Biological control of Scarabeaidae by improving the ecological fitness of insect pathogenic fungi

Major scarab pests were controlled biologically with virulent, ecologically competent strains of insect pathogenic fungi. Physiological methods were developed to monitor fungal virulence and nutritional requirement. First steps to make easier quality control of products were made. Depending on culture conditions the red pigment oosporein is excreted by the fungus Beauveria brongniartii. It could be confirmed that oosprein is no suited as a virulence determinant. Oosporein is not toxic if it is fed by cockchafer larvae. Second generation formulations of B. brongniartii and Metarhizium anisopliae, used to fight the garden chafers, were found to be ecologically effective and economic in several field tests. In this project two major scarab pests, namely the cockchafer Melolontha melolontha and the garden chafer Phyllopertha horticola were controlled with biological agents on the basis of insect-pathogenic fungi. In a first step some of these fungal strains were morphologically characterised and their aggressiveness was assured in bioassays using Melolontha larvae (white grubs). Physiological studies with the insect pathogenic fungus B. brongniartii showed that successive subculturing, which is essential for the characterisation and production process of the biocontrol agent (BCA), can cause attenuation of virulence. Measures to slow down or even prevent this attenuation were found by using a special nutrient medium which contained chitin as the sole carbon source. When attenuated sub-cultures of the insect pathogenic fungus were tested for their carbon utilisation patterns with the commercially available Biolog-system inter- and intra-specific variation of substrate utilisation was recorded. For example, attenuated B. brongniartii metabolised less L-erythritol than the highly virulent isolates, whereas M. anisopliae, which was found to be aggressive against the garden chafer P. horticola remained unaffected. Such results will help to replace or supplement costly bioassays and thus to accelerate a rapid quality control of BCAs in production process and storage. Some of the favoured substrates for B. brongniartii included monosaccharides (glucose) and polymers (tween 80), disaccharides (sucrose) and sugar alcohol`s (xylitol). HPLC studies on the secondary metabolite spectrum of the insect pathogenic fungus showed that oxalic acid and the red pigment oosporein are excreted in considerable amounts depending on culture conditions. Nevertheless, the quantity of oosporein, which was thought to represent a virulence determinant did not correlate with high aggressiveness or attenuation. Furthermore, oosporein showed neither acute toxicity nor any negative effect on M. melolontha larvae in feeding bioassays. Melocont-Pilzgerste, the commercial product which is based on barley kernels colonised by B. brongniartii, was tested against the cockchafer in large field trials over a period of more than six years. Highest efficacy of the product was achieved by incorporating the inoculum into the soil at a depth of 3 to 10 cm. The results of the field trials indicated that the density of B. brongniartii increased continuously after each of the applications. Molecular biological methods (microsatellite marker analysis) allowed to demonstrate that the applied strain and reisolated strains were identical. The application of the B. brongniartii barley kernel product resulted in a sufficient suppression of cockchafer populations after only 2 years of application. The garden chafer is a smaller relative of the cockchafer and is of considerable economic importance, as the larvae feed on roots of various plants and can cause severe damage especially in meadows, sports fields, golf courses, gardens and parks. During this project two Metarhizium anisopliae strains were identified that are highly pathogenic to P. horticola in the laboratory. Metarhizium was formulated as a wettable conidia-powder for spraying and as colonised barley kernels for soil application. First field tests on steep alpine meadows were conducted and the results indicate to accelerate further studies to fight biologically P. horticola.