Seeds of the root parasitic plants of the genus Orobanche germinate specifically in response to host-derived germination signals, which enables parasites to detect and attack preferred hosts. The best characterized class of germination stimulants is the strigolactones (SLs), although some species respond to non-SL compounds, such as dehydrocostus lactone (DCL). Recent work indicates that SLs are perceived by members of the KARRIKIN-INSENSITIVE2 (KAI2) gene family, and suggests that within parasitic Orobanchaceae the KAI2 genes have undergone duplication and specialization. The "diverged" clade of these genes, termed KAI2d, has been shown to bind SL germination stimulants in model system assays, but the precise role for KAI2d in regulating germination specificity in a parasitic plant has not been demonstrated. To address this issue, we used genetic and genomic approaches involving two closely related species, Orobanche cernua and O. cumana, which differ primarily in host range and stimulant preference. Orobanche cernua parasitizes tomato (and other Solanaceous crops) and responds to orobanchol, the major SL from tomato roots, whereas O. cumana specifically parasitizes sunflower and responds to DCL. Crosses between O. cernua and O. cumana produced hybrid populations that segregate for stimulant specificity, creating a tractable genetic system. Orobanche cernua contains four KAI2d genes (numbered OrceKAI2d1-4), while O. cumana contains six genes (OrcuKAI2d1-6). The DNA from 94 F2 hybrids was genotyped to identify the KAI2d gene composition and these were correlated with germination phenotype. The pattern of segregation indicated that the KAI2d genes are linked, but pointed to OrceKAI2d2 as a likely orobanchol receptor. Response to DCL was associated with inheritance of all O. cumana KAI2d genes together. Each KAI2d gene was expressed in the Arabidopsis thaliana kai2 mutant background and tested for ability to recover the mutant phenotype when exposed to SLs (including orobanchol, 5-deoxystrigol and GR24) or DCL. One O. cernua gene, OrceKAI2d2, responded to all SLs, but not DCL in this system. No DCL-specific KAI2 genes were identified. In summary, we have identified the likely SL receptor in O. cernua, and show evidence that the DCL receptor is either not a KAI2d protein, or uses KAI2d in combination with other signaling pathway components. / Ph. D.

Macrophomina phaseolina (Mp) and Fusarium oxysporum f. sp. fragariae (Fof) are emerging soilborne pathogens causing crown rot and Fusarium wilt, respectively, in commercial strawberry production in California. Fungicides representing eight active ingredients from four different mode of action groups (FRAC groups 1, 3, 7 and 12) were evaluated for their efficacy against each pathogen in vitro and each disease in planta. Fungicide active ingredients were evaluated for their ability to inhibit mycelial growth of both pathogens in vitro. Half-strength potato dextrose agar was amended with six different concentrations (0.01, 0.1, 1.0, 5.0, 10, 50 µg a.i./ml) of seven fungicides in FRAC groups 3, 7 and 12. Concentrations that inhibited fungal growth by 75% (EC75) compared to unamended media were determined for two different isolates each of Mp and Fof and were used to determine fungicide rates for subsequent in planta studies. Tebuconazole strongly inhibited the mycelial growth of both pathogens (average EC75 for Mp was 2.4 ppm; average EC75 for Fof was 7.48 ppm), as did metconazole (average EC75 for Mp was2.53 ppm; average EC75 for Fof was 1.28 ppm). Fludioxonil strongly inhibited mycelial growth of Mp, but had no impact on the growth of Fof. Penthiopyrad, fluopyram, flutriafol, and flutolanil were less effective at inhibiting fungal growth of either fungus. Greenhouse in planta studies evaluated twenty-four fungicide treatments (eight fungicides at low, med and high rates) that were drench applied to infested potting media two days prior to planting of pathogen susceptible strawberry cultivars (San Andreas for Mp and Monterey for Fof) and again at day 21. Controls were a non-inoculated and an inoculated water-drench treatment. Buried inoculum was recovered at days 2 and 23 and plated on selective media for colony forming unit (CFU) quantification. Plant disease assessments were made each week for 11 weeks. An analysis of variance (ANOVA) of CFUs revealed no significant differences (p > 0.05) among treatments and when compared to the non-treated control for both Mp and Fof, but showed significant decreases (p < 0.05) in CFUs between weeks 1 and 3 for both Mp and Fof. An ANOVA for disease assessments in the form of area under the disease progress curve (AUDPC) showed significant decreases of disease severity in treatments with penthiopyrad only (low, medium and high rates) (p < 0.05). There were no significant differences (p > 0.05) in AUDPC among treatments and when compared to the non-inoculated and no-fungicide controls for Fof. The data indicates that these fungicides used alone are not effective against these pathogens in planta. A strawberry plant extract (germination stimulant) was assessed for its ability to stimulate germination of Mp microsclerotia in vitro and in planta. The germination stimulant was applied as a drench at six different concentrations (0, 10, 100, 1,000, 10,000 and 30,000 ppm) to soil containing filter disk packets of microsclerotia of Mp at day 0 and 14. Filter disk packets were retrieved three days after the drench and microsclerotia were observed microscopically for germination. Results showed that the number of germinating microsclerotia was significantly higher after the application of the germination stimulant compared to non-drench and 0 ppm controls (p < 0.001). An integrated container trial was also conducted using the germination stimulant at 10,000 ppm applied three days prior to a fungicide drench with tebuconazole or thiophanate-methyl to determine the effect of fungicides on the germinated microscleotia. The use of the germination stimulant with label rates of the fungicides lowered the number of germinated intact microsclerotia significantly (p < 0.001) especially after two drench applications. The use of the germination stimulant with fungicides could be investigated further as one method for controlling soilborne diseases of strawberry.

Earnest carbohydrate research was initiated in the nineteenth century by several talented organic chemists. Carbohydrates, now known to play essential roles in a range of fundamental biological processes, are presently studied by a throng of scientists from many fields, including: biochemistry, molecular biology, immunology, structural biology, medicine, agriculture, pharmacology and, of course, chemistry. Organic chemistry remains as relevant to carbohydrate research as it has ever been; its practitioners, with their skills in synthesis and fundamental understanding of molecules, are truly indispensable. This thesis details various synthetic endeavours within the field of carbohydrate chemistry. It describes four projects with goals as diverse as natural product synthesis, rational inhibitor design and the development of new reagents in organic synthesis. The first chapter provides an account of the synthesis of compound 1, a potent germination stimulant present in smoke, from D-xylose. Many analogues of 1 were prepared from carbohydrates and evaluated as germination stimulants, which permitted the dissemination of several structure-activity relationships. Subsequent chapters describe the design and preparation of inhibitors for various carbohydrate-processing enzymes. Compounds 55 and 56 were sought after as putative synergistic inhibitors of a Vitis vinifera (grape) uridine diphospho-glucose:flavonoid 3-O-glucosyltransferase (VvGT1). It was hoped that crystallographic investigations of VvGT1-UDP-2/3 complexes by a collaborator, structural biologist Professor Gideon Davies, would aid in clarifying mechanistic aspects of this enzyme.Compounds 114, 115 and 118 were prepared as putative arabinanase inhibitors. Once again, this work was undertaken to assist in crystallographic studies that might provide a better understanding of how these enzymes operate. The thesis concludes by describing the development of compound 152.HCl, a novel reagent for the diazotransfer reaction. Previously, this reaction utilised trifluoromethanesulfonyl azide (TfN3), an expensive and explosive liquid with a poor shelf-life, to convert a primary amine directly into an azide. Reagent 152.HCl was developed to replace TfN3 in this useful synthetic transformation. A one-pot procedure enabled the simple and inexpensive preparation of 152.HCl, which was demonstrated to be shelf-stable, crystalline and, crucially, effective in the diazotransfer reaction.