Apple hammerhead viroid (AHVd) was detected in the apple cultivar ‘Šampion’ and in mixed infection with Solanum nigrum ilarvirus 1 (SnIV-1) in the cultivars ‘Selena’ and ‘Jonagored Supra’, using a high-throughput sequencing method. Experiments were conducted to eliminate both pathogens in apples using meristem tip cultures in combination with the antivirotics ribavirin, rimantadine, and zidovudine. Elimination of both pathogens was verified by repeated RT-PCR and qRT-PCR assays after 7–11 months. Elimination of SnIV-1 from all cultivars was successful with each of the three antivirotics at concentrations of 20, 40, and 80 mg L⁻¹. Elimination of AHVd was also achieved, although less effectively and only with ribavirin in the concentration range of 20–160 mg L⁻¹.

Systemic infections with phytoplasmas and viruses threaten the production of healthy plant material under the fruit species certification system. We tested the possibility of sanitation using in vitro culture and cryotherapy. The starting material of the cultivars Golden Delicious (clones A and B), Virginia Crab, and Panenské zlepšené was taken from in vivo plants that tested positive for apple proliferation phytoplasma. The Táborita cultivar was obtained from already established in vitro cultures that had tested positive for apple proliferation phytoplasma, apple mosaic virus, and apple chlorotic leaf spot virus. Cultivars Golden Delicious A, Virginia Crab, and Panenské zlepšené were sanitated from the phytoplasma in the first step, i.e., by sterilization and a subsequent transfer to in vitro conditions. Golden Delicious B remained infected with the phytoplasma, and both viruses, after the in vitro culture phase and together with Táborita, were subjected to cryotherapy by vitrification. In Golden Delicious B, three out of thirteen initial shoot tips regenerated after a liquid nitrogen treatment. Four mericlones were regenerated from 10 initial cryopreserved shoot tips of Táborita. None of the three pathogens were detected by PCR in the regenerated Golden Delicious B mericlones. On the contrary, in the case of Táborita, infection with all the pathogens was detected after regeneration. The results obtained indicate the potential applicability of in vitro cultivation techniques or, if necessary, subsequent cryopreservation as a method for sanitizing against systemic microbial contamination. However, further research on the relationship between pathogens and specific genotypes is needed.

Apple proliferation, caused by ‘Candidatus Phytoplasma mali’, is one of the most important economic threats in the field of apple production. Especially at a young age, infected trees can be affected by excessive bud proliferation and general decline. The fruit quality is also significantly reduced by this disease. To investigate treatment options, we applied a clarithromycin chemotherapy to infected in vitro cultures of ‘Golden Delicious’. With increasing concentrations of clarithromycin in the media, the phytoplasma load decreased rapidly after one month of treatment, but phytotoxicity led to a pronounced mortality at 40 mg/L, which was the highest dose used in our experiment. Out of 45 initial explants, we obtained one negative mericlone and two mericlones with a concentration of phytoplasma DNA at the detection limit of PCR. The culture propagated from the mericlone that tested negative remained phytoplasma-free after 18 months of subculturing. Our results suggest the applicability of macrolide antibiotics against phytoplasma infections in vitro; however, it might be challenging to find the threshold zone where the concentration is sufficient for pathogen elimination, but not lethal for the plant material of different cultivars.

Black elder (Sambucus nigra L.) is a temperate shrub with flowers and fruits that are edible after processing. This species is not yet widely known in the global agricultural sector, but its adaptability and drought tolerance may generate more interest in this crop. Our study aimed to find suitable micropropagation techniques for the black elder ‘Albida’ and compare suitable statistical methods for evaluating multiplication and rooting. For micropropagation, we tested the Murashige and Skoog (MS) growth medium with selected auxins and cytokinins. Five proliferation MS media containing 1, 2, and 4 mg/L BAP or 0.5 and 1 mg/L TDZ were tested. To induce root formation, three types of auxins were tested at a concentration of 1 mg/L in a 50% MS medium: IBA, IAA, and NAA. Data analysis was performed using different parametric and nonparametric tests to robustly capture the effects of treatments across varying distributional scenarios in developing explants subjected to the interactions of internal native and externally added plant growth regulators. The average multiplication rate ranged from 1.6 to 2.0 shoots per explant. High multiplication was recorded on the MS medium with 1 mg/L 6-benzylaminopurine. The root number per rooted explant was highly variable, ranging from 3.0 to 12.0 roots per explant. The highest average root number result was observed when 1 mg/L α-naphthalenacetic acid was used. All rooted plants were successfully acclimated to normal growing conditions. This in vitro propagation protocol allows for the production of hundreds to thousands of rooted plants from one initial explant within one year, enabling faster introduction to the agronomic sector.