Development of techniques for quick and easy RNA extraction for virus detection and molecular identification of yellow leaf pathogens of phalaenopsis orchids

• Main Authors: Shu-Jun Chen, 陳淑君
• Other Authors: Chin-Chih Chen
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/69260273304731986004

碩士 === 朝陽科技大學 === 生物技術研究所 === 97 === Phalaenopsis is currently the most important exporting item for the ornamental industry in Taiwan. In recent years, companies from the Netherlands have been taking advantage of their virus-free planting materials propagation system to promote their phalaenopsis; that apparently has threaten the competitiveness of Taiwan’s orchid industry in the international market. Therefore, improving the quality and virus control status of our phalaenopsis products has become the key issue for maintaining the competitiveness of our orchid industry, and for this reason, a decent virus detection technology is inevitable to accomplish this goal. Fifty-nine viruses have been known to infected orchids. The Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are recognized as the most widespread and economically important viruses for orchid industry worldwide. For plant RNA viruses, development of a RT-PCR assay would provide a significant improvement over current detection technology. Nucleotide sequences for the design of PCR primer are readily available for many viruses but nucleic acid extraction from plant tissues is a laborious and time-consuming step in RT-PCR procedures. The TPS buffer have been reported to apply on quickly release virus nucleic acid from plant tissue for detection (Thomson et al. 1995). In this study, we demonstrated an applicable quick and easy protocol for the extraction of total RNA from phalaenopsis orchid sample before amplification of ORSV and CymMV in RT-PCR. In this protocol, virus-infected leaves were groud 1:4 (g/ml) in TPS buffer, the resulting crude saps were incubated at 80℃ for 10 min, diluted 10 fold with sterilized H2O and subsequently used as RNA template in RT-PCR. Similar procedure with a lower (60℃) temperature treatement was found to be feasible for the detection of Capsicum chlorosis virus, (CaCV). We also tested the feasibility of some potential buffers and found that a CarTPS buffer is comparable to TPS buffer for the extraction of plant total RNA with decent quality for RT-PCR. However, after careful and repetitive comparison tests it was realized that our developed quick and easy RNA extraction protocol could not detect virus infection on certain orchid samples especially when they accumulated minor virus concentration. These samples could only be detected by purifying the total RNA for RT-PCR by the use of commercialized plant total RNA extraction column. Nevertheless, our protocol was confirmed in this study to be applicable for the identification of viruses in samples with normal accumulation of viruses. Another goal in our study is to develop the molecular method for the differential identification of Fusarium fungus infecting phalaenopsis orchids. Fusarium solani and F. oxysporium have been reported to infect orchidaceous plant causing yellow leaf and wilting diseases. Traditionally, Fusarium spp are difficult to be identified because of their diversified morphological characteristics and virulence. For this reason, in this studies are attempted to test the feasibility of using molecular techniques to conduct a fast and precise differentiation and identification of F. solani and F. oxysporium by the use of random amplification of polymorphic DNA (RAPD), which is one of the powerful molecular tools for fungal pathogen identification. Upon testing varius RAPD primers, the OPA02 was found to amplify differentially F. solani and F. oxysporium producing specific DNA fragments the the genomic DNA of F. solani and F. oxysporium isolated from orchid. The differentially amplified specific DNA fragments was then recovered, cloned and sequenced. Furthermore the specific primer set FS239 and FO520 were designed to the known nucleotide sequents of the special DNA fragment of F. solani and F. oxysporium, respectively. The predicted 239 bp and 520 bp DNA fragment were amplified from the primer set of FS239 and FO520, respectively. Therefore, The two specific DNA fragment were not amplified in the genomic DNA of the Phytophthora parasitica Dastur,Colletotrichum gloeosporioides Penzig, Botrytis cinerea, Sclerotium rolfsii Sacc. but were amplification some different formas speciales isolates of F. solani and F. oxysporium. The results showed that the two specific primer set developed can successfully detect F. solani spp and F. oxysporium spp genomic DNA for PCR. And used FS239 primer that the minimal DNA concentration could be detected is about 10 ng/ml; used FO520 primer could be detected is about 10 pg/ml. FS239 and FO520, was used to test naturally infected orchid tissue collected from the field. It appeared that the FS239 and FO520 DNA fragment could easily be amplified in gDNA of symptomatic orchid pseudostems, root and leaf. The primer set FS239 and FO520 can be efficiency in auxiliary identifying F. solani and F. oxysporium and accurately and rapidly detecting diseases caused in the field.