Potential of a Microbial Co‐Culture Composed of <i>Bacillus vallismortis</i> <scp>TU</scp> ‐Orga21 and <i>Bacillus subtilis</i> <scp>TU</scp> ‐Orga1 to Improve the Efficacy of Controlling Damping‐Off Caused by <i>Pythium aphanidermatum</i> in Kale

ABSTRACT This research aimed to examine the co‐culture of two Bacillus species in order to enhance damping‐off disease control in kale plants. The microbial co‐culture of Bacillus vallismortis strain TU‐Orga21 and B. subtilis strain TU‐Orga1 significantly inhibited Pythium aphanidermatum strain DOA compared to single strains, reducing mycelium growth by up to 39.26% and causing deformations in hyphal structures. Synchrotron radiation‐based Fourier‐transform infrared (SR‐FTIR) microspectroscopy was used to investigate biochemical changes in the P. aphanidermatum mycelia and showed the lowest polysaccharide content (cellulose and β‐glucans) after being treated with the co‐culture. In addition, the use of matrix‐assisted laser desorption ionisation dual time‐of‐flight tandem mass spectrometry (MALDI‐TOF/TOF MS) and high‐performance liquid chromatography (HPLC) to evaluate lipopeptides of TU‐Orga21, TU‐Orga1 and their co‐culture revealed that interactions between the two microorganisms resulted in an approximately 350% increase in the production of iturin A, which has plant pathogen‐inhibiting ability. Additionally, both strains produce surfactin, with quantifiable levels detected in all treatments, further supporting their potential as a biocontrol consortium. A greenhouse study confirmed that treating kale plants with the co‐culture, applied twice before P. aphanidermatum inoculation, effectively controlled damping‐off, achieving results equal to metalaxyl treatment, with a 100% reduction in disease incidence, significantly more effective than using either strain alone. Moreover, the application of the co‐culture to kale plants promoted greater canopy width and plant height compared to the positive and negative controls. Together, these findings indicate that the co‐culture of two beneficial bacteria can be used to enhance the production of anti‐oomycete lipopeptides and promote plant growth for enhanced biological control.