Isolation, bioformulation of bacteriophages specific to bacterial cancer and wilth (Clavibacter michiganensis subsp. michiganenis) disease from tomato production areas and investigation of their potential to use as biocontrol agent

Clavibacter michiganensis subsp. michiganensis (Cmm) is a bacterial wilt and canker pathogen that causes yield losses in tomato cultivation. The inadequacy of existing methods to control the pathogen has increased interest in phage control. For this purpose, soil and plant samples were collected from tomato growing areas with symptoms of the disease in the provinces of Canakkale, Izmir and Bilecik. Cmm isolations and phage isolations were performed from the collected samples using biochemical and molecular methods. Phages with high tolerance to abiotic factors were identified and their synergistic-antagonistic properties were determined. Molecular and morphological characterisation of the phages whose activity was determined was performed and their potential for use as biocontrol agents was investigated. The oil-in-water formulation of the phage was optimised using the Taguchi method. An oil-in-water formulation was prepared with the optimal conditions determined and the inhibition rate on Cmm-DB3 was determined in vitro. The efficacy of the formulation was verified by pot experiments. With the study performed, 22 lytic phages were obtained and their activity on 8 Cmm isolates and Cmm-DB3 was determined. The complete genome analysis of phage33, which has a high tolerance to abiotic factors, was compared with the phages in the NCBI database and it was found that there was no match on a species basis. An emulsion formulation of Phage33 was prepared under optimal conditions, and the inhibition rate of the emulsion on Cmm-DB3 was determined to be 80.7% in vitro. At the end of the 6th month, the Cmm-DB3 inhibition rate of the emulsion was determined to be 78.12% at room temperature and 79.23% at 4 °C. Furthermore, when analysing the droplet size, it was found that the emulsion had a polydisperse structure. As a result of the pot trials with the prepared emulsion, it was found that the disease symptoms were 78% in the pots where Cmm was applied and 17% in the pots where the emulsion was applied. In order to increase the tolerance of Phage33 to abiotic factors, the emulsion formulation was optimised for the first time using the Taguchi method and a local prototype was introduced that can be used in against the pathogen.