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Title: AN IMPROVED RT-PCR ASSAY FOR THE DETECTION OF TWO CHERRY FLEXIVIRUSES IN PRUNUS SPP

Author
item Li, Ruhui
item Mock, Raymond

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/31/2005
Publication Date: 11/1/2005
Citation: Li, R., Mock, R.G. 2005. An improved rt-pcr assay for the detection of two cherry flexiviruses in prunus spp. Journal of Virological Methods. 129(2)pp 162-16.

Interpretive Summary: Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV) have potential to cause disease in certain sour, flowering, and sweet cherry varieties despite symptomless infection in most Prunus spp. The current method to detect CGRMV is to graft the material onto a susceptible indicator plant, Kwanzan cherry (Prunus serrulata), in early spring. CGRMV induces disease symptoms in the indicator plant six to eight weeks after grafting. To ensure accuracy, the grafting assay has to be done in two consecutive years. This process is laborious, space consuming and takes four months each year. Therefore, it is necessary to develop a rapid and sensitive virus detection assay suitable for testing large numbers of plants. We have developed a molecular technique to detect these two cherry flexiviruses in Prunus spp. This method is sensitive and reliable, and can detect both viruses simultaneously. It can detect viruses in different tissues at different times, extending detection seasons. The method is more cost efficient and space saving, and is completed in two days instead of four months. The method will be of value of scientists for the routine diagnosis of cherry flexiviruses in Prunus spp.

Technical Abstract: A one-step reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed to detect Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV) in woody indicators and naturally-infected Prunus spp. Viral RNA suitable for RT-PCR was obtained by a simple trapping method that did not require either extraction of dsRNA or total RNA, availability of virus antibodies, or purification of viral particles. Consensus primers, degenerate primers and virus-specific primers, whose designs were based on alignments of available cherry flexivirus sequences, were tested to amplify viral genomic fragments of six CGRMV isolates and one CNRMV isolate. RT-PCR allowed CGRMV detection in total RNA and viral RNA preparations equivalent to 400 'g and 4 'g of infected leaf tissue, respectively. CGRMV was detected in tender shoots, leaves, bark and root tips whereas the strongest bands were obtained using young leaves. Detection was less consistent in summer when the temperature was elevated and plant tissues were old. The direct comparison of RT-PCR and grafting assay indicates that the RT-PCR assay is sensitive, rapid and reliable. The method will improve the routine diagnosis of cherry flexiviruses in Prunus spp.