Journal of Apicultural Research 45(3): 219–220 (2006) © IBRA 2006 NOTES AND COMMENTS Evaluation of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycota: Hyphomycetes) strains isolated from varroa mites in southern France W G Meikle 1* , G Mercadier 1 , V Girod 2 , F Derouané 1 and W A Jones 1 1 European Biological Control Laboratory, USDA – ARS, Campus International de Baillarguet, CS 90013 Montferrier sur Lez, 34988 St. Gely du Fesc, France 2 ADAPRO LR-CRALR, Maison des Agriculteurs, Mas de Saporta CS 30012, 34875 Lattes, France Received 30 March 2006, accepted subject to revision 16 May 2006, accepted for publication 16 August 2006. * Corresponding author. Email: wmeikle@ars-ebcl.org Keywords: Apis mellifera, Varroa destructor, Beauveria bassiana, Metarhizium anisopliae, bioassay Introduction Varroa mites (Varroa destructor Anderson and Trueman) are an increasingly important pest of honey bees (Shaw et al. 2002). Several species of entomopathogenic fungi have been found to infect varroa mites in the laboratory (Shaw et al. 2002) and have been tested in the field (Kanga et al. 2003). The objectives of this study were to isolate and characterize a strain of hyphomycete fungus that occurs naturally in domesticated bee colonies and is virulent against varroa. A total of 112 hives in 12 apiaries were visited in the departments of Gard, Aude, Hérault, and Pyrénées Orientales in France during 2005. Varroa mites were sampled by placing a metal tray underneath the hive, opening the hive and pouring approximately 150 g of powdered sugar (Sucre Union, Paris, France) into the brood box, making an effort to divide it evenly among the spaces between frames. The hives were then closed and about 20 min. later all the material that had collected on the tray was poured into a sealed plastic box. The boxes were transported to the European Biological Control Laboratory (EBCL), and all varroa mites were placed on water agar (6 g/L) with chloramphenicol (0.4 g/L) to reduce bacterial growth, incubated for 2 weeks at 23°C and observed for at least 3 weeks for sporulation. Entomopathogenic fungi collected in this way were used to inoculate 2 types of media: Sabouraud dextrose agar with yeast (SDAY) (Goettel and Inglis,1997) and semi-synthetic INRA media (cf. Meikle et al. 2005), both with chloramphenicol (0.5g/L), in plastic petri dishes (3.5 cm diam.). The most vigorous cultures were re-isolated two to three times and then grown as pure cultures. Two isolates from this exploration, as well as five isolates of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycota: Hyphomycetes) from the collection of entomopathogenic fungi at EBCL, were evaluated in bioassays. Cultures of candidate isolates were grown on SDAY with chloramphenicol and harvested after 12–14 days. Four experiments were conducted, each experiment including two fungal isolates and the control treatment. Prior to each experiment, conidial viability was assessed by plating a suspension sample onto SDAY,incubating the plates at 23ºC for 24 h, and examining 200 conidia for germ tubes under a light microscope. Conidial viability always exceeded 90%. The day of the bioassay,white bee pupae were collected from hives at EBCL by cutting sections of brood comb from the frame, placing the pieces in a sterile plastic box and, in the lab, carefully extracting the pupae and placing them individually in small (2x2x1 cm) plastic boxes with screen covers. Any varroa mites found on the pupae upon their removal from the comb were destroyed. Varroa mites used in the bioassays were collected en masse the day of the bioassay using the powdered sugar treatment described above. While the mites and bee pupae were being collected, a 10 7 conidia/ ml suspension was prepared by adding conidia to 10 ml of a 0.1% aqueous solution of Tween 80®, pouring the mixture into a 50 ml bottle filled with glass beads, and agitating the bottle for 2 min. using a Vortex Genie 2 (Scientific Industries, Bohemia, NY, USA). Thirty varroa mites were then placed in a petri dish (9 cm. diam.) lined with moist filter paper. Care was taken to place mites as far as possible from each other. The dish was placed in a spray tower (Burkard Manufacturing Ltd., Herts., England) and sprayed with either an aqueous Tween 80® solution (control), or the conidial suspension. The spray tower had an air pressure of 0.74 kg/cm 2 and delivered on average 714 conidia/ mm 2 using suspensions of 10 7 conidia/ ml (Meikle et al. 2005). After exposure, two varroa mites were placed on each