Short communication. Viability and pathogenicity of Ascosphaera apis preserved in integral rice cultures

Abstract

Chalkbrood is a fungal disease of honeybees (Apis mellifera L., Hymenoptera:Apidae) caused by Ascosphaera apis Maassen ex Claussen (Olive et Spiltoir). Ascosphaera apis is a heterothallic fungus that affects larvae of the honeybee. Diseased brood becomes mummified and mummies are white, gray, or black depending on the predominance of the sexual stage of the fungus. If spore-cysts are formed as a result of mating, infected larvae become gray or black in color. Larvae infected only by mycelia, without spore-cysts, are white (Gilliam et al., 1988;Gilliam and Lorenz, 1993).Larvae ingest the fungal spores when they are fed by adult bees that carry the spores. AbstractInoculation of Apis mellifera colonies with chalkbrood is a good way to test for hygienic behavior. The use of compatible pure strains of Ascosphaera apis, the causative agent of chalkbrood, could maintain homogeneity among experiments. Mycelium aging, viability, purity and capacity to produce spore-cysts were evaluated in MY20 and integral rice kernels (IRK) medium. Ascosphaera apis strains on MY20 developed aging symptoms by the 30 th day and developed pure colonies until the 77 th day. The same strains on IRK showed aging symptoms by the 30 th day of growing, but they produced pure colonies for 360 days. Spore-cysts obtained from pure strains preserved in IRK and spore-cysts from wild black mummies obtained from honeybee hives were used to inoculate fifth instar A. mellifera larvae. Ascospores from black wild mummies or spore-cysts obtained from pure strains preserved in IRK were equally effective in causing clinical symptoms of the disease, which has main advantages: permanent inoculum availability and genetic homogeneity.Additional key words: Apis mellifera, chalkbrood, culture media, honey bees, hygienic behavior, larvae inoculation, preservation. ResumenComunicación corta. Viabilidad y patogenicidad de Ascosphaera apis preservado en cultivos de arroz integral

Standard methods for fungal brood disease research

“…In addition, A. apis isolates, as seen for many other microbes, can lose virulence when kept in culture with repeated transfers, thus it is important to utilize a long term storage method soon after an isolate is obtained. Two different culture preservation methods have been successfully used: cryopreservation (Jensen et al ., 2009a) and propagation on Integral Rice Kernels (IRK) (Palacio et al ., 2007). Contamination during storage seems to be less risky during cryopreservation at −80°C, however for larger quantities and for laboratories not equipped with cryopreservation capacity, the use of rice kernels is a great alterative.…”

First Record of Ascosphaera apis Isolated from Commercial Pollen from Northern Argentinian Provinces

“…These assays were performed with a concentration of 1,250 spores/hives. In this work, a concentration of 5x10 6 spores/mL was used and the percentage of mummies was lower than that found by Flores et al (2004) and Palacios et al (2007), when they used sugar syrup. Both authors warned that the percentage of mummification depends on the stress conditions of the larvae when they receive the ascospores or on their susceptibility to contract the ectoparasite according to the period of the year.…”

“…The pathogenicity also depends on the origin of the inoculum with ascospores. Flores et al (2004) performed the infection techniques with ascospores from macerated bee mummies, while Palacios et al (2007) maintained ascospores on integral rice kernels (IRK) medium and MY20. The lower percentages of infection of the larvae achieved by the direct method of feeding than those obtained in other works could be attributed to the ascospores coming from the cultivation of commercial pollen.…”

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

“…We found, as did Palacio et al (2007), that such spores were still infective to honey bee larvae. In our bioassays, 65% (SE = 0.1064) of the larvae exposed to the 500-spore dosages were killed by A. apis infections.…”

“…An alternative for A. apis is to store cultures on rice kernels (Ruffinengo et al, 2000). Palacio et al (2007) found viability to be 73% for A. apis stored in this way for one year. Thus, the viability over time was not as stable with this method as for the cryogenic storage used in our study.…”