Fusarium verticillioides and F. proliferatum are fungal pathogens of maize that cause ear rot and contaminate maize with fumonisin. The first objective was to investigate the relationship between Fusarium ear rot and fumonisin contamination. Two populations, BC1F1:2 families created from the cross of GE440 × FR1064 (GEFR population) and recombinant inbred lines created from the cross of NC300 × B104 (NCB population) were studied. Moderate to high heritabilities and strong genetic correlations between ear rot and fumonisin concentration were estimated and suggest that selection for reduced ear rot should frequently identify low fumonisin lines. Quantitative trait loci (QTL) mapping was then used to study genetic relationships between the two traits and to investigate consistency of QTL across populations. Eight QTL in the GEFR population and five QTL in the NCB population affected both traits. At least three ear rot and two fumonisin contamination QTL mapped to similar positions in the two populations. Two QTL appeared to be consistent for both traits across both populations. To investigate the relationship between resistance and agronomic utility in the GEFR population, yield and agronomic performance were measured in line testcrosses. Correlation and QTL analyses were employed to study these relationships. QTLs identified included 7 yield, 5 grain moisture, 8 plant height, 6 ear height, 3 silk date, and 4 tassel date QTLs. If backcrossing were utilized to move resistance alleles into the FR1064 background, our results suggest that correlated responses would include an increase in grain moisture and decrease in stalk lodging. However, marker-assisted selection may facilitate breaking linkages between resistance alleles and alleles reducing agronomic performance. The second objective was to investigate the resistances to Fusarium and Aspergillus ear rots and fumonisin and aflatoxin contamination in selected lines. Based on the NCB study, the 24 highest and 24 lowest mean fumonisin concentration lines were selected. The low fumonisin group had significantly lower levels of both mycotoxins and ear rots. All four traits were significantly correlated, suggesting that at least some of the genes involved in resistance to ear rots and mycotoxin contamination by these fungal species are identical or genetically linked.

Phylogenetic analysis is a powerful tool for elucidate evolutionary relationships of organisms and genes and for testing taxonomic and evolutionary hypotheses. I conducted phylogenetic analyses of DNA sequences from five gene regions to evaluate the classification scheme of Philadelphus and used phylogenetic analysis to provide a framework for examining molecular evolution of the LFY gene in plants. Results from my study suggested that Philadelphus is a paraphyletic group with the single species genus Carpenteria nested within. Three evolutionary distinct clades were identified in this large Carpenteria-Philadelphus complex, the subgenus Deutzoides clade, the genus Carpenteria clade, and the remaining Core-Philadelphus clade, each merits the recognition of a genus. Our results mostly agreed with the most recent treatment of genus Philadelphus on the placement of Deutzoides, with the exception of P. hirsutus. However, our result does not support the classification scheme proposed for the rest Philadelphus species. Biogeographic analysis using the Statistical Bayes-DIVA method (S-DIVA) and divergence time dating with the BEAST method resolved the origin of Philadelphus s. l. in southwestern North America in the Oligocene. Several intercontinental migrations from North America to Asia and to Europe occurred at the different times of the later Tertiary to reach a worldwide distribution of the genus. For the molecular evolution study of LFY gene, our results revealed that the evolution of LFY was under strong functional constraint, with the C domain under the strongest selection force and the intervening domain being the most relaxed. Our study also showed that the detection of positive selection using the Branch Site Model was robust to taxon sampling density, but sensitive to sequence length and alignment ambiguity. Our analyses under various conditions consistently detected positive selection in Fabaceae, where FLO/LFY evolved a role of the KNOX1 gene function in regulating compound leaf development. Under the best alignment, we detected adaptive selection at several sites in Asterales, Brassicaceae, and Fabaceae where gene duplication and/or novel function of LFY have been reported.

Black shank of tobacco, caused by the oomycete Phytophthora nicotianae, is an important disease of tobacco. Emergence of race 1 has led to loss of major gene resistance and to questions on the genetic diversity in the pathogen in North Carolina. Race 3 is reported for the first time in NC. Race 3 is virulent on plants with the Phl gene and not the Php gene and causes severe disease symptoms on mature plants. Isolates of race 3 were recovered from locations where the Phl gene was deployed and in fields characterized as the wild-type race, race 0, with no history of single-gene resistance. In order to determine whether races 1 and 3 can develop as natural variants from race 0, and to track loss of Php and Phl virulence in races 1 and race 3, soil was infested with one race of P. nicotianae and planted with tobacco varieties with multigenic resistance. Isolates were recovered after five months and screened for race. Additionally, zoospore isolates were derived from progenitor zoospore isolates representing the three races. Zoospore progeny were screened to identify changes in virulence during asexual sporulation. A subset of zoospore progeny was subjected to Fluorescent Amplified Fragment Length Polymorphism analysis to investigate genetic diversity generated through clonal sporulation. Results showed a gain and/or loss of virulence within all race progeny in soil and single-zoospore isolates. Race 1 was the most stable phenotype, with 91% in infested soil and 99.7% of the zoospore progeny retaining the virulence phenotype. The race structure in soil infested with races 0 and 3 were similar after five months. Races were recovered in a 2:1 ratio (race 0: race 3) with a small percentage of race 1. Races 0 and 3 zoospore progeny also segregated. Race 0 progeny were 67% race 0 and 33% gained virulence to the Phl gene (race 3). Similarly, 68% of the race 3-derived progeny retained the parental virulent phenotype, 31% lost the virulent phenotype (race 0), and 1% gained virulence to the Php gene (race 1). Estimates of genetic diversity within each group of related zoospores ranged from 0.17013 to 0.44196. Phenotypic and genotypic investigations revealed that asexual sporulation may be a major source of variation in natural populations. A state-wide survey of P. nicotianae populations was conducted in NC tobacco-producing regions. Isolates were obtained from 76 tobacco fields in 23 counties and screened for race and mating type. Race 1 was predominant in most regions, with 59% of fields consisting of 90 to 100% race 1. The occurrence of race 1 within fields was positively correlated with the history of monogenic resistance deployment. Race 3 was identified in low frequency throughout the state, primarily in wild-type populations where no monogenic resistance was deployed. The A1 and A2 mating types were found throughout NC and were recovered concurrently from multiple fields. Pairings of isolates from within fields yielded viable oospores, indicating for the first time, the potential for sexual reproduction by P. nicotianae. A subset of the survey isolates were screened for sensitivity to the fungicide mefenoxam. All isolates were sensitive, with a mean EC50 value of 0.4 μg/ml mefenoxam, indicating fungicide applications are still a reliable method of black shank management. Results reveal a rapid state-wide shift toward race 1, correlating with the deployment of monogenic resistance and indicate that sexual recombination may be important in generating variation within the pathogen population.

Nitric oxide (NO) and reactive oxygen species are two key components in the induction of the hypersensitive response (HR) during plant defense against pathogen infection. In animal cells, the production of NO is catalyzed by nitric oxide synthase (NOS). Although NOS activity has been documented in plants, the process of NO synthesis in plants is not well understood. Isolation of the NOS protein and/or cloning of the corresponding gene will greatly facilitate the understanding of NO synthesis and its role in plant defense. The objectives of this research were to analyze the physiological and biochemical properties of a NOS-like protein (peaNOS) of pea (Pisum sativum L.), to purify and characterize peaNOS, and to clone the gene(s) encoding peaNOS and relate its expression to NOS activity in pea-bacteria interactions. The application of abiotic agents that induce systemic defense in plants [copper chloride, ActiguardÒ, Triton-X100 and salicylic acid (SA)] to pea leaves did not induce NOS activity and verified reports that NO and NOS function upstream of SA in the signaling pathway of defense responses. Maximum (two-fold) NOS activity was detected three hours before the onset of HR in pea leaves infiltrated with incompatible bacteria (Ralstonia solanacearum), which is consistent with the effect of NO in the activation of HR after interaction with H2O2. The compatible bacteria (Pseudomonas syringae pv pisi) induced NOS activity significantly, suggesting that NO generation may also be a general response to biotic stress in plants. Antibodies raised against mammalian NOS did not have apparent specificity and utility for isolating peaNOS and should be used with caution in non-mammalian systems. The peaNOS protein was most efficiently extracted under alkaline conditions (pH 8.5 and 9.0) as compared to the neutral conditions (pH 7.4-7.5) in animal systems. Precipitation of the peaNOS protein with various concentrations of ammonium sulfate, sodium citrate and sodium chloride caused rapid loss of NOS activity. The peaNOS protein did not bind to 2',5'-ADP-Sepharose and calmodulin (CaM)-agarose indicating that the protein lacks binding sites for NADPH and CaM. Cloning of a peaNOS gene based on mammalian NOS was unsuccessful suggesting that the structure of peaNOS gene may be significantly different from mammalian NOS. Analysis of the Arabidopsis thaliana genome database identified two gene sequences related to animal NOS, i.e., accessions At4g09680 (similar to NOS of Rattus norvegicus) and At3g47450 (similar to NOS of Helix pomatia). Gene At4g09680 is probably not expressed since attempts to clone cDNA of this gene using reverse transcription-polymerase chain reaction (RT-PCR) consistently failed, even when RNA of Arabidopsis was used as a template. A potential expressed peaNOS gene was successfully cloned using RNA template of pea HR tissues in RT-PCR. The 784-bp peaNOS cDNA sequence had 50% nucleotide identity to the At3g47450 coding sequence and had no other significant match in the database. The correlation of the gene expression of P protein of glycine decarboxylase complex (GDC) of pea (peaP) and NOS activity during HR in pea was not demonstrated here but peaP gene was highly expressed concomitant with NOS activity during disease development. The NOS-like protein involved in NO production during HR in pea appears to be more related to At3g47450 sequence, and is possibly encoded partially by the cloned 784-bp pea cDNA.

The tropical savanna-forest boundary is commonly characterized by an abrupt transition in vegetation structure and in tree species composition. It has been hypothesized that differences in architecture between savanna and forest trees have an important role in determining the contrasting structural differences between savanna and forest ecosystems. Because of the importance the vegetation structure in determining the ecosystem properties of these systems, I performed a comparative study of tree architecture to examine differences in plant structure of savanna and forest species. To eliminate the potential bias from phylogenetic relatedness, I used congeneric species pairs containing trees of both habitat types that occur sympatrically in savannas of the Brazilian cerrado habitat at IBGE Ecological Reserve (RECOR). I found that relative to savanna species, forest species have larger crown volumes with more apical meristems and greater height for a given stem diameter. Other traits that influence patterns of light interception also differed, with savanna species exhibiting more convoluted leaf blades and shorter petioles. There was evidence that allometry and other traits are convergent in savanna and forest tree species across lineages, providing strong support for adaptive functions of these traits. Furthermore, the larger canopies of forest species imply that they play a role in reduced light in the understory and the exclusion of grasses, which potentially facilitates further expansion of forest tree species in the absence of fire.

Secondary metabolism in some members of the genus <I>Aspergillus</i> includes the production of aflatoxin, a secondary metabolite that is both immunosuppressive and carcinogenic to animals and humans. <I>A. flavus</i>, in particular, is well known for its ability to contaminate stored food products and to accumulate high levels of aflatoxin in these products. The fungus is also able to infect plants such as corn, cotton, peanuts, and tree nuts long before harvest. Extensive research has been conducted on <I>A. flavus</i> in areas such as population biology, ecology, pathogenicity, genetics, and genomics. The aflatoxin biosynthetic cluster has been sequenced, and researchers have identified regulatory mechanisms involved in the control of aflatoxin production. In spite of the large volume of research in this area, however, many unanswered questions remain concerning the genetic regulation of aflatoxin production and the molecular signals that intimately associate the synthesis of aflatoxin with specific environmental and nutritional conditions. In an effort to identify genes whose expression is affected by growth and aflatoxin production, a wild type strain of <I>A. flavus</i> was compared to a <I>fadA^G42R</i> mutant strain using a cDNA microarray enriched for genes temporally differentially expressed with respect to aflatoxin production. HPLC measurements confirmed that the wild type strain produced high levels of aflatoxin, while the <I>fadA^G42R</i> mutant strain was unable to synthesize any detectable level of the toxin. Hierarchical clustering of differentially expressed genes produced three clusters of genes induced in the wild type strain that contained known aflatoxin genes as well as other genes with no established role in secondary metabolism. A large cluster of genes induced in the <I>fadA^G42R</i> mutant strain included 15 ribosomal genes as well as genes showing homology to an alkyl hydroperoxide reductase (Ahp1p) from <I>Saccharomyces cerevisiae</i> involved in protection against oxidative stress. In order to further characterize this gene in <I>A. flavus</i>, designated <I>ahpA</i>, and its corresponding homolog in <I>A. nidulans</i>, the two genes were overexpressed in a <I>S. cerevisiae AHP1Ä</i> mutant strain. Transformants were tested against a range of concentrations of tert-butyl hydroperoxide (TBHP) to determine whether the genes from <I>A. flavus</i> and <I>A. nidulans</i> could protect against the sensitivity to TBHP observed in the mutant strain. Results indicated that although the protection was not as effective as that conferred by the native <I>AHP1</i> gene, <I>ahpA</i> and the <I>A. nidulans AHP1</i> homolog were able to protect the yeast cells against toxicity induced by exposure to TBHP.

An interdisciplinary approach was utilized to study the toxicity of aluminum (Al) to soilborne plant pathogens with the goal of developing a pathogen-suppressive potting medium containing non-phytotoxic, Al-organic matter complexes. Toxicological studies addressed the toxicity of monomeric Al species to <i>Thielaviopsis basicola</i> and <i>Phytophthora parasitica</i> and documented the sensitivity of these organisms to the metal. Until recently, research on Al-toxicity to fungi has only focused on the trivalent Al cation (Al³⁺) which is also considered the most phytotoxic Al ion. The toxicity of Al-hydrolysis species to fungi were tested by modeling in vitro test solution equilibria using GEOCHEM-PC and correlating the predicted values of Al-species activities with reduction in spore production of the two pathogens. Chlamydospore production of <i>T. basicola</i> was negatively correlated with Al³⁺ activity, whereas inhibition of sporangia production of <i>P. parasitica</i> was related to the activity of multiple monomeric Al species. Toxicity of Al to <i>T. basicola</i> was observed in solutions containing &#8805 20 micromolar Al. Sensitivity of <i>P. parasitica</i> to Al was observed at &#60 1.0 micromolar Al, suggesting that <i>P. parasitica</i> is more sensitive to Al than <i>T. basicola</i>. Using fluorescence microscopy, the localized accumulation of Al in pathogen tissues was detected using lumogallion, an Al-specific, fluorescent stain. Accumulation of Al was observed under various chemical conditions, ranging from salt solutions to more complex systems containing Al-peat complexes. An ecological approach was applied to study the dynamic interactions of soil chemical and physical properties with soil microflora for the suppression of <i>P. parasitica</i> in a medium amended with Al₂(SO₄)₃ and composted swine waste (CSW). Abiotic and biological mechanisms of pathogen suppression were incorporated into the CSW-amended medium. Al-mediated suppression resulted in reduction of sporangia production in medium exhibiting K-exchangeable Al levels &#62 2 micromolar Al. Biological suppression also resulted in reduction of sporangia production and this suppression was maintained after Al levels dropped below the threshold necessary for abiotic suppression. The incorporation of abiotic and biological control mechanisms into a potting media may facilitate suppression of a wide range of soilborne pathogens and enhance applicability of disease-suppressive media in a disease management strategy.

Deployment of tobacco cultivars with single-gene (Ph), complete resistance to race 0 of the tobacco black shank pathogen has resulted in a rapid increase in the occurrence of race 1 in N.C. A four-year cultivar rotation study was conducted in three fields to assess how different levels and types of resistance affected the race structure and population dynamics of the pathogen. In a mixed race field, the high level of partial resistance in ?K 346? was most effective in reducing disease and race 1 populations decreased. The deployment of complete resistance in ?NC 71? resulted in intermediate levels of disease, and race 1 increased. ?K 326?, with a low level of partial resistance, had the highest levels of disease, and race 0 was dominant. In a field where no race 1 was detected initially, disease incidence was high with the use of partial resistance. Complete resistance was very effective in suppressing disease, but race 1 was recovered after only one growing season. By the end of the third growing season, race 1 was recovered from most ?NC 71? treatments. In a field where race 1 was predominant, a high level of partial resistance was most effective in controlling disease and race 0 increased rapidly. A rotation of single-gene resistance and a high level of partial resistance was the most effective rotation for disease management and it minimized race shifts in the pathogen. This may serve to prolong the usefulness of the Ph gene. Populations of race 1 decreased relative to race 0 when cultivars with partial resistance were rotated with complete resistance, suggesting that race 1 isolates are not as fit as race 0 isolates. Experiments were conducted to compare their pathogenic and ecological fitness. Forty isolates of race 0 and 20 isolates of race 1 were used to inoculate tobacco cultivars with low, moderate, and high levels of partial resistance. Race 0 isolates were more aggressive than the race 1 isolates; incubation period was shorter and root rot severity greater with race 0 isolates than with race 1 isolates. Isolates of race 1 caused greater stunting of plants than race 0 isolates. Field microplots were infested with either a single race or an equal mixture of each race. Soil samples were collected and populations determined at the end of each growing season and again the following spring. There were no statistical differences in survival between races, but over both years of the study there was a trend for race 0 to survive better than race 1. One-hundred ninety five isolates of P. parasitica var. nicotianae were subjected to amplified fragment length polymorphism (AFLP) analysis to characterize the genetic diversity among isolates and within pathogen races 0 and 1. Isolates included 20 diverse isolates and an additional 175 isolates obtained over years from a field in Duplin Co., N.C. From all isolates evaluated, 256 of 304 markers (85%) were polymorphic and provided 106 AFLP profiles. The AFLP phenotypes initially detected within each plot were maintained throughout the study but additional phenotypes were recovered over years. At least 6 race 0 and race 1 isolates collected from a single test plot were similar and clustered together in the unweighted pair-group mean analysis phenogram. Examination of the AFLP profiles showed race 0 and race 1 isolates differed by only 2 to 4 markers. Results indicated that P. parasitica var. nicotianae is diverse and that the multiple occurrences of race 1 that were recovered throughout this field over years were independent events where race 1 was selected from within the pathogen population.

Isolates of Glomerella cingulata, Colletotrichum gloeosporioides and C. acutatum, obtained from symptomatic fruit and leaves collected from apple orchards in the US and Brazil, were characterized based on morphological and cultural characteristics, vegetative compatibility groups (VCGs), mtDNA RFLP haplotypes, and the sequence analysis of a 200 bp intron of the glyceraldehyde 3-phosphate dehydrogenase (GDPH) gene. The isolates were also tested for pathogenicity on leaves and fruit. The population structure of the species associated with bitter rot of apples in two orchards of cv. Granny Smith was also studied. Multiple VCGs and mtDNA RFLP haplotypes were found within each of the species tested. Phylogenetic trees constructed based on Neighboring-Joining and Maximum Parsimony methods, using the intron sequence, produced similar topologies. Each species was separated into distinct groups. All isolates tested were pathogenic on fruit. Only isolates with haplotypes G1, G1.1, G3, and G4 and VCGs 1, 4, and 5 were capable of causing Glomerella leaf spot (GLS). G. cingulata was the predominant species associated with bitter rot in the two orchards of cv. Granny Smith. Vegetative compatibility was a better indicator than molecular characterization for distinguishing isolates of G. cingulata pathogenic on both leaves and fruit from the ones pathogenic only on fruit. Isolates of G. cingulata from the US and Brazil which cause GLS were included in different haplotypes and phylogenetic groups. Therefore, our results suggest that isolates of G. cingulata from the US capable of causing both GLS and bitter rot arose independently of Brazilian isolates of G. cingulata, and may have arisen from isolates of G. cingulata from the US that originally were capable of causing bitter rot only. Slower growth, lower optimum growth temperature, and less sensitivity to benomyl distinguished isolates of C. acutatum from isolates of G. cingulata and C. gloeosporioides.

Poor stands are a constraint on organic peanut production because stand losses of 50% or more are possible with untreated seed. Biological, other novel seed treatments, and soil amendments were tested for efficacy against pre- and post-emergence damping-off in greenhouse, microplot, and field plot trials. Seed of the lines Perry, GP-NC 343, and N03081T were utilized in all trials. Nine treatments were tested in natural soil in the greenhouse. Treatments included Bacillus subtilis (Kodiak), B. pumilus (Yield Shield), Trichoderma harzianum (T-22 PB and Plantshield HC), Muscodor albus, Coniothyrium minitans (Contans), activated charcoal, two separate soil amendments of dried herbage of Monarda didyma cultivars, a commercial fungicide check (Vitavax PC), and an untreated control. Vitavax PC and Kodiak were the only seed treatments with higher percentage emergence and survival than untreated seed. A separate greenhouse experiment was conducted for seed treatments using natural soil or soil infested with field isolates of Aspergillus niger. Seed were treated with Kodiak, copper hydroxide (Champion), Plantshield HC, Kodiak and Plantshield HC combined, Streptomyces griseoviridis (Mycostop), hot water, Vitavax PC, or were left untreated. Seedling emergence and survival was much lower in inoculated versus uninoculated plots. In all plots, treatment with Kodiak increased percentage emergence and survival compared to untreated seed, but was not as effective as Vitavax PC. In uninfested plots, treatment with Champion also increased emergence and survival compared to the untreated check. Field microplot studies in Clayton, NC evaluated seed treated with Kodiak, T. harzianum, activated charcoal, Vitavax PC, or untreated seed on the three peanut lines following wheat, oat, or triticale cover crops, soil amendment with M. albus, or no cover as a control. In both years, the percentage emergence and survival was highest for Perry seeds treated with Vitavax PC. Cover crops did not affect emergence, but M. albus treatment suppressed emergence. In field studies at Lewiston, NC, the three peanut lines were treated with M. albus, Kodiak, T. harzianum, or were untreated. In the 2007 trial, none of the treatments improved stands compared to the untreated check. In 2008, the highest stand counts were produced by seed treated with Kodiak. In both years, the largest stands were N03081T. The most commonly observed pathogen was A. niger. Confounding effects of seed line and seed source prevent assessment of performance from individual cultivars. However, regardless of seed line, in most trials Kodiak seed treatment consistently increased emergence and survival over untreated seed.