The present study described a combining thermotherapy with meristem culture for improved eradication of onion yellow dwarf virus (OYDV) and shallot latent virus (SLV) from co-infected in vitro-cultured shallot shoots. In vitro-cultured shoots infected with OYDV and SLV were thermo-treated at a constant temperature of 36 C for 0, 2 and 4 weeks, and then meristems (0.5 mm) containing 1-2 leaf primordia were excised and cultured for shoot regrowth. Meristem culture without thermotherapy produced much higher levels of survival (100%) and shoot regrowth (55%) than those (62% survival and 32% shoot regrowth) produced by the procedure combining 4 weeks of thermotherapy with meristem culture. However, much higher virus-free frequencies (70% for OYSV, 80% for SLV and 50% for both viruses) were obtained in the latter than those (10% for OYSV, 15% for SLV and 10% for both viruses) obtained in the former. Histological and subcellular studies showed that thermotherapy imposed stress or damage to the cells of meristems, thus resulting in reduced meristem survival and shoot regrowth. Studies on virus location revealed considerable alternations of virus distribution patterns in the thermo-treated meristems. The results of histological and subcellular studies and analysis of virus distribution pattern added valuable experimental data in the combining thermotherapy with meristem culture for virus eradication. These data provided explanations as to why combining thermotherapy with meristem culture improved the eradication of OYDV and SLV from the virus-infected in vitro shallot shoots.

Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.

Shallot (Allium cepa var. aggregatum) is a small-bulb onion crop mainly grown in subtropical to temperate regions of the world (Block, 2010;Fritsch & Friesen, 2002). It is consumed for its unique flavour, pungency, and health-enhancing properties (Block, 2010;Fritsch & Friesen, 2002;Shahrajabian et al., 2020). Shallots are predominantly propagated by bulbs, resulting in the accumulation and dissemination of viruses in commercially grown fields and germplasm collections (Katis et al., 2012;Smékalová et al., 2017).The main viruses prevailing in Allium crops are from the genera Potyvirus, Carlavirus, and Allexivirus, which form viral complexes (Katis et al., 2012). Virus infections, particularly by potyviruses like onion yellow dwarf virus (OYDV) and leek yellow stripe virus

In May 2021, tomato (Solanum lycopersicum L.) plants with necrosis and ringspot symptoms were observed in a farm greenhouse at Sundbyfoss, Norway. This greenhouse production focused on ecological growing of several tomato varieties (i.e., “Blush Tiger”, “Sailor’s Luck”, “Evil Oliver”, etc.) with compost for local customers. The presence of tomato brown rugose fruit virus (ToBRFV) was suspected due to the symptoms in the diseased plants. Sap inoculation was carried out with extracts from 100 mg symptomatic tomato fresh leaves in 0.03 M PBS (phosphate buffered saline, pH 7.0), and inoculation on three Nicotiana tabacum cv. Xanthi plants at the 4-6 leaf stage grown in an experimental greenhouse. The test plants showed local necrotic lesions after 3-5 days. Two symptomatic leaf samples, one from tomato and the other from a test plant, were analysed by transmission electron microscope (TEM) with the help of negative staining. The samples were treated with 0.1 M PBS (pH 7.3) first and placed on carbon-coated EM grids and stained with 2% uranyl acetate before TEM observation. Rigid rod-shaped viral particles, typical of tobamovirus particls (around 300 nm) were observed. Total RNA was isolated from two tomato leaves showing symptoms using a Norgen Plant/Fungi RNA kit (Norgen Biotek, Canada). One-step reverse-transcription (RT)-PCR with specific primers ToBRFV-F/ToBRFV-R for ToBRFV (Alkowni et al. 2019), which amplified a 560-bp fragment, was performed. The sequence obtained by Sanger sequencing from the amplicon (535 nt) showed 99.8% nt identity with ToBRFV isolate PV-1241 (NCBI accession no. MZ202349) from DSMZ (Leibniz Institute, German) and was deposited in the GenBank database under the accession number OK358628. The infection was also confirmed with duplex real-time RT-PCR test for ToBRFV using CaTa28 and CSP1325 primers and probe (ISF 2020; EPPO, PM7/146 2021). In addition, eight tomato samples from the same farm greenhouse were collected according to cultivars, location in the greenhouse and symptoms before eradication and disinfection: four of the tomato samples were confirmed positive for ToBRFV with one-step RT-PCR as described above. A surveillance program for ToBRFV in commercial greenhouse production has been carried out in 2021 in Norway. Around 4000 tomato plants from 18 commercial tomato growers were tested. No positive ToBRFV samples have been detected. However, unregulated tomato seeds from abroad and self-propagation of tomato by private gardeners and hobby growers is a potential threat to commercial production of tomato in Norway. To our knowledge, this is the first report of ToBRFV associated with tomato in Norway and in the Nordic countries.

The estimated global production of raspberry from year 2016 to 2020 averaged 846,515 tons. The most common cultivated Rubus spp. is European red raspberry (Rubus idaeus L. subsp. idaeus). Often cultivated for its high nutritional value, the red raspberry (Rubus idaeus) is susceptible to multiple viruses that lead to yield loss. These viruses are transmitted through different mechanisms, of which one is invertebrate vectors. Aphids and nematodes are known to be vectors of specific raspberry viruses. However, there are still other potential raspberry virus vectors that are not well-studied. This review aimed to provide an overview of studies related to this topic. All the known invertebrates feeding on raspberry were summarized. Eight species of aphids and seven species of plant-parasitic nematodes were the only proven raspberry virus vectors. In addition, the eriophyid mite, Phyllocoptes gracilis, has been suggested as the natural vector of raspberry leaf blotch virus based on the current available evidence. Interactions between vector and non-vector herbivore may promote the spread of raspberry viruses. As a conclusion, there are still multiple aspects of this topic that require further studies to get a better understanding of the interactions among the viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better pest management strategies.

Post-prints are subject to Springer Nature re-use terms Key message Rooting, vegetative growth, bulb production, genetic stability and biochemical compounds were maintained in cryopreserved plants of shallot. Our results support use of cryopreservation for long-term preservation of shallot germplasm.

Red raspberry, Rubus idaeus, is known to be infested by at least six species of eriophyid mites. Among them, the raspberry leaf and bud mite, Phyllocoptes gracilis, (Figure 1, A–B) is the only known vector of a raspberry virus, namely the raspberry leaf blotch virus (RLBV) (Dong et al. 2016; McGavin et al. 2012; Tan et al. 2022). Raspberry leaf blotch (Figure 1, C), a leaf disorder displaying as leaf chlorosis, distortion and patchy necrosis, yellowing and thinning on lateral branches, has been attributed to the feeding of P. gracilis until RLBV was also found to be associated with these symptoms (McGavin et al. 2012). Previous sampling of eriophyid mites was often based on the presence of the leaf blotch symptom, and there is a reasonable doubt if the symptom was caused by RLBV infection or mite infestation. It could also be hypothesized that eriophyid mites are attracted to RLBV-infected plants as viruses could make host plants more attractive to vectors (Donnelly & Gilligan 2020; Shi et al. 2019). It is therefore important to improve the detection of both mites and RLBV to efficiently manage the virus. In addition, knowledge on the dominant infestation area of eriophyid mites on raspberry canes is essential to develop an effective pest management approach. Gordon and Taylor (1976) reported that P. gracilis on primocanes during late summer (mid-August) was dominantly found on the upper leaves, due to the presence of predatory mites on the lower and middle leaves. But mite behavior on floricanes is also important to study if the goal is to prevent mite migration and RLBV transmission to primocanes. This study aimed to investigate the population density of eriophyid mites on raspberry floricanes and the association of eriophyid mites, RLBV, and leaf blotch symptoms.

Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to ‘Candidatus Phytoplasma rubi’ strain RS (GenBank Accession No. CP114006). To further characterize the ‘Ca. P. rubi’ strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with ‘Ca. P. rubi’ RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % ‘Ca. Phytoplasma’ strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference ‘Ca. P. rubi’ RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of ‘Ca. P. rubi’ from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.