S. Karimpour¹, G.H. Davarynejad¹, M. ZakiAghl², M.R. Safarnejad³, P. Martínez-Gómez⁴ and M. Rubio^4
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
⁴ Department of Plant Breeding, CEBAS-CSIC, Espinardo, Murcia, Spain

SUMMARY Production of virus-free plants is crucial for vegetative propagation. In in vitro conditions, thermotherapy treatments are the most common method for virus elimination. However, shoot-tip necrosis and hyperhydricity are the most common physiological problems in thermotherapy-treated shoots, causing an important loss of virus-free plants. In the present study Apple chlorotic leaf spot virus (ACLSV, Trichovirus genus), Apple stem pitting virus (ASPV, Foveavirus genus) and Apple mosaic virus (ApMV, Ilarvirus genus) infected ‘Abbasiʼ apple (Malus domestica Borkh.) shoots were treated through two in vitro thermotherapy strategies, constant (37°C) and alternating (32/38°C) cycles. Plant growth, shoot-tip necrosis, meristem establishment, hyperhydricity and total chlorophyll content (SPAD-502 meter readings) were recorded in virus-infected and virus-free shoots. Results showed that duration of high temperatures exposure caused shoot-tip necrosis decreasing shoot-tip survival. Meristem hyperhydricity showed two different trends related to length of treatment, increasing up to 20 d and decreasing up to 70 d applying heat treatments at constant and alternating cyclic temperatures, respectively. Treatment at constant 38°C for 5–15 d led to 100% eradication of ACLSV and ASPV, while all treated plants were still infected with ApMV. The alternating cycles of 32/38°C treatment was effective in eliminating ApMV, as well as ACLSV and ASPV. Virus-free plants had 3.9-fold growth in comparison with virus-infected plants. In addition, SPAD-502 meter readings (total chlorophyll index) showed significant differences between virus-infected and virus-free plants. Suggesting that rapid assessment of physiological, growth and sanitary state of apple shoots after thermotherapy treatments, should be performed by chlorophyll content evaluation using SPAD-502 meter. Higher SPAD-502 meter readings in apple virus-free shoots correlated with better growth and reduction of shoot-tip necrosis and hyperhydricity, confirming that photosynthetic rate is affected by virus infection via decreasing leaf chlorophyll content. Keywords in vitro culture, shoot-tip necrosis, hyperhydricity, RT-PCR, healthy plant

Significance of this study What is already known on this subject? Thermotherapy in vitro treatments are the most common method for virus elimination in plant species. However, shoot-tip necrosis and hyperhydricity are the most common problems associated to these thermotherapy-treated shoots, causing important loss of virus-free plants. What are the new findings? This work describes the development of a rapid protocol to assess the sanitary and physiological state of thermotherapy-treated apple shoots through chlorophyll content evaluation by using SPAD-502 meter readings. What is the expected impact on horticulture? The obtained results will allow the development of a rapid and routine-applied methodology for evaluation of physiological and sanitary state of thermotherapy-treated apple shoots to optimize in vitro protocols for virus elimination in infected apple trees.

E-mail: pmartinez@cebas.csic.es  davarynej@um.ac.ir  

References

• Abtahi, F.S., Shams-Bakhsh, M., and Safaie, N. (2013). First report of Apple stem pitting virus infection of apple trees in Iran. Iranian J. Plant Pathol. 50, 143–147.

• Abtahi, F.S., Shams-Bakhsh, M., Safaie, N., Autonell, C.R., and Ratti, C. (2017). Occurrence, distribution, and molecular characterization of Apple stem pitting virus in Iran. J. Agric. Sci. Technol. 19, 217–230.

• Alemzadeh, E., Katsiani, A.T., Efthimiou, K., and Katis, N.I. (2016). Occurrence of Apple chlorotic leaf spot virus in apple and quince in Southern Iran. J. Plant Pathol. 98, 171–185.

• Almasi, A., Apatini, D., Boka, K., Boddi, B., and Gaborjanyi, R. (2000). BSMV infection inhibits chlorophyll biosynthesis in barley plants. Physiol. Mol. Plant Pathol. 56, 227–233. https://doi.org/10.1006/pmpp.2000.0266.

• Almasi, A., Harsanyi, A., and Gaborjanyi, R. (2001). Photosynthetic alterations of virus infected plants. Acta Phytopathol. Entomol. Hungarica 36, 15–29. https://doi.org/10.1556/APhyt.36.2001.1-2.3.

• Barba, M. (1998). Virus certification of fruit tree propagative material in Western Europe. In Plant Virus Disease Control, A. Hadidi, R.K. Khetarpal, and H. Koganezawa, eds. (St. Paul, MN, USA: APS Press), p. 288–293.

• Barba, M., Ilardi, V., and Pasquini, G. (2015). Control of pome and stone fruit virus diseases. Advanc. Virus Res. 91, 47–83. https://doi.org/10.1016/bs.aivir.2014.11.001.

• Benson, E. (2000). Special symposium: In vitro plant recalcitrance. Do free radicals have a role in plant tissue culture recalcitrance? In Vitro Cell. Develop. Biol. Plant 36, 163–170. https://doi.org/10.1007/s11627-000-0032-4.

• 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. https://doi.org/10.17660/ActaHortic.1998.472.13.

• Borraz-Martínez, S., Simó, J., Gras, A., Mestre, M., and Boqué, R. (2019). Multivariate classification of Prunus dulcis varieties using leaves of nursery plants and near infrared spectroscopy. Sci. Rep. 9, 19810. https://doi.org/10.1038/s41598-019-56274-5.

• Brzezinka, K., Altmann, S., Czesnick, H., Nicolas, P., Górka, M., and Benke, E. (2016). Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling. Elife Sci. 5. https://doi.org/10.7554/eLife.17061.

• Campbell, A.I. (1963). The effect of some latent virus infections on the growth and cropping of apples. J. Hortic. Sci. 38, 15–19. https://doi.org/10.1080/00221589.1963.11514054.

• Cho, I.S., Choi, G.S., and Kwon, S.J. (2015). Molecular diagnostic assay for the simultaneous detection of pome fruit viruses. Int. J. Appl. Sci. Technol. 5, 39–46.

• Cieslinska, M. (2002). Elimination of Apple chlorotic leaf spot virus (ACLSV) from pear by in vitro thermotherapy and chemotherapy. Acta Hortic. 596, 481–484. https://doi.org/10.17660/ActaHortic.2002.596.80.

• Cooper, V.C., and Walker, D.G.A. (1978). Thermal inactivation of Cherry leaf roll virus in tissue culture of Nicotiana rustica raised from seeds and meristem tips. Ann. Appl. Biol. 88, 273–326. https://doi.org/10.1111/j.1744-7348.1978.tb00706.x.

• Da Camara Machado, A., Mendonca, D., Lopes, M.S., Knapp, E., Hanzer, V., Arthofer, W., Katinger, H., and Laimer de Camara Machado, M. (1998). Phytosanitary improvement of fruit tree species: Diagnostic strategies in virus-indexing of in vitro plants. Acta Hortic. 472, 511–516. https://doi.org/10.17660/ActaHortic.1998.472.64.

• Dries, N., Giannì, S., Czerednik, A., Krens, F.A., and De Klerk, G.J. (2013). Flooding of the apoplast is a key factor in the development of hyperhydricity. J. Exp. Bot. 64, 5221–5230. https://doi.org/10.1093/jxb/ert315.

• Goodman, R.N., Király, Z., and Wood, R.K. (1986). The Biochemistry and Physiology of Plant Disease (Columbia: University of Missouri Press).

• Hadidi, A., Khetarpal, R.K., and Koganezawa, H. (1998). Plant Virus Disease Control. (St. Paul, MN, USA: APS Press).

• Hu, G., Honga, N., Wang, 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 Prot. 37, 20–25. https://doi.org/10.1016/j.cropro.2012.02.017.

• Hu, G., Zhang, Z., Dong, Y., Fan, X., Ren, F., and Zhu, H. (2015). Efficiency of virus elimination from potted apple plants by thermotherapy coupled with shoot-tip grafting. Australasian Plant Pathol. 44, 167–173. https://doi.org/10.1007/s13313-014-0334-3.

• Kassanis, B. (1957). Some effects of varying temperature on the quality and quantity of Tobacco mosaic virus in infected plants. Virol. 4, 187–199. https://doi.org/10.1016/0042-6822(57)90056-9.

• Keshavarz, T., and Shams-Bakhsh, M. (2015). Incidence and distribution of Apple chlorotic leaf spot virus in the main fruit growing areas of Iran. Arch. Phytopathol. Plant Prot. 48, 306–312. https://doi.org/10.1080/03235408.2014.886852.

• 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. Acta Hortic. 386, 386–418. https://doi.org/10.17660/ActaHortic.1995.386.56.

• Koiwa, H., Kojima, M., Ikeda, T., and Yoshida, Y. (1992). Fluctuations of particles on chloroplast thylakoid membranes in tomato plants infected with virulent or attenuated strain of Tobacco mosaic virus. Ann. Phytopathol. Soc. Japan 58, 58–64. https://doi.org/10.3186/jjphytopath.58.58.

• Lämke, J., Brzezinka, K., Altmann, S., and Bäurle, I. (2016). A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory. EMBO J. 35, 162–175. https://doi.org/10.15252/embj.201592593.

• 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. https://doi.org/10.4236/ajps.2017.810168.

• Menzel, W., Jelkmann, W., and Maiss, E. (2002). Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. J. Virol. Meth. 99, 81–92. https://doi.org/10.1016/S0166-0934(01)00381-0.

• Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x.

• Naderpour, M. (2013). Assessment of virus and virus-like diseases of apple trees in Iran and standardization of detection methods for apple certification schemes. Project PVR, Seed and Plant Certification and Registration Institute. 77 pp..

• 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. https://doi.org/10.1007/s11240-008-9342-8.

• Posnette, A.F., Cropley, R., and Ellenberger, C.E. (1963). The effect of virus infection on the growth and crop of apple, pear, and plum trees. Phytopathol. Mediterr. 2, 158–161.

• Ranjeet, R., Kumar, L., Suneha, G., Sushil, K., Sharma, L., Khushboo, S., Kritika, L., Gadpayle, A., Narender Kumar, L., Gyanendra, K., Manorama Singh, L., and Raj, D. (2012). Protection against heat stress in wheat involves change in cell membrane stability, antioxidant enzymes, osmolyte, H₂O₂ and transcript of heat shock protein. Int. J. Plant Physiol. Biochem. 4, 83–91. https://doi.org/10.5897/IJPPB12.008.

• SAS Institute Inc. (1989). SAS/STAT User’s Guide, v. 6, 4^th edn., Vol. 2. (Cary: SAS Institute, Inc.).

• Schmidt, H. (1972). The effect of ‘latent’ virus infections on the yield of maiden trees on 20 apomictic apple seedling rootstocks. J. Hortic. Sci. 47, 159–163. https://doi.org/10.1080/00221589.1972.11514451.

• 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. https://doi.org/10.1007/s11240-010-9681-0.

• Tu, J.C., Ford, R.E., and Krass, C.J. (1967). Comparison of chloroplasts in maize dwarf mosaic virus-infected and healthy corn, sorghum, and Johnson grass. Phytopathol. 57, 834.

• 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 lea spot virus for in vitro-cultured pear shoot tips. HortScience 41, 729–732. https://doi.org/10.21273/HORTSCI.41.3.729.

• 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, 283–291. https://doi.org/10.1007/s11240-016-0948-y.

• Wu, Z., Chen, L.J., and Long, Y.J. (2009). Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoot. In Vitro Cell. Dev. Biol. Plant 45, 483–490. https://doi.org/10.1007/s11627-008-9180-8.

• Zilka, S., Faingersh, E., Rotbaum, A., Tam, Y., Spiegel, S., and Malca, N. (2002). In vitro production of virus-free pear plants. Acta Hortic. 596, 477–479. https://doi.org/10.17660/ActaHortic.2002.596.79.

Received: 22 January 2020 | Accepted: 6 April 2020 | Published: 3 May 2019 | Available online: 3 May 2019