Australian Ganoderma: identification, growth and antibacterial properties

Ganoderma species are one of the most widely researched fungi because of their reported potent bioactive properties. Although there is much information related to American, European and Asian isolates, little research has been conducted on Australian Ganoderma isolates. Ganoderma may only be imported into Australia under strict quarantine conditions, therefore, the isolation of a native strain that possesses bioactivity may be industrially and commercially significant. Three Australian species of this wood-decomposing fungus were isolated in northern Queensland. In this study, they have been identified as three separate species. Further, they have been studied to determine their optimal growth conditions in liquid culture and assessed for their antibacterial properties. Phylogeny inferred from the Internal Transcribed Spacer Regions (ITS) from the DNA sequences resolved the three Australian Ganoderma species into separate clades. Two isolates were identified to be isolates of Ganoderma cupreum (H1) and Ganoderma weberianum (H2). The third isolate could only be identified to the genus level, Ganoderma species, due to the lack of informative data that could be used for comparison. The effects of short term and long term storage on the viability of the fungi were investigated on agar plates, agar slants and balsa wood at varying temperatures ranging from 10 to 45°C. The most appropriate storage conditions were determined to be -80°C on balsa wood chips for periods of up to 2 years without subculture, and on agar slants at 4°C for up to a maximum of eight weeks. Light was observed to be detrimental to the survival of Ganoderma H1 and Ganoderma H2 during storage. Growth trials using potato dextrose agar plates determined the optimal temperature and pH for mycelial growth to be 30°C and a pH of 6, for all isolates. Subsequent growth trials in liquid media found that glucose, as the carbohydrate source, supported the greatest mycelial growth of Ganoderma H1 and Ganoderma H2 and that galactose and fructose supported the greatest growth of Ganoderma H3. Abstract ii Aqueous (hot water) and organic (hexane (HEX), dichloromethane (DCM), ethyl acetate (EtOAc), methanol (MeOH)) extracts from the liquid cultivated mycelium were assessed for their antibacterial activity using disc diffusion assays. Extracts from the mycelium of Ganoderma H1 exhibited activity against a greater number of Gram positive bacteria than those from Ganoderma H2 and H3. Subsequent studies on the DCM and EtOAc extracts from Ganoderma H1 determined the MIC and MBC against a number of Gram positive bacteria, including Bacillus cereus, B. subtilis, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus aureus, S. epidermidis and Listeria monocytogenes, as well as Clostridium species, including Clostridium perfringens, C. sporogenes and C. difficile, and some methicillin resistant Staphylococcus aureus (MRSA) strains. Time course growth assays confirmed that the DCM and EtOAc extracts predominantly exhibited bactericidal activity. Finally, the active compounds were determined to be terpenoid in structure with some phenolic groups attached.