Use of an entomopathogenic, endophytic Metarhizium brunneum isolate (Cb15III) to manage wireworm and Colorado potato beetle

Potatoes (Solanum tuberosum, L.) are one of the most important crops worldwide and beside of rice, wheat and maize of an essential importance for the nourishment of the world population. However, potato plants are sensitive to a broad range of pests and pathogens and cultivation without a proper plant protection management can be difficult or impossible. Over the past decade, quality issues caused by wireworm (Agriotes spp.) tuber feeding became more and more relevant. Due to the soil born character of this pest species complex management is far more complicated, particularly because efficient protection agents are missing. Moreover, well managed pests like the Colorado potato beetle (Leptinotarsa decemlineata, SAY) developed resistances against often used insecticides e.g. pyrethroids. The development of new biological control agents against these and other pests is therefore of high importance, to overcome disadvantages of chemically synthetic plant protection products, which can be highly efficient but with indeterminate environmental effects. Metarhizium brunneum PETCH is known to comprise many different isolates each specialized to one or more insect hosts. This makes M. brunneum a promising biological agent against several, relevant agricultural pests. However, field-scale, spray applications are difficult, not only because of a high sensitivity against UV- radiation and limited efficacy under arid conditions but also a low competitive competence against nematodes or microorganism like bacteria or other fungi can reduce the activity of M. brunneum in field. An option to protect the fungus from environmental influences and exploit its entomopathogenic nature is to introduce the fungus into crop plants as an endophyte to build up a systemic protection against pests. As a consequence, M. brunneum isolates must be entomopathogenic to target organisms, endophytic in the specific crop plant and an industrial propagation and formulation should be possible. To outline how a novel biological control agent is introduced to modern, biological plant protection management, this dissertation is structured in three chapters and focused on finding a new M. brunneum isolate, which can be systemically used to control potato pests.  1. Chapter: To identify a broad acting endophytic entomopathogen, M. brunneum isolates from soil samples were collected and tested against a model organism (Tenebrio molitor L.), important potato pests and inoculated into potato tubers. • General pathogenicity test: Seven M. brunneum isolates were tested against the larvae of T. molitor due to the known high sensitivity against entomopathogenic fungi. Two isolates were identified as highly pathogenic with mortalities up to 100 % within 7 days after inoculation. • Pathogenicity test against Agriotes spp.: The two most promising isolates (Cb15III and Gc2II) from the “general pathogenicity test” were tested against the larvae of Agriotes sputator (L.), A. obscurus (L.) and A. lineatus (L.). Isolate Cb15III was able to significantly reduce the number of vital A. obscurus and A. lineatus larvae compared to control groups within a time span of 70 days. • Pathogenicity test against CPB: The M. brunneum isolate Cb15III was tested against the larval and adult stages of the CPB. 100 % of the inoculated larvae died within 21 days and 80 % of those showed a characteristic M. brunneum mycosis. Adult CPB beetles were not susceptible to the M. brunneum treatment. • Endophytism test in potato: M. brunneum Cb15III was tested for its endophytic potential to colonize potato tuber tissue. Therefore, 20 potato varieties were selected and M. brunneum Cb15III was inoculated into tubers. Re-isolation of M. brunneum from new developed shoots was successful, however with very low re-isolation rates. 2. Chapter: Potato tubers were inoculated with the M. brunneum isolate Cb15III and used to evaluate the effects of colonized plants on the development of CPB. For the inoculation, dipping and injection methods were tested under greenhouse conditions and the more promising injection method was evaluated in two field experiments in southern Lower Saxony. It was shown that larval development was influenced by the endophyte and significantly less beetle emerged from treated plants in the greenhouse experiment. In the field experiments the number of laid CPB eggs, L1 – L3 and L4 larvae was significantly reduced when tubers inoculated with M. brunneum. 3. Chapter: To improve technical feasibility of field scale applications, M. brunneum Cb15III mycelium was imbed in a biopolymer matrix and formulated into dry microcapsules. Capsules with varying formulation adjuvants were tested in field in two consecutive years. Field trials were set up following the EPPO guideline for wireworms. Treatments were applied as spot applications beneath each tuber to enhance mycelium growth into the tuber tissue. Quantity and quality of potato yield was assessed and wireworm holes counted during harvest to assess quality losses. None of the tested treatments reduced the damage caused by wireworms; also no effects on plant grow or yield were observed.