Quantitative Analysis Of Puccinia Striiformis F. Sp. Tritici In Latent In The Fields Using Duplex Real-time PCR

Wheat stripe rust(caused by Puccinia striiformis f. sp. tritici, PST) is one of the most important wheat diseases in China. Effective monitoring of the PST in latent is a key to early prevention. For this reason, related researches have been performed and results are summarized as follows:1. Based on two pairs of published primers (PST and Wheat), we designed specific probes respectively and developed a duplex real-time PCR approach for the quantification of PST in latent period for the first time. In order to determine that the primers would not interact in duplex system, we designed two real-time PCR reaction systems with a single pair of primers from PST and wheat respectively for comparison. The liner regressions analysis of Ct values showed that duplex real-time PCR was hardly affected by the interaction. Serial ten-fold dilutions of samples with determined concentration were performed for sensitivity test; the minimum concentrations of PST and Wheat that could be accurately quantified were 0.4 pg and 0.5 ng respectively. In the mean time, two standard curves of PST and Wheat were also set up. This method could be applied to detect samples from two different areas(Xiangyang, Hubei; Gangu, Gansu) and to calculate molecular disease index(MDI). The results demonstrated that duplex real-time PCR is a reliable, efficient and low error calculation method. Therefore it is an optimized molecular method for detecting PST in latent period.2. In Xiangyang Area which PST could overwintering successfully, a study was conducted in 2 growing seasons to determine the relationship of latent PST in overwinter wheat leaves and the disease index(DI) of the next spring. This systemic study was combined with field sampling, DI investigation, molecular PST detection, correlation analysis and spatial analysis. In winter, sampling of wheat leaves was conducted simultaneously with GPS positioning. Duplex real-time PCR given above was used to quantify the latent PST in the sampled leaves. In spring, disease investigations were conducted simultaneously in the same sampling positions. In 2013～2014 growing season, study was conducted in Xiangyang and Yicheng. In Yicheng, an interesting result was found that the sample point had high MDI often close to Han river and so did investigation points in DI. In 2014～2015 growing season, a new experimental region was added in Zaoyang. All the areas were divided into different districts based on town/suburb district. The MDI and DI of each district were obtained by calculating the average of all sampling points and investigation points in the district. Each MDI of district was converted to risk index (RI) by a formula, and then RI was calculated for correlation with DI. The results showed that the quantities of PST in latent were significantly correlated with the disease in spring. High/low clustering analysis of all sampling points and investigation points was conducted by GIS software. The results showed that the yellow rust occurs earlier and more concentrated in the regions near river.3. In order to examine the accuracy that predict disease base on quantity of PST in latent, we carried out the verified experiments in the Gangu and Xichuan in 2 growing seasons. The result in Gangu showed that this method is suit for forecast of disease in fall, then quantify the quantity of pathogen, and then the possibility of early prediction for quantity of pathogen in overwintering areas. While the results in Xichuan showed that there was PST in latent during the winter, but occurrence of disease in spring was rely on meteorological conditions.