基于双重Reai-time PCR对潜育小麦条锈菌的田间宏观定量分析

发布时间：2018-05-04 17:33

本文选题：小麦条锈菌 + 潜伏侵染　；参考：《中国农业大学》2016年博士论文

【摘要】：小麦条锈病,由Puccinia striiformis f. sp. tritici (PST)引起,是我国小麦生产上的重要病害之一。有效监测处于潜伏阶段的条锈病菌,是早期预防的关键。为此开展了相关研究,结果如下：1.为高效准确地定量检测处于潜伏侵染阶段的小麦条锈菌,本研究根据已发表的小麦条锈菌和寄主小麦的2对特异性引物,分别设计了探针,首次建立了小麦条锈病菌潜伏侵染的双重real-time PCR检测方法。为排除多个引物互作造成的干扰,对小麦条锈菌引物探针体系、小麦体系以及二者的双重real-time PCR体系进行了比较。CT值相关线性回归分析证明,引物之间互作很小,对定量检测无影响。已知浓度样品经梯度稀释,进行灵敏度检测,确定了双重real-timePCR对小麦条锈菌DNA和小麦DNA准确定量测定的最小检测限为0.4 pg和0.5 ng。同时建立了小麦条锈菌和小麦各自的标准曲线。用此方法检测来自湖北襄阳、甘肃甘谷两个不同地区的田间样本,得到的分子病情指数(MDI)与随后的发病趋势一致。本研究证明建立的双重real-time PCR分析方法可靠、高效、低误差,是对小麦条锈菌潜伏期分子检测优化后的方法。2.结合了田间采样、病情调查、PST分子检测、相关性分析和空间分析,在PST可以越冬的湖北襄阳地区,开展了2个生长季的试验来确定越冬潜伏菌源量与春季病情(DI)的关系的研究。在冬季,对小麦叶片进行采样,同时记录采样点的GPS坐标。使用上述自建的双重real-timePCR法确定采集的小麦叶片中PST的菌量。在春季,在采样点相应的位置进行病情调查。在2013～2014生长季中,试验只在襄阳与宜城进行。宜城试验中,发现MDI值高的采样点经常靠近汉江,调查点的也有这个规律。在2014～2015生长季中,增加了在枣阳试验。所有试验区域按照镇/区划分成不同的区域。通过区域内各个点取平均值计算方法得到此区域的MDI与DI值。由MDI转换成风险指数(RI),并进行RI与春季病情指数的相关性分析。结果显示,PST潜伏侵染量与春季病情呈显著相关。使用GIS软件对2014-2015生长季所有采样点和调查点进行高/低聚类分析。结果显示,靠近河流的区域发病更早更集中。3.为检验上述依据潜伏菌量预测病情的准确性,在条锈病核心菌源区甘肃的甘谷和病害流行区的河南淅川地区也进行了验证试验,并且持续2个生长季进行。甘谷地区试验结果说明该方法适合对当地秋苗病情进行预测,进而可明确该地区秋季流行菌源量,这为冬繁区菌源量早期预测提供了可能。而淅川的试验结果说明PST虽然可以以潜育期的状态进行越冬,但是否发病更需要的当地早春的气象条件。
[Abstract]:Wheat stripe rust, caused by Puccinia striiformis F. sp. tritici, is one of the most important diseases in wheat production in China. Effective surveillance of stripe rust in latent stage is the key to early prevention. A related study was carried out, and the results are as follows: 1. In order to detect wheat stripe rust in latent infection stage efficiently and accurately, two pairs of specific primers of wheat stripe rust and host wheat were designed. A double real-time PCR method for the detection of latent infection of wheat stripe rust was established for the first time. In order to eliminate the interference caused by the interaction of multiple primers, the primer probe system of wheat stripe rust, wheat system and their double real-time PCR systems were compared by linear regression analysis of CT value. The results showed that the interaction between primers was very small. It has no effect on quantitative detection. The minimum detection limits of double real-timePCR for accurate quantitative determination of wheat stripe rust DNA and wheat DNA were determined to be 0.4 PG and 0.5 ng by gradient dilution and sensitivity detection. At the same time, the standard curves of wheat stripe rust and wheat were established. This method was used to detect the molecular disease index (MDI) from two different areas of Xiangyang and Gangu in Hubei Province. This study proved that the established double real-time PCR analysis method is reliable, efficient and low error. It is an optimized method for molecular detection of wheat stripe rust. Combined with field sampling, disease investigation, molecular detection, correlation analysis and spatial analysis, two growing seasons were conducted in Xiangyang area of Hubei Province, where PST can overwintering, to determine the relationship between overwintering latent bacteria quantity and disease status in spring. In winter, wheat leaves were sampled and the GPS coordinates of the sampling points were recorded. The double real-timePCR method was used to determine the amount of PST in wheat leaves. In spring, the disease was investigated at the corresponding location at the sampling point. The experiment was conducted only in Xiangyang and Yicheng during the 2013 / 2014 growth season. In the Yicheng test, it is found that the sampling sites with high MDI value are often close to the Hanjiang River. In the 2014-2015 growth season, Zaoyang trial was increased. All test areas are divided into different areas according to town / district. The MDI and DI values of the region are obtained by using the average value of each point in the region. The risk index was transformed from MDI to RI, and the correlation between RI and disease index in spring was analyzed. The results showed that the latent infection amount of PST was significantly correlated with the disease in spring. The high / low clustering analysis of all sampling sites and survey sites in 2014-2015 growth season was carried out by using GIS software. The results showed that the area near the river developed earlier and more concentrated. 3. In order to test the accuracy of the prediction of disease based on latent bacteria quantity, the experiments were carried out in Ganggu of Gansu Province, the source region of stripe rust, and Xichuan area of Henan Province, where the disease was endemic, and it lasted for two growing seasons. The results of Ganggu area experiment showed that this method was suitable for predicting the condition of local autumn seedlings, and could be used to determine the amount of bacterial source of autumn epidemic in this area, which provided the possibility for early prediction of the amount of bacterial source in winter propagation area. The results of Xichuan experiment show that although PST can overwintering in the stage of incubation, the weather conditions of local early spring are needed more for the onset of the disease.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.121.42

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