Thermo-stability, dose effects and shelf-life of antifungal metabolite-containing supernatants produced by Xenorhabdus szentirmaii Selcuk Hazir & David I. Shapiro-Ilan & Clive H. Bock & Luis G. Leite Accepted: 19 June 2017 /Published online: 4 July 2017 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2017 Abstract Entomopathogenic nematodes in the genus Steinernema are associated with Xenorhabdus spp. bacte- ria. When steinernematid colonise an insect host the nematode-bacterium association overcomes the insect im- mune system and kills the host within 48 h. Xenorhabdus spp. produce secondary metabolites that are antifungal to protect nematode-infected cadavers from fungal coloniza- tion. The concentrated, or cell-free metabolites of X. szentirmaii exhibit high toxicity against various fungal plant pathogens and show potential as natural bio-fungi- cides. In the current study, we determined 1) thermo- stability, 2) dose-response, and 3) shelf-life of antifungal metabolites of X. szentirmaii against Monilinia fructicola (cause of brown rot of peach and other stone fruit) and Glomerella cingulata (cause of antharacnose). Thermo- stability was determined by autoclaving bacterial culture broths (121 °C and 15 psi for 15 min) and measuring fungal growth on in potato dextrose agar (PDA) contain- ing 10% of the supernatants. Autoclaving had no impact on the antifungal activity of the secondary metabolites. Over a test period of 9 months, the activity of both extract types did not decline when stored at 4 or 20 °C. A dose- response study (10, 20, 40, 60, 80 and 100% supernatant- containing metabolite) using both phytopathogens dem- onstrated that a greater dose of supernatant increased antifungal activity. The antifungal-metabolite containing supernatant of X. szentirmaii has potential as a bio-fungi- cide. These results demonstrate the metabolite(s) are ther- mo-stable, they have a long shelf-life and require no stabilizing formulation, even at room temperature. Keywords Xenorhabdus szentirmaii . Entomopathogenic nematodes . Biological control . Bio- fungicide . Monilinia fructicola . Glomerella cingulata Introduction Entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae are used for aug- mentative biological control of insects. Different species of EPNs have mutualistic and obligate associations with bacteria (Xenorhabdus spp. and Photorhabdus spp.) that reside in the gut of infective juvenile (IJ) nematodes (Hazir et al. 2003; Lewis and Clarke 2012). The IJs enter the host insect through natural openings (oral cavity, anus, and spiracles) or, in some cases, through the intersegmen- tal membranes. After penetrating the hemocoel, the IJs release their symbiotic bacteria (Shapiro-Ilan et al. 2017). Inside the host, the mutualistic bacteria colonise the insect, kill the host, and digest host tissues, allowing the IJs to feed. The mutualistic bacteria not only provide nutrition to Eur J Plant Pathol (2018) 150:297–306 DOI 10.1007/s10658-017-1277-7 S. Hazir (*) Faculty of Arts and Sciences, Department of Biology, Adnan Menderes University, 09100 Aydin, Turkey e-mail: selcuk.hazir@gmail.com D. I. Shapiro-Ilan (*) : C. H. Bock Southeastern Fruit and Tree Nut Research Laboratory, USDA-ARS, Byron, GA 31008, USA e-mail: david.shapiro@usda.ars.gov L. G. Leite L. G. Leite Instituto Biologico, APTA, CP 70, Campinas, SP 13001-970, Brazil