Plant Pathology Masters Theses

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Scanning Electron Microscope Examination of Sugarbeet Flowers and Fruits Infected with Phoma Betae
(North Dakota State University, 1980) El-Nashaar, Hossien Mahmoud
There are three natural openings in a mature sugarbeet fruit which serve as avenues of entry for microorganisms: 1) the basal pore which contains dried parenchyma and vascular tissue and is the point where the flower was connected to the stalk; 2) the apical pore where the style was inserted; and 3) the peripheral zone of dehiscence where the operculum separates from the fruit cavity wall during germination. The apical pore was first described in this study. Scanning electron microscopy of the naturally infected fruits showed, for the first time, hyphal penetration through both the basal pore and the peripheral zone. Examination of sugarbeet flowers artificially infected with Phoma betae also showed fungal penetration through the apical pore. Dense hyphal growth was associated with stigmal lobes and ungerminated pollen grains. Fungal growth apparently was stimulated by excretions from the stigma. Penetration of the fruit cavity wall and the operculum would render the fungus inaccessible to protectant fungicides and explains why the most successful seed treatments for P. betae have included volatile mercury fungicides or seed soak in thiram. Such treatment allows direct contact between the toxin and the pathogen.
The Impact of Plant Age, Fungicide Application Methodology and Timing, and Depth of Soil Inoculation on Infection by Rhizoctonia solani on Sugarbeet.
(North Dakota State University, 2010) Pooran-DeSouza, Scmwattie
Rhizoctonia root rot caused by Rhizoctonia solani (Kuhn), is the most important problem faced by sugarbeet (Beta vulgaris L.) grm.vers in North Dakota and Minnesota. Research was conducted that may be used to manage the disease. Six cultivars from 2 to 8- leaf stage were evaluated for their ability to withstand infection after soil inoculation by R. solani AG 2-2 IIIB. All leaf stages of the cultivars evaluated were susceptible to R. solani. Sugarbeet plants at the 2-leaf stages were most susceptible and had significantly higher root rot severity than plants at the 4, 6 and 8-leaf stages. Cultivars Crystal 454 and Hillshog 3035 had the lowest root rot severity of the cultivars evaluated, but root rot severity was still greater than 50%. The cultural practice of planting early in soils when the temperature is about 10°C at the 10 cm depth may result in plants being older and more tolerant by the time the pathogen becomes infective at warmer soil temperatures. However, the fact that all plant stages were susceptible to R. solani may require additional protective measures in highly infested fields with a knovm history of severe Rhizoctonia root rot. During greenhouse research, it was observed that azoxystrobin fungicide applied as a hypocotyl drench provided excellent control of Rhizoctonia root rot. It is recommended that growers use a foliar banded application of azoxystrobin at the 4-leaf stage to control Rhizoctonia root rot. Research was conducted to compare and evaluate the effect of foliar band and hypocotyl drench applications of azoxystrobin to control R. solani. The study showed that foliar banded and hypocotyl drench applications of azoxystrobin provided significantly similar disease control under conditions that were ideal for disease development However, disease from plants with a hypocotyl drench application was not significantly different than the non-inoculated control suggesting that further testing should be done to determine the utility of this application methodology in field conditions. Research was conducted to determine the best time to apply azoxystrobin fungicide for effective R. solani control relative to timing of soil inoculation. Sugarbeet hypocotyls were drenched at the 4-leaf stage at 0, 7, 14, 21, and 28 days pre-inoculation and at 0, 3, 10, 14, and 21 days post-inoculation. Azoxystrobin applications prior to inoculation resulted in significantly lower root rot compared to fungicide applications at post inoculation. Among the post inoculation applications, treatments where the fungicide was applied within 2 hours provided the best disease control. Fungicide application at pre-inoculation provided effective control at all timings evaluated: This research reinforces the need for azoxystrobin application before infection to control the disease in field conditions. The depth at which R. solani caused root rot infection of sugarbeet was studied after burying R. solani AG 2-2 IIIB inoculurn at depths of 2.54, 7 .62, and 12. 7 cm. R. solani AG 2-2 IIIB infections occurred at all depths of inoculation. However, inoculum buried at 2.54 cm depth had significantly higher root rot severity than inoculum buried deeper. Root rot symptoms were prevalent on the upper portion of the sugarbeet root just below the soil line irrespective of the depth of inoculurn placement. This suggests that the upper part of the root below the soil line is most vulnerable to R. solani infection. Consequently, in the soil fungicide application should target the root area just below the soil line for effective disease control.
Screening of Germplasm Accessions from the Brassica Species for Resistance against PG3 and PG4 Isolates of Blackleg
(North Dakota State University, 2011) Marino, Dante
Blackleg is a disease of canola and rapeseed cultivars that is caused by the fungus Leptosphaeria maculans (Desm.) Ces. & de Not., and it is by far the most destructive pathogen of canola in North America. In recent years, blackleg strains belonging to pathogenicity groups (PG) 3 and 4 have been discovered in North Dakota. Recent outbreaks of the disease have added a sense of urgency to characterize the risk these new strains represent for the canola industry and to identify sources of resistance against them. Thus, the objectives of this study were to screen germplasm collections of Brassica rapa, B. napus. and B. juncea for their reaction to PG3 and PG4 and to evaluate the reaction of a sample of currently used canola commercial cultivars grown in North Dakota to PG3 and PG4 as means to estimate the risk these new strains represent. All canola germplasm and commercial cultivars were evaluated in replicated trials in greenhouse conditions using cotyledon bioassays. In 2009 and 2010, the effect of these strains, using five inoculation sequences, on the reaction of canola seedlings was also evaluated. Field trials were not conducted because of the limited geographical distribution of the new strains. No adequate sources of resistance were identified among the 277 B. rapa and 130 B. napus accessions evaluated; however, 22 of the 406 accessions of Brassicajuncea evaluated were considered to have moderate levels of resistance. B. juncea seedlings that survived these inoculations were self-pollinated and their progeny (F1) were also screened. As before, surviving seedlings were self-pollinated. These F2 seeds are the elite materials that could be used in future breeding programs. The complementary study evaluating the role of sequence inoculations in reaction of canola seedlings to blackleg indicated that an increased susceptibility to PG3 occurred when seedlings were first inoculated with PG4; however, reaction to PG4 was not enhanced by a prior inoculation with PG3. All 75 commercial cultivars evaluated were susceptible to PG3 and PG4, indicating that the risk these new strains represent to the canola industry of the region is serious. Further, when a subsample of 16 cultivars were challenged with PG2, they were either resistant or moderately resistant, suggesting the ratings the industry are using relate to reaction of those cultivars to PG2 but not to the new strains; thus, growers should use caution when using these ratings while deciding on which cultivars to plant.
Characterization of Cytochrome B from European Field Isolates of Cercospora Beticola with Quinone Outside Inhibitor Resistance
(North Dakota State University, 2012) Birla, Keshav
Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugar beet worldwide. Control strategies for CLS rely heavily on fungicides including quinone outside inhibitor (QOI) fungicides. We collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed their sensitivity to the QOI fungicide pyraclostrobin. To gain an understanding of the molecular basis of QOI resistance, we cloned the full-length coding region of Cbcytb. All tested QOI-resistant isolates harbored a point mutation in Cbcytb at nucleotide position 428 that conferred an exchange from glycine to alanine at amino acid position 143 (G143A). A PCR assay was developed to discriminate QOI-sensitive and QOI-resistant isolates based on the G143A mutation. Our results indicate that QOI resistance has developed in some European C. beticola populations in Italy and monitoring the G143A mutation is an essential fungicide resistance management strategy.
Identification of Multiple Virulence Loci in Pyrenophora Teres F. Teres Associated with Net Form Net Blotch in Barley
(North Dakota State University, 2013) Shjerve, Rachel Anne
The necrotrophic fungal pathogen Pyrenophora teres f. teres causes the foliar disease net form net blotch (NFNB) on barley (Hordeum vulgare). To investigate the genetics of virulence in the barley- P. teres f. teres pathosystem, we used 118 progeny derived from a cross between the isolates 15A and 6A. The barley lines, chosen based on their different reactions to 15A and 6A, were evaluated for NFNB caused by the 15A × 6A progeny. Genetic maps generated with SSR and AFLP markers in the fungal population were scanned for quantitative trait loci (QTL) associated with virulence in P. teres f. teres. Two QTL were identified in Rika, two in Kombar and PI356715 had a single unique QTL. Therefore, a total of five virulence loci were identified in this pathogen population based on inoculation on three different barley lines.
Understanding and Managing Rhizoctonia Solani in Sugarbeet
(North Dakota State University, 2013) Noor, Afsana
Rhizoctonia crown and root rot of sugarbeet (Beta vulgaris L.) caused by Rhizoctonia solani Kühn is one of the most important production problems in Minnesota and North Dakota. Greenhouse studies were conducted to determine the efficacy of azoxystrobin to control R. solani at seed, cotyledonary, 2-leaf and 4-leaf stages of sugarbeet; compatibility, safety, and efficacy of mixing azoxystrobin with starter fertilizers to control R. solani; and the effect of placement of azoxystrobin in control of R. solani. Results demonstrated that azoxystrobin provided effective control applied in-furrow or band applications before infection at all sugarbeet growth stages evaluated; mixtures of azoxystrobin and starter fertilizers were compatible, safe, and provided control of R. solani; and azoxystrobin provided effective control against R. solani when placed in contact over the sugarbeet root or into soil close to the roots.
Characterization of Sensitivity of Sclerotinia Sclerotiorum Isolates from North Central U.S. to Thiophanate-Methyl and Metconazole
(North Dakota State University, 2013) Ameen, Gazala
Sclerotinia sclerotiorum (Lib.) de Bary causes Sclerotinia stem rot on canola and many other crops of economic importance in the U.S. SSR is primarily controlled with fungicides applied at flowering time. Most fungicides currently used to control SSR can promote resistance buildup in their target populations making monitoring of sensitivity important. In this study the reaction of S. sclerotiorum to thiophanate-methyl (TM) and metconazole (MTZ) was characterized. Samples collected in several states of north central U.S. were used. Three and ten isolates were considered to be moderately insensitive to TM and MTZ, respectively. Greenhouse trials indicated, however, that diseases caused by these isolates could be effectively controlled using currently recommended doses of each compound. In vitro sensitivity to TM was temperature dependent. A previously unreported mutation at codon E111D in the β-tubulin gene of a TM-moderately insensitive isolate was identified.
Characteristics and Host Range of a Novel Fusarium Species Causing Yellowing Decline of Sugarbeet
(North Dakota State University, 2013) Villamizar-Ruiz, Johanna Patricia
In 2008, a novel and distinct Fusarium species was reported in west central Minnesota causing early-season yellowing and severe decline of sugarbeet. This study was conducted to (i) establish optimum conditions for fungal growth and (ii) determine the host range of the novel Fusarium. The optimum temperature for fungal growth is 24°C and root injury is not needed to penetrate, infect, and cause disease of sugarbeet plants. Of the fifteen common crops and weeds tested for susceptibility to the new Fusarium sp. in field and greenhouse trials, disease symptoms were only observed in sugarbeet. Host range plants were tested for the presence of latent infection by root isolations and PCR. The pathogen was only present in canola and sugarbeet. The results suggest that canola has implications in the sugarbeet production system and management strategies for the novel Fusarium species. The name and description of the new Fusarium sp. is pending.
Impacts of Soybean Cyst Nematode (Heterodera Glycines) on Sugarbeet (Beta Vulgaris) and Interactions with Rhizoctonia Solani
(North Dakota State University, 2014) Rudolph, Kurt Donald
Soybean cyst nematode (SCN), Heterodera glycines is in the same genus as the sugarbeet cyst nematode (SBCN) Heterodera schachtii, there has been concern that SCN could also penetrate sugarbeet roots. The objectives were: 1) determine if SCN eggs hatch in the presence of sugarbeet seedlings and the larvae penetrate the roots, 2) determine at what age sugarbeet seedling are most susceptible to SCN penetration, 3) determine if SCN can penetrate sugarbeet roots under field conditions and 4) and determine if SCN could increase incidence of Rhizoctonia root rot in sugarbeet seedlings. This study demonstrated that SCN can penetrate the roots of a variety of sugarbeet. The results from the field demonstrated that SCN could penetrate sugarbeet seedlings under normal field growing conditions, and that penetration occurred in both SBCN susceptible and resistant sugarbeet cultivars. Penetration by SCN increased sugarbeet seedling root damage by R. solani under controlled conditions.
Genetically Dissecting the Recessive Rpg4-Mediated Wheat Stem Rust Resistance Locus in Barley
(North Dakota State University, 2014) Arora, Deepika
The rpg4-mediated resistance locus (RMRL) in barley provides recessive resistance against wheat stem rust (Puccinia graminis f. sp. tritici) races including the virulent race TTKSK (a.k.a Ug99). Three genes (HvAdfF3, Rpg5, and HvRga1) at the RMRL are required together for resistance. Allele characterization of these three genes identified the dominant rye stem rust resistance gene Rpg5 gene as the polymorphic R-gene conditioning the recessive rpg4-mediated wheat stem rust resistance. The Rpg5 gene contains an atypical R-gene structure encoding a nucleotide-binding site, leucine rich repeat, and serine/threonine protein kinase (STPK) domains. Genetic analysis of crosses between the resistant cultivar Q21861 with a functional Rpg5 allele and different susceptible varieties determined that most susceptible cultivars contain the susceptibility gene HvPP2C.1 in place of Rpg5 STPK domain. Genetic analyses determined that HvPP2C.1 conditions the recessive nature of the reaction, possibly negatively regulating the signaling pathway initiated by the Rpg5 kinase domain.
Evaluation of Winter Wheat Germplasm for Resistance to Stripe Rust and Leaf Rust
(North Dakota State University, 2014) Kertho, Albert Okaba
Wheat leaf rust, caused by Puccinia triticina (Pt), and wheat stripe rust caused by P. striiformis f. sp. tritici (Pst) are important foliar diseases of wheat (Triticum aestivum L.) worldwide. Breeding for disease resistance is the preferred strategy of managing both diseases. The continued emergence of new races of Pt and Pst requires a constant search for new sources of resistance. Winter wheat accessions were evaluated at seedling stage in the greenhouse with races of Pt and Pst that are predominant in the North Central US. Association mapping approach was performed on landrace accessions to identify new or underutilized sources of resistance to Pt and Pst. The majority of the accessions were susceptible to all the five races of Pt and one race of Pst. Association mapping studies identified 29 and two SNP markers associated with seedling resistance to leaf rust and stripe rust, respectively.
Resistance to Septoria Musiva in Hybrid Poplar
(North Dakota State University, 2014) Qin, Ruqian
Septoria leaf spot and stem canker, caused by the fungal pathogen Mycosphaerella populorum Thompson (Anamomorph = Septoria musiva Peck). An efficient greenhouse disease resistance screening is essential for the development of resistant clones. Fourteen clones of hybrid poplar were inoculated with spore suspension. A regression model with parameters (lesion number and proportion necrotic area) is consistently and accurately predicted field resistance categories of the most resistant and susceptible clones. In second experiments, the infection biology of S. musiva was examined at several time points by scanning electron microscopy (6 h, 12 h, 24 h, 72 h, 1-week and 3-week) and histological analysis (3-week, 5-week and 7-week). Results indicated that there are differences occur following penetration between the resistant and susceptible. Those differences provide the first clues elucidating resistance mechanism in hybrid poplar stems. The results from this thesis will be used to improve resistance to Septoria canker in breeding programs.
Identification of Host Resistant Effective against the Barley Spot Form Net Blotch Pathogen
(North Dakota State University, 2014) Neupane, Anjan
Spot form net blotch (SFNB) caused by Pyrenophora teres f. maculata is a major foliar disease of barley (Hordeum vulgare L.) worldwide. SFNB epidemics have recently been observed in major barley producing countries, suggesting that the local barley cultivars are not resistant and that virulence of the pathogen populations may have changed. Here we attempt to identify sources of resistance effective against diverse isolates of the SFNB pathogen. A total of 2062 world barley core collection accessions were phenotyped using isolates of this pathogen collected in the United States (FGO), Australia (SG1), New Zealand (NZKF2), and Denmark (DEN 2.6). Isolate-specific susceptibility was identified in several of the barley accessions tested, indicating variability in both pathogen virulence and host resistance/susceptibility. Collectively, only 15 barley accessions were resistant across all isolates tested. Future research will involve the characterization of host resistance, pathogen virulence, and the host-pathogen interaction associated with SFNB of barley.
Evaluating Chemical Seed Treatments for Fusarium Root Rot Control in Dry Beans and Field Peas
(North Dakota State University, 2014) Hegde, Namratha Prakashchandra
This study evaluated commercially available seed treatment products for their ability to control Fusarium solani and F. avenaceum, causal agents of root rot in dry edible bean and field peas, respectively, through in-vitro, growth chamber and field trials. Disease severity was assessed using a 0 to 5 scale, and root health parameters were recorded. The in-vitro tests conducted were not considered good predictors of fungicide performance in growth chamber or field trials in case of dry beans for management of F. solani. In case of field peas, in-vitro and growth chamber studies provided consistent results and allowed the identification of fludioxonil, trifloxystrobin and pyraclostrobin as the most effective products to manage F. avenaceum. Overall, integration of chemical seed treatments along with cultural practices; crop varieties partially resistant to root rot, and drench application is necessary to effectively manage Fusarium root rot of dry beans and field peas in field conditions.
Characterization of Effector Encoding Genes from the Novel Sugar Beet Pathogen Fusarium Secorum
(North Dakota State University, 2015) Bian, Zhuyun
A new disease of sugar beet, named Fusarium yellowing decline, was recently found in in the Red River Valley of MN and ND. This disease is caused by a novel pathogen named Fusarium secorum. Pathogens such as F. secorum secrete proteins during infection called ‘effectors’ that help establish disease. Since pathogenicity and disease development may depend on effector proteins produced by F. secorum during infection, effector protein identification furthers our understanding of the biology of this important pathogen. A list of 11 candidate effectors was generated previously. In this study, to characterize putative effectors, we developed a transformation system using polyethylene glycol–mediated transformation. Several mutant lines were created with an effector deleted from the genome using a split-marker knock-out strategy. To explore their role in pathogenicity, mutant strains have been inoculated to sugarbeet and compared to WT F. secorum.
Seed to Seedling Transmission of Colletotrichum lindemuthianum in Dry Edible Pinto Bean
(North Dakota State University, 2015) Halvorson, Jessica Marie
Dry bean anthracnose, caused by Colletotrichum lindemuthianum, is a damaging disease that can cause significant reductions in seed quality and yield. The purpose of this study was to determine the races of C. lindemuthianum isolates collected in North Dakota and to develop a real-time qPCR assay to quantify the pathogen in seed and stem tissue. Race 73 continues to be the most common in North Dakota across 33 isolates collected in 2012. The qPCR assay using primer pair ClF1527/ClR1609 was proven useful for quantifying C. lindemuthianum, and was more specific than a previously developed method. Results from greenhouse and field studies indicated that as seed infection increased, emergence decreased and foliar symptoms and pathogen detection increased. Additionally, field studies demonstrated daughter seed infection increased and yield decreased as seed infection severity increased. Using primer pair ClF1527/ClR1609, the pathogen was detected ten days after planting in greenhouse and field studies.
Spatial and Temporal Sensitivity of Alternaria Species Associated with Potato Foliar Diseases to Demethylation Inhibiting and Anilino-Pyrimidine Fungicides
(North Dakota State University, 2015) Fonseka Gunawardena, Arthur Dimitri Lakshan
Early blight and brown spot, caused by Alternaria solani and Alternaria alternata, respectively, are important foliar diseases of potato, affecting both tuber yield and quality. Most of the commercial cultivars lack resistance; therefore, application of foliar fungicides remains a primary management strategy. Correlation coefficients comparing EC50 values for conidial germination and mycelial growth of A. solani and A. alternata in response to boscalid and fluopyram, respectively, were low, indicating that the association between these two in vitro assays was very weak. Baseline sensitivities of Alternaria spp. to difenoconazole, metconazole, and pyrimethanil using mycelial growth assays demonstrated high intrinsic activity against the two pathogens. Six out of 245 A. solani isolates exhibited reduced-sensitivity to pyrimethanil in in vitro assays and reduced-sensitive isolates were not controlled except at 100 μg/ml in greenhouse efficacy tests. The DMI chemistries and pyrimethanil remain valuable options for fungicide rotation programs in areas of high disease pressure.
Characterization of Programmed Cell Death Responses Involved in Disease Resistance/Susceptibility Responses in Barley
(North Dakota State University, 2015) Sager-Bittara, Lauren Paige
The plant innate immune system relies on regulated programmed cell death (PCD) responses which provide resistance against biotrophic fungal pathogens and are utilized by necrotrophic pathogens. The gene-for-gene interactions leading to resistance against biotrophs has been co-evolving over millions of years and provides many targets for necrotrophic pathogens to subvert the plants PCD for their benefit. Two PCD pathways involved in plant immunity were characterized. The first, nec3, appears to control runaway PCD in response to infection by many pathogens who mainly elicit pathogen or damage associated molecular pattern (PAMP or DAMP) triggered immunity. The nec3 mutant was mapped to chromosome 6H and a pool of candidate genes were identified by RNAseq. The second, Rcs5, is a dominant susceptibility gene or a putative necrotrophic effector triggered susceptibility (NETS) target that confers susceptibility to Cochliobolus sativus. The candidate Rcs5 genes are three Wall Associated Kinases (WAKs) found on chromosome 7H (1).
Evaluation of Genetic Resistance to Common Bacterial Blight in Dry Edible Bean
(North Dakota State University, 2015) Maniruzzaman
Common bacterial blight (CBB) is an economically important disease of dry bean worldwide caused by Xanthomonas axonopodis pv. phaseoli, (Xap). The objectives of this research were to determine the frequency of CBB resistance in NDSU breeding materials and to evaluate the effectiveness of two SCAR markers, SAP6 and SU91, linked with major QTL for CBB resistance, across this host population. A total of 593 advanced and preliminary lines were phenotyped in the greenhouse and genotyped using SAP6 and SU91. Phenotyping revealed CBB resistance in 310 lines, with a higher frequency of resistant lines in the pinto, great northern and small red market classes. A total of 188 lines were phenotyped under field condition and only 23 lines were found resistant. The presence of the SU91 marker, and both markers in combination, more effectively identified CBB resistance than did the SAP6 marker alone. Identification of resistant lines should accelerate breeding efforts.
Rhizoctonia Solani Damping-Off of Sugarbeet: Effect of Plant Growth Stage on Disease Severity and Management Using Penthiopyrad
(North Dakota State University, 2015) Liu, Yangxi
Rhizoctonia solani is the most damaging pathogen on sugarbeet (Beta vulgaris L.) in North Dakota and Minnesota. Research was conducted to evaluate penthiopyrad for controlling R. solani and determine when the plants are most susceptible to infection. Penthiopyrad applied in-furrow and as a soil drench resulted in significantly higher percent survival than the positive control whereas penthiopyrad applied in a band was ineffective at controlling R. solani. Penthiopyrad can also be used as a seed treatment at the 14 g rate to provide effective control of R. solani. Sugarbeet plants, irrespective of their inherent level of resistance, were easily infected by R. solani up to three weeks after planting, even longer for susceptible varieties, highlighting the need for additional protection in the form of seed treatment or fungicide application that may be required to protect vulnerable sugar beet planted in fields with a history of the disease.

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