Evaluating the efficacy of the entomopathogenic fungi brunneum against , a common pest Jennifer Han1, Nick Naeger2, Brandon Hopkins2, Paul Stamets3, Steve Sheppard2, Lori Carris1 1Department of Plant Pathology, Washington State University, 2Department of Entomology, Washington State University, 3Fungi Perfecti

Introduction Results Normalized Mite Fall The European honey bee (Apis mellifera) is the most important agricultural crop pollinator in Control Pre-Conidial Sporulating 0.16 the United States, contributing over $15 billion to the agricultural economy. Honey bees are * 0.14 facing a variety of dangers, however, the most common reported cause for colony loss is Varroa * destructor, an ectoparasitic mite. Although synthetic chemical acaricides are available for 0.12 varroa control, there are documented cases of mites resistant to these chemicals. 0.1 0.08 Metarhizium brunneum is a common soil-borne entomopathogenic fungi. It is commercially 0.06 available, EPA registered, and determined safe for human contact. Although previous studies 0.04 have shown M. brunneum can infect and kill varroa without harming the honey bee, virulence 0.02 varies between strains and application methods. Additionally, conidia, the infectious agent, 0 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 quickly loses viability at high temperatures; bees maintain an internal hive temperature around P-value Control vs. 35℃ which is much higher than the optimum growth temperature for M. brunneum. 0.27 0.15 *0.03 *0.01 0.14 0.37 Sticky board with mites and MEA agar Sporulating Control vs. debris 0.93 0.32 0.51 0.22 0.59 0.67 Objective Pre-Conidial 1. Test the virulence of M. brunneum F52 against Varroa destructor % Varroa Mycosis ℃ Control Pre-Conidial Sporulating 2. Evaluate the thermotolerance of M. brunneum and the longevity of conidia at 35 0.7

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Plated Varroa mites on antibiotic PDA 0 plates displaying Metarhizium growth Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Burnt Ridge, ID Palouse Clearwater Environmental Institute, ID Pre-Conidial MEA plate Sporulating MEA plate Spores stored at 4℃ Spores stored at 35℃ Materials & Methods Objective 1 Thirty colonies of A. mellifera infested with Varroa mites were placed in grassy fields at Burnt Ridge, ID and Palouse Clearwater Environmental Institute, ID. Hives were treated with either: 1. Two sporulating M. brunneum on malt extract agar (MEA) plates 2. Two pre-conidial M. brunneum on MEA plates 3. Two MEA plates without . Spore germinated at 25℃ Spore germinated at 35℃ Spore germinated at 25℃ Spore germinated at 35℃ Sticky boards were placed under each hive and collected every other day. Conclusion Future Work Mite drops were counted and surface sterilized with 95% ethanol wash • Conidiating Metarhizium brunneum is an • Utilize minimal media and H2O2 to induce before plating on antibiotic PDA plates. Mites showing Metarhizium effective biological control agent against stress response in M. brunneum growth were considered to have died from mycosis. Varroa destructor • Apply directed evolution techniques to stress • Peak virulence between day 5-7 induced conidia to develop thermotolerance Objective 2 • Pre-conidial strains are not effective BC. • Increase virulence through repeated passage Conidia were harvested from fully grown M. brunneum plates and stored • There is a decline in virulence starting at through an insect host ℃ ℃ at 4 or 35 in sterile water. Conidia were germinated on water agar day 9. This is most likely due to the loss Acknowledgments for 24 hours and evaluated. Typical treatment set-up of viability of conidia at 35℃ Funding provided by Whittier Family Foundation, and Fungi Perfecti