In the Northeast, powdery mildew (PM), caused by Uncinula necator (Schewein.) Burrill is one of the most important grape diseases in terms of economic loss. It has been established that cultural practices, including proper sanitation, are the first step in preventing disease, and fungicide sprays are regularly applied to manage the disease. Currently, fungicides that successfully control PM have a strong potential to develop pathogen resistance, and alternatives with low risk of initiating resistance are not as effective in disease control. Our approach to this emerging resistance dilemma is to combine a systemic acquired resistance inducer (salicylic acid and potassium phosphate) with a topical fungicide, potassium bicarbonate. To determine each treatment’s level of efficacy, multiple aspects of infection and defense were quantified and qualified, including germination rate, lethality, lignin formation, callose formation, and vine and leaf growth. The first trial showed potassium bicarbonate and the standard fungicide (Pristine) inhibited the most germination and was most lethal against PM conidia. Potassium phosphate had little effect on germination and conidia death, and when combined with bicarbonate there was no different than the water control. In the second trial, the biorational mixture was able to reduce the level of powdery mildew infection significantly more than the other compounds, including the commercial standard. The salicylic acid and potassium bicarbonate mix may be successful enough to use in the vineyard to determine if the compound can tolerate field conditions with the same level of efficacy.

Nectria haematococca mating population (MP) VI has a cytochrome P450 which confers tolerance to the pea phytoalexin pisatin. This enzyme, termed pisatin demethylase (PDA), detoxifies pisatin and is a virulence factor on pea. PDA is on a 1.6 Mb conditionally dispensable chromosome, and PDA is in a cluster of three other genes involved in pea pathogenicity. Analysis of the PEP cluster suggests it may have been acquired by horizontal gene transfer (HGT). Isolates lacking PDA are still more tolerant of pisatin than other closely related fungi and a "nondegradative" tolerance mechanism was demonstrated previously that might be responsible for this tolerance.ABC transporter, NhABC1, was identified as the gene responsible for this tolerance, fulfilling the major goal of this dissertation. NhABC1 is induced by pisatin and NhABC1 mutants are reduced in virulence on pea to a similar degree as PDA mutants. However, isolates lacking both PDA and NhABC1 are essentially non-pathogenic on pea and are more sensitive to pisatin than either single mutant, demonstrating these two proteins are the major mechanisms responsible for pisatin tolerance. A second ABC transporter in N. haematococca (NhADP1) was also shown to be involved in virulence on pea, however its function in planta remains unknown. The final part of this dissertation concerns a partial analysis of the genome sequence of N. haematococca MPVI. The genome encodes 68 ABC transporters, some of which were in multiple copies when compared to other fungal genomes. This finding led to a whole genome approach to identify extra copies of genes, which are in single copies in the most closely related sequenced fungus, Gibberella zeae. When a comparison between the orthologs found in both genomes and the unique genes found in N. haematococca was made, the results suggest HGT may have shaped the genome of N. haematococca. Several lines of evidence supports this: the large genome size of N. haematococca, the unexpected phylogenetic relationship of the extra copies of genes, the enrichment of the unique genes on dispensable portions of the genome, and a difference between codon usage and GC content of the orthologs versus the unique genes.

Grain crops are often afflicted with a disease commonly referred to as root rot or foot rot. Because root rot diseases are very variable in their symptom expression, they are frequently overlooked as the abnormal condition is often attributed to drought, wind, frost or other causes. For these reasons it is difficult to estimate the damage caused exclusively by root rots. It is generally agreed that the fungus Helminthosporium sativum P.K. & B. is of prime importance in the root rot complex affecting barley and wheat. By plating tests of seed samples of barley and wheat grown in representative areas of Canada, Greaney (1944) showed that the most important pathogen present was H. sativum. Oswald (1950) in California found the four primary pathogenic fungi attacking barley and wheat, in their order of apparent economic importance, to be Helminthosporium sativum, Fusarium roseum f. cerealis, Ophiobolus graminis and Fusarium nivale. Simard and Ludwig (1946) found that, in the years 1942 and 1943, 65 per cent of the Quebec grown barley seed samples examined by them carried H. sativum. The significance or Helminthosporium sativum in the barley and wheat growing areas is indicated by estimates of the damage caused by this fungus. Maohaeek (1943) concluded from the results of a three year survey in Manitoba that barley and wheat yields are reduced by as much as 12 per cent because of root rot.[...]

No description available.

In the eastern U. S. and other wheat growing areas of the world, powdery mildew is a major constraint to the production of wheat. Currently, the most effective and economical means of control is to use resistant cultivars. Unfortunately, the diversity of the pathogen population enables the pathogen to overcome new resistance genes, often within several seasons of commercial deployment. Surveys to determine the virulence of the population to a set of resistance genes have been performed in many areas of the world. Often, differential frequencies of virulence at varying geographic areas are extended to infer a putative population structure. Due to selective pressure, virulence frequencies are poorly suited to high-resolution analysis of population structure and results may reflect regional cultivar choices and not underlying population phylogeny. A collection of 206 single-ascospore derived powdery mildew isoaltes were utilized to determine virulence frequencies at varying locations in the eastern U. S. Primers were developed to amplify coding sequences in order to assess single nucleotide polymorphisms within this population, which allowed allowed analyses of the distribution of presumably neutral genetic variation. Dendrograms based on Nei?s standard genetic distance (Gst) indicated clustering of virulence frequencies into northern and southern subpopulations, with North Carolina as the putative boundary between groups. DNA sequence based AMOVA analysis using groups derived from Hudson?s sequence based subdivision test (Snn) support this conclusion. Tests for population richness indicate greater haplotype diversity in Virginia and Delaware; a result with several possible explanations based on the known history of North American wheat cultivation.

Three root-colonizing fungi, binucleate Rhizoctonia (BNR) isolate BNR621, BNR isolate P9023, and Trichoderma hamatum 382 (T382) were studied for suppression of Botrytis blight in geranium and demonstration of induced systemic resistance (ISR) as a mechanism of biocontrol. Root isolations of the BNR fungi confirmed that BNR621 and P9023 colonized the geranium root system. Root colonization is considered a requirement for biocontrol. Induction of resistance to Botrytis blight was observed in geraniums transplanted into potting mix amended with formulations of P9023 and T382 2 weeks prior to inoculation with B. cinerea when grown under environments either highly or less conducive to disease development. In the less disease-conducive environment, P9023 and T382 provided protection equivalent (P<0.0001) to a weekly rotation of fenhexamid and chlorothalonil fungicides. The effects of cellular and biochemical changes of ISR on germination of conidia of B. cinerea and Botrytis blight lesion area and expansion rates were tested in vitro. No differences in conidia germination were observed between treatments at any sampling time, with a mean germination rate of 80% across all treatments. The size of the leaf lesion area was dependent upon the length of time between topdressing of the geranium plants with the biocontrol agents and detachment of the leaves for inoculation. In geranium leaves detached and inoculated 7 days after topdressing with a Pesta formulation of either BNR621 or P9023, AUDPC calculated from lesion area was reduced (P<0.0001). In contrast, leaves detached and inoculated 14 days after topdressing with a formulation of T382 had a smaller (P<0.0001) AUDPC from lesion area than plants treated with a Pesta formulation of BNR621. Based on the overall suppression of Botrytis blight in geranium and the reduction in lesion size observed in the detached leaf assays, a role for restriction of lesion development is suspected in the control of B. cinerea in geranium. In a comparison study, induction of resistance to Rhizoctonia stem rot in geranium cuttings taken from stock plants treated with formulations of BNR621, P9023, or T382 was evaluated. No consistent control of Rhizoctonia stem rot in geranium cuttings by a biocontrol treatment was observed. However, cuttings taken from geraniums topdressed with a Pesta formulation of BNR621 14 days prior to propagation and inoculation had a greater probability (P=0.03) of rooting and growing out to healthy plants as compared to inoculated cuttings taken from untreated stock plants.

Peronospora tabacina Adam. is the causal agent of blue mold or downy mildew of tobacco. The pathogen is a fungus-like organism and is a member of the Oomycota. P. tabacina is an obligate parasite restricted to the genus Nicotiana spp. Identification of the pathogen is difficult since symptoms and signs generally occur 6-12 days post inoculation. The spread of the pathogen occurs through aerial long distance dispersal of inoculum and severe epidemics occur yearly in tobacco growing areas of the world. One objective of this work was to develop a real-time Taq Man assay for the detection and quantification of P. tabacina. Optimization of the assay was established at a final concentration of 450nM of primers and 125nM of probe. The assay was useful for detection of the pathogen down to a lower limit of 1fg of DNA. The pathogen could be detected after 4 days post inoculation. The real-time PCR assay was useful for the specific detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings and could be used as a tool for regulatory agencies interested in the detection of the pathogen. A second objective was to examine the genetic structure of the pathogen in North America, Central America, the Caribbean and Europe and determine the direction of migration of the pathogen. The intergenic spacer Igs2 region of the nuclear ribosomal DNA (rDNA) and the Ras-related protein (Ypt1) gene, and the mitochondrial cytochrome c oxidase subunit 2 (cox2 gene) were used. Populations of P. tabacina were characterized by high nuclear diversity, low population division and a possible mixed sexual and asexual reproductive system. Large population size, the mechanism of dispersal, mutation rate and genetic diversity indicate that this pathogen is a high evolutionary risk plant pathogen. An isolation with migration (IM) model was used to study genetic diversity in the U.S./Central America and the Caribbean (CCAM) and the European subpopulations. Results support migration from the CCAM region, Florida and Texas into the northern part of the U.S. including North Carolina. These data validate previous migration reports of the pathogen by the North American Plant Disease Forecasting Center at NCSU. In Europe estimates for the migration of the pathogen from North Central to Western Europe and both these regions to Lebanon support migration reports for the first introductions of the pathogen into Europe. Mitochondrial sequences of P tabacina and Hyaloperonspora parasitica genome were generated using bioinforrmatics approaches and PCR methodology. One quarter of the mitochondrial genome of P. tabacina has been annotated and compared with that of Phytophthora infestans and Hyaloperonospora parasitica. Similarities in direction, arrangement and number of genes and regions have been found. Results from this research will be useful in understanding the evolutionary history of the Oomycetes as well as future population genetics research to investigate how these organisms adapt, survive and attack plants in a changing environment.

Cyst nematodes in the genus Heterodera are sedentary endoparasites that induce elaborate feeding cells within host roots by secreting proteins produced within nematode esophageal glands into plant cells. Functional analyses of selected cyst nematode parasitism genes that encode such secreted proteins was the objective of this dissertation. Homologs of four parasitism genes initially isolated from Heterodera glycines, including Hg4F01 (annexin-like protein), HgSYV46 (CLAVATA3-like plant peptide mimic), Hg4E02 and Hg5D08 (novel proteins with putative host nuclear localization) were isolated from Heterodera schachtii, which can infect Arabidopsis thaliana. Greater than 90% nucleotide and predicted amino acid identity existed between the four parasitism genes homologs of H. glycines and H. schachtii. mRNA in situ hybridization and immunolocalization confirmed the expression of each gene product exclusively within the nematode esophageal gland cells. Since eukaryotic annexins affect many cellular processes involving calcium-dependent membrane association, the potential function of the Hs4F01 secreted into plant cells was analyzed. Similar to annexin mutants in Arabidopsis, transgenic Arabidopsis expressing Hs4F01 produced no observable plant phenotype, but were more susceptible to nematode infection. Hypersensitivity to osmotic stress in an Arabidopsis annAt1 annexin mutant was reduced (complemented) in mutants that expressed Hs4F01, suggesting a functional similarity of nematode and plant annexins within plant cells. Host derived RNA interference (RNAi) to silence Hs4F01 transcripts significantly reduced the number of H. schachtii females developing on roots that express dsRNA to Hs4F01. Expression of Hs4E02 and Hs5D08 in Arabidopsis produced no observable plant phenotype and susceptibility to H. schachtii was not altered in plants that expressed Hs4E02. Silencing of HsSYV46 using host-derived RNAi demonstrated a significant reduction in the development of nematode females on Arabidopsis roots that expressed double-stranded RNA to HsSYV46. Expression of dsRNA to Hs4E02 and Hs5D08 in Arabidopsis roots did not affect nematode susceptibility. In summary, parasitism gene products confirmed to have cellular functions similar to their plant homologs, including Hs4F01 (annexin-like protein) and HsSYV46 (CLAVATA/ESR-like peptide) were demonstrated by RNAi to have a significant biological role in cyst nematode parasitism of host plant roots.

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

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