Next-generation phage biocontrol in crop production

According to the World Bank, the global population is expected to reach over 9.6 billion people by 2050, inevitably leading to an increasing food demand by an estimated 59% to 98%. In sharp contrast, emerging pests and pathogens reduce crop yields. In local disease hotspots, these yield losses reach up to 20%. The most significant losses are associated with regions characterized by food deficiencies and fast growing populations. These losses impact both the local household level and entire regions. Among these pests and pathogens bacterial phytopathogens such as Agrobacterium spp., Pseudomonas spp., Xanthomonas spp. have a significant impact. Moreover, control measures in Europe to reduce crop losses caused by these pathogens are limited due to resistance development, their effects on the environment and human health. This has led to the devaluation of chemical bactericides by the European Commission, fueling the demand for sustainable disease control measures that fit into the framework of integrated pest management as currently promoted by the European Union. In this regard, bacteriophages, viruses of bacteria, could be an interesting biocontrol strategy. These lytic viruses parasitize bacteria, killing their host during their replication process with high selectivity. Nevertheless, a major hurdle towards the commercial implementation of phage-based biocontrol strategies concerns aspects of regulation and intellectual property protection. This dissertation elucidates the current state-of-the-art on these IP aspects, based on two datasets which have been compiled to analyze both scientific publications and patent documents using a systematic abstract and claim analysis. A total of 137 papers and 49 patent families were selected from public databases, showing a gradual increase over time. The majority of the patent documents were filed by non-profit organizations in Asia. There seems to be a good correlation between the research papers and patent documents in terms of targeted bacterial genera. Furthermore, granted patents appear to have rather broad claims and cover methods of treatment. In the experimental part of this PhD research, we designed a data-driven platform which allows us to efficiently isolate, characterize and test the applicability of phages, focusing on three distinct pathosystems, inspired by the principles of integrated pest management. Using high-throughput sequencing data, we characterized representative bacterial and phage collections, studied in to the phage-host interaction and finally designed application strategies relevant each individual pathosystem in an attempt to raise the standards of phage biocontrol and gain new insights. The pathosystems used to optimize the platform were crazy roots caused by rhizogenic Agrobacterium biovar 1 species in tomato (Ab1, specialized pathogen), bacterial blight caused by Pseudomonas syringae pv. porri in leek (Pspo, biotroph) and black rot disease in brassica crops caused by Xanthomonas campestris pv. campestris (Xcc, necrotroph). Building on an existing Ab1 bacterial collection, a set of six bacteriophages was isolated and characterized. OLIVR1, OLIVR2 and OLIVR3 belong to the Schitoviridae, while OLIVR4, OLIVR5 and OLIVR6 are members of the Myoviridae. The phages were taxonomically classified as new genera within the different families. A phage cocktail consisting of OLIVR1, OLIVR4 and OLIVR5 infects 75% of the bacterial collection which contains ten genomospecies of the Agrobacterium species complex. By transposon mutagenesis, we demonstrated that the flagellum appears to play a role in the infection of OLIVR1 and OLIVR4. Unfortunately, the receptor of OLIVR5 remains elusive. We evaluated the stability of the phages in hydrogen peroxide as a potential combinatorial approach. OLIVR4 and OLIVR5 showed to be stable in concentrations relevant for today's application. Finally, the efficacy of the phage cocktail was evaluated in greenhouse trials. An infection assay with Agrobacterium strain ST15.13/040 was optimized in greenhouse conditions, showing the development of disease symptoms in Maxifort rootstock. Infection via the roots and nutrient solution seemed both valid strategies to infect the plants. However, when phages were applied using either infection protocol, disease symptoms could not be reduced even though the phages are amplifying in the nutrient solution in presence of the bacterial host and the titers of virulent Agrobacterium were significantly lower compared to the controls. Within the Pspo pathosystem, we built on the bacterial and phage lab collections established at the Laboratory of Gene Technology (PhD, Dr. S. Rombouts). In this study, we characterized the infection strategy of two Pspo phages, KIL3b and KIL5. Both phages recognize lipopolysaccharide (LPS) moieties on the surface of the bacterium, an important pathogenicity factor of Pspo. Our data also suggest that KIL5 requires an additional protein in the bacterial cytoplasmatic membrane to efficiently infect its host. Virulence tests indicate that this protein also contributes to Pspo virulence. Furthermore, a cocktail of both phages was applied in a seed bioassay. A combination of KIL3b and KIL5 reduced the bacterial concentration 100-fold. However, in vitro Pspo resistance against phage infection developed quite rapidly. The impact of this phage resistance might be mitigated as is suggested by the fact that those resistance mutations preferably occur in genes involved in LPS metabolism, and that the virulence of those mutants is possibly reduced. A bioassay based on the natural infection strategy of Pspo further demonstrated the potential of the phage cocktail to prevent infection. These findings suggest that the phage cocktail is promising to reduce the prevalence of Pspo in the environment of plant nurseries. The final research project in this dissertation focuses on Xanthomonas campestris pv. campestris (Xcc), a vascular pathogen that invades the xylem of Brassica crops causing blackrot disease. A representative bacterial collection was sequenced combining Illumina and nanopore sequencing to ensure complete genomes for each strain. A phylogenetic analysis of the core genome shows that the current collection consists of strains from three genomic clusters. We isolated and characterized bacteriophages from two distinct genomic clades and assessed their potential in phage-based biocontrol. The most promising phages, FoX2 and FoX6, specifically recognize (lipo)polysaccharides, associated with the wxc gene cluster, on the surface of the bacterial cell wall. Next, we determined and optimized the applicability of FoX2 and FoX6 in an array of complementary bioassays, ranging from seed decontamination to irrigation- and spray-based applications. While FoX2 showed promising results in each of the bioassays, FoX6 was not able to reduce symptoms when applied during irrigation. In a final proof-of-concept, a CaCl2-formulated phage cocktail was shown to control the outbreak of Xcc in the open field by up to 29%. This comprehensive approach illustrates the potential of phage biocontrol of black rot disease in Brassica and serves as a reference for the broader implementation of phage biocontrol in integrated pest management strategies. We could show that our phage cocktails to tackle Pseudomonas syringae pv. porri and Xanthomonas campestris pv. campestris have potential as biocontrol strategies to prevent disease in leek and cabbage, respectively. Basic understanding of the triangular interaction between phages, bacteria and plants will be critical to launch phage biocontrol to the European market. How phages shape the microbial community, what effect they have on plant health and what effect phage isolates have on the microbial community are important questions to demonstrate the safety of bacteriophages. Targeted research to investigate these environmental effects is necessary and will determine whether phages are indeed low risk products, eventually determining the fate of phages as biocontrol agents.

Jaar van publicatie:2021

Toegankelijkheid:Closed