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.

Bitter rot, caused by the fungus <i>Greeneria uvicola</i> (Berk. & Curtis) Punith., is one of the most important fruit rot diseases that threaten the burgeoning winegrape (<i>Vitis vinifera</i> L.) industry in the southeastern United States. Epidemiological studies were conducted to examine the variation in aggressiveness among isolates, period of fruit susceptibility in <i>V. vinifera</i>, relative susceptibility of cultivars to bitter rot, and influence of temperature and duration of wetness on infection. Detached <i>V. vinifera</i> ?Chardonnay? fruit were inoculated with 10 isolates of <i>G. uvicola</i> obtained from fruit of <i>V. vinifera</i>, <i>V. rotundifolia</i> (muscadine grape), and a French-American hybrid. Isolates HCMD1 and HCMD5, obtained from <i>V. vinifera</i> grapes from a Maryland vineyard, were the most aggressive. Severity of disease on fruit inoculated with isolates collected from <i>V. vinifera</i> was significantly higher than with isolates collected from <i>V. rotundifolia</i>. The period of fruit susceptibility was distinguished by inoculating intact clusters of grapes in vineyards in Alamance Co. and Rockingham Co., NC, every 2 weeks from bloom until 2 weeks before harvest. Susceptibility of <i>V. vinifera</i> ?Merlot,? ?Chardonnay,? and ?Cabernet Franc? fruit increased from bloom until véraison in 2003 and from bloom until 2 weeks before véraison in 2004. The relative susceptibility of 38 cultivars and selections, including 23 <i>V. vinifera</i> cultivars and 5 French-American hybrids, was determined by inoculating and incubating detached fruit at 26°C. Fruit of <i>V. vinifera</i> were significantly more susceptible to infection by <i>G. uvicola</i> than French-American hybrids. <i>V. vinifera</i> ?Petite Sirah,? ?JB97-8-0-7,? ?MissBlanc,? ?Roussanne,? ?Mourvédre,? and ?Petit Verdot? were among the most susceptible to the bitter rot pathogen. <i>V. aestivalis</i> ?Cynthiana Norton,? <i>V. vinifera</i> ?Arkansas 1271? and ?Riesling,? and French-American hybrid ?Traminette? and ?Chardonel? were among the most resistant. Growth chamber studies also were conducted to examine the influence of temperature and duration of wetness on infection. Detached fruit of <i>V. vinifera</i> ?Cabernet Sauvignon,? ?Cabernet Franc,? and ?Chardonnay? were inoculated and incubated at 14, 18, 22, 26, or 30°C for 6, 12, 18, or 24 hours. The optimal conditions for infection of fruit by <i>G. uvicola</i> were a temperature of 23.7°C and 9 hours of wetness.

Mannitol is purported to have role in fungi as a storage carbohydrate and has been shown to quench reactive oxygen species (ROS) both in vitro and in vivo. Mannitol metabolism in fungi is thought to occur through the mannitol cycle, which was proposed in the late 1970?s from studies of cell free extracts of the fungus Alternaria alternata. In this cycle, mannitol 1-phosphate 5-dehydrogenase (MPDH; EC 1.1.1.17) reduces fructose 6-phosphate into mannitol 1-phosphate, which is dephosphorylated by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in mannitol and inorganic phosphate. Mannitol also can be made through the enzyme mannitol dehydrogenase (MtDH; EC 1.1.1.138), which reduces fructose to mannitol. Here we report confirmation of these enzymes in the fungus A. alternata, the isolation of the genes, and the generation of strains mutated in MPDH, MtDH, or both genes. PCR confirmed gene replacement and enzyme assays using these mutants showed no activity for MtDH or MPDH. GC-MS analysis showed that double mutants did not produce mannitol, while single mutants had reduced mannitol production. Mannitol, as a quencher of ROS, may also have a role in host-pathogen interactions, by allowing the fungus to suppress ROS-mediated plant defense responses. To assess the contribution of mannitol in plant-pathogen interactions, wild type, single and double mutants were used in pathogenicity assays on tobacco plants. Severity of lesions caused by the MtDH disruptant was not significantly different from that of the wild type. By contrast, the MPDH disruptant and the double mutant caused significantly less disease. Microscopy analysis and histochemical staining for H2O2 showed that both the wild type strain and the double mutant were able to germinate, produced appressoria, and elicited a defense response from the host. Quantitative PCR studies showed that genes for both enzymes were upregulated in the presence of tobacco extracts, with MPDH having a stronger response. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for normal spore germination either in vitro or in planta or for initial infection.

<p>Weather based models help time fungicide applications to the periods when the diseases are most likely to occur. The first objective of this work was to compare and adapt weather-based advisories developed for the control of Cercospora arachidicola on peanuts for resistant cultivars. It was achieved by comparing the disease progress curves of the 1997-1999 growing seasons in Lewiston NC, to spray schedules simulated by the Virginia Advisory, the Parvin, Smith and Crosby Advisory (PSC), NC Advisory, and AU-Pnuts Advisory and their adaptations for resistance. Field trials were conducted in 1997, 1998 and 1999 to test adaptations for resistant genotypes based on the NC Advisory. In all three years the leaf spot epidemics started late in the season (September). There was no yield difference due to leaf spot control except in 1999 in Lewiston for the susceptible genotypes (NC 7 and NC 11). All the advisories had a tendency to overspray at the beginning of the season, this might be due to a lack of inoculum at this time. The resistant genotype used for the study, GP-NC 343, did not lose any yield due to leaf spot in any of the tests and therefore did not need to be sprayed. The model that had the best fit to the disease progress curve of the susceptible genotypes was the AU-Pnuts 12/4. The AU-Pnuts advisory 7/3, currently used in the Southeastern US, started spraying to early in the season for NC. The Virginia advisories also oversprayed. The NC advisory and the PSC were considered almost equivalent, and the adaptations for the PSC did not differ from the PSC itself.The second objective was to develop a simulation model to predict epidemics of Stagonospora nodorum on winter wheat. The CERES-Wheat model was used to simulated leaf area indexes (LAI) for the wheat plant throughout the season. The disease model developed in this work simulated the spread of spores onto the plant leaves and heads, infection, the latent period and, lesion extension. The model equations were inferred from the literature and were calibrated with disease assessments made on Coker 9904 during the spring of 1998 in Plymouth NC. For 1998 and 1999, disease increase in the lower leaves took place 20 days after the disease increase was simulated by the model both years. The most effective spray timing corresponded to a period when disease was first observed in the lower leaves, no disease was seen on the flag leaf, and simulated onset of disease on the flag leaf had occurred. A sharp simulated disease increase in the flag leaf compartment may be a very good indicator for a spray recommendation. Combining a disease model to an already existing crop growth model facilitated modeling disease progress. Further work will be needed to fully validate both the CERES-wheat and the S. nodorum models in North Carolina Coastal Plains.<P>

<p>The United States has experienced a recent boom in pork production. Associated with this growth has been a shift from traditional small family farm units to large confined housing operations. North Carolina, with 9.5 million swine, has been the leader in the development of large, efficient swine operations and is second only to Iowa in pork production. This change has resulted in production of more swine on less land and an increase in animal waste application to adjoining farm land. The repeated application of swine waste may result in increased accumulation of copper and zinc in soils. There is concern that these two metals, which are added to swine feed, will accumulate to phytotoxic levels in agricultural soils. The objective of the research described in this thesis was to investigate the ability of fungi to remove copper and zinc from swine wastewater. The imperfect fungus Aspergillus niger was found to be the most resistant (of six fungi examined) to copper, and the one best able to remove copper from culture media and swine wastewater. A. niger was able to remove as much as 91% of the copper and 70% of the zinc from hog wastewater collected from an aerobic/anaerobic swine waste treatment facility. Interestingly, the majority of the copper and zinc removed by the fungus was by absorption. Absorption of metal by fungi has not been reported as a useful method for bioremediation. These studies show that A. niger is a promising candidate for the removal of copper and zinc from swine wastewater.<P>

<p> Anastomosis group 3 (AG-3) of Rhizoctonia solani is frequently associated with diseases of potato and tobacco. Although isolates from the two hosts are taxonomically related, previous studies have shown differences in their biology, fatty acid composition, pathogenicity and ribosomal DNA. However, the genetic diversity of populations of R. solani AG-3 from potato and tobacco are not known. In this study, the genetic diversity of field populations of R. solani AG-3 from potato and tobacco in North Carolina were examined using somatic compatibility and amplified fragment length polymorphisms (AFLP). A sample of 32 isolates from potato and 36 from tobacco were paired in all possible combinations on PDA plus activated charcoal and glass slides and examined for somatic interactions. Approximately 5% of tobacco isolates and less than 0.5% of potato isolates were somatically compatible. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the potato and tobacco samples, respectively. AFLP analyses indicated that the potato sample of R. solani AG-3 was more genetically diverse (32 AFLP patterns) than the tobacco sample (26 AFLP patterns). None of the potato or tobacco isolates were somatically compatible or shared a common AFLP pattern. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP pattern) were identified only in the tobacco sample. Eight clones of R. solani AG-3 from tobacco represented 22% of the total sample. All eight SCG of R. solani AG-3 from tobacco were associated with more than one AFLP pattern. Compatible interactions between potato isolates only occurred between isolates from the same field (two isolates in each of four different fields), with similar but not identical AFLP pattern. For analysis of the population structure of R. solani AG-3 from potato, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify and differentiate genotypes. A population sample of 104 isolates from five counties in eastern NC was analyzed using polymorphic codominant single-locus PCR-RFLP markers identified after sequencing and screening anonymous DNA from a fungal genomic library. Multilocus genotypes were determined screening isolates using combinations of PCR product/polymorphic enzyme for each locus, generating seven PCR-RFLP markers. There was evidence for high levels of gene flow between populations of R. solani AG-3. The five samples were genetically similar with one another. When data was clone-corrected and samples pooled into one single population from NC, random associations of alleles within and between loci were found for all the loci or pairs of loci, indicating random mating. However, when all genotype were analyzed the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 on potato that includes both recombination and clonality. This study describes the application of a population genetics-based statistical method for detecting migration in populations of R. solani AG-3 from potato using multilocus PCR-RFLP genotypes. The effect of migration from source populations of the pathogen on potato seed tuber on the magnitude of gene flow with a recipient (soil population) in NC was also examined. Analysis of genetic data indicated that the NC population of R. solani AG-3 has experienced recent migration. There was also an indication of high levels of gene flow between the source and the recipient population. Unidirectional migration from source population(s) followed by establishment of migrant genotypes in the recipient population, through colonization, is postulated to explain the high level of gene flow observed.<P>

<p>Fusarium ear rot and fumonisin contamination are serious problems for North Carolina maize growers. With the discovery of fumonisin toxicity to animals and humans, and the finding that no maize genotypes are resistant to Fusarium verticillioides infection or fumonisin contamination, management strategies for limiting fungal and toxin contamination of harvested grain are necessary. Maize ears were harvested weekly for 14 or 15 weeks after pollination and assayed for percent kernel infection and fumonisin contamination. Kernel infection and fumonisin contamination occurred before kernel maturity and increased throughout the season, with kernel infection peaking 7 to 10 weeks after pollination. Data from this experiment and data from grower?s fields indicate that early harvest is necessary to limit rotten kernels and fumonisin in harvested grain.Difficulty in identifying resistant genotypes has limited the development of more resistant hybrids. Many inoculation techniques have been employed to reproduce Fusarium ear rot with marginal results, primarily because differentially resistant and susceptible hybrids were not used to identify promising inoculation techniques. In my study, ears were treated with different inoculation techniques to reproduce ear rot and fumonisin contamination in hybrids of known resistance to Fusarium ear rot. Two inoculation techniques, Pinbar and Silk Channel, were able to separate hybrids on visible ear rot and fumonisin contamination. Addition of inoculum to ears appears important for screening hybrids for resistance to Fusarium ear rot and fumonisin contamination.<P>

Imidacloprid, a neonicotinoid insecticide, is commonly applied on eastern hemlock to reduce populations of Hemlock woolly adelgid (HWA). A large number of other herbivorous and transient insects also are associated with eastern hemlock. These herbivorous insects may acquire imidacloprid through feeding on treated plants. Predatory insects may acquire imidacloprid when they feed on insecticide-contaminated prey. To investigate this phenomenon, a study was conducted at Indian Boundary Campground, Cherokee National Forest, 2005-2007. This study was conducted to: 1) ascertain the effect of imidacloprid used against HWA on the predatory guild associated with eastern hemlock, 2) determine seasonal abundance of the predatory guild on eastern hemlock, and 3) assess influence of vertical stratification on spiders and other predators. During this study, 4,917 predators representing 75 families and 10 orders were collected. Spiders were the most dominant predator group, and the most abundant spider families were Mimetidae (1,038), Salticidae (736), Araneidae (733), Gnaphosidae (517), Philodromidae (330), Theridiidae (168), Tetragnathidae (161) and Thomisidae (142). The most abundant insect predator families were Vespidae (132), Ichneumonidae (50), Braconidae (31), Pentatomidae (25), Reduviidae (24), Coccinellidae (15), and Elateridae (15). Predator densities were not significantly different between pesticide application times (Fall and Spring). In both years, predator densities in control treatments and horticultural oil treatments were significantly (p<0.05) greater than those in imidacloprid treatments. However, predator densities were not significantly (p<0.05) different among soil drench, soil injection, and tree injection treatments or between control and horticultural oil treatments. Predator densities were at least 1.5-3X greater in the imidacloprid-treated plots in 2007 than in 2006, possibly suggesting a rebound in predator densities 1-1½ years after treatment. Predator densities were significantly (p<0.05) greater in the top and middle canopy than in the lower canopy. Imidacloprid concentration level declined progressively from the bottom stratum to the top stratum of the tree canopy. Highest levels were observed in the bottom stratum which shows that higher concentrations of imidacloprid lead to lower numbers of predators and lower concentrations of imidacloprid lead to higher numbers of predators.

Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa and also one of the more common mucosal conditions mostly affecting middle aged individuals. Even though OLP is well investigated the etiology of this disease is still unknown, even if autoimmunity as a possible etiologic factor has been suggested. WHO classifies OLP as a pre malignant condition but malignant transformation of OLP is a matter of great controversy. The p53 protein is a tumour suppressor with the potential to induce apoptosis or cell cycle arrest of DNA damaged cells. Another member of the p53 family, p63, comprises six different isoforms, and plays a crucial role in the formation of oral mucosa, salivary glands, teeth and skin. p63 has also been suggested to be involved in development of squamous cell carcinoma of the head and neck (SCCHN). β-catenin, E-cadherin and epidermal growth factor receptor (EGFR) are p63 related proteins and abnormalities in their expression are suggested to be involved in development of SCCHN. Methods. Using immunohistochemistry and antibodies directed against p53 and those distinguishing between the p63 isoforms we analysed biopsies of OLP, SCCHN and normal oral tissue. We also mapped levels of p63 and p53 isoforms using RT-PCR technique. Furthermore expression of the p63 related proteins β-catenin, E-cadherin and EGFR was studied using immunoblot analysis. In an attempt to investigate autoimmunity as a causative factor of OLP we analysed sera from patients diagnosed with OLP and matched control individuals in order to see if there were autoantibodies directed against the p53 family. Results. When mapping p53 and p63 protein status decreased expression of p63 and increased expression of p53 was seen in OLP compared to normal tissue. In accordance with these results, levels of p63 RNA were also lower in OLP lesions compared with normal tissue. Concerning p53 isoforms, the “original” p53 isoform was expressed in all OLP lesions and normal control tissue. Of the other isoforms, p53β and Δ133p53 were expressed in the majority of samples. Our results regarding p63 related proteins showed a generally lower expression of these proteins in OLP lesions compared to normal control tissue. When studying sera from patients with OLP we found circulating autoantibodies against all six p63 and four p73 isoforms in two patients. Conclusions. The potential for malignant transformation of OLP is still a subject of discussion and rather controversial. While some of our results regarding status of p53 and p63 both at protein and RNA levels support this theory, other results concerning for example p63 related proteins point in the opposite direction. Based on our studies it is thus not possible to either support nor contradict the statement that OLP is a clear-cut premalignant condition. In our effort to understand the etiology of OLP we were the first to demonstrate autoantibodies against p63 and p73 in what could be a subgroup of OLP patients. OLP could thus be suggested to be not one distinct disease, but based on our data a disease comprising different subgroups.