| Sugarcane is the most important sugar crop, which produces more than90%of total sugar production in China. The Leifsonia xyli subsp. xyli (Lxx) which causes the ratoon stunting disease is one of the most important bacterial diseases in sugarcane planting areas of the world and can only be detected from the sugarcane until now. Planting the healthy seedcane without virus and Lxx, is the most efficient and widely adopted way to control the disease in the world, which has also been proved to be an effective measure to increase sugarcane production. Nine demonstration sites have been set in the four main sugarcane producing provinces in China, Guangxi, Yunnan, Guangdong and Hainan. It indicates that the large-scale producing plan of sugarcane healthy seedlings has been officially launched. Accordingly, an accurate, efficient and low-cost technology for detecting the Lxx is the supporting technology for the industrial application of the healthy seedcane, which is also indispensable in the breeding process for the RSD resistance sugarcane varieties. Therefore, the research conducted in this study has great practical significance and a good prospect for application.The study has been carried out to meet the need of sugarcane industry. This study involved several aspects of the Lxx detection, the isolation of Lxx, polymerase chain reaction technique (PCR), the production of polyclonal antibody, serological detection technology and the loop-mediated isothermal amplification detection technology (LAMP). The main results and conclusions are as follows.First, using the sugarcane juice from sugarcane stalk suffering the ratoon stunting disease as the material, the Lxx was successfully isolated and cultured. The bacteria were recognized as the pure culture of the Lxx through morphological identification, PCR identification and purity testing. The result of the successful separation and culture of Lxx laid the necessary foundation for the establishment of technology for the Lxx detection.Second, the polyclonal antibody, which was produced in New Zealand white rabbit immunized with the protein of Lxx, was detected by DB-EIA (dot blot enzyme immunoassay). It was determined that the most suitable concentration of the polyclonal antibody in DB-EIA test was1:100. The result was tested by gradient test, in which the concentration of the polyclonal antibody was1:2000,1:500and1:100, respectively. The bacterial liquid of Lxx, the internode juice and the leaf midrib with Lxx all had immunereaction in the DB-EIA test under the polyclonal antibody concentration of1:100. In specificity test, this antibody could react with the model strain of Leifsonia poae and with some Leifsonia spp. isolated from the internode juice, which indicated the antibody specificity was not ideal though the serological detection is low-cost.Third, using the total DNA from the internode juice and the leaf midrib of the ROC22, YG18and YG26as the templates, the effectiveness and sensitivity of PCR detection were tested. In the study of sensitivity, the minimal detectable Lxx for variety ROC22in a25μL PCR reaction volume was0.2ng for the internode juice and2.0ng for the leaf midribs, respectively. For variety Yuegan18, it was1.0ng for the internode juice and10ng for the leaf midribs, respectively. For variety Yuegan26, it was2.0ng for the internode juice and100ng for the leaf midribs, respectively. The results suggested that the internodes and leaf midribs were both feasible to be used as the sampling for the Lxx detected by PCR. Although the efficiency and sensitivity of Lxx detection were comparable between internodes and leaf midribs that met the requirement for Lxx inspection in sugarcane by PCR, the minimum level of total DNA for detection was lower for internodes than midribs. It indicated the higher concentrations of Lxx in internodes than in leaf midribs according to the above results. Four types of amplified bands had been found in the PCR detection of the total DNA from the internode juice and the leaf midrib. They were the single band which was in the same size as that of the positive control, the dual-band in which one was at the same location as that of the positive control while the other was smaller than that of the positive control, dual-band which were both smaller than that of the positive control, and the single band which was smaller than that of the positive control. According to the results of cloning and sequencing of these fragments, this study suggested that as long as the PCR products appeared to be at the same location with the band of the positive control, it could be determined to be the specific amplification, and thus the samples were determined to be positive with Lxx.Fourth, for the first time, a novel and low-cost detection method for Lxx named LAMP with a rapid, simple, sensitive and practical characteristic and without the use of a PCR instrument was established. According to the specific sequence of the Lxx, four primers were designed and synthesized. One reaction could be completed in60min under a constant temperature at65℃by LAMP. The inside and outside primer concentration ratio and the Mg2+concentration had been optimized, which showed the most appropriate ratio of inside and out primer concentration was4:1while the optimal concentration of Mg2+was5.75mmol/L. Under these conditions, the LAMP reaction was proved to be highly specific and sensitive. In the sensitivity comparision test of PCR and LAMP, the total DNA from the internode juice and the leaf midrib of the Yuegan18were used as the templates. The results demonstrated that, the sensitivity of LAMP was10times higher than that of PCR using the total DNA from the internode juice as the template, while the sensitivity of the LAMP reaction was the same as that of PCR using the total DNA from the leaf midrib as the template. It was also indicated that in the LAMP detection of Lxx, the sensitivity using the total DNA from the internode juice as the template was100times higher than that using the total DNA from the leaf midrib. It was easy to discriminate the result in LAMP detection. If using the SYBR Green I (1000x) as the indicator, it was determined to be positive when the color of the reaction solution was green. If using gel electrophoresis as the detection method, it was determined to be positive when there was a ladder-type feature bands. The successful application of LAMP technology in the detection of Lxx also provides a reference for the detection of other pathogens and even GMO detection in sugarcane. |
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