Thermotherapy Followed by Shoot Tip Cryotherapy Eradicates Latent Viruses and Apple Hammerhead Viroid from In Vitro Apple Rootstocks

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 a combination of both 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 one day followed by 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. Following treatments, shoot tips were 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 treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting 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.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies

“…To guard against possible false negatives, the virus status of plants that underwent in vitro therapies should be assessed for a second time after the plants have grown in the greenhouse: one should not rely just on the test result for in vitro plants. Virus titer may have been reduced and virus particles may occur in tissues that are not sampled for virus testing at the in vitro stage in some treatments, resulting in false negative, as we have shown in two “V500” plants (PVM-infected) derived from thermotherapy and chemotherapy treatments in this research and previously in raspberry and apple plants infected with Raspberry bushy dwarf virus and Apple hammerhead viroid , subjected to chemotherapy treatment ( Mathew et al, 2021 ), and combined thermotherapy with cryotherapy ( Bettoni et al, 2022 ), respectively. Therefore, repeat virus testing on progenies growing in the greenhouse and later in the field is recommended as standard best practice to confirm virus-free status of the plant materials.…”

Elimination of Solanum nigrum ilarvirus 1 and Apple Hammerhead Viroid from Apple Cultivars Using Antivirals Ribavirin, Rimantadine, and Zidovudine

“…Retests for AHVd were carried out after 7–11 months and will be repeated during weaning and multiplication of the plants. The recommended interval in the literature for verifying the elimination of virus-like pathogens is at least 4–6 months [ 5 ].…”

“…Each crop is affected by a number of economically important virus-like pathogens. Apple cultivars grown in orchards can be infected by at least 21 known viruses and 8 viroids [ 5 , 6 ], and once infected, trees remain infected throughout their life. Preference should be given to prevention of infection rather than implementing costly measures for treatment or eradication.…”

Eradication of PPV and PNRSV Viruses from Three Peach Cultivars Using Thermotherapy In Vitro, Including Optimization of Microshoots’ Multiplication and Rooting Medium

“…Among the established thermotherapybased methods typically used for virus elimination, combined in vitro thermotherapy with shoot tip culture, micrografting, electrotherapy, chemotherapy (especially using ribavirin), and shoot tip cryotherapy has been reported to be efficient for the eradication of different viruses from plants [8,[13][14][15][16]. The efficiency of thermotherapy-mediated elimination of viruses from plant materials is very diverse and ranges from 20 to 100% [11,15,[17][18][19][20].…”