|dc.description.abstract||The insect-killing fungus Beauveria bassiana produces a wide range of metabolites, which presumably contribute to the pathogen’s virulence and infection process. Some of these metabolites showed negative effects when injected into the insect’s body but toxicity after topical application was hardly reported. Interestingly, the B. bassiana isolate K4B3 (owned by a New Zealand company, Biotelliga Ltd) produces insecticidal metabolites that are capable of killing via contact when cultivated under specific conditions. This project aimed to identify the insecticidal components and the key parameters for their production. Furthermore, this study provided baseline bioassay data, chemistry profiles and enhancement strategies for isolate K4B3.
Aphids, mosquito larvae and brine shrimps were the most susceptible to both mycelial extract and supernatant of K4B3 after contact and within 24 h of application. Thrips and all stages of diamondback moth (eggs, caterpillars and pupae) were unaffected when treated (contact or ingestion) with cell-free supernatant but were killed by mycelial extracts. Artificial diet containing freeze dried supernatant material stunted growth and killed Spodoptera litura armyworms within 48 h. All unprocessed daily supernatants of K4B3 showed no inhibition against microbes. Only concentrated supernatants at 100 mg ml-1 inhibited the growth of Rhizoctonia solani (fungus), Bacillus subtilis (Gram-positive bacterium) and Pseudomonas aeruginosa (Gram-negative bacterium). The mycelial extract of K4B3 at 50 mg ml-1 showed strong inhibition against R. solani, B. subtilis and Salmonella typhi.
The metabolites of K4B3, guided mostly by aphid bioassays in each step, were fractionated and purified using analytical High Performance Liquid Chromatography (HPLC). The metabolites were analysed based on their mass and fragmentation patterns using Mass Spectrometry (MS). A reference of potential metabolites produced by K4B43 was established as a result. Two major nonpolar contact insecticidal metabolites from K4B3 were identified as the previously described beauvericin and bassianolide. Beauvericin and analogues of K4B3 were demonstrating weak contact insecticidal activity. Bassianolide was the most aphicidal via contact. Both were detected in the mycelia rather than the supernatant. Apart from beauvericin and bassianolide, the insecticidal activity from the mycelial extract was also attributed to a few unidentified metabolites. Oosporein and a variety of beauverolides were also detected in the mycelia and supernatant but they were not investigated further for lack of insecticidal activity via contact. A group of UV-detectable, polar and heat-stable metabolites in the supernatant were found to be aphicidal. These hydrophilic metabolites were not retained on conventional reverse phase C18 column and were eluted in the ‘void volume’. No conclusive mass analysis could be performed on these metabolites.
Enzymatic metabolites of the fungus, representing known chitinases and a protease (Pr1) were also observed in the supernatant. Heat and EDTA treatment of the supernatant, which generally inactivate enzymes, suggested that these enzymes were not responsible for the insecticidal activity observed. A protein of 55 kDa in size, but of unknown function, was identified to be potentially insecticidal. Enhancement of polar organic acids (oxalic and dipicolinic acid) produced by K4B3 did not increase the insecticidal activities of the supernatant despite high yield of the organic acids. While oxalic acid appeared insecticidal, there was poor correlation between hyperproduction and insecticidal effect.
K4B3 requires liquid media with a start pH above 5.5, preferably neutral or slightly alkaline, at 27 degree Celsius for the production of insecticidal metabolites. K4B3 cultured with start pH 3 or at 35 degree Celsius failed to produce insecticidal supernatant. Supplementing vitamin B, insect materials and minerals did not enhance direct insecticidal activity. Supplementing K4B3 culture with citric acid at 200 mM and adjusting the start pH back to pH 8 increased the insecticidal activity of the supernatant. Glucose and starch were the preferred carbon source in the production of insecticidal metabolites. Mycological peptone could substitute for bacteriological peptone. In a time course study, the strongest insecticidal activity was found in the supernatant of a three-day-old culture. In comparison, reduced activities were observed in five- and seven-day-old cultures. This work thus provides a blueprint for commercial production.
Overall, the encouraging findings reported here demonstrate for the first time the contact insecticidal activity of beauvericins and bassianolide produced and contained in the mycelia of K4B3, of which traces were also detected in the supernatant. The rapid direct toxicity of the mycelial extract and supernatant was also attributed to a group of unknown UV-detectable metabolites. Other known metabolites (beauverolides and oosporein) and organic acids (oxalic acid and dipicolinic acid) in the supernatant lacked strong insecticidal activity. The potential synergy of all metabolites produced was not tested and therefore needs to be explored in future studies.||en