• Research in Plant Disease
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• v.23 no.1
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• pp.75-81
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• 2017

In May 2016, 24 peach samples showing abnormal and virus like symptoms were collected in one of major peach producing area, Yeongcheon-si, Gyeongsangbuk-do, Korea. We performed RT-PCR diagnosis for confirmation of viral infection. The diagnostic targets are 17 species of viruses and viroids that quarantine and high risk pathogens when it occur. As a results, seven species of viruses and viroids, including an unreported (Apricot pseudo-chlorotic leaf spot virus, APCLSV) and a quarantine (Peach latent mosaic viroid, PLMVd) species in Korea, were detected. For the sequence analysis of unreported virus, APCLSV, the sequence of coat protein gene were amplified and cloned. The sequence showed 97% nucleotide identity with other APCLSV isolates and compared with other seven species of reported Trichoviruses. This virus was classified as APCLSV based on the sequence and phylogenetic analysis. This isolate was named Yeongcheon. As patterns of APCLSV occurrence, all samples that APCLSV detected were co-infected with Apple chlorotic leaf spot virus (ACLSV). As properties of ACLSV, APCLSV has high possibility of wide spread disease in fruit tree farms in Korea. Therefore, it is necessary to do related researches, such as infection route and influence of disease in commercial orchards.

• The Plant Pathology Journal
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• v.22 no.2
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• pp.168-173
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• 2006

To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the N-terminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RT-PCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor $1{\alpha}(EF1{\alpha})$. This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.

• The Plant Pathology Journal
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• v.33 no.6
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• pp.608-613
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• 2017

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.222-229
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• 2022

Apples are the most grown fruit crops in the fruit industry of Korea. However, virus or viroid infection such as apple mosaic virus (ApMV), apple stem grooving capillovirus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple scar skin viroid (ASSVd) causes fruit yield reduction and poor fruit quality. Therefore, in this study, we examined to established an efficient virus-free system to eliminate the most infected ASGV virus in domestic apple orchard. We investigated that the shoot growth rate and the virus removal rate in ASGV infected potted apples that were treated with heat treatment in a growth chamber (constant temperature/humidity device) maintained at 36℃, 38℃ and 40℃ for 4 weeks. Here we found that the shoot growth rate was the highest in the heat treatment group (36℃) and the virus was removed in the middle and top of the shoot but not in the bottom. The virus was did not removed in the 38℃ and 40℃ heat treatment group in all section of shoots, and the heat treatment group (40℃) died after 4 weeks of heat treatment without growth of shoots. We performed in vivo shoot-tip grafting using the shoot-tip of potted apple heat-treated at 36 ℃, and we also investigated the viability and virus removal rate, which showed 94% viability and 20% virus removal rate. Collectively, our results suggest that it would be possible to produce the virus-free apple plants through heat treatment and shoot-tip grafting.

• Research in Plant Disease
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• v.19 no.4
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• pp.288-293
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• 2013

In worldwide, viral diseases of apple plants has caused the serious problems like reduced production and malformation of fruits. Also, the damages of apple plants by virus and/or viroid infection (Apple chlorotic leaf spot virus, Apple stem grooving virus, Apple mosaic virus, and Apple scar skin viroid) were reported in Korea. However there is few report about the protection approach against the infection by apple viruses. Therefore, this paper introduced the experimental protocol for the development of virus-free apple cultivars (Danhong, Hongan, Saenara, Summerdream). Apple plants were treated at $37^{\circ}C$ for 4 weeks and shoot tips were cultured in vitro. After heat treatment, the detection of apple viruses was performed by RT-PCR using virusspecific detection primers in new apple cultivars. With the heat treatments followed by in vitro shoot tip culture, the proportion of virus-free stocks of 'Danhong', 'Hongan', 'Saenara', and 'Summerdream' was 28%, 16%, 12%, and 12%, respectively. Taken together, this approach can be a good tool for production of virus-free apple stocks.

• Research in Plant Disease
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• v.26 no.2
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• pp.95-102
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• 2020

The investigation of the infection rate of domestic apple orchards by four types of apple viruses (Apple chlorotic leaf spot virus [ACLSV], Apple stem pitting virus [ASPV], Apple stem grooving virus [ASGV], Apple mosaic virus [ApMV]) and one type of viroid (Apple scar skin viroid, ASSVd) found that most apple trees were infected with viruses and viroid at the rate of 97.3%. By region, the infection rate in Jeongseon stood at 98.8%, Danyang at 100%, Yesan at 100%, Jangsu at 89.1%, and Muju at 98.1%. By each virus and viroid, the infection rate of ASGV was the highest at 93.4%, followed by ASPV at 85.7%, ACLSV at 59.0%, ASSVd at 6.7%, and ApMV at the lowest 3.6%. In addition, 84.8% of the cases were infected with two or more types of viruses and viroid, nearly seven times the single type infection rate of 12.4%, and the cases infected with three viruses, ASPV, ACLSV, and ASGV accounted for 56.2%, more than the half the total number of trees investigated.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.379-387
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• 2017

Various sizes (0.2 ~ 1.2 mm) and developmental stages (referred to as Stage 1 ~ 3) of apical and lateral meristems were excised, together or separately, directly from dormant buds of apple 'Hongro'. They were mixed infected by Apple scar skin viroid (ASSVd), Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV), which are major viruses attacking apples. A total of 31 callus lines (> 10 mm in diameter) were obtained by culturing the explants on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 3.0 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-butyric acid (IBA), and they were subjected to RT-PCR analysis for virus detection. A high rate of virus elimination (expressed as the percentage of calli that did not amplify during RT-PCR, i.e., RT-PCR negative calli per total number of calli obtained) was achieved for ACLSV (100%), ASSVd (93.7%), and ASPV (93.7%), whereas it was only 25.8% for ASGV. ASPV was detected in the presence of 2 ~ 3 bracts. Simultaneous virus elimination of ASSVd, ASPV, ACLSV, and ASGV occurred during the meristem culture, in which the early stages of the dormant buds (Stage 1) were used, because ASGV was mostly eliminated during that stage. The results of the present study will be valuable for the production of virus-free apple trees.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.142-146
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• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• Research in Plant Disease
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• v.27 no.2
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• pp.79-83
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• 2021

The aim of the present study was to develop a sensitive and specific detection method for the rapid detection of apple scar skin viroid (ASSVd) in apple leaves. The resulting reverse transcription recombinase polymerase amplification (RT-RPA) assay can be completed in 10 min at 42℃, is 10 times more sensitive than conventional reverse transcription polymerase chain reaction, and can specifically amplify ASSVd without any cross-reactivity with other common apple viruses, including apple stem grooving virus, apple stem pitting virus, and apple chlorotic leaf spot virus. The reliability of the RT-RPA assay was assessed, and the findings suggested that it can be successfully utilized to detect ASSVd in field-collected samples. The RT-RPA assay developed in the present study provides a potentially valuable means for improving the detection of ASSVd in viroid-free certification programs, especially in resource-limited conditions.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.52-52
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• 2018

Apple (Malus domestica) is one of the most economically important fruits in Korea. But virus infection has decreased sustainable production of apple and caused the serious problems such as yield loss and poor fruit quality. Virus or viroid infection including Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV) and Apple scar skin viroid (ASSVd) has been also reported in Korea, furthermore, its damages and economic losses have increased constantly. In our research, we tried to survey virus infection for commercial nursery trees of major apple cultivars, especially dwarfing rootstocks 'M.9' and 'M.26' as well as scions. Trees were collected from 11 locations which have produced a great amount of apple nursery stocks in Korea. Infection degree was investigated in apple cultivars, 'Hongro' and 'Fuji' using RT-PCR method. In the scion of cultivar 'Hongro', infection ratio of ACLSV, ASPV and ASGV were 100%, 81.8% and 100% respectively. In the rootstock of cultivar 'Hongro', infection ratio of ACLSV, ASPV, ASGV and ApMV were 90.9%, 81.8%, 100% and 9.1% respectively. In the scion of cultivar 'Fuji', infection ratio of ACLSV, ASPV and ASGV were 81.8%, 90.9% and 100% respectively. In the rootstock of cultivar 'Fuji', infection ratio of ACLSV, ASPV, ASGV and ApMV were 81.8%, 90.9%, 100% and 9.1% respectively. Infection of ASSVd was not detected in both cultivars. From our results, it was found that most of apple rootstocks and scions had multiple infections by apple viruses which have caused economic damage in fruit production.