Pearson, Thomas
(2021)
Fungal sex for disease control and strain improvement.
PhD thesis, University of Nottingham.
Abstract
The overarching theme of this thesis was to explore if and how sexual reproduction in fungi could be exploited for disease control and strain improvement. Work focussed on two distantly related members of the Pezizomycotina; Pyrenopeziza brassicae, an important pathogen of Brassica napus (oilseed rape) and other Brassica species; and Penicillium roqueforti, the famous species used for commercial production of blue cheeses.
First, investigations were undertaken to characterise a putative lipid hormonal compound, “Sex Factor” (SF), involved in controlling the switch between asexual and sexual reproduction in Pyrenopeziza brassicae, the causative agent of light leaf spot in Brassica plant species. SF shows promise as a novel disease control agent because of its reported ability to repress asexual sporulation, an important factor in the spread of disease. Crude SF was extracted from mated cultures of P. brassicae and it was confirmed via bioassays that extracts were able to repress asexual sporulation in this species. By contrast, asexual sporulation of North American “Lineage 2” isolates of P. brassicae was unaffected by SF. Biochemical purification work including the use of LC-MS then identified 16-(β-D-Glucopyranosyloxy)-2,15-dihydroxyhexadecanoic acid as a putative candidate for being an active component of crude SF. Finally, progress was made towards identifying key genes which may be involved with regulation of asexual and sexual development. Overall, this work has provided new insights into the asexual and sexual biology of P. brassicae, and laid the groundwork for developing new methods of disease control against this important fungal pathogen.
Secondly, investigations were made to characterise sexual progeny of Penicillium roqueforti. A sexual cycle has only recently been discovered in this species, and therefore it was of academic and of commercial interest to determine whether sex can lead to the production of progeny with novel characteristics relevant to blue cheese production. Four pairs of parental isolates and respective progeny were screened for proteolytic activity and lipolytic activity, and production of certain secondary metabolites. It was found that up to 91 % of progeny had altered proteolytic activities and up to 55 % had altered lipolytic activities, compared to parental isolates. No progeny were found to have novel secondary metabolite production capacity, although significant variation was detected, suggesting novelty may be found with further screening. Thus, it was demonstrated that strains of P. roqueforti with alterations in desirable characteristics can be generated via the sexual cycle. These results may encourage the use of sexual reproduction for strain improvement of this species and other species used in industrial settings.
Third, work was undertaken to characterise lipase genes involved in blue cheese maturation given that the genetic basis of the lipolytic system of P. roqueforti, the most important factor in determining the flavour of blue cheese, was largely unknown. Nine putative lipase genes were identified in silico, five of which were found to be more than two-fold upregulated in model blue cheese conditions. Concurrently, a “laboratory strain” of P. roqueforti (ku70::pyrG) was developed which allowed for more efficient genetic manipulation than would be possible using a wild type strain. This laboratory strain was then used to generate lipase knockout strains which led to the identification of two genes, lipD and lipI, which encode for lipases that likely constitute the main extracellular lipolytic activity of P. roqueforti. Finally, the expression of these key genes was determined in parental isolates and sexual progeny of P. roqueforti. Novel expression profiles were found for lipD, indicating that sexual reproduction can lead to variation in lipolytic activity by alteration of gene expression. The development of a laboratory strain more amenable to genetic manipulation will be of interest to researchers working on this species who can now conduct genetic characterisation studies more easily. In addition, identification of lipD and lipI will allow more specific screening for strains which show desired expression levels of or mutations within these genes, and could therefore be of interest to blue cheese producers.
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