Biological Characterization of the Biocontrol Agent Bacillus amyloliquefaciens CPA-8: The Effect of Temperature, pH and Water Activity on Growth, Susceptibility to Antibiotics and Detection of Enterotoxic Genes.

This work focuses on the biological understanding of the biocontrol agent Bacillus amyloliquefaciens CPA-8 in order to accomplish the characterization required in the registration process for the development of a microorganism-based product. The tolerance of CPA-8 to grow under different pH-temperature and water activity (a w)-temperature conditions was widely demonstrated. Regarding the pH results, optimum growth at the evaluated conditions was observed at 37 °C and pH between 7 and 5. On the contrary, the slowest growth was recorded at 20 °C and pH 4.5. Moreover, the type of solute used to reduce a w had a great influence on the minimum a w at which the bacterium was able to grow. The lowest a w values for CPA-8 growth in media modified with glycerol and glucose were 0.950 and 0.960, respectively. Besides, the lowest a w for CPA-8 growth increased when the temperature decreased to 20 °C, at which CPA-8 was not able to grow at less than 0.990 a w, regardless of the type of solute. Antibiotic susceptibility tests were carried out to determine which antibiotic could affect the behavior of the bacteria and revealed that CPA-8 was clearly resistant to hygromycin. Finally, a PCR amplification assay to detect the presence of enterotoxic genes from Bacillus cereus in CPA-8 was also performed. CPA-8 gave negative results for all the genes tested except for nheA gene, which is not enough for the toxicity expression, suggesting that fruit treated with this antagonist will not be a potential vehicle for foodborne illnesses.

Environmental fate and behaviour of the biocontrol agent Bacillus amyloliquefaciens CPA-8 after preharvest application to stone fruit.

BACKGROUND: Bacillus amyloliquefaciens strain CPA-8 has been described as an effective biocontrol agent to control brown rot in stone fruit for both preharvest and postharvest applications. However, no information about the environmental fate and behaviour of this strain under field conditions is available. RESULTS: The dispersion of the CPA-8 application was evaluated using water-sensitive papers, and complete coverage was observed on the leaves of treated trees, while <1% of non-treated tree leaves had CPA-8. CPA-8 persisted on the fruit of treated trees during preharvest and postharvest conditions, while a significant decrease on leaves and weeds was observed 21 days after treatment. On non-treated trees, CPA-8 was detected on leaves until 180 days after treatment, and on weeds, the CPA-8 population was dependent on the distance from the treated trees. A high persistence of CPA-8 was detected on inert materials, such as clothes and gloves worn by handlers and plastic harvesting boxes. More than 99% of the samples with a CPA-8 phenotype were confirmed as CPA-8 using polymerase chain reaction (PCR). CONCLUSION: This work demonstrated a good distribution, persistence and adaptation of the CPA-8 strain to field and postharvest conditions. Monitoring of dispersion and persistence is an excellent tool to determine the time of application and provides valuable information for registering issues. © 2017 Society of Chemical Industry.