Phytophthora capsici is a destructive pathogen of tomato, pepper and cucurbits worldwide. Its genetic variation has a significant impact on disease management. Morphological analysis, fungicide resistance, isozyme analysis, internal transcribed spacers (ITS) and random amplified polymorphic DNA (RAPD) analysis were used to investigate the genetic variation in the genome of Phytophthora capsici from Western Massachusetts, Long Island, New York and Quebec, Canada. Isolates from the first two locations contained both mating types: Al and A2. Only one mating type, A2, was present in Quebec, Canada. The morphological characters used in this study included length and width of sporangia, length of pedicel, length and width of antheridia, and diameters of oospore and oogonia of P. capsici. All morphological characters had significant variation among the three collections. Within each collection, there were significant differences between isolates and between characters. There were no significant differences in resistance to the fungicide metalaxyl among the three collections, but more fungicide resistance was observed in the collection from Massachusetts. Crosses between the most resistant and sensitive isolates were performed. The banding patterns of the isozymes: glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate isomerase (GPI), Malate dehydrogenase (MDH) and Malate dehydrogenase NADP (ME) were examined. The results from isozyme analysis showed no difference among isolates. ITS analysis revealed no difference among isolates, and suggested that the isolates may have a very close genotype. In RAPD analysis, twenty 10-mer random primers were tested and the banding patterns were analyzed with MacClade 3.5.1. The results suggested that (1) isolates of the same mating type tended to cluster together; (2) Massachusetts isolates were spread throughout in tree, suggesting more genetic variation; (3) New York and Canadian isolates were relatively close, implying less variation; (4) progenies of metalaxyl resistant and susceptible isolates tended to cluster with one parental isolate, although some were widely divergent indicating that genetic variation arose from sexual reproduction. Canadian isolates, which lacked sexual reproduction, had less genetic variation, and less metalaxyl resistance. This research demonstrated that DNA recombination during sexual reproduction can generate genetic variation that can lead to increased opportunity of occurrence of fungicide resistance. The RAPD technique was used to develop a Phytophthora capsici specific probe and primers for pathogen identification and detection.

Many plant RNA viruses provide replication and encapsidation functions for one or more subviral RNAs that can modulate the symptoms of the associated helper virus. In this dissertation, I report my studies on symptom modulation in Arabidopsis thaliana by subviral RNAs associated with turnip crinkle virus (TCV), a single-stranded, positive-sense, plant RNA virus. Satellite RNA C (satC) is a virulent satRNA that normally intensifies symptoms of wild-type (wt) TCV but can attenuate symptoms if the TCV coat protein (CP) is either replaced with that of cardamine chlorotic fleck carmovirus (TCV-CPCCFV) (Kong et al., 1995) or if TCV contains an alteration in the CP initiation codon (TCV-CPm) (Kong et al., 1997b). I found that TCV-CPm produced reduced level of a CP (10∼20% of wt level) that contained two additional amino acids at its N terminus and did not form virions in infected protoplasts. SatC did not substantially affect the accumulation of TCV-CPm genomic RNA in protoplasts. These results, along with data reported previously (Kong, 1996), led to the conclusion that satC-mediated symptom attenuation of TCV-CPm involves a reduction in virus long-distance movement (Kong et al., 1997b). By characterizing the promoter for the CP mRNA, i.e., the 1.45-kb sgRNA promoter and defining the sequence and structural elements required for promoter activity, I was able to construct TCV variants expressing a level of wt CP similar to TCV-CPm (10∼20% of wt). I found that these mutants also have their symptoms attenuated by satC, indicating that the level of viral CP, not the mutation in the N terminus, is the crucial factor in determining whether satC is going to attenuate or exacerbate symptoms. Another normally virulent subviral RNA, namely defective interfering RNA G (diG) exhibited different symptom modulation of TCV-CPm compared with satC, and the determinants for this differential symptom modulation were previously localized to the T-terminal 100 bases of the subviral RNAs containing six positional differences (Kong et al., 1997a). In this dissertation I report the further characterization of the determinants in these six positions and the possible mechanism underlying the differential symptom modulation by these two subviral RNAs. My results revealed that two positions located in the 3′-terminal stem-loop structures of satC and diG, which also serve as promoters for complementary strand synthesis, are critical for symptom modulation. Furthermore, the hairpin CP binding capacity correlates with the symptom modulation. Several models for symptom modulation by the subviral RNAs associated with TCV are proposed.

The banana weevil, Cosmopolites sordidus (Germar), is the most important constraint to banana production in East African highlands. For most resource-poor farmers in the region, biological control is the only viable option because it requires little or no cash investment. I investigated patterns of predation by natural enemies of the banana weevil in its presumed native range in Indonesia to determine if there were natural enemies that could be imported to control the pest in Africa. Work was also done in Uganda to determine if predatory insects already present in local banana farming systems could be better conserved to produce banana weevil control. Through farm surveys in Indonesia, I confirmed existing reports that banana weevil damage is low in Sumatra and Java. However, I found no evidence of parasitism from 25,000 eggs and 3600 larvae collected from seven diverse geographical locations. The adult and larvae of the histerid, Plaesius javanus Erichson, were found to be important predators of C. sordidus. P. javanus larva entered tunnels of plants, presumably in search of banana weevil stages. This predator should be imported to Uganda for establishment as a classical biological control agent of the banana weevil. Experiments in Uganda showed that destruction of crop residues in bananas, as recommended for weevil control and practiced by some farmers, reduced predator numbers on farms, reduced predator: prey ratios and had no benefit to the plant with respect to damage prevention. Instead, I demonstrated that residue presence, through maintaining high predator: prey ratios, offsets damage to plants that would otherwise occur from increased numbers of banana weevils. Ants are the major predatory group now present in banana farming systems in Uganda. Surveys in banana farms found 55 species from pitfall traps, 17 from banana pseudostem residues and 34 from banana corms. Eleven species of ants came to banana weevil egg and larvae exposed in the field as baits and thirteen species were tested in the laboratory for their potential to attack the banana weevil. Two species—Pheidole sp. 2 (Myrmicinae) and Odontomachus troglodytes (Santschi) (Ponerinae)—caused significant banana weevil larvae mortality in crop residues and significant egg mortality in living plants in microcosm experiments and in the field.

Tropospheric ozone is considered the most damaging gaseous air pollutant worldwide to which plants are exposed. Tissue injuries, reductions in plant growth and productivity, and changes in crop quality are some of the effects of ozone on plants. Ozone causes more damage to vegetation than all other air pollutants combined. The economic impacts of ozone on crop growth and productivity continue to be significant. Over the past several decades, researchers have focused on quantifying ozone-related damage on plants and establishing methods to explain plants' reaction to ozone. Increasingly, however, it has been shown that plant species react to ozone in many different ways: some are excessively susceptible and their development suffers or is severely reduced; others adapt themselves to new environmental conditions and become more tolerant. In some cases, certain cultivars even show resistance. The studies presented here focus on the impacts of ozone injury on the development of plants with varying degrees of sensitivity to ozone exposure and how growth rate and morphological characteristics affect plant responses to ozone at various concentrations. The degree of plant sensitivity to ozone was determined by comparative field studies, or by using open top field chambers where plants were exposed to ozone at various concentrations. In addition to crop yield evaluations from plants with differences in response to ozone, stomatal conductance was measured as a method of determining potential physiological changes. Stomatal density and aperture size were also assessed. The present studies assess the relationship between morphological characteristics of ozone-sensitive and ozone-tolerant genotypes of bush bean (Phaseolus vulgaris L.) and ozone exposure under different environmental conditions. The results support the expectation that genotypes within same species may show differences in response to ozone. Morphological characteristics, especially that of leaves, seem to influence individual plant responses to various atmospheric treatments.

In this dissertation, I report the functions of the TCV coat protein in TCV viral and subviral RNA replication using in vivo and in vitro systems. In chapter 2, I present my results on construction of a biologically active, full-length cDNA of cardamine chlorotic fleck virus (CCFV), a new carmovirus. In chapter 3, I show that p28 and p88 of TCV viral RdRp components can be expressed in insect cells using a baculovirus expression system. In addition, I demonstrate that p28 and p88 are present in infected plant cells using antiserum against p28 fusion protein expressed in E. coli. In Chapter 4, using a TCV derivative with a deletion of coat protein ORF (TCV$\Delta$CP) and several other TCV derivatives that alter the TCV coat protein ORF, I show that TCV coat protein up-regulates the accumulation of sat-RNA C and DI RNA G, but not sat-RNA D, in protoplasts, suggesting that TCV coat protein plays a role in regulation of subviral RNA replication in vivo. In chapter 5, I show that TCV coat protein functions in TCV viral and subviral RNA transcription and TCV RdRp expression. The immunochemical analysis of cellular proteins obtained from protoplasts infected with TCV$\Delta$CP, TCV, TCV-CP$\rm\sb{CCFV}$ (a TCV derivative containing the CCFV coat protein ORF, or TCV-CPm (a TCV derivative producing low amounts of TCV-related coat protein) suggests that TCV coat protein might be involved in the repression of TCV viral RdRp synthesis and regulation of readthrough of p28, leading to a change in the stoichiometry of p28 and p88. I show evidences that TCV RdRp prepared in the absence of coat protein has template dependency and specificity with small, subviral RNA sized-templates. However, with genomic RNA sized (3-4 kb) templates, no strict template specificity is observed. Using an in vitro RdRp assay system free of coat protein, I demonstrate that TCV coat protein not only enhances the transcription of minus- and plus-strand RNA of sat-RNA C and DI RNA G at low concentrations, but also inhibits the transcription of both strands of sat-RNA C and DI RNA G at higher concentrations. Using sat-RNA D minus-strand template, no inhibition of transcription is observed even in the presence of higher than 10-fold molar ratio of TCV coat protein to template. Similar stimulatory and inhibitory effects of TCV coat protein on the transcription of TCV plus- and minus-strand genomic RNA are observed, and the inhibitory effect is more pronounced during minus-strand synthesis. These in vitro RdRp assay results suggest that the TCV coat protein may act as an important viral factor involved in regulation of viral and subviral RNA replication during virus infection in plants.

Fruit ethylene was manipulated by harvest maturity, 2-dichloroethylphosphonic acid (ethephon), aminoethoxyvinylglycine (AVG), and storage conditions. Advanced maturity or ethephon reduced scald on 'Delicious'. AVG-treated fruit scalded like controls in a commercial room (high ethylene), but developed no scald in a low-ethylene room. Free phenolics were present in wax scraped from apples, and in enzyme-isolated fruit cuticle. Free phenolics were 8 to 45 $\rm\mu g{\cdot}g\sp{-1}$ cuticle. Advanced maturity and ethephon increased, and AVG decreased, cuticular phenolics. They increased during early storage, then remained unchanged. Bound phenolics were 50 to 110 $\rm\mu g\cdot g\sp{-1}$ cuticle, and were unaffected by fruit maturity, AVG or ethephon, remaining constant during storage. In a linoleic acid system, antioxidant activity of standard flavonoids was higher than that of phenolic acid standards. Antioxidant activity of free cuticular phenolics was equivalent to that of flavonoid standards, and activity of bound phenolics approximated phenolic acid standards. Both were lower than diphenylamine and higher than $\alpha$-tocopherol. In hexane, cuticular phenolics inhibited $\alpha$-farnesene oxidation more than diphenylamine. Free cuticular phenolics correlated negatively with conjugated trienes (CT281) and scald. At harvest epidermal and hypodermal cells contained 50-fold higher phenolics than did cuticle, correlating positively with scald. Advanced maturity or ethephon reduced cellular phenolics and scald. AVG increased them, but fruit did not scald in a low-ethylene room. In a linoleic acid system, cuticular lipid-soluble antioxidant activity was 10-15% of total activity. Cuticular lipid-soluble antioxidants did not reduce $\alpha$-farnesene oxidation in hexane. Bagging fruit mid-July until harvest reduced cuticle thickness and phenolic contents, but did not affect ethylene, $\alpha$-farnesene or cellular phenolics. Bagging increased CT281 and scald. Waxing at harvest did not inhibit $\alpha$-farnesene oxidation or reduce scald. Conclusions. Cuticlar free phenolics are relatively stable, protecting $\alpha$-farnesene from oxidation and reducing scald susceptibility. Minimally, four factors are involved in scald development: cuticular $\alpha$-farnesene, phenolics in vacuole, cuticular phenolics, and antioxidants in living cells. The former two are enhancing; the latter two, protective. With low ethylene, fruit synthesize little $\alpha$-farnesene and develop no scald. Ethylene increases $\alpha$-farnesene, stimulates cuticular phenolic accumulation and lipid-soluble antioxidant activity, and reduces free phenolics in vacuoles. The balance determines scalding.

Listronotus maculicollis (Dietz) is a major pest of golf course turf in the northeastern United States. Because the larval stage of this insect causes considerable damage to short-mowed turfgrass, such as tees, greens, fairways, golf course superintendents rely on chemical applications for control. I investigated physiological, ecological, and behavioral characteristics of L. maculicollis to enhance management strategies that will lead to reduced insecticide inputs in golf course turf. Among the cool season turfgrass species, L. maculicollis larvae are reportedly found primarily in Poa annua L., annual bluegrass, among cool-season turfgrass species. To confirm this observation, I conducted a quantitative investigation to determine how abundant L. maculicollis larvae were in P. annua compared with other grasses. I also investigated the influence of mowing height and fertilization on the abundance of larvae. L. maculicollis larvae were present in highly maintained grasses (P. annua and Agrostis palustris Huds., creeping bentgrass) in field studies; no differences in numbers of larvae were detected between P. annua and two types of creeping bentgrass. However, in choice and no-choice tests among five grass types, L. maculicollis were significantly abundant in P. annua. Additionally, one study showed L. maculicollis larvae collected from P. annua weighed more than larvae from other grass types. I found a significant effect of fertilizer application. More L. maculicollis were collected in non-fertilized turf compared to fertilized turf, and more larvae were collected in short-mowed plots than from long-mowed plots. I also investigated adult spring emergence and the distribution of adults and larvae on turfgrass hosts. Golf course superintendents observe primary damage on perimeters of short-mowed areas. In an attempt to corroborate their observations, I examined the distribution of adults and larvae across the width of a golf course fairway, but no differences were detected. Although no more larvae were detected at fairway edges, we determined by visual assessment that the turf on the edge of the fairway was poorer quality, and substandard turf quality is often a result of pest, mechanical, or environmental damage. Therefore, from our results, larval feeding alone does not account for the increased damage in edge areas. I also established that adult L. maculicollis emerge from overwintering sites and walk onto host plants in the spring. These results will be utilized to develop perimeter treatment. (Abstract shortened by UMI.)

The objective of this study was to determine the role of Phomopsis vaccinii in upright dieback disease of cranberry (Vaccinium macrocarpon). Specifically, the goals were to complete Koch's Postulates for P. vaccinii as a pathogen of cranberry, determine infection courts, evaluate the pathogenicity of various isolates of P. vaccinii and isolates of non-P. vaccinii Phomopsis from cranberry and blueberry (Vaccinium corymbosum), and determine which cranberry tissues P. vaccinii infects and colonizes. Koch's Postulates were completed using tissue-cultured and rooted cuttings of two cultivars. It was therefore concluded that P. vaccinii is a causal agent of upright dieback disease. Various infection courts were tested by conducting inoculations of different tissue using different wounding techniques. It was determined that, while non-wounded plants occasionally developed symptoms, stem-pierce wounds resulted in infection of more plants and typically greater tissue death than other wound techniques. A higher percent of plants on which current-year growth was inoculated developed symptoms compared to plants on which 1-yr-old growth was inoculated. It was concluded that current-year growth in spring was the most susceptible growth stage, although plants can be infected throughout the season if wounded. It was observed that only current-year growth was affected when infection occurred in the current-year growth, and infection did not progress to adjacent runners or uprights if the infection occurred in the 1-yr-old growth. It was determined that isolates of P. vaccinii and non- P. vaccinii isolates of Phomopsis could result in symptom development on tissue-cultured cranberry plants and rooted cuttings of cranberry. More P. vaccinii isolates resulted in disease development than other Phomopsis sp. isolates. A few isolates did not result in symptom development on any inoculated plant, or resulted in symptom development on only a low percent of plants. Since these isolates were regularly isolated from symptomless tissue, it is probable that these isolates are non-parasitic endophytes of cranberry plants. P. vaccinii-inoculated tissue-cultured plants were examined microscopically, and P. vaccinii was observed throughout necrotic leaf tissue and in vascular stem tissue. These observations indicate that P. vaccinii is a vascular pathogen. It is expected that the fungus infects succulent growth and progresses from leaf tissue into the stem tissue, or infects through stem wounds, eventually colonizing vascular tissue.

Several members of the soft rot Pectobacteriaceae family such as Pectobacterium brasiliense 1692 (Pb1692) attack a wide range of crops worldwide. P. brasiliense is a major cause of black leg and soft rot disease in several important plants including beetroot, sugarbeet, cabbage, pepper, cucumber, tomato and potatoes. Currently, no chemical control exists to fight the spread of this disease only methods based on avoiding the pathogen. Despite all the research on Pectobacterium species, little is known on how P. brasiliense is able to colonise and cause disease in susceptible hosts. Therefore, as part of advancing knowledge on this organism, the objective of this study was to determine the role of two transcriptional regulators SlyA and PhoP in the biology of Pb1692. In order to better understand the role of the PhoP regulon during the infection process, transcriptional profiles of the wild type Pb1692 strain with its isogenic phoP deletion mutant were analysed using RNA-sequencing and phenotypic assays. Analysis of the transcriptome data demonstrates that the PhoP regulon is substantially broader than previously suspected affecting the differential expression of more than 400 genes. Additionally, the transcriptome data revealed the repression of T6SS and carbapenem genes, two of the most important traits involved in antibacterial competition, indicating that PhoP negatively regulates T6SS and carbapenem in planta. Apart from a role in antibacterial competition, PhoP was shown to play a role in the production of plant cell wall degrading enzymes (PCWDEs), with the repression and activation of some important PCWDEs, PELI/B/Z (pectate lyases) and PEH (polygalacturonase). The broad PhoP regulon included other transcriptional regulators such as EXPR (quorum sensing regulator). In addition to RNA-seq, the study functionally characterized phoP mutants based on virulence in susceptible potato tubers and their ability to outcompete Dickeya dadanti and attenuated motility. Furthermore, by implementing a time-course RT-qPCR analysis, the study revealed the importance of phoP gene in the survival of Pb1692 under acidic conditions encountered in the apoplast. The findings of the RT-qPCR revealed that the relative expression of phoP gene was low in the wild type when conditions are still acidic and gradually increased in expression over time when the environment supposedly becomes alkaline. The data demonstrates that PhoP functions as a global regulator in the survival and adaptation of Pb1692 in planta. Bioinformatics analysis was used to understand the evolutionary history and distribution of SlyA in different Enterobacteriaceae. In this thesis, multiple sequence alignments of SlyA revealed a 90-100% sequence similarity between the different Enterobacteriaceae, indicating that SlyA is conserved among closely related species. Furthermore, the transcriptional profiles of the wild type Pb1692 strain with its isogenic slyA mutant were analysed using RNA-sequencing and phenotypic assays. Findings from this study revealed a number of genes encoding important virulence factors such as, PCWDEs, biofilm formation as well as other important physiological processes, such as oxidative stress response, assimilation of carbohydrates, iron uptake are all SlyA regulated. The transcriptome data identified genes that are either downregulated or upregulated by slyA in planta, suggesting that SlyA activated and/or repressed a number of essential genes to adapt and proliferate in a stressful environment. Thus, highlighting that SlyA plays a key role in the adaptive responses of Pb1692. This study demonstrated that a functional SlyA is essential for full pathogenicity of Pb1692 in planta. Overall, the thesis sheds insights into the regulation of virulence by PhoP and SlyA transcriptional regulators in Pb1692 in planta. / Thesis (PhD (Microbiology))--University of Pretoria, 2020. / Microbiology and Plant Pathology / PhD (Microbiology) / Unrestricted

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