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.

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.

Forty-two Phytophthora cinnamomi isolates from Camellia spp., Ilex spp., Juniperus spp., and Rhododendron spp. were characterized for mating type, mefenoxam fungicide sensitivity, and aggressiveness on Rhododendron âHino de Giriâ. Isolates collected from Camellia spp. were of the A1 mating type, while isolates from the other host plants were A2. All isolates were sensitive to mefenoxam at 100 ppm and all but one was sensitive at 1 ppm. Isolates from Rhododendron spp. scored higher average foliar disease and root rot ratings, while A1 isolates from Camellia spp. had the lowest average foliar disease and root rot ratings. The population sample of 42 isolates was also examined for DNA sequence polymorphisms in two nuclear loci, beta-tubulin (Btu) and a portion of the intergenic spacer (IGS) region of the nuclear rDNA repeat, and one mitochondrial DNA locus, cytochrome c oxidase subunit 1 (COX 1). Six base substitutions were found among the 42 isolates with a multi-locus data set. Isolates grouped into four haplotypes. Haplotype grouping corresponded to isolate mating type, plant host, and heterozygosity in the Btu locus. Our inferred multilocus rooted gene genealogy revealed a putative ancestral lineage representing the most frequently sampled haplotype in the population. This haplotype contained A2 isolates collected from Ilex spp., Juniperus spp., and Rhododendron spp.. Isolates of the A1 mating type diverged more recently in the genealogy. There is an increase in heterozygosity at the Btu locus that coincides with the appearance of the A1 mating type. These findings increase our understanding of the population structure of P. cinnamomi.

Foliar nematodes (Aphelenchoides fragariae) infect ornamental crops in greenhouse and nursery production. The objectives of this research were to study A. fragariae population dynamics in a woody ornamental, Lantana camara, during the growing season and during overwintering in a commercial nursery, and to determine the dispersal gradient of A. fragariae in a nursery with overhead irrigation. In the 2006, 2007, and 2008 growing seasons symptomatic, asymptomatic, and defoliated leaf samples were taken throughout a study plot of 30 lantana plants (Lantana camara) infected with foliar nematodes at a commercial nursery in North Carolina. Air temperature, relative humidity, and rainfall data were recorded at the nursery. Over the growing season, nematode densities per gram of fresh weight leaf tissue were low in May and June, and then reached a peak in July, with 122 nematodes/g in July 2006, 406 nematodes/g in July 2007, and 180 nematodes/g in July 2008. Nematode densities decreased over the rest of the summer, except for October 2007 when a second peak occurred. Nematode densities in symptomatic leaves were positively correlated with daily high temperatures and daily low temperatures, while nematode density in asymptomatic leaves were positively correlated to daily low temperatures and relative humidity. Nematode densities in defoliated leaf samples were positively correlated to relative humidity, daily low temperatures, and daily high temperatures. Leaves were also collected during the 2006-7 and 2007-8 overwintering seasons, when the 30 lantana plants were moved to a polyhouse. During overwintering nematode counts remained low in the three different types of leaf tissue, but nematodes were still detected throughout the overwintering season. In 2007 and 2008 a dispersal gradient for foliar nematodes was examined during the summer at a research nursery by spacing healthy plants at a distance of 0 (touching), 30, or 100 cm from an A. fragariae-infected source plant. After 11 weeks in 2007, 100% of the plants at the 0 cm from the inoculum source were infected, while only 10% of the plants at the 30 cm distance and 5% of the plants at the 100 cm distance were infected. In 2008 100% of the plants at the 0 cm spacing became infected after 12 weeks, and 5% of the plants at the 30 cm spacing became infected. No plants at the 100 cm spacing became infected in 2008.

Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato and is one of the world?s most devastating plant diseases. P. infestans left its footprint in human history when, in the 19th century, it was responsible for the Irish Potato Famine. Nuclear and mitochondrial DNA variability was used to examine the population history of P. infestans. DNA sequence data from three nuclear regions (Intron Ras, Ras, and â-tubulin) and two mitochondrial regions (P3 and P4) were obtained from ninety isolates from various locations including Brazil, Bolivia, Ecuador, Peru, Costa Rica, Mexico (Toluca Valley), the USA and Ireland. Population summary statistics show that the Mexican population from the presumed center of origin of P. infestans, harbored less nucleotide and haplotype diversity than South American populations, and was genetically differentiated from other populations, particularly at the mitochondrial loci. Coalescent-based genealogies of mitochondrial (rpl14, rpl5, tRNAs, cox1) and nuclear (Intron Ras+Ras) loci were congruent and demonstrated the existence of two lineages leading to the present day haplotypes of P. infestans associated with potatoes. A third lineage, associated with a group of isolates from Solanum tetrapetalum collected in the Andean Highlands of Ecuador was also found. In the mitochondrial genealogy the two potato lineages corresponded to the mitochrondrial haplotypes Type I and Type II described elsewhere. Mitochondrial haplotypes were associated with different nuclear backgrounds. Haplotypes found in the Toluca Valley population were derived from only one of the two lineages in both mitochondrial and nuclear genealogies, whereas haplotypes found in South American populations (Peru and Ecuador) were derived from both lineages. Haplotypes found in USA and Ireland populations were also derived from both lineages and these populations were not genetically differentiated from the Peruvian populations, suggesting a common ancestry among these populations. Evidence for recombination was found for Mexican and USA populations. Solanum tetrapetalum isolates were highly polymorphic within the regions analyzed and may be a new species. The results support a South American origin of P. infestans and are discussed in relation of previous hypotheses regarding the geographic origin of this plant pathogen.

Root-knot nematodes (Meloidogyne sp.) are sedentary endoparasites that infect roots of a wide range of plant species and cause considerable economic loss to many crops. Root-knot nematodes (RKN) transform selected root vascular cells into enlarged, multinucleate feeding sites called giant-cells that arise from repeated karyokinesis without cytokinesis. Giant-cells undergo extensive modifications of the cell wall architecture including cell wall thickening and the formation of ingrowths that act to increase the surface area of the plasma membrane to facilitate solute uptake by the nematode. Extensive cell division is stimulated around the giant-cells to give rise to the root gall that is characteristic of RKN infection. Extensive cell wall modifications taking place in feeding cells are hypothesized to be mediated by both cell wall-loosening and cell wall biosynthetic enzymes of plant origin based on evidence that nematodes alter gene expression in plants during formation of feeding cells. Ten members of the cellulose synthase (CesA) gene family of Arabidopsis thaliana were analyzed to monitor cell wall deposition in RKN infection sites. CesA gene promoter::GUS constructs and developmental quantitative RT-PCR indicated that CesA genes responsible for both primary and secondary cell wall synthesis were temporally and quantitatively expressed in the same pattern, with peak activity in RKN infection sites at five days post-inoculation. Sections of RKN infection sites in CesA promoter::GUS roots indicated that upregulated secondary cell wall CesA genes were localized within the central giant-cells and primary cell wall CesA genes were primarily localized to the surrounding dividing cells (gall tissue) of the infection site. The number of galls and RKN female development were decreased in Arabidopsis mutants in eight of the CesA genes, and complementation studies with the constitutive 35S promoter restored the mutant phenotypes of CESA4, CESA5, and CESA7 (involved in secondary cell wall synthesis) and also restored normal RKN infection levels. Mutant complementation of the CESA4, CESA5, and CESA7 genes with the giant-cell-inducible NtCel7 promoter had limited effects on mutant plant phenotype and RKN infection rates, but the development of successful infective RKN females was increased dramatically. The combined data support a critical role for plant CESA gene activity working in consort to generate the proper root morphology to promote nematode infection and for the development of feeding cells to support nematode growth and reproduction.