Vitrification Cryo-Foil for Apple Cryopreservation and the Seesaw Effect Between Shoot Recovery and Virus Eradication

Improvements of existing cryopreservation protocols are necessary to facilitate long-term preservation of plant germplasm and the cryotherapy-effect of pathogen eradication. This study reported a vitrification (V) cryo-foil/plate methods for cryopreservation of shoot tips and cryotherapy effect in ‘Pink Lady’ apple. In V cryo-foil/plate protocols, shoot tips were first attached onto aluminum foils/plates using calcium alginate before other procedures. Shoot tips cryopreserved by V cryo-foil required 6.1 weeks to fully recover and 53% of shoot regrowth was obtained, comparable to the Dv cryopreservation. Similar regrowth levels were produced between applying V cryo-foil and Dv cryopreservation to another 4 Malus genotypes. Histological observations in shoot tips cryopreserved by Dv and V cryo-foil found only those with surviving apical dome and leaf primordia (LPs) could recover after cryopreservation. In apical meristem of shoot tips cryopreserved by Dv and V cryo-foil, higher surviving probability was detected from the V cryo-foil protocol, and the young LPs showed the highest level of surviving. Virus detection in cryo-derived plants showed apple stem grooving virus and apple chlorotic leaf spot virus were all preserved after cryopreservation, and higher eradication efficiency of apple stem pitting virus (70%) was produced by Dv than the 55% of V cryo-foil. These results supported applying V cryo-foil as an improvement to the widely applied Dv method in shoot tip cryopreservation, and also revealed a seesaw mode between shoot recovery and cryotherapy effect. Once the seesaw moves to increase the recovery after cryopreservation, the cryotherapy-effect on the other side would be decreased.

First Detection and Molecular Characterization of Apple Stem Grooving Virus, Apple Chlorotic Leaf Spot Virus, and Apple Hammerhead Viroid in Loquat in Spain

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date.

First report of Cnidium officinale as a natural host plant of Apple stem grooving virus in South Korea

Cnidium officinale is a perennial plant in the family Apiaceae. It is native to China and cultivated in China, Japan, and Korea for its roots for medicinal purposes. In August 2019, 63 C. officinale plants showing symptoms of vein chlorosis, yellowing and chlorotic spots (Supplementary Fig. 1) were collected from commercial farms in Bonghwa and Youngyang, Gyeongsangbuk-do, South Korea. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to confirm the presence of apple stem grooving virus (ASGV), cnidium vein yellowing virus 1, cnidium vein yellowing virus 2, lychnis mottle virus, and Cnidium virus X with specific primers (Supplementary Table 1). Forty-one out of the sixty-three samples were positive for ASGV in mixed infection with one or more of the other four viruses. Nicotiana benthamiana plants mechanically inoculated with the crude sap of one of the ASGV-infected C. officinale plants showed mosaic symptom on upper leaves 10 days post inoculation (dpi). Infection was confirmed by RT-PCR and Sanger sequencing. N. benthamiana plants systemically infected with ASGV-CO-kr1 isolate alone were used for subsequent sequencing and host range test. Twenty-day old seedlings of 23 species of plants (two to 14 species for each family) from the families Solanaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae and Amaranthaceae (Supplementary Table 2) were mechanically inoculated with sap of ASGV-CO-kr1-infected N. benthamiana plants. ASGV-CO-kr1 infected all tested 23 species as confirmed by symptomology, RT-PCR, and Sanger sequencing at 10 to 20 dpi. The MP and CP genes of ASGV-CO-kr1 were amplified by RT-PCR with specific primers 4300-4325F/5642-5666R and 5592-5612F/6475-6499R, respectively (Supplementary Table 1). The amplicons were cloned and sequenced (GenBank accession numbers: MP = MW889883 and CP = MW889884). Multiple sequence alignment using the MegAlign program in DNASTAR showed that the complete CP and MP genes of ASGV-CO-kr1 shared 89.9%-99.7% and 83.1%-99.5% identities, respectively at the nucleotide (nt) level and they shared 92.4%-99.6% and 93.8%-99.4% identities, respectively at amino acid (aa) level with corresponding sequences of 34 other ASGV isolates from various host plants and countries. Phylogenetic analysis with the Maximum Likelihood method using the MEGA X program (Kumar et al., 2018) showed that ASGV-CO-kr1 grouped with isolates Cuiguan (KR185346), BH (LC480456), and YY (LC480457) based on the CP aa sequences, while it grouped with isolates SG (LC475148) and TL101 (MH108976) based on the MP aa sequences. ASGV is known to naturally infect apples, European pear, Asian pear, citrus, apricot, cherry, kiwifruit, loquat, lily, and lotus (Clover et al., 2003; He et al., 2019; Hu et al., 2017; Liu et al., 2017; Yanase et al., 1975). To the best of our knowledge, this is the first report of the natural infection of ASGV in C. officinale. C. officinale plants are propagated by root division, so they are susceptible to infection with viruses. The result of this study is important for generating virus-free seedlings to produce C. officinale.

Alleviation of Postharvest Skin Dimpling of MN55 Apple Fruit and a Possible Association with Apple stem pitting virus

MN55 is an apple (Malus ×domestica Borkh.) cultivar recently released by the University of Minnesota apple breeding program, with fruit marketed in the U.S. as Rave®. When stored for 4 months at 0 to 4 °C, MN55 fruit can develop several storage disorders, including skin dimpling. Skin dimpling incidence was greater for fruit harvested 1 week later than those harvested earlier. Dimpling was not alleviated by prestorage treatments of 1-methylcyclopropene or diphenylamine or by holding fruit at room temperature for 1 day before long-term cold storage. However, dimpling incidence was very low when fruit were stored at 6 to 7 °C. Because viruses have been implicated in other fruit dimpling disorders, the presence of viruses in MN55 leaves and fruit was studied. Apple stem pitting virus (ASPV) was detected by microscopy, reverse transcriptase polymerase chain reaction (RT-PCR) methodology, and high throughput sequencing (HTS) in peel of fruit from MN55 trees that exhibited skin dimpling after 4 months of storage at 0 to 1 °C. ASPV was also detected in supermarket-purchased fruit of other cultivars with noticeable skin dimpling. Although ASPV was not conclusively demonstrated to cause skin dimpling in our work, its prevalence indicates that further investigations are warranted to determine the relationship between viruses and skin deformities in stored apples.

Adaptation and Codon-Usage Preference of Apple and Pear-Infecting Apple Stem Grooving Viruses

Apple stem grooving virus (ASGV; genus Capillovirus) is an economically important virus. It has an approx. 6.5 kb, monopartite, linear, positive-sense, single-stranded RNA genome. The present study includes identification of 24 isolates—13 isolates from apple (Pyrus malus L.) and 11 isolates from pear (Pyrus communis L.)—from different agricultural fields in South Korea. The coat protein (CP) gene of the corresponding 23 isolates were amplified, sequenced, and analyzed. The CP sequences showed phylogenetic separation based on their host species, and not on the geography, indicating host adaptation. Further analysis showed that the ASGV isolated in this study followed host adaptation influenced and preferred by the host codon-usage.