New promising strains of entomopathogenic fungi isolated from organic orchards and natural spaces in Geneva Etienne Laurent, Julien Crovadore, Pauline Deneux, Bastien Cochard, Pegah Pelleteret, Romain Chablais & François Lefort Groupe Plantes et pathogènes, Institut Terre Nature et Environnement, hepia, HES-SO//Genève 150 route de Presinge, 1254 Jussy, Suisse. E-mail: francois.lefort@hesge.ch Genetic identification Agriculture has become during the past century very dependent of the chemical control, which indeed contributed very efficiently to crops consistency and to the increase in yields. But the disadvantages and environmental impacts had been early documented. They are now more and more viewed by the public as unacceptable. The chemical residues in food, the agricultural micropollutants in soils and waters, their impact on human health and the biodiversity without forgetting the selection of resistance of pathogens and pests, all these disadvantages plead for the search of alternative solutions. In this context, the entomopathogenic fungi represent an interesting potential for protecting crops against phytophagous insects and could be the basis of a sustainable phytosanitary control Following the DNA extraction of the pure cultures and PCR amplification of the ITS region with the ITS4/5 primer pair, DNA sequencing of the PCR products allowed to identify 71 single isolates, among which 40 belonged to well documented entomopathogenic species (Tab. 1). Entomopathogenic fungi species Number of isolates Beauveria bassiana 17 Mucor hiemalis 9 Metharizium sp. 7 Fusarium solani 3 Paecilomyces sp. 2 Bionectria ochroleuca 1 Metacordyceps chlamydosporia Total 40 Table 1. Fungal isolates of entomopathogenc fungi. Challenge tests against the aphid Myzus persicae In the frame of this work, fungi have been isolated from the soils of fruit trees orchards, prairies and forests of the Canton of Geneva. After genetic identification, the isolates of species known as entomopathogens were used in challenge tests against aphids in order assess their infectious or toxic potentials. Twelve isolates were tested against the aphid Myzus persicae. Two preparations were tested from the axenic liquid cultures for each isolate: FS: Sterile filtrate without any spores (membrane filtration at 0.22 µm) FC: Axenic filtrate with spores Leaf disks of Chinese cabbage were soaked in the filtrates, and installed in Petri plates (Fig. 4) with 5 aphids per plate in climatic chambers. The mortality was recorded (Fig.5) Figure 4. Soaked leaf disks of Chinese cabbage with 5 aphids. Baiting method of Zimmermann (1986) Soil samples (200 g) were collected in 11 different locations : 5 untreated apple tree orchards 2 treated apple tree orchards 1 semi-treated apple tree orchards 3 other ecosystems (forest, grassland, biodynamic vineyard). The baiting method used larvae of the wax moth Galeria mellonella (Lepidoptera) as preys. (Fig. 1). Figure 5. Aphids mortality at 3 days (A) and 6 days (B) when challenged by the filtrates of the twelve isolates . A B Figure 1. Each soil sample was put in plastic trays with avec 5 larvae of Galeria mellonella. Challenge tests against the aphid Acyrthosiphon pisum Seven strains of Metarhizium (Fig. 6) were then tested against the aphid Acyrthosiphon pisum, according the same methodology Figure 2. Mortality of larvae in the plastic trays. The Mortality increased at 10 days. No marking differences were found between treated and untreated orchards All samples Untreated orchards Treated orchards Average mortality of aphids (in numbers of deads) Figure 6. Average mortality of aphids A. pisum when challenged by the filtrates of 7 Metharizium strains. The dead larvae were removed every day from the plastic trays, surface disinfected with ethanol 70% and sodium hypochloride and then kept in Petri dishes for one week at room temperature, in order to allow the sporulation of entomopathogenic fungi (Fig.3). Day 0 Day 3 Day 4 Day 5 Day 7 Day 10 Results and Conclusion The baiting method used has been very efficient, particularly in natural and untreated environments, which were much richer in species and isolates. The first experiment showed that 4 isolates were highly virulent on Myzus persicae with high mortalities after 6 days : Beauveria bassiana 2.1. with 100% mortality Beauveria bassiana 6.3. with 86.67% mortality Metarhizium sp. 10.1. with 100% mortality Paecilomyces sp. 28.2. with 93.33% mortality The second experiment highlighted a strong entomotoxicity of the Metarhizium isolates resulting in 100% mortality at 7 days. The isolates 10.1 and 32.1 showed an early entomotoxicity at 3 days. Figure 3. Dead larvae coated with mycelium after 1 week in Petri plate. The fungi which sporulated on dead larvae were sampled and isolated in axenic cultures on Potato Glucose Agar (PGA) medium. 11th Annual Biocontrol Industry Meeting ABIM Congress Center Basel, Basel, Switzerland 24 - 26 October 2016