Assessing the cytotoxic and mutagenic effects of secondary metabolites produced by several fungal biological control agents with the Ames assay and the VITOTOX(®) test.

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites.

Mass spectrometry as a tool for the selective profiling of destruxins; their first identification in Lecanicillium longisporum.

Mass spectrometry was applied to the identification of the destruxins (dtxs), cyclic peptides that are commonly produced by the fungal insect-pathogen, Metarhizium anisopliae. The aim of the study was to optimise a methodology in order to firstly determine whether these compounds were present in other species and to determine the effect of differing growth conditions upon the dtx content detected. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) was initially used to analyse the dtxs, but limitations were indicated. Nano-scale high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) and automated 'data-dependent' tandem mass spectrometric (MS/MS) analysis were also applied, utilising characteristic neutral losses during fragmentation to confirm the presence of the dtxs. This latter approach distinguished the dtx E and B isoforms by retention time and diagnostic neutral losses during fragmentation allowing extraction of the destruxin data from a complex dataset. This process revealed the presence of a number of dtxs in the fungal species Lecanicillium longisporum, a species previously not known to produce dtxs, and dtx production in this species was shown to be significantly higher in aerated cultures compared with still cultures.

Evaluation of different biological test systems to assess the toxicity of metabolites from fungal biocontrol agents.

The development of fungal biocontrol agents (BCAs) as alternatives to chemical pesticides is of increasing public interest. Tools to assess the toxicity of the secondary metabolites that these BCAs produce are often not available or existing methods have not yet been evaluated for these compounds. This study compares five different test systems, which include a representative bacterium, protozoan, arthropod and insect and human cell lines, as regards their sensitivity. It also compares the cost in time and resources for conducting the tests. Pure metabolites and crude extracts from two fungal BCAs as well as two chemical pesticides (hoestar and chlorpyrifos) and the mycotoxin patulin were employed as test compounds. All tests systems proved to be suitable for toxicity studies of metabolites from fungal BCAs and showed different grades of sensitivity to the different substances. The possibility of employing an array of test systems to determine ecotoxicological properties is discussed.

Toxicity testing of destruxins and crude extracts from the insect-pathogenic fungus Metarhizium anisopliae.

Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.

Mass spectrometric studies on the intrinsic stability of destruxin E from Metarhizium anisopliae.

Destruxins are of current interest as bioactive agents. They are cyclic hexadepsipeptides produced by fungi, the most common destruxins, A, B and E, differing in the structure of a side chain. Before they can be widely used, the potential risk of destruxins and their metabolites entering the human food chain must to be assessed; thus, knowledge of the structures of their degradation products is essential. Here we report a study aimed at identifying, by tandem mass spectrometry and accurate mass analysis, the products resulting from thermally and temporally induced degradation of destruxin E. The degradation products fell into two groups: those with relatively simple modifications of the side chain and those involving much more complex rearrangements. The structures of most of the degradation products were deduced from the MS data, with the major product being destruxin E diol: significantly, this compound had previously been reported to have only been produced as a metabolic product of enzyme action rather than as a simple degradation product as demonstrated here.

Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae.

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.

Concurrence of losing a chromosome and the ability to produce destruxins in a mutant of Metarhizium anisopliae.

In a previous study, a spontaneous subtilisin pr1A and pr1B gene-deficient mutant of the entomopathogenic fungus Metarhizium anisopliae strain V275 has been identified [Wang, C.-S. et al. (2002) FEMS Microbiol. Lett. 213, 251-255]. The insecticidal metabolites of this mutant were studied further. High-performance liquid chromatography (HPLC) analysis indicated that the mutant isolate lost the ability to produce cyclic peptide toxins, destruxins, both in vitro and in vivo. Pulsed-field gel electrophoresis revealed that the mutant concurrently lost a 1.05 Mb (approximately) chromosome, demonstrating for the first time that a conditionally dispensable (CD) chromosome exists in the insect pathogenic fungus, M. anisopliae. Concurrence of losing the ability to produce destruxins and a CD chromosome in the mutant suggests that the toxin synthetase genes of M. anisopliae are located on this CD chromosome, as similarly described for plant pathogenic fungi. Semi-quantitative api ZYM analysis showed more biochemical disparities between the mutant and the wild-type strain.