Breeding Port-Orford-cedar for resistance to Phytophthora lateralis, a causal agent of root disease, begins by screening, through artificial inoculation, phenotypically resistant trees selected from natural stands. The successful program selected tolerant or resistant POC parent trees for the purpose of disease management. Candidate resistant POCs were used in my dissertation to: 1. validate screening methods such as stem- and root-dip inoculation; 2. test for increased virulence of P. lateralis; and 3. evaluate detection techniques. The results showed that the established screening methods were appropriate, and no evidence of changed virulence was found. A PCR technique was more reliable than other techniques for detection of P. lateralis in seedlings. An additional test for foliar infection showed that initial penetration through wounds and natural openings was possible. POC seedlings and rooted cuttings from resistant and susceptible families were used to demonstrate resistance mechanisms. In order to explain the mechanisms at the cellular level, the susceptible response of POC seedlings to P. lateralis was first observed with light microscopy. Zoospores encysted on lateral roots, germinated, and penetrated by means of appressoria. Direct penetration between epidermal cells was common but penetration through epidermal cell walls was also observed. The hyphae colonized the root cortex inter- and intracellularly. Wound inoculation on stems resulted in inter- and intra cellular hyphal growth in cambial, sieve, and parenchyma cells in the secondary phloem. Several resistance mechanisms were observed: 1) there was a difference in zoospore attraction between susceptible and certain resistant POCs revealed by microscopic observation, direct count of encysted zoospores, and quantitative real-time PCR; 2) the frequency of encystment, penetration, and colonization of resistant seedlings was much lower than susceptible seedlings, but no differences in infection pathway were observed by means of light or electron microscopy; 3) collapsed cell walls were present in resistant POCs showing increased cell wall thickness, wall appositions, and electron dense materials. / Graduation date: 2005

Graduation date: 2002

Molecular tools, coupled with unique germplasm stocks and rigorous phenotyping, are useful for developing a better understanding of qualitative and quantitative disease resistance genes in plants. The identification of molecular markers linked to all types of resistance genes provides opportunities for implementing a range of resistance breeding strategies, ranging from gene pyramiding to gene deployment. This thesis consists of two chapters. The first describes a disease resistance gene mapping effort and the second describes a disease resistance gene introgression effort. The number, location, and effects of genes determining resistance to stripe rust, leaf rust and Barley Yellow Dwarf Virus were determined using a population of doubled haploid (DH) lines from the cross of Shyri x Galena. Resistance to leaf rust was qualitatively inherited, and the locus was mapped to the long arm of chromosome 1. Resistance to stripe rust and BYDV was quantitatively inherited. Multiple QTLs were detected for each type of resistance. The principal stripe rust resistance QTL was on the short arm of chromosome 5 and the principal BYDV resistance QTL was on the long arm of chromosome 1, linked in repulsion phase with the leaf rust resistance gene. Additional QTLs and QTL x QTL interactions were detected. The majority of the qualitative and quantitative resistance loci detected in the Shyri x Galena population coincided with Resistance Gene Analog Polymorphisms (RGAPs) mapped in the same population. These RGAPs were based on degenerate primers derived from cloned resistance gene sequence motifs. These associations should be useful for efficient resistance gene mapping and provide an approach for ultimately isolating and describing quantitative and qualitative resistance genes. The second chapter describes a molecular marker assisted selection (MMAS) effort to introgress stripe rust resistance QTLs on chromosomes 4 and 7 into susceptible germplasm. DH lines were derived form a MMAS backcross-one (BC-1) population, extensively phenotyped for stripe rust resistance, and genotyped for the introgressed QTLs and background genome. The resistance QTLs that were introgressed were significant determinants of resistance in the new genetic background. Additional resistance QTLs were also detected. Together, these chapters describe an integrated approach to disease resistance gene characterization and utilization. / Graduation date: 1999

The level of polymorphisms of many biochemical and DNA markers are low in cultivated sunflower (Helianthus annuus L.). The number of mapped public DNA markers is limited. Molecular markers have not been developed for the most important diseases of sunflower, such as downy mildew. The objectives of this study were (i) to help alleviate the problem of low DNA marker polymorphisms by developing simple sequence repeat (SSR) markers, (ii) to build an integrated AFLP-RFLP linkage map by using previously described probes and newly developed AFLPs, and (iii) to clone and characterize candidate disease resistance genes. Forty-four polymorphic SSR markers were developed from a genomic DNA library. Diversity analysis of these SSRs for variability among 10 public inbred lines produced an average of 1.86 alleles per locus and mean heterozygosity of 0.21. The number of alleles ranged from 1 to 5. Trinucleotide SSRs were less polymorphic than dinucleotide and mononucleotide SSRs. Cluster analysis and multidimensional scaling separated elite inbred lines from wild species. There was more polymorphism in wild species than in elite lines. Three hundred and six AFLP markers were developed using 18 primer combinations. Two sets of previously mapped RFLP markers were tested for segregation in an F��� mapping population. A total of 401 markers were assigned to 17 linkage groups covering 1326 cM with a mean spacing of 3.3 cM between adjacent markers. The RFLP markers were well spaced and well distributed throughout the genome. Some linkage groups are sparsely populated with common markers. There were two gaps of 30 or more cM in two linkage groups. We cloned candidate disease resistance genes for downy mildew resistance based on sequence homology among resistance genes in other species. Eleven unique nucleotide binding sequence (NBS) containing clones were isolated and showed similarity to the corresponding domains of cloned disease resistance genes in other plant species. Seven clones mapped to four linkage groups and identified nine loci. A cleaved amplified polymorphic sequence (CAPS) marker that was 3.7 cM from the Pl1 resistance gene was developed by analysis of NILs. This CAPS marker should facilitate marker-assisted selection in sunflower. / Graduation date: 1999

Cephalosporium stripe of wheat (Triticum aestivum), caused by the soilborne fungus Cephalosporium gramineum, results in significant yield reductions in dryland winter wheat crops of the U.S. Pacific Northwest. The development of resistant cultivars offers the best hope for disease control. Breeding for resistance is hampered by the long trial times inherent in screening adult plants, and by cultivar x environment interactions in field tests. The principal objective of this research was to develop and test a procedure for screening wheat seedlings in controlled environments for resistance to Cephalosporium stripe. Wheat seedlings were raised hydroponically in growth chambers, and the fungus was increased in large fermentation tanks. The seedlings were inoculated at about 12 days post-germination. Disease severity was assessed approximately seven days later using a chlorophyll meter to measure the symptoms of chlorosis and striping. In three trials, five soft white cultivars from the Pacific Northwest and four hard red cultivars from the Southern Great Plains with known levels of field resistance were tested with a Pacific Northwest fungal isolate. With one exception, chlorophyll readings ordered the cultivars appropriately, with moderately resistant cultivars ranking above susceptible cultivars. Three other moderately resistant cultivars from the Pacific Northwest also appeared in one or two trials, and were ranked properly by chlorophyll level. Chlorophyll levels of uninoculated plants were assayed to determine if differences in chlorophyll content were innate in the cultivars. The chlorophyll levels of uninoculated and inoculated seedling treatments were only significantly correlated when the cultivar Madsen, which ranks high both in resistance and in chlorophyll content, was included. In adult plants, flag-leaf chlorophyll level corresponded to intensity of Cephalosporium stripe symptoms where disease was present, and was independent of known field resistance in undiseased cultivars. The seedling screening technique was used to investigate pathogenic variability in C. gramineum. In two experiments, a total of eight cultivars from the Pacific Northwest and the Southern Great Plains were tested with three fungal isolates from each region. No evidence of virulence/vertical resistance was found. There was also no significant adaptation of isolates to greater virulence on cultivars from the same region. / Graduation date: 1998

Strawbreaker foot-rot is a major limiting factor to cost efficient winter wheat production in the Pacific Northwest. Development of resistant cultivars has been hindered by the lack of adequate levels of genetic resistance and screening techniques which can consistently detect desired genotypes. Studies were conducted to determine if the reported strawbreaker foot-rot resistance of the cultivar "Rendezvous" is effective on isolates of Pseudocercosporella herpotrichoides found in the Pacific Northwest. Protected, naturally infected and artificially inoculated treatments were employed to determine the level of resistance of 10 cultivars including Rendezvous. Different concentrations of inoculum and stages of development were also used to determine if observations on leaf sheath penetration of seedlings obtained in the greenhouse were related to disease severity index readings taken in the field for selected cultivars. In addition, the nature of inheritance of strawbreaker foot-rot was studied in two crosses involving Rendezvous. Experiments were conducted at three locations and over two years at one location. Despite cultivar x treatment interaction, consistent levels of infection were observed in all experiments at each location. Significant differences were found for treatments and cultivars for most attributes. Yield losses, including the components of yield spikes per square meter, 1000 kernel weight, and kernel number per spike were proportional to the severity of the disease. Losses were greater when lodging occurred, which was also associated with disease severity. However, even in the absence of lodging losses were recorded in the naturally and artificially inoculated plots. Traits measured involving Rendezvous and Vpm/Mos 95//*2Hill were only slightly influenced by the treatments. Under greenhouse conditions, it was possible to distinguish the level of resistance of Rendezvous from susceptible cultivars at concentrations of 100 spores/ml, two weeks after inoculation at the seedling stage. Leaf sheath penetration of seedlings was found to be closely associated with the disease severity index obtained under field conditions. Generation means analysis performed in crosses involving Rendezvous indicated that additive and additive x additive gene action were responsible for most of the genetic variability associated with resistance. Narrow-sense heritability estimates also confirmed these fmdings. It would appear that Rendezvous has at least two major genes for resistance to strawbreaker foot-rot. / Graduation date: 1992

This study quantified the frequency of simple versus complex races of Puccinia striiformis Westend. in mixtures of wheat cultivars possessing different race-specific resistance genes. A simple race of a pathogen can infect only one component, and a complex race of the pathogen can infect two or more components of an intraspecific plant mixture. The treatments were designed so that the race that was complex changed depending on the host mixture, thus enabling us to observe the influence of pathogen complexity in different host genetic backgrounds. Six cultivar mixtures and one pure stand of winter wheat were inoculated with three races of P. striiformis (CDL 27, CDL 29, and CDL 41) at two locations for two seasons. Potted plants of three winter wheat cultivars (Paha, Tres, and Tyee) that were each susceptible to one of the three races of the pathogen were used to sample the pathogen during the field epidemics. Disease incidence on the differential cultivars was used to calculate the proportion of the three races in each treatment. The specific cultivars included in the mixtures influenced the frequencies of the three races. Increasing the number of virulent races in a mixture reduced the frequency of the complex race relative to the other two races. When two of the races (races 29 and 41) were complex on the same mixture, location had an effect on which of the races was more frequent. When race 29 was the complex race in the mixture, it was more frequent than when race 41 was the complex race. The results suggest that environmental interactions, genetic background of the pathogen race, host composition, and interaction among pathogen races may be as important in determining race frequencies in mixtures as is stabilizing selection sensu Vanderplank (1968). / Graduation date: 1993

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published_or_final_version / Botany / Master / Master of Philosophy

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