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.

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.

(+)-Pisatin, ([+]-[6aR,11aR]-6a-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan) is the major phytoalexin of the garden pea (Pisum sativum L.). Despite being the first phytoalexin to be chemically characterized, its biosynthesis remains to be fully elucidated. RNA-mediated genetic interference (RNAi) was used to gain further insights into the (+)-pisatin biosynthetic pathway. The expression of three genes, isoflavone reductase (IFR) catalyzing the reduction of 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone to (-)-sophorol, sophorol reductase (SOR) involved in reducing (-)-sophorol to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol and hydroxymaackiain-3-O methyltransferase (HMM) involved in methylation of (+)-6a-hydroxymaackiain to (+)-pisatin was silenced. The genes are transcriptionally co-regulated during (+)-pisatin biosynthesis, with the IFR and SOR proposed to function upstream of the HMM gene.Hairy roots expressing the HMM RNAi construct, deficient in (+)-pisatin biosynthesis were identified. However, these did not accumulate (+)-6a-hydroxymaackiain, precursor to (+)-pisatin. Instead they accumulated 2,7,4'-trihydroxyisoflavanone, daidzein, liquiritigenin and isoformononetin. The amino acid sequence of HMM is very similar to that of another methyltransferase, hydroxyisoflavanone-4'-O-methyltransferase (HI4MOT), found in most legumes. HI4?MOT catalyzes the methylation of 2,7,4'-trihydroxyisoflavanone (THI) to 2,7-dihydroxy-4'-methoxyisoflavanone, one of the earliest enzymatic steps in isoflavonoid biosynthesis. In pea, HI4OMT may be the same enzyme as "HMM" catalyzing the methylation of both THI and (+)-6a-hydroxymaackiain. Preventing the methylation of THI could divert pea intermediates to the production of daidzein and isoformononetin instead of (+)-pisatin.None of the transgenic hairy roots expressing the IFR RNAi construct were totally deficient in (+)-pisatin biosynthesis. However, all produced reduced amounts of (+)-pisatin, with one culture accumulating 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone, the substrate for IFR. Hairy roots expressing the SOR RNAi construct deficient in (+)-pisatin biosynthesis were identified. These accumulated (-)-sophorol, the substrate for SOR. These data provide evidence for the involvement of these genes and the intermediates with (-)-optical activity in (+)-pisatin biosynthesis.The elicitation of the biosynthesis of secondary metabolites in plant cell and tissue cultures by electric current was explored. Electric current was demonstrated to elicit the biosynthesis of secondary metabolites in pea hairy and intact roots, seedling, root and cell suspension cultures of various plant species. Electric current has potential for use as an elicitor of secondary metabolites in basic and commercial research ventures.

The nature of host susceptibility to phytopathogenic parasites, especially the obligates, remains one of the major problems in plant pathology even though it has been investigated extensively from the early part of the present century until the late thirties. Studies on the genetics of the pathogens and breeding for resistant varieties have predominated since that time. There is at present a revival of interest in the physiology of disease resistance. [...]