Mode of action of Bacillus subtilis ATCC 55466 as biocontrol agent of postharvest diseases of avocados

Abstract

Avocados are an economically important crop in South Africa and are mainly exported to Europe. As with any other tropical and subtropical crop, avocados are prone to pre- and postharvest diseases. Until recently, chemical control was the only effective measure to control fungal avocado pathogens In 1987, a Bacillus subtilis isolate was found that showed promise as a biocontrol agent in both pre- and postharvest applications to control postharvest diseases. However, over time variable results has been obtained in semi commercial trials. From the original B. subtilis isolate several subcultures have been made and used over a 15 year period in various experimental trials. The dual culture technique was used to compare the biocontrol activity of the subcultures against postharvest pathogens (Colletotrichum gloeosporioides, Phomopsis perseae, Dothiorella aromatica and Lasiodiplodia theobromae). The subcultures differed significantly in their effectiveness and genetic stability. No difference between the subcultures could be found when DNA fingerprinting using RISA PCR was used. The most effective subculture, MI-14, was used in further studies. The mode of action employed by a biocontrol agent is of utmost importance and can be used to enhance its efficacy. In a previous study it was hypothesized that antibiosis as well as competition for nutrients and space is the modes of action involved in biocontrol of B. subtilis against postharvest pathogens of avocado. The direct interaction between B. subtilis and C. gloeosporioides on avocado fruit were observed using scanning electron microscopy. Cells of B. subtilis were observed to colonize the hyphae of C gloeosporioides. In some instances, hyphal walls were lysed in the presence of B. subtilis and may be due to the presence of enzymes or antibiotic substances. Conidia of C. gloeosporioides did not germinate in the presence of B. subtilis. Diffusible inhibitory metabolites active against C. gloeosporioides were produced in vitro by B. subtilis. Inhibitory volatile substances were also produced by B. subtilis and were found to be active against P. perseae, D. aromatica and L. theobromae but not C. gloeosporioides. Siderophores production as well as chitinase, amylase, lipase and proteinase activity were also observed and may play a role in antagonism. Antibiotic production by B. subtilis is a well-known phenomenon. Most antibiotics are polypeptides and lipopeptides. The involvement of phenolic metabilites in biocontrol by B subtilis is less known. A seven-day-old culture of B. subtilis in a minimal medium was analyzed for the presence of free acid phenolic compounds active against fungi. Free acid phenolic metabolites were found and separated using layer chromatography. TLC plates containing the separated spots were sprayed with Clasdosporium cladosporioides and plates were observed for inhibition zones. The phenolic substances were present at 7.06 ± 0.95 mg gallic acid ml-1. The phenolic substances fall in the hydroxycinamic acid group due to their fluorescent coloring under UV at 350 nm. The mode of action involved is also influenced by environmental factors. The effect of temperature and carbon- and nitrogen sources of the in vitro inhibitory activity of B. subtilis against C. gloeosporioides, P. perseae, D. aromatica and L. theobromae were investigated using the dual culture technique. The most effective temperature range for B. subtilis was found to be between 20 and 37°C. At temperatures lower than 15°C, B. subtilis was found to be not very effective, suggesting why postharvest applications followed directly by cold storage do not always work effectively. D-arabinose and D-(+)-mannitol evaluated as carbon source as well as L-glutamic acid, L-glutamine and L-(+)-asparagine used as nitrogen sources support in vitro antagonism against the pathogens most effectively. They also do not support the growth of C. gloeosporioides, P. perseae, D. aromatica and L. theobromae. These nutrients can potentially be the most effective ones to incorporate in commercial B. subtilis formulations. The study showed the potential role of antagonistic free acid phenolic substances, volatiles and siderophores on inhibition of fungal avocado pathogens. Further studies to confirm their in situ activity are required. In conclusion, various factors affect the efficacy of B. subtilis against postharvest pathogens of avocado. These factors should be kept in mind when applying the commercial product in order to achieve the best results.