• The Plant Pathology Journal
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• v.38 no.4
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• pp.417-422
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• 2022

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.

• Research in Plant Disease
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• v.29 no.2
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• pp.193-199
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• 2023

The apple stem grooving virus (ASGV) is one of the most harmful latent viruses infecting pear orchards worldwide. To examine the genetic diversity of ASGV in Korean pear orchards, the complete coat protein (CP) gene of five ASGV isolates collected from various regions were identified. The five Korean ASGV isolates showed 88-96% nucleotide identity with the 11 isolates worldwide occurring elsewhere in the world. Phylogenetic analysis of five isolates, as well as the previously sequenced isolates, indicated that the ASGV clusters had no correlation with the host or geographical regions of origin. Recombination analysis showed that one of the five Korean isolates is a recombinant, with a recombination site in the CP gene region (nt 532-708). This study is the first report of natural recombination within the CP gene of ASGV isolates from pears grown in Korea.

• Korean Journal of Agricultural Science
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• v.43 no.1
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• pp.21-27
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• 2016

Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) have been known to induce top working disease causing economical damage in apple. Occurrences of these three viruses in pome fruit trees, including apple, have been reported around the world. The transmission of the three viruses was reported by grafting, and there was no report of transmission through mechanical contact, insect vector, or seed except some herbaceous hosts of ASGV. As RNA extraction methods for fruit trees, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and multiplex RT-PCR techniques have been improved for reliability and stability, and low titer viruses that could not be detected in the past have become detectable. We studied the seed transmission ability of three apple viruses through apple seedling diagnosis using RT-PCR. Nineteen seeds obtained from commercially grown apple were germinated and two of the resulting plants were ASGV positive. Seven clones of the amplified ASGV coat protein (CP) genes of these isolates were sequenced. Overall sequence identities were 99.84% (nucleotide) and 99.76% (amino acid). Presence of a previously unreported single nucleotide and amino acid variation conserved in all of these clones suggests a possible association with seed transmission of these 'S' isolates. A phylogenetic tree constructed using ASGV CP nucleotide sequences showed that isolate S sequences were grouped with Korean, Chinese, Indian isolates from apple and Indian isolates from kiwi.

• 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.28 no.2
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• pp.92-97
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• 2022

Apple stem grooving virus (ASGV) is a high-risk viral pathogen that infects many types of fruit trees, especially pear and apple, and causes serious economic losses across the globe. Thus, rapid and reliable detection assay is needed to identify ASGV infection and prevent its spread. A reliable reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed, optimize, and evaluated for the coding region of coat protein of ASGV in pear leaf. The developed RT-LAMP facilitated the simple screening of ASGV using visible fluorescence and electrophoresis. The optimized reaction conditions for the RT-LAMP were 63℃ for 50 min, and the results showed high specificity and 100-fold greater sensitivity than the reverse transcription polymerase chain reaction. In addition, the reliability of the RT-LAMP was validated using field-collected pear leaves. Furthermore, the potential application of paper-based RNA isolation, combined with RT-LAMP, was also evaluated for detecting ASGV from field-collected samples. These assays could be widely applied to ASGV detection in field conditions and to virus-free certification programs.

• 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.34 no.6
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• pp.575-579
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• 2018

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

• 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.22 no.3
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• pp.255-259
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• 2006

The pathogenicity to pear trees and other experimental hosts of the Apple stem grooving virus Korean isolate (ASGV-K) carried by a fungal vector, Talaromyces flavus was examined. ASGV-harboring T. flavus induced mild symptoms on virus-free pears. Symptom severity was intermediate between pears showing typical PBNLS and virus-free pears. Ten cultivars of Phaseolus vulgaris showed 35%-90% infectivity by direct infiltration into leaves and roots by ASGV-harboring T. flavus. Application of fungal cultures to soils showed 0%-70% infectivity depending on the P. vulgaris cultivar. Sap extracted from ASGV-infected Chenopodium quinoa induced similar symptoms on P. vulgaris at 25 days after inoculation. Similar symptoms were also detected on P. vulgaris which were inoculated with ASGV-harboring T.flavus. When healthy P. vulgaris leaves were challenged with sap extracted from P. vulgaris leaves infected with ASGV-harboring T. flavus, typical symptoms were observed. These data suggest that T. flavus mediates the transfer of ASGV to host plants.