| Eucalyptus, an important component of the forestry resources, originated in Australia, then was introducted worldwide, including China. Prior to the early nineteenth century, with the increasing introduction of this tree species, Eucalyptus dieback, caused by Cylindrocladium scoparium (teleomorph:Calonectria morganii), were frequent in most parts of China, and it has historically been an important disease of Eucalyptus, which was defined as a key national plant quarantine disease in China, on January 5,1996. In Southern China, the disease affects Eucalyptus throughout its range of cultivation and causes severe yield losses and tree mortality. C. scoparium, especially in late infection, can cause large damage to the growth of Eucalyptus, and occurs dieback, all branches withered severely. Even in early infection, it can easily lead to reddish-brown spots, wilting leafs, and droping branches. However, thus far, it is still a great constraint to the cultivation and production expansion of Eucalyptus in many provinces of China, such as Sichuan, Fujian, Guangxi, and so on. Therefore, to establish rapid and effective detection technologies have important significance for control and prevention of Eucalyptus dieback. Based on different target gene sequences, double PCR, nested PCR and LAMP, these three different kinds of four molecular biology diagnostic techniques were established respectively, which aimed to provide strong technical supports for forestry production and construction of eucalyptus dieback. The result of this study as follows:1. The double PCR technique based on genera and species, for rapid detection of eucalyptus dieback disease pathogen C. scoparium, was established with the total DNA as templates, two pairs of specific primers (CYS1/CYS2 and EF-S-1/EF-A-1) for Calonectria de Not and C. scoparium on their ITS sequence and factor 1-alpha gene were designed, respectively. The results showed that all tested strains belong to Cylindrocladium genera were amplified a stripe of 351 bp, while only these C. scoparium were amplified a stripe of 197 bp, and the double PCR with these two pairs of primers was also amplified two stripes of 351 bp and 197 bp. Furthermore, under the annealing temperature at 53℃, the minimum amount of DNA could be detected was 450 fg/μL. The infected eucalyptus tissue with different levels was accurately detected, the result showed that it fits entirely in the field test. This was the first report about the rapid detection technique on eucalyptus dieback caused by C. morganii.2. Based on its conserved domain beta-tubulin gene, two parirs of specific primers BT-S-1/BT-A-1 and universal primers BT-T1-S/BT-CYLTUBIR-A were severally designed by the aid of the software Premier 4.5 and were synthesized respectively, and the specificities and sensitivities of these two pairs were tested with the method of universal PCR and nested PCR. Meanwhile, the field tests were conducted. The results showed that all tested strains were amplified a stripe of 500 bp with the universal primers BT-T1-S/BT-CYLTUBIR-A, while only these six isolates of C. scoparium were amplified a stripe of 148 bp, and the nested PCR with these two pairs of primers was also amplified the stripe of 148 bp. The difference was that the sensitivity of Nested PCR was 4.5 fg/μL, which was significantly 103 times higher than universal PCR detection. The filed tests showed that nested PCR was much accurater and sensitiver than universal PCR. In conclusion, detection technique of nested PCR of C. scoparium proves to be quickly and specific in monitoring of C. scoparium and has great significance for the prevention and control of Eucalyptus dieback. Furthermore, the result of the study this nested PCR can play an effective role in the early diagnosis of Eucalyptus dieback and also could provide a new basis for the detection of other diseases.3.Based on the species-specific conserved region of beta-tubulin gene on Cylindrocladium scoparium, a set of primers (inner primer:FIP/BIP, outer primer:F3/B3, Loop primers: LooPF/LooPB) were designed, a LAMP assay for rapid detection of C. scoparium was developed, evaluated and optimized. The results showed that the LAMP reaction system (50 μl) consisted of 8 U Bst DNA Polymerase 1 μl,5 M Betaine solution 3.0 μl; 2.5 mM dNTP 3.5 μl; 10×Thermopol Ⅱ 2.5 μl; 100 mM MgCl2 2 μl; 40 μM FIP/BIP 1 μl; 10 μM F3/B3 0.5 μl; 10 μM LooPF/LooPB 1 μl; DNA template 2 μl; with ddH2O up to the volume of 50 μl. Response procedures acted as the mixture was in 65 ℃ water bath pot for about 45 min first, then inactivated 5 min under the 90 ℃ to end of reaction. Specificity assays showed that the amplification results of 22 test isolates can be distinguished though visual inspection, ultraviolet light, add fluorescent dye SYBR Green I, and electrophoresis test strip, and the 4 pathogens were positived, and that of the other 8 pathogens were negative. The detection limit of LAMP was 40 pg pure genomic DNA per 45 μl reaction, which fits entirely in the field test. The wild field effectiveness showed that the different physiological small kinds different regions of C. scoparium can be detected with the method of LAMP, while mean the ultraviolet light and electrophoresis test strip were significantly. Summing up the above, the LAMP rapid detection can be applied in filed effectively.Summing up the above, double PCR, nested PCR and LAMP rapid detection all can be applied in filed effectively. While nested PCR has the highest sensitivity than the two detection technologies, which is up to 4.5 fg/μL and is higher than ordinary conventional PCR detection limit of 4.5 pg/μL. Then, the detection limit of double PCR is at the amount of 450 fg/μL, which is higer than LAMP. As for LAMP detection sensitivity, although lower than the other one, but the visibility observations potential to become the most productive means of detection. |
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