Challenges in the development of a microbial fungicide based on a strain of Lysobacter capsici

Introduction: Lysobacter capsici AZ78 (AZ78) is a Gram-negative bacterium that effectively controls oomycetes, in particular Plasmopara viticola. Non-spore forming bacteria populations quickly decline when exposed to harsh enviroments as phyllosphere and formulation can play an important role to achieve consistent efficacy in field applications. Limited information is available in literature on formulation of Gram-negative bacteria. Survival and fate in the environment are also important traits to be assessed for the registration as a biopesticide. Objectives: The main aim of this work was to set up and apply a stepwise flow diagram to design performing formulations for AZ78 and follow its fate in the environment (Figure 1). Materials and methods: Cell mass production was maximized in a benchtop fermenter and shelf life of the harvested cells was assessed. We screened compounds capable to protect the bacterial cells against desiccation, UV irradiation and wash-off. We tested the ability of combinations of selected compounds to preserve the efficacy of AZ78 against P. viticola and to establish populations on grapevine. A specific primer pair was developed starting from REP-PCR fingerprinting and subsequently used in a qPCR procedure for monitoring the fate of AZ78 in vineyards. Results: The optimised fermentation protocol gave a harvest of at least 1010 AZ78 cell/ml. Viability of cells decreased only one order of magnitude after one year of storage at 4°C. The use of a combination of polyethyleneglycol, corn steep liquor and lignosulfonate in the formulation improved the survival of AZ78 cells in response to environmental stresses and the efficacy against P. viticola on grapevine in field conditions. Moreover, the qPCR procedure showed that the AZ78 population reached 106 cell/gram of leaf after its application and revealed that the use of additives in the tank mix enhanced the persistence of AZ78 cells in vineyards. Conclusion: The stepwise flow diagram allowed us to achieve high biocontrol efficacy of AZ78 in field conditions and fulfill some of the requirement for the registration of AZ78. The same approach could be extended to other members of the genus Lysobacter for the development of biofungicides.