Biologically inspired nanoformulations for the control of bacterial canker pathogens Clavibacter michiganensis subsp. michiganensis and subsp. capsici

Clavibacter michiganensis subsp. michiganensis (Cmm) and C. michiganensis subsp. capsici (Cmc) are phytopathogenic bacteria that cause bacterial canker disease in tomatoes and peppers, respectively. Bacterial canker disease poses serious challenges to solanaceous crops, causing significant yield losses and economic costs. Effective management necessitates the development of sustainable control strategies employing nanobiotechnology. In this study, the antibacterial effects of four Aspergillus sojae-mediated nanoformulations, including cobalt oxide nanoparticles (Co₃O₄ NPs), zinc oxide nanoparticles (ZnO NPs), cobalt ferrite nanoparticles (CoFe₂O₄ NPs), and CoFe₂O₄/functionalized multi-walled carbon nanotube (fMWCNT) bionanocomposite, were evaluated against Cmm and Cmc. The diameters of the zone of inhibition of A. sojae-mediated Co₃O₄ NPs, ZnO NPs, CoFe₂O₄ NPs, and CoFe₂O₄/fMWCNT bionanocomposite against Cmm and Cmc were 23.60 mm, 22.09 mm, 27.65 mm, 22.51 mm, and 19.33 mm, 17.66 mm, 21.64 mm, 18.77 mm, respectively. The broth microdilution assay was conducted to determine the minimal inhibitory and bactericidal concentrations. The MICs of Co₃O₄ NPs, ZnO NPs, CoFe₂O₄ NPs, and CoFe₂O₄/fMWCNT bionanocomposite against Cmm were 2.50 mg/mL, 1.25 mg/mL, 2.50 mg/mL, and 2.50 mg/mL, respectively. While, their respective MBCs against Cmm were 5.00 mg/mL, 2.50 mg/mL, 5.00 mg/mL, and 5.00 mg/mL. The respective MICs of Co₃O₄ NPs, ZnO NPs, CoFe₂O₄ NPs, and CoFe₂O₄/fMWCNT bionanocomposite against Cmc were 2.50 mg/mL, 1.25 mg/mL, 5.00 mg/mL, and 5.00 mg/mL. While, their respective MBCs against Cmc were 5.00 mg/mL, 2.50 mg/mL, 10.00 mg/mL, and 10.00 mg/mL. The morphological and ultrastructural changes of Cmm and Cmc cells were observed using field-emission scanning and transmission electron microscopy before and after treatment with sub-minimal inhibitory concentrations of the nanoformulations. Nanoformulation-treated bacterial cells became deformed and disrupted, displaying pits, deep cavities, and groove-like structures. The cell membrane detached from the bacterial cell wall, electron-dense particles accumulated in the cytoplasm, cellular components disintegrated, and the cells were lysed. Direct physical interactions between the prepared nanoformulations with Cmm and Cmc cells might be the major mechanism for their antibacterial potency. Further research is required for the in vivo application of the mycosynthesized nanoformulations as countermeasures to combat bacterial phytopathogens.