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Title: A multiple reverse transcription-polymerase chain reaction assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees

item HAO, LU - China Agricultural University
item XIE, JIPENG - China Agricultural University
item CHEN, SHANYI - China Agricultural University
item WANG, SHAOJIE - China Agricultural University
item GONG, ZHUOQUN - China Agricultural University
item Ling, Kai-Shu
item GUO, LIYUN - China Agricultural University
item FAN, ZAIFENG - China Agricultural University
item ZHOU, TAO - China Agricultural University

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2016
Publication Date: 4/28/2016
Citation: Hao, L., Xie, J., Chen, S., Wang, S., Gong, Z., Ling, K., Guo, L., Fan, Z., Zhou, T. 2016. A multiple reverse transcription-polymerase chain reaction assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees. Journal of Virological Methods. 234:16–21.

Interpretive Summary: Apple is one of the most important and widely grown fruit crops in the world. Apple trees are susceptible to infection by several pathogens, especially viruses and viroids, including apple chlorotic leaf spot virus, apple stem grooving virus, apple stem pitting virus, apple scar skin viroid, and apple dimple fruit viroid. In any single virus or viroid infection, no visible disease symptoms could be observed on trees or fruits. But, a mixed infection of two or more viruses and/or viroids could lead to some serious diseases with significant impact on apple fruit production and quality. Therefore, the ability to allow for a simultaneous detection and differentiation of multi viruses and viroids is an important concept in health management of apple orchards worldwide. In this study, an ARS scientist cooperating with others at the China Agricultural University developed a simple and sensitive multiplex molecular assay to detect and differentiate the above mentioned viruses and viroids in apple. To our knowledge, this is the first time that such technology has been successfully applied to simultaneously detect the multiple apple viruses and viroids in a single reaction. This concept of detecting multi-viruses and/or viroids in a crop health screen could be applied to other agricultural production systems. This research is of special interest to plant pathologists working to combat viral diseases.

Technical Abstract: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1' (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100 bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs.