Functional Genomics of Bacterial and Fungal Biocontrol Agents for Biotechnological Applications
Creators: Rosana, Albert Remus R.
Publisher: name: University of Alberta Library
Year: 2022

<p>Microbial-based biotechnologies were developed using combinations of functional genomics, biochemical tools, and a tripartite pine tree-beetle-fungal infection model. Several bacterial and fungal species were probed for natural product biosynthesis potential using whole genome sequencing while mycoinsecticides formulations were developed for the biocontrol of the epidemic of invasive mountain pine beetle, Dendroctonus ponderosae. <br>
The novelty of six environmental Bacillus isolates were established using whole genome sequencing and comparative genomics. The isolates were phylogenetically classified as Bacillus thuringiensis DNG9, Bacillus velezensis F11, Bacillus cereus E41, Bacillus anthracis F34, Bacillus paralicheniformis F47, and Bacillus licheniformis SMIA-2. Functional genomics revealed an arsenal of gene inventories supporting the production of putative bioactive molecules such as lanthipeptides, sactipeptides, lassopeptides, thiopeptides, and lipopeptides. The antimicrobial potential of the organic extracts of the isolates revealed potent activity against several indicator pathogenic microorganisms, especially relevant members of the WHO’s ESKAPE bacterial group associated with antimicrobial resistance. Targeted antibacterial activity-guided purification and tandem mass spectrometry revealed two fengycin lipopeptides, supported by the fenABCDE operon in B. velezensis F11. Evolutionary comparative genomics further supported the phylogenetic placement of the six isolates in either the B. cereus or B subtilis sensu lato group.<br>
The extremophilic novel actinobacterium, Streptomyces sp. AI-08, was isolated from volcanic soils. The antimicrobial assay of the ethyl acetate extract from spent fermentation medium supports a wide spectrum of antibacterial, antifungal and anti-colorectal cancer activity. The draft genome sequence was established and whole genome sequence-based phylogenetic analysis supported a novel species closely related to Streptomyces olivaceus NRRL B-3009T. Functional genomics revealed a total of 48 biosynthetic gene clusters were predicted which may support the observed biological activity. The speciation of Streptomyces sp. nov. A1-08 was proposed.<br>
Morphological and biochemical characterization of ~93 strains of Beauveria bassiana, representing Canada- and world-wide collections, was established for evaluation of virulence against the mountain pine beetle (MPB). The fungal strains were screened for UV-light resistance, monoterpene tolerance, and desiccation tolerance to account for the major abiotic factors that could potentially limit the efficacy of the fungal species in the environment. The strains were categorized based on virulence factor production, conidial density, and myceliation rate. Although the fungus can colonize other non-target insects, in-vivo honeybee infection model revealed ~5% mortality, representing the natural death rate of the hive population. Conversely, laboratory results indicated 100% killing effect and mycosis against laboratory-reared and field-collected MPBs. <br>
Beauveria bassiana-based mycoinsecticide formulations were tested under relevant in planta and in natura conditions. An industrial level biphasic liquid-solid fermentation was employed to access suitable conidial biomass for large scale MPB infection assays. Greenhouse-based MPB in planta infection assays supported a strain- and concentration-dependent killing and mycosis while in natura field pine forest application significantly reduced the reproductive success of MPB, perturbing the life cycle for at least one season. The mechanism of efficacy of the mycoinsecticides, under in planta and in natura conditions, was probed using functional genomics and in vivo fungal community interaction challenges. This is the first account supporting the efficacy of field application of B. bassiana as a biological control agent of MPB in Western Canada.</p>