In vitro studies of biofilm-forming Bacillus strains, biocontrol agents isolated from the maize phyllosphere

Abstract

We aimed to assess how biofilm formation by three Bacillus isolates was affected by changes in temperature, water potential, growth media, time, and the combinations between these factors. The strains had been selected as potential biological control agents (BCAs) in earlier studies, and they were identified as B. subtilis and B. velezensis spp. through 16 rRNA sequencing and MALDI-TOF MS. Maize leaves (ML) were used as one of the growth media, since they made it possible to simulate the nutrient content in the maize phyllosphere, from which the bacteria were originally isolated. The strains were able to form biofilm both in ML and biofilm-inducing MSgg after 24, 48, and 72 h. Biofilm development in the form of pellicles and architecturally complex colonies varied morphologically from one strain to another and depended on the conditions mentioned above. In all cases, colonies and pellicles were less complex when both temperature and water potential were lower. Scanning electron microscopy (SEM) revealed that changing levels of complexity in pellicles were correlated with those in colonies. Statistical analyses found that the quantification of biofilm produced by the isolates was influenced by all the conditions tested. In terms of motility (which may contribute to biofilm formation), swimming and swarming were possible for all strains in 0.3 and 0.7% agar, respectively. A more in-depth understanding of how abiotic factors influence biofilm formation can contribute to a more effective use of these biocontrol strains against pathogens in the maize phyllosphere.