenn was the least susceptible while Steele-ND was the most susceptible to one F. culmorum isolate and one B. sorokiniana isolate tested. Evaluation of 20 spring wheat genotypes for reaction to CRR at the adult plant stage showed that Freyr and RB07 were more resistant while Len and Briggs were more susceptible to CRR compared to other wheat genotypes evaluated.
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.
From Bioinformatics to Identifying R-genes, Enhancers, Signaling Pathways and Pathogen Elicitors in the Barley-Stem Rust Pathosystem
(North Dakota State University, 2018) Sharma Poudel, Roshan
Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt) is a threat to wheat and barley. Rpg1 is the only deployed stem rust resistance gene in barley that provides resistance to the predominant races in North America, except to the local race QCCJB and the widely virulent race TTKSK (aka Ug99) and its lineages. The barley rpg4-mediated resistance locus (RMRL) confers resistance against the majority of Pgt races, including TTKSK and its lineage. With the goal of identifying Pgt effectors/suppressors that elicit/suppress RMRL resistance responses twenty-four Pgt isolates showing differential infection types were genotyped utilizing in planta RNAseq. The RNAseq experiment identified 114K SNPs within genes that resulted in predicted nonsynonymous amino acid changes and were utilized to identify genes associated with virulence/avirulence. Twenty-two genes were identified that were associated with RMRL virulence that represent candidate suppressors of resistance. Host differential gene expression analysis comparing virulent vs avirulent isolates identified virulent isolate specific down regulation of stress response genes, genes involved in chloroplastic ROS, and non-host resistance responses, suggesting that Pgt isolates may contain a conserved virulence factor that elicits RMRL responses and virulent isolates contain suppressors of virulence rather than dominant avirulence genes. The second chapter focused on the observations that introgression of RMRL into the elite malting variety Pinnacle (Rpg1+) resulted in susceptibility to Pgt race QCCJB (RMRL) and HKHJC (Rpg1) suggesting the presence of a gene required for rpg4/Rpg5 and Rpg1 resistance. Utilizing a Pinnacle RMRL-NIL X Q21861 derived RIL population and PCR-GBS genotyping, the required for rpg4- and Rpg1-mediated resistance 1, Rrr1 gene was mapped ~5cM proximal to RMRL on barley chromosome 5H. A second gene required for Rpg1- mediated resistance 2, Rrr2, complimentary to Rrr1 was mapped to the telomeric region of the short arm of barley chromosome 7H. A novel Pgt race TTKSK resistance gene designated RpgHv645 was identified in an unimproved swiss landrace Hv645. Utilizing a RIL population developed from a Hv645 X Harrington cross and Pgt race TTKSK phenotyping data generated at the adult plant stage in Njoro, Kenya, RpgHv645 was mapped distal of RMRL and delimited to an ~11cM region.

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