Plant pathology

Methylobacterium spp., a dominant and functionally conserved group of plant-associated bacteria, have long been recognized for their roles in promoting host growth, stress tolerance, and phytohormone modulation. This body of work collectively repositions Methylobacterium not only as a plant growth-promoting genus but also as a promising agent of microbiome-mediated crop protection. Across several investigations, the ecological, biochemical, and functional attributes that underpin this potential were examined, with specific focus on hormone production, compatibility with agrochemical inputs, and antifungal activity.

A comprehensive inventory of 46 Methylobacterium strains revealed widespread production of cytokinins – including highly active forms such as trans-zeatin – and variable capacities to synthesize indole-3-acetic acid. Cytokinin output increased under carbon-limiting conditions, highlighting the genus's adaptive hormonal response. Parallel investigations demonstrated that commercial glyphosate-based herbicide formulations significantly inhibited the growth of most Methylobacterium strains, whereas pure glyphosate alone showed negligible toxicity. Key findings of experiments indicate that non-active formulation components participate in the disruption of beneficial bacteria by facilitating higher intracellular glyphosate concentrations and subsequent toxic effects. This introduces a novel link between agrichemical formulation practices and the selective disruption of keystone microbial taxa.

Contrastingly, fungicide compatibility testing showed that Methylobacterium strains tolerate key fungicides such as azoxystrobin, fludioxonil, and metalaxyl-M, supporting their inclusion in integrated pest management frameworks. Subsequent functional antagonism assays further revealed that specific Methylobacterium isolates inhibit phytopathogenic Fusarium species in vitro and in planta. Notably, M. organophilum enhanced soybean seedling vigor and reduced disease severity when co-inoculated with F. graminearum by preserving the integrity of the seed coat, demonstrating protective activity with unique mechanics.

Finally, differential hormone profiling at the pathogen-antagonist interface revealed that biocontrol-effective Methylobacterium strains not only produce higher levels of auxin and salicylic acid but also induces jasmonic acid production – likely derived from Fusarium – suggesting complex cross-signalling and interference with fungal development and sensing pathways. Together, these findings advance our understanding of Methylobacterium as a keystone genus in the phytobiome, capable of contributing to both plant vigor and pathogen suppression and reinforce its relevance in the design of next-generation biocontrol strategies.

Author Keywords: agrochemical interactions, biological control, Fusarium antagonism, Methylobacterium, phytobiome, phytohormone signalling

Cytokinin biosynthesis in organisms aside from plant species has often been viewed as a byproduct of tRNA degradation. Recent evidence suggests that these tRNA degradation products may actually have a role in the development of these organisms, particularly fungi. This thesis examines the importance of cytokinins, a group of phytohormones involved in plant cell division and differentiation as well as the phytohormone abscisic acid, involved in plant response to environmental factors, and their presence and role in fungi.

An initial survey was conducted on 20 temperate forest fungi of differing nutritional modes. Using HPLC-ESI MS/MS, cytokinin and abscisic acid were detected in all fungi regardless of their mode of nutrition or phylogeny. The detection of the same seven CKs across all fungi suggested the existence of a common CK biosynthetic pathway and dominance of the tRNA pathway in fungi. Further, the corn smut fungus Ustilago maydis is capable of producing CKs separate from its host and different U. maydis strains induce disease symptoms of differing severity. To determine if CK production during infection alters disease development a disease time course was conducted on cob tissue infected with U. maydis dikaryotic and solopathogenic strains. Dramatic changes in phytohormones including an increase in ABA followed by increases in cisZCKs were detected in tumour tissue particularity in the more virulent dikaryon infection, suggesting a role for CKs in strain virulence. Mining of the U. maydis genome identified a sole tRNA-isopentenyltransferase, a key enzyme in CK biosynthesis. Targeted gene deletion mutants were created in U. maydis which halted U. maydis CK production and decreased pathogenesis and virulence in seedling and cob infections. CK and ABA profiling carried out during disease development found that key changes in these hormones were not found in deletion mutant infections and cob tumour development was severely impaired. These findings suggested that U. maydis CK production is necessary for tumour development in this pathosystem. The research presented in this thesis highlights the importance of fungal CKs, outlines the dominant CK pathway in fungi, identifies a key enzyme in U. maydis CK biosynthesis and reveals the necessity of CK production by U. maydis in the development of cob tumours.

Author Keywords: abscisic acid, cytokinins, high performance liquid chromatography-electrospray ionization tandem mass spectrometry, tRNA degradation pathway, Ustilago maydis, Zea mays

The emergence of fungal hybrid pathogens threatens sustainable crop production worldwide. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they infect a common host (Zea mays), can hybridize, and tools are available for their analysis. Hybrid dikaryons exhibited filamentous growth on plates but reduced virulence and limited colonization in Z. mays. Select virulence genes in the hybrid had similar transcript levels on plates and altered levels during infection of Z. mays relative to each parental dikaryon. Virulence genes were constitutively expressed in the hybrid to determine if its pathogenic development could be influenced. Little impact was observed in hybrids with increased expression of effectors known to modify host response and metabolism. However, increased expression of transcriptional regulators of stage specific pathogenic development increased the hybrid's capacity to induce symptoms. These results establish a base for investigating molecular aspects of fungal hybrid pathogen emergence.

Author Keywords: effectors, hybrid pathogenesis assays, Sporisorium reilianum, transcription factors, Ustilago maydis, virulence factors