Data sets for evaluation of the virulence of honey-derived bacteria, <i>Paenibacillus</i> sp. J27TS7, <i>Paenibacillus</i><i> azoreducens</i> J34TS1, and <i>Paenibacillus</i> <i>melissococcoides </i>J46TS7 to honeybee larvae under continuous dosing conditions (Bioassay I).

Data sets (cumulative number of dead broods/number of larvae used and survival rate [%]) for evaluation of the virulence of honey-derived bacteria, <i>Paenibacillus</i> sp. J27TS7, <i>Paenibacillus</i><i> azoreducens</i> J34TS1, and <i>Paenibacillus</i> <i>melissococcoides </i>J46TS7 to honeybee larvae.The virulence was evaluated by feeding spores of these <i>Paenibacillus</i> strains to <i>Apis mellifera</i> larvae reared <i>in vitro</i>. For preparation of the spores, bacteria grown on BHI agar (J27TS7) and Columbia<b> </b>blood agar containing 5% sheep blood (CSA) (the other strains) were suspended in saline, and appropriately diluted bacterial suspensions were spread onto BHI agar (J27TS7) and CSA (the other strains) plates. The plates were incubated at 35°C for 11–16 days under aerobic conditions. After sporulation of more than 90% cells was confirmed by staining with malachite green, spores were collected from 10 or more agar plates, suspended in cold sterile H₂O, and washed four times with cold sterile H₂O by collecting spores via centrifugation (12,000 x g, 15 min, 4°C), discarding the supernatant, and suspending the spore pellet in 30 mL of cold sterile H₂O. Washed spores were suspended in 10 mL of cold sterile H₂O and stored at 4°C. One week or more later, the spores were washed several more times with 30 mL of cold sterile H₂O to remove organic matter from the lysed vegetative cells. The re-washed spores were suspended in 5–20 mL of cold sterile H₂O and stored at 4°C until use. Clinically healthy<i> A. mellifera</i> colonies maintained in the Research Institute for Animal Science in Biochemistry and Toxicology, Sagamihara, Japan, were used in this bioassay. From the colonies, less than 24-h-old larvae were grafted onto royal jelly in sterile Petri dishes using a grafting tool and randomly divided into test groups. Each larva was reared in grafting cells placed in a 48-well cell culture plate. The culture plates were kept in a desiccator (relative humidity, 95% ± 5%) and incubated at 34 ± 0.5ºC until day 6 post-grafting (pg). Larvae were infected by feeding them a diet containing spores (5,000,000 spores/ml) from day 0 pg to day 5 pg. Larvae in the non-infected control group were fed artificial diets containing the same components as the inocula except the spores. On and after day 7 pg, larvae in the culture plates were incubated in a desiccator at 34 ± 0.5ºC (relative humidity, 80% ± 5%). On day 14 pg, each plate was transferred into an emergence box in a desiccator and incubated at 34 ± 0.5ºC (relative humidity, 80% ± 5%) until day 21 pg. In this bioassay, larval survival was monitored until day 21 pg.