Lamellicorn beetles, such as the cockchafer (Melolontha melolontha) and the wood chafer (Melolontha hippocastanl), and especially their larvae (grubs) can cause serious damage to crops in agriculture and forestry. Since their control by means of chemical insecticides is difficult and environmentally harmful, attempts are increasingly being made to control the reproduction and spread of those insects using biological means. For example, EP 633 936 A1 describes a method in which vegetative cells, spores or protein crystals of certain strains of Bacillus thuringiensis are used to control lamellicorn beetles. The effectiveness of that method is not satisfactory, however.
WO 87 05928 proposes the use of Bacillus popilliae (B. popilliae) spores in the biological control of scarabaeid larvae.
B. popilliae is the causative organism of so-called milky disease in larvae of may bugs and other lamellicorn beetles. The larvae infested with the bacillus have high concentrations of vegetative cells and sporangia of B. popilliae in their haemolymph, which result in a milky-white discolouration of the grub.
B. popilliae was first described by Dutky as a cause of milky disease in the Japanese beetle (Popillia japonica) in the U.S.A. (in: Journal of Agriculftural Research 61 (1940) pages 57-68, "Two new sporeforming bacteria causing milky disease of the Japanese beetle") and was later identified by Hurpin and Vago and by Wille also in grubs of the cockchafer (Melolontha melolontha) (B. Hurpin and C. Vago in: Entomophaga 3 (1958) pages 285-330, "Les maladies du hanneton commun (Melolontha melolontha L. (Col., Scarabaeidae)"; H. Wille in: Mitteilungen. Schweizerische Entomologische Gesellschaft 29 (1956) pages 271-282: "Bacillus fribourgensis n. sp., Erreger einer "milky disease" im Engerling von Melolontha melolontha L.").
Characteristics of the B. popilliae bacterium are inter alia that it does not form catalase and that most isolates are resistant to the antibiotic vancomycin and during sporulation form a distinctive protein crystal which is arranged inside the spindle-shaped sporangium next to the actual spore.
In its capacity as a pathogen for scarabaeids, B. popilliae has a high degree of specificity. The B. popilliae subspecies isolated from different species of scarabaeids differ in some cases considerably in their growth characteristics, in the composition of the protein crystal and in their plasmids.
The infestation of beetle larvae with B. popilliae is effected by peroral ingestion of the sporangia. The spores germinate in the gut of the larvae and the vegetative bacterial cells penetrate through the gut epithelium and the basal membrane into the haemolymph where they multiply during the subsequent three to four weeks. The B. popilliae cells then sporulate, which ultimately leads to the death of the beetle larva.
In contrast to other Bacillus species, however, under in vitro conditions B. popilliae forms predominantly vegetative cells and forms spores only exceptionally. Although WO 87 05 928 describes a method of obtaining the spores in vitro, in which the vegetative cells of B. popilliae are cultured in a defined medium and are finally stimulated to sporulate by the addition of a specific adjuvant, that method achieves a sporulation rate of only about 80%. In order to obtain quantities of infectious spore material sufficient for biological control it is therefore necessary to invest considerable resources in terms of equipment, personnel and financial expenditure, which make that method economically unviable and therefore unsuitable for practical purposes.
The aim of the present invention is therefore to provide biological means for controlling scarabaeids that enable those pests to be controlled satisfactorily and that are technically simple and economical to produce also on a large scale. Furthermore, means whose application minimizes the burden on the environment are desirable.
USPTO Backgrounds
USPTO Backgrounds