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The 5 apple viruses, apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple mosaic virus (ApMV) and prunus necrotic ring spot virus (PNRSV) and 1 viroid, apple scar skin viroid (ASSVd) are known major viral/viroid pathogens of apple in India. For identification of virus-free elite mother plants, 267 proposed mother plants from Trans-Himalayan region of India were indexed through DAS-ELISA and RT-PCR. Only 20% plants showed typical symptoms of virus/viroid, where as indexing though DAS-ELISA and RT-PCR revealed that, about 95% of apple plants were found infected with the virus/viroid. ASPV was most prevalent virus with 74.91% incidence followed by ACLSV (73.41%), ASGV (45.32%), PNRSV (1.5%) and ApMV (1.5%). Whereas, incidence of ASSVd was 2.62%. In the present study, it was recorded that 24.71% proposed mother plants were infected with a single virus, 38.95% with at least 2 viruses and 31.46% with more than 3 viruses. Only 4.86% plants out of 267 proposed mother plants were found free from virus/viroid infection. For sustainable supply of quality planting material, these virus free elite mother plants were multiplied and distributed to the farmers for establishment of mother blocks.

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 42oC, 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.

Apple scar skin viroid (ASSVd), the type member of the genus Apscaviroid, induces symptoms only on fruits, which include colour dappling, russeting, cracking and distortion. Studies on viroid incidence were conducted during past five years and incidence up to 90.0 percent was recorded in commercial apple cultivars (Royal Delicious, Red Delicious, Red Gold, Golden Delicious). From 2015 to 2018, forty apple orchards were surveyed in different blocks of Shimla district of Himachal Pradesh and fruits in six orchards were found with typical symptoms of apple scar skin viroid infection without symptoms on leaves. The presence of ASSVd in symptomatic fruits and symptomless leaves were confirmed by RT- PCR. It was also revealed that ASSVd infection not only deteriorate the quality of apple fruits but cause large losses in term of yield, as 17.80 quintal less fruit production was recorded in the affected orchards with loss in net return up to Rs. 3,91,102/- per hectare (72.68% loss in net return per hectare).

Viroids are naked non-coding RNAs that can infect plants and cause severe disease. Apple scar skin viroid (ASSVd) is the causal agent of apple scar skin disease, which can infect apple trees and cause dappling and scar skin symptoms. In this study, we investigated the incidence of apple scar skin disease in apple-grafted small apple tree (Malus pumila ‘Saiwaihong’) in North China. Of 1322 tested trees, 90 exhibited obvious dapple symptoms in fruit skin (disease incidence of 6.8%). Dot-blot hybridization analysis proved that the peel from 25 diseased small apple tree were all positive for ASSVd, confirming that disease was associated with ASSVd infection. RT-PCR, cloning and sequencing were performed, and 12 complete sequences of ASSVd variants from Malus pumila ‘Saiwaihong’ were obtained. Sequence comparison showed that the 12 sequence variants shared 90.6–99.7% sequence similarity, phylogenetic analysis, performed based on the 12 ASSVd Malus pumila ‘Saiwaihong’ variants and other ASSVd variants reported from different hosts, did not show any clustering according to the respective host species. This is the first report of ASSVd infection in small apple tree (Malus pumila ‘Saiwaihong’).

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with real-time detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of 1 × 104 to 1 × 1012 ASSVd RNA copies/μl with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from 1 × 107 to 1 × 1012 copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), and Apple scar skin viroid (ASSVd) are economically important viruses that infect pear tree species worldwide. To evaluate the prevalence of these viruses in Korea, we investigated infection degree of three viruses and one viroid for the commercial nursery trees of the pear cultivars, Niitaka, Chuwhang, Wonwhang, and Whasan in 2017 and 2018. The results showed that the infection ratio of ACLSV, ASPV, ASGV, and ASSVd for the scion of pear cultivar Niitaka was 10%, 45%, 77%, and 50%, respectively. From the scion of pear cultivar Chuwhang, infection ratios of ASPV, ASGV, and ASSVd were found to be 70%, 50%, and 60%, respectively. From the scion of pear cultivar Whasan, infection ratios of ACLSV, ASPV, ASGV and ASSVd were found to be 40%, 60%, 93%, and 20%, respectively. From the root stock of pear cultivar Wonwhang, infection ratios of ACLSV, ASPV, ASGV, and ASSVd showed 28%, 57%, 100%, and 14%, respectively. ASGV had the highest recorded infection rate, and ACLSV was characterized by the lowest infection rate. The mixed infection ratio of Niitaka, Chuwhang, Whasan, and Wonwhang was 45%, 60%, 70%, and 85%, respectively.

Apple dimple fruit viroid was detected from an apple tree (‘Jonagold’) bearing apples with mild dapple apple symptom. The isolates in Japan were distinct from those in apples in Italy and China and in fig in Italy. Graft-inoculation experiments showed that the symptoms were variable depending on the cultivar, and the symptom on ‘Starking Delicious’ was virtually similar to those reported in Italy. Symptoms induced by apple dimple fruit viroid were similar in part to those by apple fruit crinkle viroid or apple scar skin viroid, indicating that they cannot be discriminated by symptoms on any specific variety.

To identify genome sequences of Apple scar skin viroid (ASSVd) isolates in Korea, the field survey was performed from ‘Hongro’ apple orchards located in eight sites in South Korea (Bongwha, Cheongsong, Dangjin, Gimchoen, Muju, Mungyeong, Suwon, and Yeongwol). ASSVd was detected by RT-PCR and PCR fragments were cloned into cloning vector. Full-length viral genomes of eight ASSVd isolates were sequenced and compared with 21 isolates reported previously from Korea, India, China, Japan and Greece. Eight isolates in this study showed 92.2-99.7% nucleotide sequence identities with those reported previously. Phylogenetic analysis showed that seven isolates reported in this study belong to the same group distinct from other groups.

A one-step multiplex quantitative reverse transcription polymerase chain reaction protocol is described, for the detection in pome trees of Pear blister canker viroid and Apple scar skin viroid, together with universal detection of phytoplasmas. Total nucleic acids extraction is performed according to a modified CTAB protocol and TaqMan MGB probes are used to surpass high genetic variability of viroids. The multiplex real-time assay is at least ten times more sensitive than conventional protocols and its features make it suitable for rapid and massive screening of pome fruit trees phytoplasmas and viroids in certification schemes and surveys.

Viroids are small, single stranded, covalently closed circular, non-encapsidated plant pathogenic RNA molecules which do not code for any protein and thus depend on the host plant for their replication and other functions. After discovery of viroids in 1971, several diseases whose etiology was not known earlier were confirmed to be caused by this new group of plant pathogen. Besides the symptomatic expression, viroids often cause latent infection with no visible symptom in one plant species but when transferred to a new species they may cause severe infection. Indian scenario on researches of viroids-induced diseases started much earlier, even before the discovery of viroids as a plant pathogen. However, due to lack of proper diagnostics and infrastructural support the progress on such diseases were confined to initial level till the end of twentieth century. During this period the research efforts were focused on recording the diseases of unknown etiology, their impact assessment, understanding on the spread of such diseases and screening of resistant sources. With the addition of new infrastructural support and ease in the access of sequencing technologies several laboratories in India and in collaboration with laboratories abroad, viroid research gets its momentum from early twenty-first century. As a result several viroids infecting citrus, tomato, apple, ornamentals, rubber and grapes have been characterized from India and their sequences have been deposited in the sequence database. Association of viroids with yellow corky vein disease of citrus, bunchy top of tomato, tapping panel dryness of rubber, deformation of apple have been established. Other viroids which have been detected in ornamentals and grapes do not produce visible symptoms. Till now no major outbreak of viroid disease in the form of epidemic has been reported in India.

Four apscaviroids (family Pospiviroidae), grapevine yellow speckle viroids 1 and 2 (GYSVd-1 and -2), Australian grapevine viroid (AGVd), and grapevine latent viroid (GLVd) infect grapevines. Apscaviroids are phylogenetically classified into two major subclades, one comprising GYSVd-1 and -2, the other comprising apple scar skin viroid and GLVd. GYSVd-1 and -2 may induce symptoms in the field in Australia, but are rarely symptomatic elsewhere. GYSVd-1 and -2 are symptomatic in cv. “Chardonnay” during hotter years, but asymptomatic in milder years. Unlike GYSVd-1 and -2, AGVd is asymptomatic in grapevine but symptomatic in tomato, cucumber, and other herbaceous experimental hosts and is apparently very stable both structurally and phylogenetically. For example, full-length sequences of GYSVd-1 and AGVd have been recovered from a 10-year old bottled Cabernet Sauvignon wine. GLVd has been discovered in China and there is no report on its occurrence in other vine growing areas of the world.

The reverse transcription-polymerase chain reaction technique has been proven to be a rapid and reliable method for amplifying viroid sequences from infected plants. From 1990, when the first amplification method was applied to apple scar skin viroid, amplification methods have increased in number with the development and application of real-time reverse transcription-polymerase chain reaction, reverse transcription-Loop mediated isothermal amplification, and the combination of different techniques.

Apple scar skin viroid (ASSVd) causes serious pome fruit diseases such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. Diseased apple and pear fruits are unmarketable. The viroid may also be latent in some pear cultivars without causing a disease. ASSVd was reported from many countries, especially those of East Asia. It naturally infects different species of apple and pear as well as apricot, peach, sweet cherry, and Himalayan wild cherry. Nucleotide sequences of several ASSVd variants from different hosts were determined and comparative sequence analyses were made. Using genetic engineering approaches, ASSVd is transmitted to herbaceous plant species. The viroid is also transmitted by the greenhouse whitefly to herbaceous hosts. Other means of transmission, detection, and control are similar to those of other temperate fruit tree viroids.

Four varieties of potted apple were subjected to thermotherapy (37 ± 1 °C) coupled with shoot tip grafting. Using reverse transcription-polymerase chain reaction assays prior to treatment, mixed infections with 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) were detected in 95.8 % of treated plants. It was found that the sprouts of Dahongrong (DHR) and Qiyuetianxian (QYT) variety buds were inhibited by high temperature, although some buds of these two varieties were able to sprout, the growth was slow. The average survival rate of shoot tips cut after thermotherapy was 41.2 % (61/148). The survival rate of DHR (63.2 %) was highest among the four varieties. Sixty-one surviving apple plants were detected over two periods, indicative of an average elimination rate of 65.6 % (40/61) for new leaves (June) and 37.7 % (23/61) for dormant branches (November). The difference of elimination rate for DHR between the two periods was the most obvious. Detections rates of viruses were also different in the two periods; ASGV in dormant branches were 27.8 % higher than those in new leaves, and ASPV (90.6 %) remained the same over the two periods.

Gunsup Lee*, Jeong Hee Kim*, Hyun Ran Kim, Il Sheob Shin, Kang Hee Cho, Se Hee Kim, Juhee Shin and Dae Hyun Kim** Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 440-706, Korea Apple Research Station, National Institute of Horticultural and Herbal Science, Rural Development Administration, Gunwi 716-812, Korea Future & Creation Strategy Team, Rural Development Administration, Suwon 441-707, Korea (Received on November 12, 2013; Revised on November 21, 2013; Accepted on November 25, 2013)

Apple scar skin viroid (ASSVd) is one of the most destructive viroids of pome fruit. We report 12 new sequence variants of ASSVd from apple and pear trees in north-eastern Iran. The Iranian variants ranged in size from 329 to 334 nucleotides. They formed a cluster distinct from other reported ASSVd isolates, and showed sequence variations in specific regions of the viroid RNA. This is the first report of the identification and molecular characterization of ASSVd in Iran.

Apple scar skin viroid (ASSVd) is one of the smallest viral pathogens infecting fruits, especially apple, and causes a significant damage to fruit trees. ASSVd usually induced the skin-dapple ring symptoms, but in `Fuji` varieties, corked spot were occurred on the fruit skin in 2009. This new symptom will be of great helpful to diagnosis ASSVd in sight. ASSVd was surveyed in apple and pear from 2009 to 2010 in Korea, and ASSVd was identified in 20 out of 1,193 trees. The infection rate was 1.7%. To screen the infectivity of ASSVd among apple cultivars, real-time RT-PCR was applied followed by designing of ASSVd specific primers based on highly conserved regions of several ASSVd isolates including Korean isolate. NADH dehydrogenase subunit 5 (nad 5) gene, which is mRNA of the mitochondrial gene, was used for internal control. In this study, ASSVd infected apples were classified into 12 groups depending on different symptoms and symptom severity (scaring, rusting or malformation). Taken together, this study suggested that real-time PCR analysis was more sensitive to detect the low copy of ASSVd on early viroid infected apple skins than regular RT-PCR method.

Apple scar skin viroid (ASSVd) infection is a major limitation to apple fruit quality and causes huge economic losses. In surveys of apple orchards in the northern Indian state of Himachal Pradesh, fruits with dappling symptoms were noticed. ASSVd was detected from these fruits and molecularly characterized. Ten clones from three isolates were sequenced, of which seven were new sequence variants of ASSVd. The clones had significant sequence variability (94-100%) with each other. Variability was more common in the pathogenic domain of the viroid genome. Four of the clones were 330 nucleotides (nt) long, and the other six had an additional nucleotide. Phylogenetic analysis showed close affinity of the present isolates with some Chinese and Korean isolates. The study reports seven new variants of ASSVd and also provides the first molecular evidence of viroid infection (ASSVd) in apple in India.

A validated RT-PCR method was used to investigate the presence of Apple scar skin viroid (ASSVd) inthe commercial apple orchards of eastern Anatolia. Among three modified and simplified silica-capturebased extraction methods, one was used for mass extraction to ascertain the presence of ASSVd. Thetest was initially performed from an ASSVd-infected source and then applied to total RNA preparationsfrom fresh leaf tissues of apple trees collected from eastern Anatolia. ASSVd was found to occur in appletrees. Among 263 apple samples, 121 were positive for ASSVd. The infected trees showed no apparentdisease symptoms on the leaves other than scarring on fruit skin. Overall incidence of ASSVd was 46%in eastern Anatolia. The presence of ASSVd reported for the first time in Turkey. Among three totalRNA preparation methods, Method II was determined to be the best procedure for large scale routineanalysis. The improved test can be used in a certification or clean stock program to contribute to theprevention of the spread of ASSVd in the eastern Anatolia.

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. (C) 2014 Elsevier B.V. All rights reserved.

Apple scar skin viroid (ASSVd), a pathogenic RNA viroid, infects apple and pear trees. To determine if ASSVd can also infect apricot, we performed reverse transcription-polymerase chain reaction (RT-PCR), spot hybridisation andin situ RT-PCR and detected ASSVd-specific sequences from apricot. We cloned and sequenced the apricot-derived ASSVd and registered it in GenBank.In situ RT-PCR further confirmed the existence of ASSVd in apricot leaf tissues. Our data indicate that apricot is a novel host of ASSVd. These findings provide the basis for the rapid identification of ASSVd in apricot.

A new distribution map is provided for Apple scar skin viroid . Pospiviroidae: Apscaviroid. Hosts: apple ( Malus spp.), pear ( Pyrus spp.), and sweet cherry ( Prunus avium ). Information is given on the geographical distribution in Europe (Denmark, France, Crete, Greece, Italy, Poland, and UK), Asia (Hebei, Liaoning, Shaanxi, Shanxi, Xinjiang, China, Himachal Pradesh, India, Iran, Honshu, Japan, Korea Republic, Turkey), North America (British Columbia, Canada, Maine, Massachusetts, Missouri, New Hampshire, Washington, USA), and South America (Argentina).

In this paper, low molecular weight RNAs were extracted from tender leaves and shoots of apple, pear, peach and apricot trees, which were collected from Yanqi, Hejing, Bohu, Heshuo, Xinhe, Korla and Aksu in Xinjiang province, China. Then the samples were detected by reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR technology. The detection results demonstrated that four kinds of fruit trees were infected by Apple scar skin viroid (ASSVd) and the detection rates were 2.1%, 2.1%, 2.8% and 3.3% on apple, pear, peach and apricot trees, respectively. I n situ RT-PCR result further confirmed that the existence of ASSVd was mainly distributed in the nucleus of the leaf tissues. And then the RT-PCR products from the samples were cloned and sequenced, and we obtained 42 ASSVd nucleic acid sequences, which were registered in GenBank and the accession numbers was from EU031455 to EU031496 by using biological software to analyze and the total detection rate was 3.0%. The homology analysis results showed that the 42 isolated ASSVd sequences had 85%~100% nucleotide sequence identity with previously published sequence NC_001340 (Puchta et al.,1990). All this results indicated that the variation of nucleic acid sequence of ASSVd isolate in each host was not obvious and was no significant difference in regions and varieties. In this present study, we established the optimized detection methods of RT-PCR and in situ RT-PCR, which would lay a good foundation for the rapid identification of ASSVd in this four kinds of fruit trees.

Apple scar skin viroid (ASSVd) has been one of the most destructive diseases in Korean apple orchards. Symptoms of the scar skin viroid disease were detected in various apple cultivars, namely, Sansa, Fuji, Chukwang, Miki-Life, Hongro, and Songbongeum cultivated in the southern part of Korea. The RNA molecules were extracted from the apples bearing dapple apple symptoms with the application of CF-11 RNA extraction method. The purified RNAs were used for the synthesis of cDNA with RT-PCR. The PCR products were cloned and sequenced. The viroid RNA molecules from the six different cultivars bearing the dapple symptos showed the same nucleotide sequences as that of the Korean strain of ASSVd（ASSVd-K). ASSVd-K was detected from apple orchards in Kunwi, Sangju, Uiseong, Yeong-yang, Andong, and Youngduk in Gyeongbuk Province in 2001, and in Muju in Jeonbuk Province in 2002. As the viroid disease could be propagated vegetatively, it can be widely transmitted gradually in Korea.