Thermotherapy followed by shoot tip cryotherapy eradicates latent viruses and Apple hammerhead viroid from in vitro apple rootstocks

Authors: BETTONI, J - Plant And Food Research; Fazio, Gennaro; CARVALHO COSTA, L - Animal And Plant Health Inspection Service (APHIS); HURTADO-GONZALES, O - Animal And Plant Health Inspection Service (APHIS); AH RWAHNIH, M - University Of California, Davis; Nedrow, Abby; Volk, Gayle

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2022
Publication Date: 2/22/2022
Citation: Bettoni, J.C., Fazio, G., Carvalho Costa, L., Hurtado-Gonzales, O.P., Ah Rwahnih, M., Nedrow, A.K., Volk, G.M. 2022. Thermotherapy followed by shoot tip cryotherapy eradicates latent viruses and Apple hammerhead viroid from in vitro apple rootstocks. Plants. 11(5). Article e582. https://doi.org/10.3390/plants11050582.
DOI: https://doi.org/10.3390/plants11050582

Interpretive Summary: The presence of viruses and viroids in apple plant genetic resources affects movement of materials into the United States, maintenance in genebanks, industry propagation, and field production. Obtaining virus- and viroid-free plants is difficult using currently available technologies. This manuscript describes the eradication of Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple hammerhead viroid (AHVd) from new apple rootstock cultivars that will be released to industry. Combinations of in vitro plant thermotherapy and shoot tip cryotherapy were tested to determine if the viruses and viroids could be removed from the plant materials. The highest percentage of clean plants were obtained when in vitro plants were treated with thermotherapy for four weeks, followed by shoot tip vitrification cryotherapy treatments. Shoot tips were recovered in vitro, transferred and grown in the greenhouse, placed into dormancy, and then grown again prior to sampling for viruses and viroids. The success of this project has resulted in the use of cryotherapy techniques in the apple rootstock breeding program in Geneva, NY and in the USDA-APHIS Quarantine program in Beltsville, MD for pathogen eradication.

Technical Abstract: Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or in combination could effectively eradicate ACLSV, ASGV and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36°C (day) and 32°C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for 1 day and then exposure to PVS2 for 60 or 75 min at 22°C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Shoot tips were then warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatments resulted in the highest percentage of virus- and viroid-free plants. This combination of procedures has great potential for producing virus and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm.