Research On Fast Detection Of Citrus Tristeza Virus

Citrus Tristeza disease, caused by Citrus Tristeza Virus, is a worldwide disease of citrus. Tristeza has seriously destroyed the citrus industry which had once been got close to the breakup in South America nations such as Brazil. In China, pumelo and sweet orange are severely infected by CTV with stem pitting symptom in Zhejiang, Sichuan and Guangxi province. Researches on the fast detection of CTV are of great importance to the control of citrus Tristeza disease. In order to develop and optimize the detection methods for CTV, establish technical protocols for CTV diagnosis, we had conducted biology indexing, inclusion bodies histological observation, immunology (serology) and molecular biology detction of CTV isolated from 8 samples: HB pummelo, Page tangelo, Gillete navel, Navelina navel, Frost navel, Bendizao mandarin, Fisher navel and Atwood navel.CTV isolate TR-L514 and isolates from Bendizao mandarin and Navelina navel samples were graft-inoculated to Mexican lime, sour orange, Duncan grapefruit and Madam vinous sweet orange. These isolates caused typical symptoms on indicator plants, thus were identified as SY strain, SP strain and mild strain, respectively. CTV inclusion bodies were observed in phloem tissue of affected Mexican lime petiole freehand section stained with Azure blue, while not found in xylem tissue and parenchyma.The spring shoots, summer shoots and autumn shoots of 8 samples were diagnosed by TAS-ELISA. Navelina navel, Fisher navel and Atwood navel were positive. The P/N values of spring shoots and autumn shoots were obviously higher than that of summer shoots, hi addition to the three samples, summer shoots of Frost navel and Bendizao mandarin were also positive detected by biotin-avidin system ELISA (BAS-ELISA). Bark and young leaf are more reliable than mature leaf in ELISA tests. Mature leaves were all negative by ELISA tests, regardless the citrus varieties or sampling periods (spring, summer or autumn). Navelina navel, Fisher navel, Gillete navel and Atwood navel were also positive in tissue print tests. The color of print on the PVDF membrane was proportional to the P/N value in ELISA tests. Tissue print results indicate that CTV distributes unevenly in citrus phloem tissue. Virus titer in the top end of twigs was higher than that in bottom end.dsRNAs extacted from Navelina navel, Frost navel, Bendizao mandarin, Fisher navel, Atwood navel and TR-L514 were eletrophoresed in 10% polyacrylamide gel. 13.3 X106 MW, 2.0X 106MW and 0.8X 106MW dsRNA segments were detected in all 6 samples.The 0.5X106MW dsRNA segment was prensent in TR-L514, Navelina navel, Fisher navel and Atwood navel but not in Frost navel and Bendizao mandarin. The 1.7 X106 MW dsRNA segment was detectable in the other 5 samples except for TR-L514. dsRNA analysis imply that dsRNA pattern and biology properties are not necessarily correlated. Results from the present study suggested that dsRNA analysis was not reliable to differentiate various CTV isolates.Total RNAs extracted from the above 8 samples ' leaves were amplified by one step RT-PCR. Spring and summer shoots of Navelina navel, Fisher navel, Atwood navel, Frost navel, HB pummelo were all positive. The spring shoot of Bendizao mandarin was positive while the summer shoots was negative. The detectable amounts of CTV in summer shoots were much more less than in spring shoots. RT-PCR results further illustrated that virus concentrations decreased markedly as the temperature rising in hot-weather summer.The autumn shoots of the above 8 samples were also detected by IC-RT-PCR. 5 out of 8 samples were positive. We compared the IC-RT-PCR and TC-RT-PCR detection of summer shoots of Navelina navel, Fisher navel and Atwood navel. Both of two methods amplified expected 354bp product. But the detectable bands of the same sample in IC-RT-PCR were brighter than in TC-RT-PCR, which demonstrated that the sensitivity of TC-RT-PCR was lower than that of IC-RT-PCR for CTV detection.The autumn shoots of HB pummelo, Frost navel and Bendizao mandarin were detected by IC Nest...