Enzyme-mediated approaches for the biological control of Phytophthora agathidicida

Phytophthora spp. are globally important pathogens of both crop and forest plants. As part of their complex lifecycle, Phytophthora spp. make oospores—long-term survival spores that are key for the success, persistence, and spread of Phytophthora diseases. Oospores are relatively understudied as a research focus and an antimicrobial target. The longevity and treatment-resistant nature of oospores are thought to be due to their thick, complex, and multi-layered cell wall, the components of which represent valuable and underexplored targets for pathogen control. The goal of this research was to explore the potential of cell wall-degrading enzymes, and microorganisms that produce them, as biological control tools against Phytophthora oospores. The causal agent of kauri dieback disease in Aotearoa New Zealand, P. agathidicida, was used as a case study.

In the first part of this thesis, I show that targeting two components of the oospore cell wall (β-1,4-glucan and β-1,3(1,6)-glucan) leads to measurable degradation of oospore cell walls and to changes in oospore cell wall permeability. This confirms the importance of these glucans in oospore cell wall integrity, which was not previously known for P. agathidicida. Based on this knowledge, I then screened environmental bacteria, isolated from a New Zealand green seaweed, for their ability to produce enzymes that target β-1,4-glucan (cellulases) and β-1,3(1,6)-glucan (laminarinases). From this, a cellulase-producer and a laminarinase-producer were chosen for further study.

Next, I bioinformatically identified and biochemically characterized two cellulases (a secreted endo-β-1,4-glucanase, CellA, and an intracellular β-glucosidase, CellB) from one seaweed-derived bacterial isolate (Rhizobium sp. C1). I show that CellA and CellB have significant effects on oospore wall permeability and oospore germination. I also bioinformatically identified and described three candidate glucanases (an endo-β-1,3-glucanase, GlucA, an endo-β-1,4-glucanase, GlucD, and a large exo-β-1,3/1,4-glucanase, GlucE), which may contribute to the degradation of intact oospore walls, from a second seaweed-derived bacterial isolate (Shewanella sp. C6).

Finally, I consider a different cohort of microorganisms—this time soil-dwelling—as producers of β-glucanases and as direct biocontrol agents of Phytophthora. I do this by exploring the relationship between genomic β-glucanase abundance, in vitro β-glucanase activity, and anti-Phytophthora effects, and comparing this with the seaweed-associated bacteria already studied. I show that the genomes of certain soil taxa encode very high numbers of β-glucanases, and that this abundance translates to higher in vitro cellulolytic activity, and to greater antagonism of P. agathidicida. Overall, the work described in this thesis lays the foundations for a biological approach to Phytophthora control, based on the actions of cell wall-degrading enzymes on oospores. The findings provide novel insights into the localization and nature of β-glucans in the walls of live, intact Phytophthora oospores, suggesting that cellulose forms an important structural and protective component of the outer oospore cell wall layers, and that the noncellulosic β1,3(1,6)-glucan is likely protected by cellulose in inner layers. Future work should focus on optimizing wall-associated β-glucan degradation, then on understanding and targeting the other unique components and properties of the oospore cell wall, in order to move toward developing highly effective, and highly specific, anti-Phytophthora tools.