Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica) (original) (raw)
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Phytoparasitica, 2009
Apple mosaic virus (ApMV) is an important virus of apple worldwide. Surveys were conducted in the states of Himachal Pradesh (HP) and Jammu & Kashmir (J&K), India, to determine the prevalence of ApMV and to develop ways for its early diagnosis. Symptomatic leaf samples, bud and bark were collected during spring and late summer. DAS-ELISA revealed the presence of ApMV in 13/198 samples from four districts in HP, indicating a disease incidence of approximately 6.5%. The sequence of amplicons produced by reverse transcription-polymerase chain reaction (RT-PCR) using virus-specific primers confirmed the ELISA results. Comparison of the sequences of three amplicons with nine complete available sequences of ApMV coat protein (CP) (from apple) at amino acid level revealed a maximum of 96% homology to a Korean isolate of ApMV (AY125977). Comparison of the amino acid sequence of the CP of the Indian isolate with the amino acid sequence of the CP from different hosts showed that the Indian isolate clustered most closely with an isolate found in pear and originating from Czechoslovakia. Cloned DNA was reliably used for diagnosis of the virus and was a useful tool for screening at nursery level using slot blot hybridization. This study confirms the presence of ApMV at the molecular level in India and reveals sequence information.
Detection of Four Apple Viruses by ELISA and RT-PCR Assays in Turkey
Plant samples were collected from the main apple growing provinces of Turkey in order to evaluate the incidence of 4 important apple virus diseases during spring 2004. Collected leaves and shoots were tested using enzyme linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) for Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Since no commercial antiserum is available, Apple stem pitting virus (ASPV) was tested only using RT-PCR and the results were compared. A total of 174 apple samples were collected from varietal collections belonging to governmental and university institutions and also from commercial orchards. Out of 174 plants, 126 were infected by at least one virus disease. The incidence of the 4 viruses in varietal collections was 70.21%, while it was 75.00% in commercial orchards. The results obtained from the comparison of ELISA and RT-PCR in this study showed that with the R...
Molecular characterization and diagnostic development for Apple chlorotic leaf spot virus
2011
Work done in the present thesis entitled "Molecular characterization and diagnostic development for Apple chlorotic leaf spot virus" includes studies on incidence of the viruses infecting pome and stone fruits, virus transmission, host range, purification, molecular characterization, diversity analysis, heterologous over expression of viral coat protein (CP) in E. coli and development of nucleic acid and ELISA based diagnostic systems. Horticultural crops play a unique role in India's economy by supplementing the income of the rural people. These crops form a significant part of total agricultural produce and have become key drivers of economic development in many of the states in the country. In the past one decade, the change in cropping pattern indicates a shift towards the fruit and commercial crops. In Himachal Pradesh (HP) apple is the major fruit accounting for more than 40% of total area under fruits and about 88% of total fruit production from the state. Commercially, apple is the most important of all the fresh fruits grown in HP, Jammu & Kashmir (J&K) and Uttarakhand. Any losses occurring would affect the economy and livelihood of some growers for whom apple is the only cash crop. Some of the economically important major viruses and viroids known to cause diseases in apple and other pome and stone fruits are Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). During extensive surveys (2007-2009) of pome and stone fruit growing areas in HP and J&K, presence of typical virus like symptoms on these fruits indicated viral infection. Symptoms of leaf mosaic, severe chlorosis, deformation, curling etc were recorded in apple orchards. While plum, cherry, peach/nectarine, almond, cherry and apricots exhibited various symptoms like yellow flecking on leaves, mosaic, mottling, necrotic ring spots and shot holes. The symptoms were not persistent throughout the year, and disappeared late in the season making the plant seem healthy. Most of the orchards surveyed had mixed plantation of pome and stone fruits.
Phytoparasitica, 2018
Majority of the apple trees are known to be infected by two latent viruses, Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV). The importance of ASGV and ASPV is due to their non expression of symptoms, worldwide occurrence and wide host range on pome and stone fruits. Due to their latent nature in apple, early and rapid diagnostics plays important role for production of virus free quality planting material. The present investigation was conducted to detect and quantify ASPV & ASGV from different plant parts (spatial) in apple trees during different seasons (temporal) for optimisation of tissue and time for their rapid and early detection. Detection and relative quantification using immuno-molecular diagnostic techniques like, Double Antibody Sandwich-ELISA, Reverse Transcription-PCR and Real Time RT-PCR in various plant parts (leaf, whole flower, sepal, petal, anther, stigma with style, bark, fruit, seed and root) during different seasons was done. The DAS-ELISA based detection revealed infection in all plant parts except root and fruit with ASGV and ASPV, showing more expression in leaves followed by bark and whole flower. Similar results were also observed on RT-PCR based detection. Quantitative real time PCR analysis showed variation in expression of ASGV and ASPV in different parts during different seasons. Results confirmed that the ASGV and ASPV expression is higher in leaves followed by bark and whole flower. Periodic detection of these viruses in different plant parts during all the four seasons revealed varied virus titer from one season to another in the same plant. During all the seasons, both ASPV and ASGV were detected in bark in measurable titer using immunomolecular detection tools, however via DAS-ELISA, ASGV remained undetected during dormant season. Hence leaves and bark except leaf during fall, can be directly used as detection material for their early and rapid detection leading to production of virus free planting material.
Quantitative detection of four pome fruit viruses in apple trees throughout the year
Phytopathologia Mediterranea, 2016
A one-step real-time RT-PCR assay (RT-qPCR) with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed to detect and quantify RNA targets from Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) in infected apple trees. Single PCR products of 87 bp (ApMV), 70 bp (ASGV), 104 bp (ASPV) and 148 bp (ACLSV) were obtained, and melting curve analyses revealed distinct melting temperature peaks for each virus. A dilution series using in vitro synthesized transcripts containing the target sequences as standards yielded a reproducible quantitative assay, with a wide dynamic range of detection and low coefficients of variance. The content of selected viruses in apple plant tissues was stable throughout the year, and their accumulation did not significantly change between different plant tissues. The only minor exceptions were for ApMV and ACLSV, in which noticeable differences in...
Molecular Detection of Latent Apple chlorotic leaf spot virus in Elite Mother Plants of Apple
Indian Journal of Virology, 2012
Apple chlorotic leaf spot virus (ACLSV; family Betaflexiviridae genus Trichovirus) is one of the economically important latent virus infecting apple (Malus 9 domestica Borkh.). Reverse transcriptase polymerase chain reaction (RT-PCR) procedures were used to amplify coat protein gene of ACLSV. Among 5 primer sets used, two primer sets (1F1R and 1F2R) amplified fragments of expected size (432 bp). Products visible on agarose gel were produced using templates extracted from apple leaves. The results were further validated by sequencing fragment of 432 bp which was amplified from leaf of apple by using primer set 1F 1R. Comparisons with published sequences indicated that the isolate have very high 91 % identity values to the corresponding region of ACLSV isolate from apple. Selected primer pair (1F1R) was further used for screening 42 elite mother plants collected from apple growing areas of Himachal Pradesh, India, where in 17 were found free from ACLSV. Use of NAD5 gene in mitochondrial mRNA of the apple as an internal control, reduced the risk of false negative results that may occur with routine RT-PCR assays.
Diversity of Apple mosaic virus Isolates in India Based on Coat Protein and Movement Protein Genes
Indian Journal of …, 2011
Apple mosaic virus (ApMV), an Ilarvirus is one of the most common pathogens of apple worldwide. During field surveys in commercial plantations of Himachal Pradesh and Jammu & Kashmir, observations of bright chlorotic mosaic like symptoms on apple trees indicated probable infection by the virus, which was later detected by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). An incidence of 24 and 28% (based on ELISA) was obtained as 6/25 and 15/53 samples from HP and J&K were positive, respectively. An amplification of approximately 700 and 850 bp was obtained for coat and movement protein genes (CP and MP), respectively. The CP was 223 amino acids in length and showed 87-99% identity when compared to 21 ApMV isolates. Whereas, MP (286 amino acids) showed 91-95% identity with other isolates. However, the gene sequences were quite conserved among Indian isolates and grouped together phylogenetically. CP of the Indian isolates showed maximum identity of 95% with Korean isolate (AY 125977) in apple and in other host these showed a maximum identity of 98% to Czech Republic pear isolate. MP showed maximum identity with Chinese isolate i.e., 95%. The diversity study will also help in analyzing variability among the isolates and also to formulate diagnostic and resistance strategies.
TURKISH JOURNAL OF AGRICULTURE AND FORESTRY, 2013
Introduction Apple (Malus domestica Borkh.), one of the most widely grown fruit crops worldwide, is a sensitive host to the infection of Apple mosaic virus (ApMV), which is an economically important and common pathogen in commercial apple cultivars (Campbell 1963; Posnette et al. 1963). The virus has no vector and is not pollen-or seedborne (Rybicki 1995). ApMV is a member of subgroup III of the Ilarvirus group (family Bromoviridae) with a positive-sense tripartite RNA genome. RNA3 codes for the movement protein and the capsid protein (CP) (Francki et al. 1991). It occurs as isometric or quasi-isometric labile particles and often produces ringspot and mosaic symptoms on hosts (DeSequeira 1967). ApMV is present worldwide, preferentially on woody hosts such as blackberry, raspberry, apple, apricot, cherry, almond, plum, peach, hazelnut, roses, and hop (Brunt et al. 1996). ApMV has also been reported in mountain ash (Sorbus aucuparia), silver birch (Betula pendula), horse chestnut (Aesculus hippocastanum), and red horse chestnut (A. × carnea) (Polak et al. 1997). The virus does not occur in seedling rootstocks and is not pollen-borne. In a recent study, ApMV was found to infect the weeds naturally (Arlı Sökmen et al. 2005). In Turkey, the molecular detection of ApMV was performed for the first time by Ulubaş and Ertunç (2003). The coat protein gene of ilarviruses is translated from RNA4, a subgenomic messenger derived from the bicistronic RNA3. The coat protein of ilarviruses forms the shell for the 3 genome components. It also plays a major role in initiation and propagation of infection (Bol 1999; Petrzik and Lenz 2002). The virus is on the quarantine list of the European and Mediterranean Plant Protection Organization. Since no information was available on genetic variability and the incidence of ApMV in East Anatolia, these issues were addressed in the present study. To determine optimal conditions for dot-blot hybridization, 3 different RNA
Determination of major viral and sub viral pathogens incidence in apple orchards in himachal pradesh
Indian journal of virology : an official organ of Indian Virological Society, 2012
Apple is the major commercial horticulture crop in Himachal Pradesh and other hill states of Jammu & Kashmir, Uttarakhand and some parts of Northeastern states of India. In order to gather data on health status and incidence of virus and virus-like pathogens in apple orchards, survey was conducted in the month of June and September, 2010 in Hatkoti, Rohru, Kuthara, Jubbal and Khadapathar areas of major apple producing Shimla district of Himachal Pradesh. A total of 250 samples were collected and analyzed by DAS-ELISA, NASH and RT-PCR. NASH results indicated that a total of 117 samples were infected with Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple scar skin viroid (ASSVd). Results showed that ASSVd is predominant in these areas with highest infection rate of 27.6% followed by ASPV (17.2%), ACLSV (16.8%), ApMV (15.2%) and ASGV (12%). Mixed infection of these viruses and viroid was frequ...