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.

Anthocyanins are one of the richest pigments, which belong to flavonoid compounds in plant kingdom. They have many biological and ecological functions. Over the past many years, numerous efforts have been made to determine the biosynthetic pathway of anthocyanins and also to identify several regulatory proteins mainly in flowers and fruits of model plants and crop plants. However, many questions concerning the metabolism of anthocyanins in foliage remains unsolved. One example is âHow can developmental processes impact on accumulation patterns of anthocyanins in leavesâ. In this study, we choose several cultivars from one of the most popular ornamental plants Acer palmatum Thunb. to understand the mechanism of developmental changes of pigmentation in leaf. Several other maple species were also analyzed. We propose that the metabolism of anthocyanins play an essential role in such changes. We use an integrated approach of phytochemistry and metabolic profiling to determine the biosynthesis and metabolism of anthocyanins and their impacts on foliage color. Proanthocyanidin analysis was carried out as well to determine their relationship to both anthocyanin production and foliar coloration. We have found that even for green leaves with no/trace amount of detectable anthocyanins, the biosynthetic pathway of anthocyanidin/proanthocyanidin is still activated. Our results indicate that metabolic channeling directing the anthocyanin pathway to the proanthocyanidin biosynthesis plays a very important role in pigmentation pattern change along developmental processes.

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>