Doctor of Philosophy / Department of Plant Pathology / John P. Fellers / Harold Trick / In Kansas, wheat (Triticum aestivum L.) is severely affected by the biotrophic fungus Puccinia triticina (leaf rust). Although resistant varieties have been developed, the fungus tends to overcome new sources resistance very quickly. Plants have evolved a single gene (R genes) defense network that can recognize specific pathogen effectors (Avr), in a gene-for-gene manor. In rusts, effectors are secreted proteins responsible for inducing the uptake of nutrients and inhibit host defense responses. Identification of secreted proteins during the infection may help to understand the mode of infection of P. triticina. Little is known about molecular interactions in the pathosystem wheat-leaf rust and no Avr genes from cereal rusts have been cloned. In order to understand pathogenicity in leaf rust and generate new alternatives for disease control, the goal of this research is identify P. triticina secreted proteins from a collection of expressed genes during the infection, and to characterize putative Avr function for three candidates. From 432 EST’s derived from haustoria and infected plants, fifteen secreted proteins were identified and 10 were selected as potential avirulence candidates. Pt3 and Pt 51 are two P. triticina (Pt) candidates expressed specifically in the haustoria and encode small cysteine-rich secreted proteins. Eight candidates are expressed at early stages of infection, during spore germination and 6 days after inoculation. They are small-secreted proteins. None are repetitive elements or have nuclear localization signals. They also do not share a conserved motif with known filamentous fungus Avr proteins. Five candidates are novel proteins, two have similarity with predicted proteins, one is homologous with Hesp-379-like protein, one is homologous with superoxide dismutase, and one has a cell glucanase predicted function. Pt3, Pt12 and Pt27 were tested by transient expression experiments using co-bombardment with GUS into leaf rust resistant isogenic lines. Reduction in the expression of reporter gene GUS co-expressed with Pt27 indicates a potential avirulence factor for Lr26 in wheat.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Jack Fry / Zoysiagrass (Zoysia spp.) is more heat and drought resistant and requires fewer cultural inputs than cool-season (C3) turfgrasses; however, its widespread use may be limited by an extended period of winter dormancy, the disease Rhizoctonia large patch (Rhizoctonia solani Kühn AG-2-2 LP), and lack of cold hardiness. Objectives of these 2012-2014 field studies were to evaluate: 1) turfgrass colorants and overseeding for color enhancement; 2) three colorant application volumes on color persistence; 3) colorants applied at two volumes, once or sequentially, on buffalograss (Buchloë dactyloides) and zoysiagrass; 4) the impact of nitrogen source and application timing on large patch severity; and 5) winter hardiness and turf quality of new experimental lines. The colorants Ultradwarf Super and Green Lawnger provided acceptable lawn-height ‘Chisholm’ zoysiagrass color for 7 to 9 and 19 to 24 weeks after treatment (WAT), respectively. Chisholm receiving a sequential application of either product in mid-winter (14 WAT) maintained an acceptable color level after that point until mid-May (28 WAT). Overseeding with annual ryegrass did not provide acceptable color for more than 4 weeks. Evaluation of the colorants Green Lawnger, Endurant, and Wintergreen Plus showed that acceptable Chisholm color at lawn-height occurred for 8 to 14 WAT at 80 gallons per acre (GPA) and 16 to 26 WAT at 240 GPA. Buffalograss at lawn-height receiving a single autumn colorant application had acceptable color for 8 to 12 WAT at 100 GPA or 8 to 14 WAT at 160 GPA. ‘Meyer’ zoysiagrass maintained at a 0.5 inch height receiving a single autumn colorant application had acceptable color for 8 to 18 WAT at 100 GPA or 14 to 18 WAT at 160 GPA. No differences in large patch occurred between spring and fall applications of ammonium sulfate and calcium nitrate, nor between those treatments and summer-applied urea. Applying fertilizer in spring when soils reached 21 ºC increased green cover on some rating dates compared to applications in fall when soil temperatures fell to 21 ºC but differences were minimal. Out of 985 experimental zoysiagrass lines planted in the field, about 25 were identified for further evaluation for cold hardiness, large patch resistance, and turf quality.

Master of Science / Department of Plant Pathology / Bernd Friebe / Bikram S. Gill / Common, bread, or hexaploid wheat, Triticum aestivum L. (2n=6x=42, AABBDD), has several relatives in the Triticum/Aegilops complex of the Poaceae family in the Triticeae tribe, which are valuable sources for broadening genetic diversity and may provide genes for disease and pest resistance and general wheat improvement. Other wild relatives of wheat also may be exploited for wheat improvement, such as Haynaldia villosa (L.) Schur. (2n=2x=14, VV). It is a diploid species with resistance to powdery mildew, wheat curl mite colonization, cereal eyespot disease, rust diseases, and wheat spindle streak mosaic virus. H. villosa may harbor many other as yet unidentified traits for wheat improvement. The polyploid nature of bread wheat allows tolerance to genomic changes, because homoeologous chromosomes from other genomes compensate for missing wheat chromosomes. In this experiment, we crossed the disomic alien addition line DA4V (2n=6x=44) with a pair of H. villosa chromosomes added to the wheat chromosome complement with wheat monosomic for chromosome 4D (2n=41) to produce 4D/4V double monosomic plants. According to centric breakage-fusion mechanisms, univalents tend to break at their centromeres at meiotic metaphase I producing telocentric chromosomes with unstable or “sticky” ends that can fuse with the sticky ends of other newly formed telocentric chromosomes. This fusion results in Robertsonian whole-arm translocations that may be compensating if a short arm of one chromosome fuses with a long arm of another. Double monosomic plants were screened cytogenetically and further visualized by genomic in situ hybridization (GISH). Five transfers were identified, including T4DS.4VL and T4VS.4DL translocations, and a T4VS-W.W transfer of unknown wheat origin. These results were confirmed by GISH. The T4DS.4VL and T4VS.4DL translocations are genetically compensating and should be exploited in wheat improvement.

Master of Science / Department of Plant Pathology / Erick D. DeWolf / Stripe rust, caused by Puccinia striiformis f. sp. tritici, historically has been a minor problem in the Great Plains. However, Kansas had significant losses due to stripe rust in 2001, 2003, and 2005. Recent research on the population of P. striiformis suggests changes in the fungal population may have been responsible for these epidemics. The objectives of this research were to determine conditions that are favorable for the infection of P. striiformis f. sp. tritici isolates from the current population and develop models to predict infection events. Two week old potted seedlings were inoculated with an isolate of P. striiformis and exposed to ambient weather conditions for 16 hours. Results of this bioassay were used to develop logistic regression models of infection. Models using hours at relative humidity >87%, leaf wetness, and mean relative humidity predicted infection with 93%, 80%, and 76% accuracy. Future research will use these results to determine weather patterns that influence the probability of stripe rust epidemics and to facilitate the development of regional prediction models for stripe rust. Foliar diseases of wheat result in an average yield loss of 7.8% in Kansas. Although it is possible to reduce these losses with foliar fungicides, the yield increases resulting from these applications may not justify the additional costs. The objective of this research was to develop models that help producers identify factors associated with disease-related yield loss and the profitable use of foliar fungicides. Data were collected for two years at three locations in central Kansas to determine yield response to fungicide application on eight varieties with varying degrees of resistance. Logistic regression was used to model the probability of a yield response >4 bushels per acre based on disease resistance of a variety, historical disease risk, and in-season disease risk. The accuracy of the resulting prediction models ranged from 84% to 71%. A model combining in-season disease risk and variety resistance was most accurate. The prediction accuracy of the model was 79% when tested with an independent validation dataset. In the future, these models will serve as educational tools to help producers maximize profit and productivity.

Doctor of Philosophy / Department of Plant Pathology / Karen A. Garrett / Late blight of potato, caused by Phytophthora infestans, is a pernicious disease of potatoes worldwide. This disease causes yield losses as a result of foliar and tuber damage. Many models exist to predict late blight risk for control purposes with-in season but rely upon fine-scale weather data collected in hourly, or finer, increments. This is a major constraint when working with disease prediction models for areas of the world where hourly weather data is not available or is unreliable. Weather or climate summary datasets are often available as monthly summaries. These provide a partial solution to this problem with global data at large time-steps (e.g., monthly). Difficulties arise when attempting to use these forms of data in small temporal scale models. My first objective was to develop new approaches for application of disease forecast models to coarser resolution weather data sets. I created metamodels based on daily and monthly weather values which adapt an existing potato late blight model for use with these coarser forms of data using generalized additive models. The daily and monthly weather metamodels have R-squared values of 0.62 and 0.78 respectively. These new models were used to map global late blight risk under current and climate change scenarios resistant and susceptible varieties. Changes in global disease risk for locations where wild potato species are indigenous, and disease risk for countries where chronic malnutrition is a problem were evaluated. Under the climate change scenario selected for use, A1B, future global late blight severity decreases. The risk patterns do not show major changes, areas of high risk remain high relative to areas of low risk with rather slight increases or decreases relative to previous years. Areas of higher wild potato species richness experience slightly increased blight risk, while areas of lower species richness experience a slight decline in risk.

Doctor of Philosophy / Department of Plant Pathology / Frank F. White / Plants have evolved sophisticated mechanisms to protect against microbial invaders of which resistance (R) genes are an important component. R genes mediate specific recognition of pathogens possessing cognate avirulence (avr) gene products, which leads to the induction of plant defense responses and the arrest of pathogen ingress. In contrast to numerous examples of R gene–avr interactions, the susceptible interaction is less well examined. Recent studies on rice and wheat indicate that host resistance to pathogens also involves genetic variability in dominant traits for susceptibility. Xanthomonas oryzae pv.oryzae (Xoo) causes bacterial blight disease in rice, a serious threat in the major rice growing regions of Asia. The pathogenicity of Xoo depends on the translocation of a cocktail of effector proteins into rice cells by a type III secretion system. The family of transcription activator like (TAL) effectors is the one of the most intriguing due to their eukaryotic features and function as major virulence determinants. The specificity of TAL effectors is determined by the nearly identical repeat units at the center of each protein. The major virulence determinant of the strain PXO99A is PthXo1, which hijacks the transcription of the host susceptibility (S) gene Os8N3, an allele of recessive resistance gene xa13. The strains that overcome xa13-mediated resistance harbor alternate major TAL effectors including PthXo2, PthXo3 and AvrXa7. Alternate effectors do not induce Os8N3. This study identified the alternate S gene Os11N3, which is dependent on the effectors AvrXa7 and PthXo3. The effectors bind to specific elements in the proximal promoter regions of the respective S genes and act as transcriptional activators. Our results indicate that rice–Xoo interactions involve gene-for-gene susceptibility to bacterial blight in addition to gene-for-gene resistance.

Master of Science / Department of Plant Pathology / Harold Trick / The object of this study was to find a target sequence for the known Heterodera glycines Y25 sequence that contained no homology to any known Glycine max genes so homologous endogenous soybean gene expression will not be effected. In addition, in attempt to improve the accuracy of SCN bioassays performed in greenhouse settings, applications of a variety of insecticides with differing modes of action were applied to screen for any detectable effects on the SCN populations. The full-length sequence of the Y25 gene was blasted against the G. max genome using the National Center for Biotechnology Information blast database and a portion of the gene was found to contained no homology to the G. max genome. A rapid hairy root assay was used to screen for resistance to H. glycines. The sequence was transformed into Agrobacterium rhizogenes using a modified heat shock method. The transformed A. rhizogenes were used to inoculate soybean seedlings. The inoculated seedlings developed hairy roots expressing the target sequence. Upon finishing the hairy root assay it was discovered that there were no detectable differences across any of the treatments or the controls. It was neither proved nor disproved that the new target sequence containing no homology to the G. max genome was as effective as the original target. Further investigation will need to be conducted to show the level of control for the new target sequence.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Jack Fry and Megan Kennelly / Rough bluegrass (RBG, Poa trivialis L.) is a difficult-to-control weed that commonly infests cool-season turfgrass swards after movement of vegetative propagules or contamination from seed lots. Rough bluegrass is less tolerant of heat stress than desirable cool-season species such as tall fescue (TF, Festuca arundinacea Schreb. Syn [italicize]Schedonorus [italicize]arundinaceus Schreb.), and often declines during mid-summer due to biotic or abiotic stresses. The objectives of these 2011-2013 controlled environment and field experiments were to: 1) observe growth and physiological differences between ‘Laser’ and ‘Pulsar’ RBG and TF; 2) differentiate between physiological and pathological contributors to RBG decline; 3) determine the effects of TF seeding rate and mowing height on TF/RBG establishment when RBG is a seed contaminant; 4) evaluate herbicide combinations for selective RBG control; and 5) evaluate seasonal timing of glyphosate for nonselective RBG control. Tall fescue was less affected by elevated temperature than RBG. At 35°C, Laser and Pulsar experienced similar reductions in quality, gross photosynthesis (Pg), shoot and root biomass, and root length density compared to when grown at 23°C, but maximum electrolyte leakage was greater for Pulsar (63%) than for Laser (49%). Cell membrane thermostability could contribute to the better heat tolerance of Laser RBG. Evaluation of RBG foliage and roots did not reveal a fungal pathogen associated with RBG decline. Still, repeated applications of azoxystrobin (610 g a.i. ha⁻¹) or pyraclostrobin (556 g a.i. ha⁻¹) increased RBG quality, cover, and Pg during summer compared to untreated RBG, possibly due to poorly understood non-target physiological effects of the fungicides. Mowing TF at 7.6 or 11.4 cm reduced RBG incidence up to 57% compared to mowing at 3.8 cm. Tall fescue seeding rate had no effect on RBG incidence. Several herbicides and herbicide combinations resulted in some RBG injury in the field, but bispyribac-sodium was the only treatment that provided RBG control (16 to 92%) in Manhattan, KS; Hutchinson, KS; and Mead, NE. Spring-applied glyphosate resulted in the lowest RBG coverage (1 to 31%) among field studies in Manhattan and Mead, followed by late-summer applications (6 to 58%), and mid-summer applications (9 to 86%).

Master of Science / Department of Plant Pathology / John P. Fellers / Allan K. Fritz / Leaf rust is caused by Puccinia triticina and is one of the most widespread diseases of wheat worldwide. Breeding for resistance is one of the most effective methods of control. Lr16 is a leaf rust resistance gene that provides partial resistance at the seedling stage. One objective of this study was to use RNA-seq and in silico subtraction to develop new resistance gene analog (RGA) markers linked to Lr16. RNA was isolated from the susceptible wheat cultivar Thatcher (Tc) and the resistant Thatcher isolines TcLr10, TcLr16, and TcLr21. Using in silico subtraction, Tc isoline ESTs that did not align to the Tc reference were assembled into contigs and analyzed using BLAST. Primers were designed from 137 resistance gene analog sequences not found in Tc. A population of 260 F[subscript]2 lines derived from a cross between the rust-susceptible cultivar Chinese Spring (CS) and a Thatcher isoline containing Lr16 (TcLr16) was developed for mapping these markers. Two RGA markers XRGA266585 and XRGA22128 were identified that mapped 1.1 cM and 23.8 cM from Lr16, respectively. Three SSR markers Xwmc764, Xwmc661, and Xbarc35 mapped between these two RGA markers at distances of 4.1 cM, 10.7 cM, and 16.1 cM from Lr16, respectively. Another objective of this study was to use genotyping-by-sequencing (GBS) to develop single nucleotide polymorphism (SNP) markers closely linked to Lr16. DNA from 22 resistant and 22 susceptible F[subscript]2 plants from a cross between CS and TcLr16 was used for GBS analysis. A total of 39 Kompetitive Allele Specific PCR (KASP) markers were designed from SNPs identified using the UNEAK and Tassel pipelines. The KASP marker XSNP16_TP1456 mapped 0.7 cM proximal to Lr16 in a TcxTcLr16 population consisting of 129 F[subscript]2 plants. These results indicate that both techniques are viable methods to develop new molecular markers. RNA-seq and in silico subtraction were successfully used to develop two new RGA markers linked to Lr16, one of which was more closely linked than known SSR markers. GBS was also successfully used on an F[subscript]2 population to develop a KASP marker that is the most closely linked marker to Lr16 to date.

Master of Science / Department of Plant Pathology / William W. Bockus / The effects of tan spot on lipid profiles in wheat leaves were quantified by mass spectrometry. Inoculation with Pyrenophora tritici-repentis significantly reduced the amount of many lipids, including the major lipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in leaves over time. These two lipids accounted for 89% of the mass spectral signal of detected lipids in wheat leaves. Reductions in amounts of lipids were at much higher rates over time for susceptible cultivars compared with resistant cultivars. Furthermore, data show that cultivars resistant to tan spot have different lipid profiles when compared with susceptible cultivars. Resistant cultivars had more MGDG and DGDG than susceptible ones, even in non-inoculated leaves. Using linear models that were fit to data, non-inoculated cultivars with a rating of 1 (highly resistant to tan spot) were calculated to have 66.1% more MGDG and 52.7% more DGDG signal than cultivars with a rating of 9 (highly susceptible). These latter findings are indirect evidence that the amounts of some lipids in wheat leaves may be determining factors in the resistance response of cultivars to tan spot.