Oxalotrophy as a biocontrol strategy. New insights in bacterial-fungal interactions
Authors:   , Estoppey Aislinn 
Published: November 2023

Phytopathogenic fungi are a major threat to crops, and cause important economic losses every year. Furthermore, intensive and systematic fungicide application contributes to select resistant pathogenic strains and it is difficult to develop non-susceptible plant cultivars. In order to ensure the future of the agriculture, there is therefore a necessity to develop alternative pathogen control strategies, such as biocontrol. Many fungal phytopathogens use low molecular weight organic acids, and in particular oxalic acid, as a factor of pathogenicity. Oxalic acid is a highly versatile molecule mainly produced by plants and fungi, and ubiquitously found in nature. Numerous bacterial species can consume oxalate, the conjugated base of oxalic acid, as carbon and energy sources, and are thus called oxalotrophic bacteria. Bacterial oxalotrophy induces a strong alkalinisation locally, therefore modifying the physicochemical properties of the environment. Oxalotrophy as a biocontrol strategy has shown a great potential against oxalogenic phytopathogenic fungi such as Botrytis cinerea and Sclerotinia sclerotiorum, two relevant pathogens for important crops. The aim of this thesis was therefore to provide new insights in bacterial-fungal interactions in the case of oxalotrophy as biocontrol strategy. For this purpose, three methods to assess the interactions occurring between fungal resting structures, such as spores or sclerotia, and bacterial cells were developed. We investigated the control of germination of B. cinerea spores by two oxalotrophic soil bacteria, Cupriavidus necator and Cupriavidus oxalaticus, with two different media, diluted malt medium and Reasoner’s 2 medium. The first medium favours fungal growth, while the second was originally developed for bacterial growth. Oxalic acid production by the fungus and growth control by the bacteria was assessed in both media. Mycelial growth control was only efficient in Reasoner’s 2 agar or broth (R2A; R2B), the medium in which B. cinerea produced oxalic acid. Conversly, no oxalic acid was detected in diluted malt broth (MB1/10) and both bacteria were able to control the mycelial growth, but not the germination of B. cinerea spores. The control was attributed to the consumption of oxalic acid, however, as oxalotrophy impacts the physicochemical properties of the environment, the next step of this study was to disentangle oxalic acid consumption from the impact of bacterial development on pH. To determine the effect of the pH change on the germination and the mycelial growth of S. sclerotiorum, a wild type (WT) strain of the pathogen and a mutant deficient in oxalic acid production (Δoah) were confronted to C. necator and C.oxalaticus on the aforementioned media. Growth control was efficient only in R2B, the medium in which oxalic acid was produced by S. sclerotiorum WT. However, S. sclerotiorum Δoah was also controlled on this medium, indicating that oxalic acid consumption was not the only mechanism controlling fungal growth. Further investigations showed that media alkalinisation driven by oxalic acid consumption or additional aspects of bacterial metabolism was responsible for the fungal growth control. Observations by microscopy of the interactions occurring during biocontrol assays also showed that C. necator and C. oxalaticus had very different behaviours towards the fungal mycelium depending of the medium used. In R2B, bacteria were attracted to the fungus, while on MB1/10, they were clearly repulsed. Further experiments showed that the bacteria were not attracted by the spent medium of S. sclerotiorum cultures in R2B and that the attraction seemed to be dependant of a metabolite physically associated to the mycelium. For the repulsion, additional experiments showed that the repulsive metabolite had antibacterial properties and could be retrieved by selective binding to poly-l-lysine. Further studies are still required to determine the nature of the metabolite and its antibacterial potential against other Gram-positive and Gram-negative bacteria. Finally, oxalotrophic endospore-forming bacteria (EFB) were isolated from a soil sample in order to assess their efficiency as biocontrol agents. Several oxalotrophic EFB were isolated. All the isolates were identical at more than 99% based on 16S rRNA gene sequencing and were closely related to Ammoniphilus oxalaticus, an obligate oxalotrophic bacterium. The isolated strain, Ammoniphilus oxalaticus strain HJ, was able to germinate and grow with calcium-oxalate (CaOx), potassium-oxalate (KOx) or oxalic acid as carbon source. However, the bacterium was only able to complete its life cycle (germination, growth, and sporulation) with CaOx. The germination and growth of A. oxalaticus strain HJ spores was also assessed in presence of actively growing mycelium of B. cinerea and S. sclerotiorum, in order to determine if the concentration of oxalic acid produced by these two oxalogenic fungi was sufficient to support the growth of A. oxalaticus strain HJ. Endospore germination and growth of A. oxalaticus strain HJ was observed only in the presence of S. sclerotiorum. Vegetative cells were found to be very active in proximity of the mycelium. Unfortunately, the potential of A. oxalaticus strain HJ as biocontrol agent could not be assessed during the course of this thesis. However, preliminary results showed that S. sclerotiorum growth was slowed down in presence of vegetative cells of A. oxalaticus strain HJ. Still, much remains to be investigated to assess the potential of A. oxalaticus strain HJ as biocontrol agent against oxalogenic phytopathogenic fungi.