The ecology, species distribution, and population structure of Armillaria was investigated in the forests of Massachusetts. From 64 plots at 16 sites, 640 isolates of Armillaria were collected from six forest types (northern hardwoods, mixed oak, pitch pine, white pine, white pine/mixed oak, and eastern hemlock). Armillaria gallica proved to be the most abundant species, making up 316/640 (52%) of all isolations. This was followed by A. solidipes (219/640; 34%), A. mellea (46/640; 7%), A. calvescens (36/640; 6%), A. gemina (16/640; 3%), and A. sinapina (7/640; 1%). Armillaria gallica was routinely encountered causing significant decay of the lower bole on living hardwood hosts, especially oaks. The population structure of 153 isolates of A. gallica collected from mixed oak forests was investigated using amplified fragment length polymorphisms (AFLPs). From a total sampling area of 4.51 ha, 38 AFLP genotypes were discovered, yielding a figure of eight genets per hectare with the average A. gallica genet occupying 0.13 ha. When the effects of hydrolyzable tannins on in vitro growth were compared between A. calvescens and A. gallica, it was A. gallica that appeared better at oxidizing and metabolizing commercial tannins (tannic acid and gallic acid) along with black oak root bark extracts. This was determined through measurements of colony area and dry biomass, and suggests that A. gallica may be a better adapted pathogen of oak. In order to more accurately distinguish between isolates of A. calvescens and A. gallica, a three-gene phylogeny was reconstructed, using partial sequences of the elongation factor 1-alpha (tef1), RNA polymerase II (rpb2) and nuclear large subunit (nLSU) genes. After comparing 12 isolates each of A. calvescens and A. gallica that originated from across northeastern North America, only the tef1 gene could accurately distinguish these two species. Five single nucleotide polymorphisms were present between the two species and maximum likelihood and maximum parsimony methods grouped A. calvescens and A. gallica into monophyletic clades.

Control of Rhipicephalus microplus is predominantly mediated by the application of acaricides, but the rapid acquisition of resistance by this species and environmental pollution resulting from discarded acaricides, necessitates the discovery of new control measures. Due to the fact that Rhipicephalus spp. are genetically diverse and often have more than one host, it has been difficult to identify a common protective vaccine candidate able to target all species of this genus. Only one anti-tick antigen, Bm86, has been commercialized to date and is sold as GAVAC® and GAVACPlus® in South America. In an attempt to identify protective antigens, a protein termed subolesin was identified using expression library immunisation. RNAi studies showed that subolesin knockdown causes the degeneration of tick guts, salivary glands, reproductive tissues and embryos. Subolesin additionally mediates tick gene expression, impacts the innate immune response and affects tick infection by Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. The R. microplus EF-1α homolog was identified as a subolesin-interacting protein via yeast two-hybrid and co-affinity purification experiments. RNAi experiments have suggested that EF-1α is another possible anti-tick vaccine candidate since it exhibits a similar phenotype as subolesin upon knockdown. The aim of the present research was to express R. microplus EF-1α in the yeast, Pichia pastoris and to exploit the yeast two-hybrid system in an attempt to identify its protein-binding partners. This will provide insight into understanding the translational machinery of this species and of ixodid ticks. Recombinant EF-1α was expressed as a 24 kDa protein, validated by western blotting. A highly representative cDNA library was produced from R. microplus mixed lifestages mRNA, fractionated and cloned into a two-hybrid prey vector. No definitive hits were obtained during the two-hybrid screen of reporter genes, as E-values attained after tblastx and PSI-BLAST analysis were higher than the required limit of 1 x 10-4. / Dissertation (MSc)--University of Pretoria, 2013. / Biochemistry / unrestricted