Plant Pathology Doctoral Work

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Pathogenic Variation, Host Specialization and Aggressiveness in Helminthosporium Sativum
(North Dakota State University, 1984) El-Nashaar, Hossien Mahmoud
Common root rot, caused by Cochliobolus sativus, is an important disease of spring wheat and barley in the northern Great Plains. While the disease has been widely studied, extensive systematic investigation of pathological variation in C. sativus populations has not been reported. Five widely separated North Dakota counties were selected for field sampling. More than 430 isolates of C. sativus were obtained from wheat and barley plants. Pathogenicity of isolates was assessed according to amount of disease on wheat test plants in randomized, replicated greenhouse trials. Data were analyzed using the UNIVARIATE procedure of the SAS computer package. Relative pathogenicities of isolates were normally distributed continuous variables. Although the range of pathogenicity in populations of C. sativus within counties was similar, the means and variances were slightly different. Repeated planting of the same crop often results in an increase of inoculum of soilborne plant pathogens, but little is known of the effect on pathogenicity in the pathogen population. I collected C. sativus from symptomatic wheat plants from a plot planted continuously to wheat for over 90 years and from normally rotated grain fields in the surrounding area. Relative pathogenicity of isolates was tested in replicated greenhouse trials using wheat plants scored for disease six weeks after inoculation. Isolates from the continuous wheat plot were more pathogenic on average than isolates from commercial fields. Long term continuous cropping to wheat appears to shift the population of C. sativus toward more aggressive types, although many less pathogenic types also remain. Variability in C. sativus has been recognized in morphology of culture and pathogenicity for many years. Variability on virulence and/or aggressiveness of C. sativus isolates is not well documented. From the original collection of 432 isolates, 50 were selected to cover the entire range of pathogenicity. In three experiments, relative pathogenicity of the isolates was evaluated according to the amount of disease on each of four durum wheat cultivars. Aggressiveness patterns in the C. sativus-wheat system were reasonably reproducible. The C. sativus isolates originally selected for differential responses to wheat cultivars were not consistent in repeated testing. It was concluded that a range of pathogenicity (= aggressiveness ) exists in the C. sativus population and the apparent host differential responses are not repeatable and are probably experimental artifacts.
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.
Studies on the Biology of Soybean Cyst Nematode
(North Dakota State University, 2011) Poromarto, Susilo Hambeg
Soybean cyst nematode (SCN), Heterodera glycines, is a threat to soybean production in North Dakota. Studies on the biology of SCN were conducted to improve my understanding and management of this plant parasitic nematode. The objectives of the research were to; (1) determine if SCN reproduces on crops commercially grown or being tested for production in North Dakota, (2) evaluate the effects of SCN on growth of dry bean, (3) determine if there could be a shift in the SCN population toward greater ability to reproduce on dry bean, and (4) characterize the spatial distribution of SCN in research size field experiments. Canola, clover, lentil, and sunflower were nonhosts while borage, camelina, chickpea, crambe, cuphea, field pea, nyjer, and safflower were poor hosts for SCN with female indices (FI) less than 8. Lupines were susceptible hosts with FI’s of 42 to 57. FI’s of dry bean cultivars varied from 5 to 117. Kidney beans averaged the highest FI at 110 followed by navy, pinto and black at FI’s 41, 39, and 16, respectively. Pod number (PN), pod weight (PW), seed number (SN), and seed weight (SW) of GTS-900 (pinto bean) were significantly less at 5,000 and 10,000 eggs/100 cm3 soil compared with the control by 44 to 56% averaged over the two years. Significant reduction in growth of Montcalm (kidney bean) and Mayflower (navy bean) was observed at 2,500 and 5,000 eggs/100 cm3 soils in 2009, but not in 2008. There was no evidence that SCN was increasing reproduction during two 11 month periods of continual reproduction on roots of dry bean cultivars Premiere and Cirrus (navy), Buster and Othello (pinto), and Eclipse and Jaguar (black). The spatial distribution of SCN in field plots was aggregated in nine of ten field sites with large differences in egg numbers between plots. Lloyd’s index of patchiness ranged from 1.09 to 3.34. Spatial distribution of SCN can be an important factor affecting the results of field experiments.
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.
Genomics and Management of Fusarium Root Rot of Field Peas
(North Dakota State University, 2012) Chittem, Kishore
Dry Pea or field pea (Pisum sativum L.) is an important cool season legume crop grown in the United States. Field peas are vulnerable to many diseases of which, soil borne diseases including wilt and root rot are of major economic importance and can cause significant reduction in yield. There is a dearth of satisfactory methods for control of root rot and no varieties with complete resistance to Fusarium root rot are currently available. Root rot disease was found to be prevalent in all the major pea growing counties of North Dakota surveyed in 2004, 2005, 2010 and 2011. Fusarium species were the most frequently isolated fungal species from the infected pea roots of which, F. oxysporum and F. avenaceum were the most common. 21 Field pea varieties were screened for resistance against F. avenaceum and F. solani f. sp. pisi, the Fusarium species traditionally associated with root rots of field pea in growth chamber experiments and field trials. Low levels of resistance were detected in a few cultivars but no variety was found to be completely resistant to any of the pathogens tested. Efficiency of precipitated calcium carbonate (PCC) in controlling Fusarium species most commonly associated with root rots was evaluated under in vitro and field conditions. Significant reduction in spore production, spore germination, and dry mycelial weight of Fusarium spp. were detected on PCC amended media in laboratory studies. In greenhouse and field experiments significant reduction in root rot disease severity was observed with PCC application compared to control. Fungal gene expression in artificially infected field pea roots and F. graminearum grown in culture was assessed using the Illumina mRNA-Seq technology. A total of 613 F. graminearum genes were found to be differentially expressed in planta on pea. Functional classes associated with amino acid metabolism, nitrogen metabolism, extracellular polysaccharide degradation, detoxification by degradation and defense related proteins were found to be significantly enriched in the up-regulated gene set as determined using FunCatDB. Expression of four up-regulated genes was confirmed by RT-PCR to validate the inferences from the sequencing results.
Inoculation Techniques, Development of Brassica Napus Breeding Lines and Identification of Markers Associated with Resistance to Sclerotinia Sclerotiorum (Lib.) De Bary
(North Dakota State University, 2012) Burlakoti, Pragyan
Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economic disease affecting canola (Brassica napus L). Since expression of sclerotinia stem rot symptoms shows much variability and the trait is quantitative in nature, reliable phenotypic evaluation methods for characterization are needed. The three major objectives of this dissertation were to: i) evaluate eight different inoculation methods to discriminate between S. sclerotiorum-resistant and susceptible B. napus germplasm; ii) develop breeding lines with resistance to multiple diseases, and; iii) to identify QTL associated with resistance to sclerotinia stem rot using association mapping (AM). The eight methods evaluated were the detached leaves, detached stems, petiole inoculation (PIT), straw-inoculation, stem-piercing with toothpick, mycelial spray (MSI), petal inoculation and oxalic acid assay. MSI and PIT can better discriminate between the isolates and germplasm. Breeding lines resistance to S. sclerotiorum, Leptosphaeria maculans, and Rhizoctonia solani were developed from a cross between two moderately sclerotinia stem rot resistant plant introductions (PI). F2 seedlings were screened for sclerotinia stem rot using PIT. Surviving plants were self pollinated and their progeny screened again. This process was repeated until the F6 generation. In addition, F5 seedlings were evaluated for their reaction to R. solani and F5 and F6 seedlings for their reaction to L. maculans. Eight lines were identified as moderately resistance to these three pathogens. The genomes of a group of 278 B. napus plant introductions were screened using Diversity Array Technology to detect QTL associated with resistance to sclerotinia stem rot. The population was classified into nine sub-populations and 32 significant markers each explaining between 1.5 and 4.6% of the variation were identified. Blastn search indicates that similar nucleotide sequences are distributed throughout the genomes of B. oleracea, B. rapa, and A. thaliana. Results of these studies suggest the PIT and MSI are reliable screening tools to evaluate materials for resistance to sclerotinia stem rot; materials identified as resistant to S. sclerotiorum were also moderately resistant against R. solani and L. maculans and could be valuable sources for canola improvement programs; and AM allowed us to identify QTL associated with resistance to sclerotinia stem rot.
Movement and Accumulation of Candidatus Liberibacter Solanacearum in Potato Plants
(North Dakota State University, 2012) Rodriguez, Juan Jose
A new disease affecting potatoes was first detected in Mexico in 1993. Affected plants had aerial symptoms similar to those caused by potato purple top and psyllid yellows, but tubers had internal brown discoloration when sliced and dark stripes and streaks when processed to produce potato chips. The disease has been found in many potato production areas in Guatemala, Mexico, Honduras, New Zealand and the United States. The disease, termed Zebra Chip (ZC), has been associated with the presence of heavy infestations of the potato-tomato psyllid (Bactericera cockerelli). In 2009, a research group in New Zealand discovered that a new disease in tomato and pepper plants was caused by Candidatus Liberibacter solanacearum (Lso) and subsequently this same bacterium was associated with ZC in potato samples from Texas. The objectives of this study were: to assess the accumulation of Lso in various potato organs, to determine the effect of plant age on detection of Lso, symptom development and plant death, and (iii) to determine the effect of phosphorous acid on the development of ZC. Results from these studies showed significant differences in Lso populations between above and below ground tissues of the potato plant, with Lso populations in stolons and tubers being three to four times higher than those of leaf tissue and over seventy times greater than in stems. Time for detection of Lso by PCR in potato leaves of different ages at the time of inoculation ranged from 21 to 26 days after inoculation, symptoms development took 23 to 36 days. Plant death, took 24 to 47 days in plants of different age groups at the time of inoculation. In plants 15 weeks old at the time of inoculation, Lso was detected after 14 days in one plant out of 18; in plants 16 weeks old at the time of inoculation, Lso was detected after seven days in two plants out of 18. Phosphorous acid applications had no effect on the populations of Lso in potato tubers, onset of symptoms or plant death. All tubers showed ZC symptoms, making them unacceptable for the market.
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.
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.
Identification and Characterization of Pythium Spp. on Glycine Max (Soybean) in North Dakota
(North Dakota State University, 2013) Zitnick-Anderson, Kimberly Korthauer
The Oomycete Pythium comprises one of the most important groups of seedling pathogens affecting soybean, causing both pre- and post-emergence damping off. Numerous species of Pythium have been identified and found to be pathogenic on a wide range of hosts. Recent research on Pythium sp. infecting soybean has been limited to regions other than the Northern Great Plains and has not included North Dakota. In addition, little research has been conducted on the pathogenicity of various Pythium species on soybean or associations between Pythium communities and soil properties. Therefore, the objectives of this research were to isolate and identify the Pythium sp. infecting soybean in North Dakota, test their pathogenicity and assess if any associations between Pythium sp. and soil properties exist. Identification of the Pythium sp. was achieved using molecular techniques and morphological features. A total of 26 described Pythium sp. and several unknown species were recovered from soybean roots collected from 138 fields between 2011 and 2012. The majority of Pythium species (P. attrantheridium, P. debaryanum, P. diclinum, P. dissotocum, P. heterothallicum, P. hypogynum, P. inflatum, P. intermedium, P. irregulare, P. kashmirense, P. lutarium, P. minus, P. oopapillum, P. perplexum, P. terrestris, P. viniferum, P. violae,and an unknown Pythium sp.) caused pre-emergence damping off on soybean seedlings with less than 50% emergence and survival. In contrast, P. orthogonon, P. nunn, and P. rostratifingens had approximately 80% or greater emergence and survival of soybean seedlings. The negative and positive controls had 100% and 0% emergence and survival of soybean seedlings. Associations between soil properties and three Pythium groups were performed using logistic regression analysis. Logistic regression analysis determined that the presence of group one characterized by P. ultimum was correlated with zinc levels. Group two was characterized by P. kashmirense and an unknown Pythium sp. and was correlated with cation exchange capacity (CEC) values. Group three was characterized by P. irregulare and P. heterothallicum and was correlated with calcium carbonate exchange and CEC.
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.
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).
Molecular Characterization and Pathogenicity of Sunflower Stem Pathogens
(North Dakota State University, 2014) Mathew, Febina
Sunflower (Helianthus annuus L.) production can be limited by several stem diseases. Among these, Phomopsis stem canker causes frequent yield reductions in Australia, Europe and North America. In the U.S., while Diaporthe helianthi was assumed to be the sole causal agent, Diaporthe gulyae was found to cause Phomopsis stem canker in Australia. In order to determine the causal agent in the U.S., 234 isolates were cultured from 275 infected sunflower stems collected from the Northern Great Plains. Phylogenetic analyses of the internal transcribed spacer region, elongation factor subunit 1-α, and actin gene sequences confirmed two species, D. helianthi and D. gulyae. Four methods were tested to assess the Phomopsis stem canker response using four D. helianthi isolates on sunflowers. Stem-wound method was adopted for subsequent experiments based on the recovery of D. helianthi and its correlation with disease severity at 14- d after inoculation. Aggressiveness of two Diaporthe species was determined in greenhouse and results suggest they did not vary significantly (p=0.0012) in their aggressiveness, except at 3-d after inoculation. Among the nine genotypes screened for resistance, USDA ‘PI 162784’ and ‘PI 219649’ were less susceptible to the two Diaporthe spp. Fusarium is commonly regarded a minor pathogen on sunflowers in most production regions of the world. A total of 110 Fusarium isolates were recovered from 1,637 stalks randomly sampled for stem diseases in the Northern Great Plains and identified to species level. Phylogenetic analyses of repetitive-sequence-based polymerase chain reaction fingerprints and the translation elongation factor 1-alpha gene revealed that Fusarium isolates from sunflowers represented clades of eight species; namely, F. graminearum, F. proliferatum, F. culmorum, F. avenaceum, F. oxysporum, F. acuminatum, F. sporotrichioides and F. equiseti. Pathogenicity studies of eight Fusarium spp. in the greenhouse suggests F. sporotrichioides and F. equiseti were most aggressive. The study comparing the aggressiveness of three Fusarium spp. and V. dahliae isolates representing six VCGs showed V. dahliae VCG4B and VCG2A were significantly more aggressive (p ≤ 0.05) than F. sporotrichioides, F. oxysporum and F. equiseti. The identification of Diaporthe spp. and Fusarium spp. on sunflowers has implications for breeding for resistance and disease management.
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.
Identification and Functional Characterization of Necrotrophic Effectors in Parastagonospora Nodorum
(North Dakota State University, 2015) Gao, Yuanyuan
The necrotrophic fungus Parastagonospora nodorum (teleomorph; Phaeosphaeria nodorum), is the causal agent of Septoria nodorum blotch (SNB) on common wheat (Triticum aestivum L.) and durum wheat (Triticum turgidum L.). SNB is a serious foliar and glume disease which causes significant yield losses in major wheat growing areas and has serious impact on grain quality. P. nodorum produces necrotrophic effectors (NEs) that are recognized by and interact with dominant host sensitivity genes in an inverse gene-for-gene manner. The NE-host interaction is critical to induce necrotrophic effector-triggered susceptibility (NETS), resulting in SNB disease. To date, nine NE-host sensitivity gene interactions, following a NETS model, have been identified in the P. nodorum-wheat pathosystem. One of the NE-host sensitivity gene interactions, SnTox6-Snn6 interaction was characterized in this study. The SnTox6-Snn6 interaction was shown to be light dependent and Snn6 was located to a major disease susceptibility QTL on wheat chromosome 6A. SnTox1, another NE first identified in our lab, interacts with the corresponding wheat sensitivity gene Snn1. SnTox1 was further characterized in this study. The SnTox1 protein harbors C-terminal domains with a high degree of structural homology to plant chitin binding proteins and was subsequently shown to bind chitin, a main component of the fungal cell wall. Therefore, SnTox1 was hypothesized to compete with wheat chitinases to bind chitin, preventing fungal cell wall degradation. To investigate this hypothesis, the SnTox1 binding affinity with chitin was tested, as well as its potential function in the protection against chitinases during fungal mycelial growth. To identify additional NE regions, genome wide association study (GWAS) technology was used. A global collection of 191 P. nodorum isolates were genotyped using a restriction-site associated DNA genotyping by sequencing (RAD-GBS) protocol to identify SNP markers. Phenotypic data including fungal inoculations and culture filtrate infiltrations were collected using 191 P. nodorum isolates across several wheat lines. GWAS analyses were performed by regressing the phenotypic data and genotypic data by running multiple GWAS models.
Studies on the Management of Potato Mop-Top Virus-Induced Tuber Necrosis
(North Dakota State University, 2015) Domfeh, Owusu
Tuber necrosis caused by the Potato Mop-top Virus (PMTV) has become one of the most important tuber necrosis diseases in the United States. PMTV is transmitted by the powdery scab pathogen (Spongospora subterranea f.sp subterranea (Sss)) and no effective methods of control are currently available for these pathogens. Potato cultivars have been reported to exhibit natural variability in their reaction to PMTV infection, making cultivar selection a viable management option. This dissertation focuses on finding short to long term strategies for managing PMTV tuber necrosis. In the first study, potato cultivars were assessed for sensitivity to PMTV-induced tuber necrosis in three separate field trials. Results of tuber assessments demonstrated that sensitivity to PMTV-induced tuber necrosis among cultivars follows a continuum of tolerant to sensitive. In the second study, advanced breeding selections of potato were evaluated for sensitivity to PMTV-induced tuber necrosis. The results revealed high variability in PMTV-induced tuber necrosis incidence and severity among selections and identified 17 of them to be tolerant, nine – moderately tolerant, eight - moderately sensitive, while six were found to be sensitive. Results of the field trials show that russet-skinned cultivars and selections are less sensitive to PMTV tuber necrosis than red-, yellow- and white-skinned types. In the third study, a growth chamber experiment was conducted to investigate the potential of using moisture regime to manage PMTV tuber necrosis. The results showed significant differences in PMTV tuber necrosis and powdery scab infection among moisture regimes. Maintaining soil moisture level at 90% field capacity throughout the season resulted in significantly higher incidence and severity of PMTV tuber necrosis and powdery scab infection than keeping soil at 60% field capacity. The results also show that the potato plant may be susceptible to PMTV-induced tuber necrosis and powdery scab infection throughout the season. The results of these investigations offer potato growers the option to plant less sensitive cultivars in areas where PMTV and powdery scab exist. This information can be utilized in future breeding efforts to obtain resistant potato cultivars. Useful information on using soil moisture regime as a management strategy for PMTV tuber necrosis has been provided.
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.
Sensitivity of Rhizoctonia Solani and Aphanomyces Cochlioides to Fungicides, and Fitness of Tetraconazole-Resistant Isolates of Cercospora Beticola after Exposure to Different Temperature Regimes
(North Dakota State University, 2015) Arabiat, Sahar Ibrahim
North Dakota and Minnesota produce 55% of USA sugarbeet production. Diseases caused by Rhizoctonia solani, Aphanomyces cochlioides, and Cercospora beticola are the major diseases affecting sugarbeet production in North Dakota and Minnesota. Growers mainly use partial resistant varieties and fungicides to manage diseases of sugarbeet. Sensitivity of R. solani and A. cochlioides to fungicides were evaluated in vitro using mycelium radial growth assay and by evaluating disease severity on inoculated plants treated with fungicides in the greenhouse. Phenotypic stability of tetraconazole-resistant isolates of C. beticola after exposure to different temperature regimes was evaluated. For R. solani, mean EC50 values for baseline isolates were 49.7, 97.1, 0.3, 0.2, and 0.9 μg ml-1 and for non-baseline isolates were 296.1, 341.7, 0.9, 0.2, and 0.6 μg ml-1 for azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole, respectively. The mean EC50 values of azoxystrobin, trifloxystrobin, and pyraclostrobin increased with a change factor of 6.0, 3.5, and 2.7, respectively. All fungicides at labeled rates effectively controlled R. solani in vivo. For A. cochlioides, tetraconazole, prothioconazole, and pyraclostrobin reduced mycelium radial growth in vitro with mean EC50 values of 3.5, 2.4, and 0.8 μg ml-1, respectively. However, these fungicides were not effective at controlling A. cochlioides in vivo. Sugarbeet plants up to three weeks old were found susceptible to A. cochlioides. Resistant isolates of C. beticola had no fitness penalty as measured by spore production, spore germination, mycelium radial growth, and disease severity after exposure to different temperature regimes. However, isolate 09-347, resistant to tetraconazole, reverted to a moderate resistance level after exposure to -20ºC, and -20ºC to 4ºC to -20ºC to 4ºC with a factor of change of 38.6 and 32.8, respectively. This research indicated that R. solani sensitivity to the evaluated QoIs had decreased, but they were still effective at labeled rates under greenhouse conditions, and rotation of different fungicide classes could be a useful strategy to manage fungicide resistance. No fitness penalty was found after exposure of C. beticola isolates to cold treatments. However, C. beticola isolates resistant to tetraconazole became more sensitive to this fungicide after exposure to cold treatments.
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.

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