Detection of Ascosphaera apis, causing chalkbrood disease in the colonies of European honey bee, Apis mellifera in West Bengal, India
DOI:
https://doi.org/10.13102/sociobiology.v70i4.9129Keywords:
18s rDNA, Bees, Necrotrophs, PhylogenyAbstract
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
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. DOI: https://doi.org/10.1093/aob/mcp076
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. DOI: https://doi.org/10.1080/00218839.1997.11100944
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. DOI: https://doi.org/10.1017/S0953756297005261
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. DOI: https://doi.org/10.1071/SB96026
Aronstein, K.A. & Murray, K.D. (2010). Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, 103: 20-29. DOI: https://doi.org/10.1016/j.jip.2009.06.018
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. DOI: https://doi.org/10.1016/B978-0-12-073481-8.50009-6
Baker, G.M. & Torchio, G.M. (1968). New records of Ascosphaera apis from North America. Mycologia, 60: 189-190. DOI: https://doi.org/10.1080/00275514.1968.12018556
Bissett, J. (1988). Contribution toward a monograph of the genus Ascosphaera. Canadian Journal of Botany, 66: 2541-2560. DOI: https://doi.org/10.1139/b88-346
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. DOI: https://doi.org/10.1080/00218839.2008.11101447
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. DOI: https://doi.org/10.1016/j.vetmic.2005.03.005
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. DOI: https://doi.org/10.3852/mycologia.97.6.1171
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. DOI: https://doi.org/10.1051/apido:19960401
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. DOI: https://doi.org/10.1038/nature12977
Gilliam, M. (1986). Infectivity and survival of the chalkbrood pathogen, Ascosphaera apis, in colonies of honey bees, Apis mellifera. Apidologie, 17: 93-100. DOI: https://doi.org/10.1051/apido:19860202
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. DOI: https://doi.org/10.1016/0022-2011(91)90077-4
Gilliam, M., Taber, S. & Richardson, G.V. (1983). Hygienic behaviour of honey bees in relation to chalkbrood disease. Apidologie, 14: 29-39. DOI: https://doi.org/10.1051/apido:19830103
Gilliam, M., Taber, S. & Rose, J.B. (1978). Chalkbrood of honey bees, Apis mellifera L., a progress report. Apidologie, 9: 75-89. DOI: https://doi.org/10.1051/apido:19780106
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. DOI: https://doi.org/10.1111/1365-2664.12134
Heath, L.A.F. (1982). Development of chalkbrood in a honey bee colony; chalk brood pathogens: a review. Bee World, 63: 119-135. DOI: https://doi.org/10.1080/0005772X.1982.11097877
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. DOI: https://doi.org/10.1080/00218839.1987.11100768
Hitchcock, J.D. & Christensen, M. (1972). Occurrence of chalkbrood (Ascosphaera apis) in honey bees in the United States. Mycologia, 64: 1193-1198. DOI: https://doi.org/10.1080/00275514.1972.12019370
James, R.R. & Skinner, J.S. (2005). PCR diagnostic methods for Ascosphaera infections in bees. Journal of Invertebrate Pathology, 90: 98-103. DOI: https://doi.org/10.1016/j.jip.2005.08.004
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. DOI: https://doi.org/10.1128/JCM.01228-07
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. DOI: https://doi.org/10.13102/sociobiology.v65i2.2685
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. DOI: https://doi.org/10.1016/j.jip.2012.10.011
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. DOI: https://doi.org/10.1093/molbev/msy096
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. DOI: https://doi.org/10.1080/00218839.2005.11101150
Nei, M. & Kumar, S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press, New York. DOI: https://doi.org/10.1093/oso/9780195135848.001.0001
Neumann, P. & Carreck, N.L. (2010). Honey bee colony losses. Journal of Apiculture Research, 49: 1-6. DOI: https://doi.org/10.3896/IBRA.1.49.1.01
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. DOI: https://doi.org/10.4137/EBO.S653
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. DOI: https://doi.org/10.1051/apido:2000139
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. DOI: https://doi.org/10.1051/apido:19940604
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. DOI: https://doi.org/10.1038/srep32023
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. DOI: https://doi.org/10.1371/journal.pone.0014357
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. DOI: https://doi.org/10.1002/j.1537-2197.1955.tb11154.x
Spiltoir, C.F. & Olive, L.S. (1955). A reclassification of the genus Pericystis Betts. Mycologia, 47: 238-244. DOI: https://doi.org/10.1080/00275514.1955.12024448
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. DOI: https://doi.org/10.1093/nar/22.22.4673
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. DOI: https://doi.org/10.1016/B978-0-12-372180-8.50042-1
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. DOI: https://doi.org/10.3852/10-047
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.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2023 Rakesh Das, Ritesh Kumar, Gautam Kunal, Suraj Goldar, Subrata Dutta, Shantanu Jha

This work is licensed under a Creative Commons Attribution 4.0 International License.
Sociobiology is a diamond open access journal which means that all content is freely available without charge to the user or his/her institution. Users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles in this journal without asking prior permission from the publisher or the author. This is in accordance with the BOAI definition of open access.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).

eISSN 2447-8067









