Plant Pathology Doctoral Work

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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.
Biology and Development of Two Wilt Fungi of Potato: Verticilliam Dahliae and Colletotrichum Coccodes
(North Dakota State University, 2012) Pasche, Julie Sherman
The demand for high quality tubers for the frozen processing industry has exerted increased pressure on producers to control diseases that can compromise tuber quality, including, C. coccodes and V. dahliae. Infection of potato plants by C. coccodes, the causal agent of black dot, can result in foliar necrosis, plant wilting, yield loss, tuber vascular discoloration and skin blemishes. Black dot of potato can originate from foliar-, seed- of soil-borne infections and, all plant tissues can be affected. The development of colonization in above- and below-ground plant tissues, from each inoculation/infestation source, was evaluated at two sites across two growing seasons. Colonization of potato tissue was detected at the first sampling date, even prior to plant emergence. While disease resulted from natural inoculum only, as inoculation/infestation sources increased, so did host colonization. Disease development also was greater in treatments with more than one site of inoculation/infestation. Overall, it was determined that stem tissue was colonized at greater frequency than root or stolon tissue. Infection of potato plants with V. dahliae, the causal agent of Verticillium wilt, occurs mainly through root contact with infested soil and can result in premature senescence as well as losses from decreased tuber yield and quality. Control of this pathogen is difficult and expensive and, therefore, efforts recently have increased towards the development of resistance. Resistance to V. dahliae in potato is thought to be multigenic, and therefore, quantification of the host:pathogen interaction is required to accurately define the level of resistance in a particular cultivar. Unfortunately, current methods used for quantification of V. dahliae in potato stems are time and labor intensive. A real-time duplex quantitative PCR assay was developed to simplify pathogen quantification to help breeding programs and researchers identify resistance in germplasm and cultivars. QPCR assays were validated using plant material from greenhouse and field trials, demonstrating specificity for V. dahliae, as well as sensitivity and accuracy when compared to traditional plating assays. Results from greenhouse and field evaluations also indicated that resistance is present among the eight russet-skinned cultivars evaluated in these studies.
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 Fusarium Head Blight Resistance in Wheat
(North Dakota State University, 2017) Zhao, Mingxia
Fusarium head blight (FHB) is a devastating fungal disease threatening wheat production worldwide. Utilization of resistant wheat cultivars is generally considered as the most effective, economic and environmental friendly approach for management of the disease. This research aimed to identify and map quantitative trait loci (QTL) for FHB resistance in two spring wheat lines (ND2710 and PI 277012) and two durum wheat lines (10Ae564 and Joppa). Using a mapping population consisting of 233 recombinant inbred lines (RILs) from a cross between ND2710 (with FHB resistance derived from Sumai 3) and the spring wheat cultivar ‘Bobwhite’ (susceptible to FHB), four QTL (Qfhb.ndwp-3B, Qfhb.ndwp-6B, Qfhb.ndwp-2A, and Qfhb.ndwp-6A) were mapped on chromosomes 3B, 6B, 2A, and 6A, respectively, in ND2710. Qfhb.ndwp-3B and Qfhb.ndwp-6B were mapped to the same genomic regions as Fhb1 and Fhb2, confirming that they originated from Sumai 3. Two FHB resistance QTL, Qfhb.rwg-5A.1 and Qfhb.rwg-5A.2, were previously identified on chromosome 5AS and 5AL, respectively, in PI 277012. In this study, Qfhb.rwg-5A.2 was delimited in a 1.09-Mbp genomic region, and DNA markers tightly linked to Qfhb.rwg-5A.2 were developed using 947 RILs from the cross between PI 277012 and Grandin (susceptible to FHB). Using a mapping population consisting of 205 RILs from the cross between the durum cultivar Joppa and the durum wheat line 10Ae564 (with FHB resistance derived from PI 277012), one QTL (Qfhb.ndwp-2A) on chromosome 2A from Joppa and two QTL (Qfhb.ndwp-5A and Qfhb.ndwp-7A) each on 5A and 7A from 10Ae564 were detected. Qfhb.ndwp-5A was mapped to the same genomic region as Qfhb.rwg-5A.2 on 5AL, and thus confirming that this QTL was derived from PI 277012. The DNA markers closely linked to the FHB resistance QTL identified in two spring wheat lines and two durum wheat lines will be useful for marker-assisted selection of FHB resistance in wheat breeding programs.
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.
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.
Characterizing Pyrenophora Teres F. Maculata in the Northern United States and Impact of Spot Form Net Blotch on Yield of Barley
(North Dakota State University, 2015) Kinzer, Kasia Marie
Pyrenophora teres f. maculata causes spot form net blotch (SFNB) on barley and was recently documented in North Dakota. The impact of SFNB on barley, the genetic diversity of the pathogen, and virulence structure are unknown for the state. Yield and quality loss in North Dakota due to SFNB was investigated over eleven year-sites, and simple linear regression of percent yield loss on adjusted percent disease using year-site means of treatments predicted a 0.77% increase in yield loss for every 1% increase in disease. When virulence of isolates of P. teres f. maculata collected from geographically diverse regions in the northern United States was evaluated on differential barley genotypes, few isolates were identical in terms of virulence patterns, and the virulence profile of a population from Idaho differed from other populations. To understand population structure and genetic diversity, SNPs of 140 isolates were generated using genotyping-by-sequencing for analysis of population genetics and structure. Evidence for sexual recombination in each population includes the ratio of mating-type idiomorphs that do not significantly differ from a 1:1 ratio; low index of association values for most populations; and high variation within and low variation among populations. Association mapping detected forty-five significant marker-trait associations of SNPs associated with virulence or avirulence across 19 P. teres f. maculata scaffolds using 82 isolates of P. teres f. maculata from diverse areas in the northern United States. The most significant marker, 01700_198, was found on P. teres f. maculata-scaffold 8 when the population was challenged with four different barley lines. This research demonstrates that SFNB causes significant yield loss; that high diversity exists in the pathogen, with respect to virulence and population genetics; and that association mapping can be used to identify virulence/avirulence marker-trait associations to fill gaps in our understanding of host-parasite genetic interactions in this pathosystem.
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.
Comparative Population Genetics of Fusarium Graminearum and Novel Sources of Resistance to Fusarium Head Blight in Spring Wheat
(North Dakota State University, 2013) Puri, Krishna Datta
Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) in cereal crops. The fungus produces several types of trichothecenes [Deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV)]. Characterization of 123 isolates collected during 1980 to 2000 (old collection), and 43 isolates collected in 2008 (new collection) from North Dakota revealed that 15ADON producing isolates were predominant (98%) in the old collection while the 3ADON producing isolates accounted for 43% in the new collection. Further, evaluation showed that the 3ADON isolates caused a higher disease severity and accumulated more DON in spring wheat than the 15ADON isolates. The 3ADON also exhibited higher DON in rice culture, and produced more spores on agar media. Population genetic analyses revealed a significant genetic differentiation between the two populations. To elucidate the transcriptomic differences between the two populations in vitro and in planta, RNA-sequencing was used. The in vitro gene expression comparison identified 479 up- and 801 down- regulated genes in the 3ADON population compared to 15ADON population. The in planta pair-wise comparisons between the two populations revealed 185, 89, and 62 unique genes to 3ADON at 48, 96 and 144 hours after inoculation (HAI), respectively. In a different study, population genetic analysis was conducted on 160 isolates collected in 2008 and 2009 from a FHB disease nursery located in China. All isolates analyzed were F. asiaticum except one (F. avenaceum). Of the 159 isolates, 79% were NIV producing, 18% were 15ADON and 3% were 3ADON. The two populations grouped based on year of collection exhibited low genetic differentiation (Fst = 0.032). To identify new sources of FHB resistance, 71 wheat accessions of diverse origins were re-evaluated for FHB severity and haplotyped using seventeen DNA markers associated with known resistance quantitative trait loci (QTL). Twenty two accessions had a haplotype different from all known sources used, suggesting that they may carry novel loci for FHB resistance. In conclusion, the information obtained in this study could have an impact on development of effective disease management measures and on improvement of FHB resistance in wheat.
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.
Developing Management Tools for Sunflower Rust (Puccinia Helianthi)
(North Dakota State University, 2013) Friskop, Andrew John
Sunflower rust, caused by Puccinia helianthi (Schwein), is an economically important disease in North Dakota. Since 2008, sunflower rust incidence and severity have increased. Therefore, a four year study was initiated to examine the management strategies for the disease. A total of 19 fungicide efficacy and timing trials were conducted from 2008-2011. Results indicated that DMI and QoI chemistries can be used effectively to manage the disease. Timing trials indicated that disease control was highest when fungicide applications were made at R5.0-R5.8. Results indicate a fungicide application is warranted when rust severities at approximately 1% are found on the upper-four leaves at R5. To observe phenotype variability in the pathogen, a P. helianthi survey was completed in 2011 and 2012. Single-pustule isolates were obtained and virulence phenotypes were evaluated on a set of nine differentials. Race characterization was assigned based on virulence phenotypes. In 2011, the most commonly detected races were 300 and 304, while the most virulent was 776. In 2012, races 304 and 324 were the most commonly detected and the most virulent was 777. To identify new sources of rust resistance, the core-set of Helianthus annuus germplasm was obtained from the USDA-North Central Regional Plant Introduction Station. The accession lines were screened both in the greenhouse and the field. The accessions were screened in the greenhouse individually to races 300, 304, 336, 337, and 777. Lines were screened in the field to a mixture of P. helianthi isolates coding to 300, 304, 336, and 337. The majority of lines were susceptible in both the field and greenhouse. At both field locations, PI 431538, PI 432512, and PI 650362 had year-end severities under 1%. Similarly, PI lines 432512 and 650362 had resistant infection types across all five races. The results of these studies provide information and tools that are being used currently to manage rust and will contribute to management in the future.
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.
Dissecting the Mystery Behind the Rpg5 Mediated Puccinia graminis Resistance in Barley Using Genetics, Molecular and Bioinformatics Approaches
(North Dakota State University, 2017) Solanki, Shyam
Barley rpg4/Rpg5 locus harbors three tightly linked genes, two NLRs Rpg5 and HvRga1, and HvAdf3, togather providing resistance against Puccinia graminis f. sp. tritici, causal agent of wheat stem rust including race TTKSK, considered a threat to global food security. The integrated decoy hypothesis proposes role for head-to-head genome architecture present in the dual plant NLR immunity receptors, where one NLR partner contains an integrated sensory domain (ISD). The ISDs represent mimics of virulence effector targets translocated to the immunity receptors and act as baits to recognize virulent effectors to initiate defense responses. Alleles of Rpg5 contain two diverse C-terminal, the Rpg5 resistance allele has a serine threonine protein kinase (STPK) ISD whereas the major class of rpg5 susceptible alleles contain a protein phosphatase 2C (PP2C) ISD. Genetic and functional analysis shows that in the heterozygous state rpg5-PP2C allele acts as a dominant susceptibility factor suppressing Rpg5-STPK mediated Pgt resistance. This is the first integrated decoy NLR gene identified that contains two distinct ISDs. Liabrary scale Y2H screeing using Rpg5-STPK as bait identified HvVoz1. HvVoz1 was also interacts with the HvRga1, Rpg5-LRR, and rpg5-PP2C domains suggesting that it may act as a scaffold to hold the R-protein complex together until effector manipulation. We identified Rpg5-STPK ISD progenitor HvGak1, ortholog of the Arabidopsis guard cell AT5G15080 and AtAPK1b, shown to function in stomatal aperture opening in response to light. We hypothesize that several forma specialis of P. graminis contain virulence effector/s, that manipulate HvGak1, mimicking the presence of light to open the stomates, allowing the pathogen to gain entry in to the plant during dark period that P. graminis spores evolved to germinate. We identified dark period pathogen penteration through stomata by deveoping a novel staining method and using confocal microscopy. To further characterize the Rpg5 immunity pathway fast-neutron irradiation was utilized to generate rpr9 mutant, compromised for rpg4/Rpg5-mediated resistance. Utilizing genetic mapping and exom capture we identified candidate genes for rpr9 mutants. Based on our understanding of this resistance mechanism it would be a good candidate system for generating synthetic resistances utilizing different ISD baits fused to the Rpg5 NLR.
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.
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.
Genetic and Molecular Characterization of Genes Involved in Barley-Cochliobolus Sativus Interaction
(North Dakota State University, 2015) Wang, Rui
Spot blotch caused by ascomycete fungus Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dastur. [anamorph: Bipolaris sorokiniana (Sacc.) Shoem.] is one of the most common and economically important diseases on barley. To better understand the molecular interaction between the different pathotypes of this pathogen and barley differential lines, fungal genes involved in virulence and barley genes for spot blotch resistance were characterized in this study. Previous studies have revealed that the virulence factor in the pathotype 2 isolate ND90Pr making cv. Bowman susceptible is a secondary metabolite peptide synthesized by two non-ribosomal peptide synthesdases (NRPSs). However, the global regulation of biosynthesis of this secondary metabolite is not well understood in this fungus. Recently, the velvet-complex proteins containing LaeA and velvet proteins (VeA, VelB, VelC and VosA) have been shown to be involved in global regulation of secondary metabolism and fungal development in many fungal pathogens. To characterize the functions of the orthologous of velvet-complex proteins in C. sativus, single and double gene knockout mutants were generated. The results indicated that the velvet-complex factors affect virulence of the fungus by regulating expression of the NRPSs involved in virulence. In addition, velvet-complex proteins were found to coordinately and distinctly regulate fungal development, such as conidiogenesis and conidia germination. To identify and characterize genes for resistance to the new pathotype, 2,062 barley accessions from a USDA barley core collection were screened for spot blotch resistance to this pathotype and 24.5% of them showed resistance or moderate resistance at the seedling stage. Genome-wide association analysis identified four QTLs associated with the seedling resistance, which were located on chromosome 1H, 2H, 3H, and 6H, respectively. A genetic analysis of the cross between a highly resistant line (PI 235186) and a highly susceptible accession (PI 356741) suggested a single dominant gene confers the resistance in PI 235186. The resistance gene was further mapped to the short arm of chromosome 6H based on bulk segregant analysis using 194 SSR markers and genotyping-by-sequencing using 20 SNP markers in a F2 population. Additional markers were developed to fine map the resistance gene to a ~6.5 cM genomic region.
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.
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.
Genetic Mapping and Characterization of Net Blotch Dominant Resistance and Dominant Susceptibility Loci in Barley
(North Dakota State University, 2017) Tamang, Prabin
Net blotch is an economically important foliar disease of barley that occurs in two distinct forms: Spot Form Net blotch (SFNB) and Net Form Net Blotch (NFNB) caused by the necrotrophic fungal pathogens Pyrenophora teres f. maculata (Ptm) and Pyrenophora teres f. teres (Ptt), respectively. The recent emergence and the identification of both Ptm and Ptt isolates virulent on popular ND malting barley varieties have warranted the identification of new resistance sources. Association mapping was conducted on 2,062 diverse barley accessions phenotyped at the seedling stage with four diverse P. teres f. maculata isolates and genotyped with the 9k Illumina barley iSelect chip. A total of 138 significant marker-trait associations (MTA; −log10P value > 3.0) corresponding to 27 resistance loci were identified of which 21 loci were novel and six corresponded to previously characterized SFNB resistance QTL. Further, two higly resistant lines PI67381 and PI84314 were crossed with the two susceptible cultivars Tradition and Pinnacle grown in the Upper Midwestern US to develop three bi-parental recombinant inbred line (RIL) mapping populations of Tradition x PI67381, Pinnacle x PI67381 and Pinnacle x PI84314. These RIL populations were phenotyped with six diverse Ptm isolates and genotyped using PCR-GBS. MapDisto and Qgene were used to analyze the data and a total of twelve QTL were identified on chromosome 2H, 3H, 4H, 6H and 7H, of which nine were previously reported and the remaining three are considered novel. These resistances and the markers delimiting the QTL are being utilized to develop prebreeding lines by introgressing SFNB resistance into the cultivars Pinnacle and Tradition utilizing marker assisted selection. The barley line CI5791 exhibits a high level of resistance to diverse Ptt isolates collected from around the world. A forward genetics approach and an exome capture-mediated mapping-by-sequencing identified a candidate HvWRKY6 transcription factor gene required for NFNB resistance on chr 3H. We hypothesize that the HvWRKY6 gene function as a component of a conserved basal defense mechanism, which regulates the expression of other defense response genes that restrict lesion growth. The resistance/susceptibility loci identified in this study will facilitate the development of net blotch resistant cultivars.
Genetic Structure of Leptosphaeria Maculans Populations in North Dakota and Identification of Genes Associated with Resistance to L. Maculans in Brassica Juncea
(North Dakota State University, 2013) Nepal, Achala
Blackleg, caused by Leptosphaeria maculans is one of the most devastating diseases of canola (Brassica napus) in North Dakota. A study was conducted to characterize prevalence of pathogenicity groups (PG), identify population structure of L. maculans and identify sources of resistance among B. juncea accessions. Approximately 56% of the isolates belonged to PG-4, 13% to PG-3, 11% to PG-T, 5% to PG-1, and 2% to PG2. The remaining 13% of isolates could not be identified. The 605 single-spore cultures used to study the population genetics of L. maculans in ND were grouped according to their county of origin in five regions (NE, NC, NW, WC, and C) and each region was considered a population. These populations were tested for genetic variation at 7 microsatellite, 4 minisatellite, and for mating type loci. High levels of genetic diversity (H = 0.63 to 0.70) and significant gametic disequilibrium (P < 0.001) was observed in all populations. The ratio of mating type idiomorphs deviated significantly (P < 0.05) from 1:1 ratio for four populations. Highly significant (P < 0.001) G''ST between pairs of populations indicated a strong population differentiation. STRUCTURE analysis identified three distinct genetic populations in which the majority of the isolates from WC and nearly half of the isolates from NC were assigned to subpopulation one. The remaining half of NC isolates were assigned to subpopulation two and rest were assigned to subpopulation three. To identify sources of resistance against PG-2, PG-3, PG-T, and PG-4, a set of 298 B. juncea accessions were screened in greenhouse trials. Six accessions were resistant to PG-2 and PG-3, nine accessions were resistant to PG-T and PG-4, and two accessions were resistant to PG-2 and PG-4. DNA extracted from these accessions was screened using 766 DArT markers to identify QTL associated with resistance. Thirteen DArT markers were significantly (P<0.05) associated with resistance to PG-2 with variability ranging from 0.9% to 6.4%. Of these, three were also found significantly (P<0.05) associated with resistance to PG-3 with variability of 4.4%. No markers were found to be significantly associated with resistance to PG-T or PG-4 at (P<0.05).

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