Detection of Ascosphaera apis, causing chalkbrood disease in the colonies of European honey bee, Apis mellifera in West Bengal, India

Rakesh Das; Ritesh Kumar; Gautam Kunal; Suraj Goldar; Subrata Dutta; Shantanu Jha

doi:10.13102/sociobiology.v70i4.9129

Authors

Rakesh Das School of IndiaAgriculture, Swami Vivekananda University, India
Ritesh Kumar Department of Plant Pathology, Centurion University of Technology and Management, India
Gautam Kunal Plant Protection (Entomology), Dr. Rajendra Prasad Central Agricultural University, India
Suraj Goldar Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, India
Subrata Dutta Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, India
Shantanu Jha Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, India

DOI:

https://doi.org/10.13102/sociobiology.v70i4.9129

Keywords:

18s rDNA, Bees, Necrotrophs, Phylogeny

Abstract

The decline of honey bee (Apis mellifera L.) populations is of great concern around the world. Among the several key drivers, dissemination of pests and pathogens is potential one. Chalkbrood is very common fungal disease of honey bee, caused due to Ascosphaera apis. In the present study, survey was conducted regarding the prevalence of diseases in A. mellifera beekeeping in Gangetic plains of West Bengal, India. Results confirmed the occurrence of chalkbrood disease in different apiaries, where dead and mummified bee larvae with cotton like chalky white or greyish-black covering were found as physical symptoms of the disease. From three surveyed apiaries, a total of 16 hives out of 113 hives were found to be infected with the diseases; and 46 frames out of 132 frames from the infected 16 hives were found to be affected by the pathogen. Microscopic examination reflected that nearly 87% of the samples collected from the infected frame were found to be positive for the spore of Ascosphaera. The fungus associated with the disease was isolated on Potato Dextrose Agar medium, pure cultured and its genomic DNA was isolated to perform PCR and based on 18s rDNA sequencing by using specific primer pair of ITS-1 and ITS-4, the fungus was identified as Ascosphaera apis.

Keywords: 18s rDNA, Bees, Necrotrophs, Phylogeny

Downloads

Download data is not yet available.

References

Aizen, M.A., Garibaldi, L.A., Cunningham, S.A. & Klein, A.M. (2009). How much does agriculture depend on pollinators? Lessons from long-term trends in crop production. Annals of Botany, 103: 1579-1588.

Anderson, D.L., Giacon, H. & Gibson, N. (1997). Detection and thermal destruction of the chalkbrood fungus (Ascosphaera apis) in honey. Journal of Apiculture Research, 36: 163-168.

Anderson, D.L., Gibbs, A.J. & Gibson, N.L. (1998). Identification and phylogeny of sporecyst fungi (Ascosphaera spp.) using ribosomal DNA sequences. Mycological Research, 102: 541-547.

Anderson, D.L. & Gibson, N.L. (1998). New species and isolate of spore-cysts fungi (Plectomycetes: Ascosphaerales) from Australia. Australian Systematic Botany, 11: 53-72.

Aronstein, K.A. & Murray, K.D. (2010). Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, 103: 20-29.

Bailey, L. (1981). Honey Bee Pathology. Academic Press, London, UK: pp. 40-44.

Bailey, L. & Ball, B.V. (1991). Honey Bee Pathology. Academic Press, London, UK: pp. 53-62.

Baker, G.M. & Torchio, G.M. (1968). New records of Ascosphaera apis from North America. Mycologia, 60: 189-190.

Bissett, J. (1988). Contribution toward a monograph of the genus Ascosphaera. Canadian Journal of Botany, 66: 2541-2560.

Borum, A.E. & Ulgen, M. (2008). Chalkbrood (Ascosphaera apis) infection and fungal agents of honey bees in north-west Turkey. Journal of Apiculture Research, 47: 170-171.

Chhuneja, P.K., Garcha, S. & Gatoria, G.S. (2001). Bee diseases – Impending threat and challenge to Indian beekeeping. Indian Bee Journal, 63: 59-63.

Chorbinski, P. (2004). Identification of Ascosphaera apis strains using ribosomal DNA sequences. Medycyna Weterynaryjna, 60: 190-192.

Chorbinski, P. & Rypula, K. (2003). Studies on the morphology of strains Ascosphaera apis isolated from chalkbrood disease of the honey bees. Veterinary Medicine, 6: 1-12.

Flores, J.M., Spivak, M. & Gutierrez, I. (2005a). Spores of Ascosphaera apis contained in wax foundation can infect honey bee brood. Veterinary Microbiology, 108: 141-144.

Flores, J.M., Gutierrez, I. & Espejo, R. (2005b). The role of pollen in chalkbrood disease in Apis mellifera: transmission and predisposing conditions. Mycologia, 97: 1171-1176.

Flores, J.M., Ruiz, J.A., Ruz, J.M., Puerta, F., Bustos, M., Padilla, F. & Campano, F. (1996). Effect of temperature and humidity of sealed brood on chalkbrood development under controlled conditions. Apidologie, 27: 185-192.

Furst, M.A., McMahon, D.P., Osborne, J.L., Paxton, R.J. & Brown, M.J.F. (2014). Disease associations between honey bees and bumblebees as a threat to wild pollinators. Nature, 506: 364-366.

Gilliam, M. (1986). Infectivity and survival of the chalkbrood pathogen, Ascosphaera apis, in colonies of honey bees, Apis mellifera. Apidologie, 17: 93-100.

Gilliam, M. & Taber, S. (1991). Diseases, pests, and normal microflora of honey bees, Apis mellifera, from feral colonies. Journal of Invertebrate Pathology, 58: 286-289.

Gilliam, M., Taber, S. & Richardson, G.V. (1983). Hygienic behaviour of honey bees in relation to chalkbrood disease. Apidologie, 14: 29-39.

Gilliam, M., Taber, S. & Rose, J.B. (1978). Chalkbrood of honey bees, Apis mellifera L., a progress report. Apidologie, 9: 75-89.

Gilliam, M. & Vandenberg, J.D. (1997). Fungi. In: Morse, R., Flottum, K. (Eds.), Honey Bee Pests, Predators, and Diseases, 3rd ed. AI Root, Ohio, OH, pp. 81-110.

Graystock, P., Yates, K., Evison, S.E.F., Darvill, B., Goulson, D. & Hughes, W.H.O. (2013). The Trojan hives: pollinator pathogens, imported and distributed in bumble bee colonies. Journal of Applied Ecology, 50: 1207-1215.

Heath, L.A.F. (1982). Development of chalkbrood in a honey bee colony; chalk brood pathogens: a review. Bee World, 63: 119-135.

Heath, L.A.F. & Gaze, B.M. (1987). Carbon dioxide activation of spores of the chalkbrood fungus Ascosphaera apis. Journal of Apiculture Research, 26: 243-246.

Hitchcock, J.D. & Christensen, M. (1972). Occurrence of chalkbrood (Ascosphaera apis) in honey bees in the United States. Mycologia, 64: 1193-1198.

James, R.R. & Skinner, J.S. (2005). PCR diagnostic methods for Ascosphaera infections in bees. Journal of Invertebrate Pathology, 90: 98-103.

Janda, J.M. & Abbott, S.L. (2007). 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls. Journal of Clinical Microbiology, 45: 2761-2764.

Jara, L., Martínez-López, D., Munoz, I. & De la Rua, P. (2018). Epidemiological Survey of Ascosphaera apis in Small-Scale Migratory Apis mellifera iberiensis Colonies. Sociobiology, 65: 285-290.

Klinger, E.G., James, R.R., Youssef, N.N. & Welker, D.L. (2013). A multi-gene phylogeny provides additional insight into the relationships between several Ascosphaera species. Journal of Invertebrate Pathology, 112: 41-48.

Kluser, S. & Peduzzi, P. (2007). Global pollinator decline: a literature review. UNEP/GRID-Europe: pp. 1-12.

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution, 35: 1547-1549.

Mehr, Z., Menapace, D.M., Wilson, W.T. & Sacket, R.R. (1976). Studies on the initiation and spread of chalkbrood within an apiary. The American Bee Journal, 116: 266-268.

Murray, K.D., Aronstein, K.A. & Jones, W.A. (2005). A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA. Journal of Apiculture Research, 44: 61-64.

Nei, M. & Kumar, S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press, New York.

Neumann, P. & Carreck, N.L. (2010). Honey bee colony losses. Journal of Apiculture Research, 49: 1-6.

Nilsson, H., Kristiansson, E., Ryberg, M., Hallenberg, N. & Larsson, K.H. (2008). Intraspecific ITS variability in the kingdom Fungi as expressed in the International Sequence Databases and its implications for molecular species identification. Evolutionary Bioinformatics, 4: 193-201.

Palacio, M.A., Figini, E.E., Ruffinengo, S., Rodriguez, E., del Hoyo M.L. & Bedascarrasbure, E. (2000). Changes in a population of Apis mellifera L. selected for higienic behaviour and its relation to brood disease tolerance. Apidologie, 31: 469-471.

Puerta, F., Flores, J.M., Bustos, M., Padilla, F. & Campano, F. (1994). Chalkbrood development in honey bee brood under controlled conditions. Apidologie, 25: 540-546.

Rose, J.B., Christensen, M. & Wilson, W.T. (1984). Ascosphaera species inciting chalkbrood in North America and taxonomic key. Mycotaxon, 19: 41-55.

Saitou, N. & Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406-425.

Schoch, C.L., Seifertb, K.A., Huhndorf, S., Robert, V., Spougea, J.L., Andre Levesque, C. & Chen, W., (Fungal Barcoding Consortium) (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America, 109: 6241-6246.

Shimanuki, H. & Knox, D.A. (1990). Diagnosis of Honey Bee Diseases. Agriculture Handbook No. AH-690, US Department of Agriculture.

Simone-Finstrom, M., Li-Byarlay, H., Huang, M.H., Strand, M.K., Rueppell, O. & Tarpy, D.R. (2016). Migratory management and environmental conditions affect lifespan and oxidative stress in honey bees. Scientific Reports, 6: 32023.

Singh, R., Levitt, A.L., Rajotte, E.G., Holmes, E. & Ostiguy, N. (2010). RNA viruses in Hymenopteran pollinators: evidence of inter-taxa virus transmission via pollen and potential impact on non-apis Hymenopteran species. PLoS ONE, 5: e14357.

Skou, J.P. (1972). Ascosphaerales. Friesia, 10: 1-24.

Skou, J.P. (1988). More details in support of the class Ascosphaeromycetes. Mycotaxon, 31: 191-198.

Spiltoir, C.F. (1955). Life cycle of Ascosphaera apis (Pericystis apis). American Journal of Botany, 42: 501-508.

Spiltoir, C.F. & Olive, L.S. (1955). A reclassification of the genus Pericystis Betts. Mycologia, 47: 238-244.

Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22: 4673-4680.

White, T., Burns, T., Lee, S. & Talyor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis, M.A., Gelfand, D.H, Sninsky, J.J., White, T.J. (Eds), PCR protocols: a guide to methods and applications. Academic Press, San Diego, USA: pp. 315-322.

Wynns, A.A. (2012). The bee specialist fungus family Ascosphaeraceae and its allies: systematics, ecology and co-evolution with solitary bees, Ph.D. thesis, Department of Agriculture and Ecology, University of Copenhagen. Denmark: 109 p.

Wynns, A.A., Jensen, A.B., Eilenberg, J. & James, R. (2012). Ascosphaera subglobosa, a new spore cyst fungus from North America associated with the solitary bee Megachile rotundata. Mycologia, 104: 108-114.

Yacobson, B.A., Elad, D., Rosenthal, K., Kmer, I., Slovecky, I. & Efrat, H. (1991). A recent chalkbrood outbreak in Israel: attempts at therapeutic intervention. The American Bee Journal, 131: 786.

Zaghloul, O.A., Mourad, A.K., El Kady, M.B., Nemat, F.M. & Morsy, M.E. (2005). Assessment of losses in honey yield due to the chalkbrood disease, with reference to the determination of its economic injury levels in Egypt. Communications in Agricultural and Applied Biological Sciences, 70: 703-714.