In Vitro Thermotherapy and Thermo-Chemotherapy Approaches to Eliminate Some Viruses in Pyrus communis L. cv. ‘Natanz’

Document Type : Original Research

Authors
1 Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran.
2 Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran.
3 Department of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural Research Education and Extension Organization (AREEO), Tehran, Islamic Republic of Iran.
Abstract
Production of virus-free stocks is crucial for efficient management of plant viruses in cultivation of pome fruits. Regarding the importance of producing the pre-basic stocks of valuable fruit trees, pear cultivar ʽNatanzʼ, an important local pear cultivar in Iran, was selected for virus eradication. In the present study, tissue culture combined with in vitro thermotherapy and thermo-chemotherapy techniques were used for elimination of Apple Stem Pitting Virus (ASPV) and Apple Mosaic Virus (ApMV). In thermotherapy approach, in vitro shoots were initially incubated for 55, 60, 65, and 70 days in alternating temperatures (32/38°C), then, meristems were cultivated on meristem medium. In thermo-chemotherapy approach, in vitro shoots were incubated for 50 days at 32/38°C, and then meristems were cultivated on a medium containing ribavirin. Virus detection by RT-PCR using specific primers was carried out after rooting and adaptation of the regenerated shoots. The percentage of survived shoots and meristem establishment were depended on thermo-duration. After 55 days, 83.33% of shoots survived, while it decreased to 33.33% after 70 days. Both ASPV and ApMV were eliminated after 60 days of thermotherapy. Ribavirin at 10 and 20 mg L-1 reduced the percentage of meristem establishment to 50 and 37%, respectively, compared to the control (88.88%). Thermo-chemothery was also effective for ASPV and ApMV eradication from pear shoots.

Keywords

Subjects


Abtahi, F., Shams-bakhsh, M., Safaie, N., Autonell, C.R., and Ratti, C. 2017. Occurrence, Distribution, and Molecular Characterization of Apple Stem Pitting Virus in Iran. JAST. 19 (1):217-230.
Bhardwaj, S.V., Rai, S.J., Thakur, P.D., and Handa A.1998. Meristem Tip Culture and Heat Therapy for Production of Apple Mosaic Virus Free Plants in India. Acta Hortic 472:65-68.
Cembali, T., Folwell, R.J., Wandschneider, P., Eastwell, K.C., and Howell, W.E., 2003. Economic Implications of a Virus Prevention Program in Deciduous Tree Fruits in the US. Crop Prot. 22, 1149-1156.
Cieślińska, M. 2002. Elimination of Apple Chlorotic Leaf Spot Virus (ACLSV) from Pear by in Vitro Thermotherapy and Chemotherapy. Proc.8th IS on Pear Eds. L. Corelli-Grappadelli et al. Acta Hort, 596: 481-484.
Cieślińska, M., and Zawadzka, B. 1999. Preliminary Results of Investigation on Elimination of Viruses from Apple, Pear and Raspberry Using Thermotherapy and Chemotherapy in Vitro. Phytopathologia Polonica, 17: 41-48.
Crotty, S., Maag, D., Cameron, C.E., and Andino, R. 2001. RNA virus error catastrophe: direct molecular test by using ribavirin. P Natl Acad Sci USA, 12: 6895-6900.
Guo, Ch., Wu, R., Shao, J., Sun, J., and Shi, X. 2014. Study on Elimination and Recrudescence of Apple Latent Virus by Alternating Heat Therapy in Vitro. Northern Horticulture, 17.
Hu, G., Honga N., Wanga L.P., Hu H.J, and Wang G.P. 2012. Efficacy of Virus Elimination from in Vitro-Cultured Sand Pear (Pyrus pyrifolia) by Chemotherapy Combined with Thermotherapy. Crop Protection, 37: 20-25.
Hu G., Dong Y., Zhang Z., Fan X., Ren F., and Zhou J. 2015. Virus Elimination from in Vitro Apple by Thermotherapy Combined with Chemotherapy. Plant Cell Tiss Organ Cult, 121:435–443.
Hu, G.J., Hong, N., and Wang, G.P. 2018. Elimination of Apple Stem Pitting Virus from in Vitro-Cultured Pear by an Antiviral Agent Combined with Thermotherapy. Australas. Plant Pathol.. https://doi.org/10.1007/s13313-018-0606-4
James, D. 2010. Confirmation of the Elimination of Apple Stem Grooving Virus from Apple Trees by in Vitro Chemotherapy. 21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops, 427: 47-50.
Knapp, E., Hanzer, V., Weiss, H., Da Camara Machado, A., Weiss, B., Wang, Q., Katinger, H., and Lamier Da Camara Machado, M. 1995. New Aspects of Virus Elimination in Fruit Trees. Fruit tree virus diseases XVI. Acta Hort, 386: 409-418.
Liu, J., Zhang Z., Zhang, F., Hong, N., Wang, G., Wang, A., and Wang, L. 2015. Identification and Characterization of microRNAs from in Vitro-Grown Pear Shoots Infected with Apple Stem Grooving Virus in Response to High Temperature Using Small RNA Sequencing, BMC Genomics, 16 (945): 1-16.
Lizarraga, A., Ascasibar, J., and Gonzalez, M.L. 2017. Fast and Effective Thermotherapy Treatment for in Vitro Virus Eradication in Apple and Pear Trees. Am. J. Plant Sci., 8: 2474-2482.
Menzel, W., Jelkmann, W., and Maiss, E. 2002. Detection of Four Apple Viruses by Multiplex RT-PCR Assays with Co-Amplification of Plant mRNA as Internal Control. J. Virol. Methods, 99: 81–92.
Murashige, T., and Skoog, F. 1962. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15:473–497.
Nacheva, L., and Milusheva, S. 2008. Preliminary Results of the Effect of Ribavirin on in Vitro Cultivated Apple Plants with the Aim of Eliminating Some Viruses. JMAB, 11(1): 129-137.
Nickel, O., and Fajardo, T.V.M. Elimination of Apple Latent Viruses by in Vitro Chemotherapy and Meristem-culturing. 2012. Congresso Brasilerio de fruticultura 22: 2217-2220.
O’Herlihy, E.A. Croke, J.T., and Cassells, A.C. 2003. Influence of in Vitro Factors on Titre and Elimination of Model Fruit Tree Viruses. Plant Cell Tiss Organ Cult., 72: 33–42.
Paprstein, F., Sedlak, J., Polak, J., Svobodova, L., Hassan, M., and Bryxiova, M. 2008. Results of in Vitro Thermotherapy of Apple Cultivars. Plant Cell Tiss Organ Cult. 94:347–352.
Parstein, F., Sedlak, J., Svobodova, L., Polak, J., and Gadiou, S. 2013. Results of in Vitro Chemotherapy of Apple Cultivar Fragrance – Short communication. Hort. Sci. (Prague), 40: 186–190.
Paprstein, F., Sedlak, J., and Talacko, L. 2014. In vitro Chemotherapy of Pear Cultivars. Proc. IInd IS on Biotechnology of Fruit Species Ed.: S.E. Gardiner, Acta Hort., 1048: 221-224.
Panattoni, A., Luvisi, A., and Triolo, E. 2013. Review. Elimination of Viruses in Plants: Twenty Years of Progress. Span. J. Agric. Res., 11(1): 173-188.
Plese, N., Hoxha, E., and Milicic, D. 1975. Pathological Anatomy of Trees Affected with Apple Stem Grooving Virus. Phytopathology, 82: 315-325.
SAS Institute Inc. 1989. SAS/STAT User’s Guide, Version 6. 4th Edition, Volume 2. SAS Institute, Inc., Cary
Sedlak, J., Paprstein, F., and Talacko, L. 2011. Elimination of Apple Stem Pitting Virus from Pear Cultivars by in vitro Chemotherapy. Proc. XXVIIIth IHC – IS on Micro and Macro Technologies for Plant Propagation Eds.: A. Fabbri and E. Rugini. Acta Hort 923: 111-116.
Shim, H.K., Min, Y.J., Hong, S.Y., Kwon, M.S., Kim, H.R., Choi, Y.M., Lee, S.C., and Yang, J.M., 2004. Nucleotide Sequences of a Korean Isolate of Apple Stem Grooving Virus Associated with Black Necrotic Leaf Spot Disease on Pear (Pyrus pyrifolia). Mol. Cell. 18: 192-199.
Stouffer, R.F. 1989. Apple Stem Pitting. In: Fridlund, P.R. (Ed.), Virus and Virus-like Diseases of Pome Fruits and Simulating Noninfectious Disorders. Washington State University, Pullman, WA, USA, 138-144.
Tan, R., Wang, L., Hong, N., and Wang, G. 2010. Enhanced Efficiency of Virus Eradication Following Thermotherapy of Shoot-tip Cultures of Pear. Plant Cell Tiss Organ Cult. 101:229–235.
Yanase, H. 1983. Back Transmission of Apple Stem Grooving Virus to Apple Seedlings and Induction of Symptoms of Apple Topworking Disease in Mitsuba Kaido (Malus sieboldii) and Kobano Zumi (Malus sieboldii var. arborescens) Rootstocks. Acta Hort. 130: 117-122.
Zilka, S., Faingersh, E., Rotbaum, A., Tam, Y., Spiegel, S., and Malca, N. 2002. In Vitro Production of Virus-free Pear Plants. Proc.8th IS on Pear Eds. L. Corelli-Grappadelli et al. Acta Hort, 596: 477-479.
Wang, L., Wang, G., Hong, N., Tang, R., Deng, X., and Zhang, H. 2006. Effect of Thermotherapy on Elimination of Apple Stem Grooving Virus and Apple Chlorotic Leaf Spot Virus for in Vitro-Cultured Pear Shoot Tips. HortScience, 41 (3): 729-732.
Wang, M.R., Li, B.G., Feng, C.H., and Wang, Q.C. 2016. Culture of shoot tips from adventitious shoots can eradicate Apple stem pitting virus but fails in Apple stem grooving virus. Plant Cell Tiss Organ Cult. 125 (2): 283–291.