Mycotoxin production by entomopathogenic fungus Conidiobolus coronatus
Abstract
Qualitative and quantitative analysis of selected mycotoxins has been performed in extracts of Conidiobolus coronatus pathogenic fungus cultivated under optimal and stress conditions. Furthermore, the analyses of these compounds in post-incubation filtrates were done. For identification purposes the analytical method allows identification and quantitation of selected mycotoxins including beauvericin , fumonisin B1, enniatin A and B and destruxin A based on high performance liquid chromatography coupled with tandem mass spectrometry was developed. Only beauvericin was detected in very low amounts in C. coronatus mycelium extract cultivated under optimal condition. In the extract of C. coronatus mycelium grown on LB 12.3 ± 0.1 µg/g of beauvericin was determined, while in the extract of C. coronatus mycelium grown on MM medium beauvericin content was lower and amounted 4.6 ± 0.1 µg/g. Also the presence of beauvericin was confirmed in postincubaction filtrate extract (MM). The content of this compound was 2.2 ± 0.1 µg/g. In other extracts beauvericin was not detected. In addition, in the tested extracts other compounds were not detected.
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Introduction
The use of chemical insecticides can cause a variety negative effect on the environment. They exhibit high toxicity, but also a low biodegradability and thus accumulate in the environment. As a result of drift by the wind or flushing them torrential rains, these compounds get into reservoirs and waterways. Therefore, it is necessary to search for alternative methods of pest control, which will not have a negative impact on the environment, including humans and animals. The solution to this problem may be use of entomopathogenic fungi. Entomopathogenic fungi are ubiquitous in the environment and plays an important role due to its ability to spontaneous infection reduce the amount of many plant pests [1]. Currently, there are about 3,000 known species of fungi that can cause diseases of living arthropods. Only 30 of them are used as biological agents to limit the number of plant pests [2]. The most of these products is based on the fungi species such as Metarhizium anisopliae, Beauveria bassiana, Beauveria brongniartii, Paecilomyces fumosoroseus, Lecanicillium longisporum and Lecanicillium muscarium [3]. These entomopathogenic fungi can be use as bioinsecticides, due to their ability to mass propagation on artificial media [1]. Entomopathogenic fungi produce a number of secondary metabolites which have a different effect on insects [4-7]. Beauveria bassiana produces bassianolides-depsipeptide which proved to be important factors in the insect infection [4]. Destruxins produced by the Metarhizium anisopliae causes paralysis and death of the infected host [5]. There are also metabolites do not cause the death of organisms, but does not exclude the importance of these compounds in the infection process, e.g. beauverolides not show the insecticidal activity and the immune response [7]. Fumonisins are produced by fungi of the genus Fusarium, for example: F. moniliforme and F. proliferatum occurring primarily in corn grain and its processing products intended for food and feed. The most important analogues found in naturally contaminated corn are fumonisin B1, fumonisin B2 and fumonisin B3. Several strains of fungi Fusarium spp produce secondary metabolites belonging to the enniatin group. They are six-membered cyclic depsipeptides having ionophoric, phytotoxic, antiparasitic and antibiotic properties. Beauvericin (BEA) is a toxic metabolite produced by entomopathogenic fungi. This mycotoxin was isolated from an entomopathogenic fungus Beauveria bassiana and several other species belonging to the family Cordycipitaceae in the Hypocreales (Ascomycota) [8-11]. Fusarium species infecting maize, rice, and wheat are also known as beauvericin producers [9]. There is only one report of BEA occurrence and co-occurrence with fumonisin B1, fumonisin B2 and ochratoxin A. BEA is cyclohexadepsipeptide fungal metabolite with a wide range of biological activities, such as insecticides, anthermintic, antibacterial, antifungal, antiplasmodial, antimycobacterial and anticancer activities. It is the most potent specific inhibitor of cholesterol acyltransferase and possesses ionophoric properties. BEA increases ion permeability in biological membranes by forming a complex with some cations (Ca2+, Na+, K+), which may affect the ionic homeostasis [8]. The insecticidal activity of BEA was first discovered by Hamill et al.[12]. BEA was confirmed as the active compound against Artimia salina, which was considered as a model organism to insecticidal activity study. Subsequently, the insecticidal effect of BEA on a microgram level was investigated on Calliphora erythrocephala, Aedes aegypti, Lygusspp., Spodoptera frugiperda and Schizaphis graminum [13-16]. BEA exhibits toxicity to bacteria: Bacillus subtiilis, Escherichia coli, Mycobacterium phlei, Sarcinea lutea, Staphylcoccus aureus and Streptococcus faecalis [73]. Furthermore, it is an effective integrase inhibitor of HIV-1 [71]. A very important feature of bEa is the antitumor effect [71]. It interferes with the motility of tumor cells which reduces the speed of many processes in the development of the disease, including the formation of new blood vessels in the tumor cells, and metastasis [53, 77]. It also inhibits the acetyltransferase and cholesterol results in programmed cell death, similar to apoptosis, as well as cytolysis [72, 73, 74].
Conidiobolus coronatus is an opportunistic pathogen with a fairly wide range of infected hosts. For the first time this species was described in 1897 by Costantin'a in France. In contrast, C. coronatus was isolated in 1961 by Chester Emmons and Charles Bridges [13, 14]. It occurs commonly in soil and decaying plant material [14]. This entomopathogenic fungus causes the disease process in many arthropods, eg. greater wax moth (Galleria mellonella), pine lappet moth (Dendrolimus pini), springtails and other [13-16]. After penetrating into the body cavity of the fungus kills the insects within 1-2 days, resulting in tissue damage, which is caused by the depletion of nutrients and the production of mycotoxins, which are considered to be the main factor causing the death of an insect. Because of their insecticidal potential C. coronatus can be used as a source of new generation bioinsecticides. However, to date, a mycotoxin produced by C. coronatus not have been identified and described.
Therefore, the main goal of this study was to assess selected mycotoxins content in extracts of pathogenic fungus C. coronatus. As first, different solvents and solvent mixtures were applied to extraction of target analytes. Then, the analytical method based on high performance liquid chromatography coupled with tandem mass spectrometry was developed for qualitative and quantitative determination of these compounds in obtained extracts. The productions of selected mycotoxins by C. coronatus grown in rich and poor media were determined. Moreover, the impact of changes in culture conditions, including temperature and pH on mycotoxins production were also assessed. The results of this study provide useful information to assess the potential use of C. coronatus as a source of new generation bioinsecticides.
Conclusion
Economic losses caused by harmful insects are a serious economic problem. Reducing of the pests are still made mainly using non-selective chemical insecticides, the accumulation of which in the environment is a serious threat to biodiversity and human health. Accompanying this process the growing resistance of harmful insects to classical insecticides, tends to reduce the use of chemical insecticides and the search for new, selective and less environmentally harmful methods of pest control. In this work the isolation and qualitative and quantitative analysis of mycotoxins in the mycelium and post-incubation filtrates of the C. coronatus using chromatographic methods were done. In the extracts C. coronatus mycelium grown in LBand MM medium the presence of BEA was confirmed (12.3 ± 0.1 µg/gand 4.6 ± 0.1 µg/g, respectively). In turn, the content of BEA in the post-incubation filtrate (MM) was 2.2 ± 0.1 µg/g. On the other hand, in the test samples of mycelium and post-incubation filtrates the presence of compounds from the group of enniatin, fumonisins and destruxin were not determined.