Detection Of Latent Infection Of Wheat Leaves Caused By Blumeria Graminis F. Sp. Tritici Using Nested PCR

Powdery mildew caused by the fungal pathogen Blumeria graminis f. sp. tritici is one of main wheat diseases. Disease forecast and risk assessment of epidemics serve as an important part in integrated disease management. Early detection of the pathogen from plant tissues to accurately estimate the possible epidemic trend is a key issue in disease forecast. Although variations in disease epidemics of wheat powdery mildew and weather patterns among different wheat-growing regions of China exist, a common key issue of disease forecast among these regions is to timely and accurately estimate and quantify the latent infection levels in overwintering seedlings and oversummering volunteer seedlings that serve as sources of initial inoculum of epidemics. However, the conventional methods to estimate latent infection level rely on wheat leaf or seedlings incubation process which is labor intensive, time consuming and inaccurate. The rapid development of bio-technology and molecular biology provide a huge potential to improve the methodologies in epidemiological research. The objective of this study was to develop a technique that can be used to rapidly and accurately estimate levels of latent infection of wheat leaves caused by B. graminis f. sp. tritici based on principles of molecular biology and bio-technology, and to accumulate experience in molecular epidemiology by improving the conventional epidemiological methods in detection of latent infection by using molecular approaches.This study designed three molecular primer pairs (F1/R1,F2/R1,F3/R1) based on the internal transcribed spacer (ITS) sequences of ribosome of B. graminis f. sp. tritici. The species specificity of these primers was confirmed by testing isolates of other species of wheat fungal pathogens. The primer pair F1/R1 demonstrated a higher sensitivity than those of the primer pairs F2/R1, F3/R1, and could detect as low as 1 pg DNA ofB. graminis f.sp. tritici. A nested PCR assay has been developed for which an internal and an external primer pair were generated based on the sequence of PCR product amplified with the primer pair F1/R1. The sensitivity of this nested PCR assay was as low as about 0.1 fg by detecting pure culture of the pathogen DNA.A technique of DNA extraction from wheat seedlings was developed. Experiments were conducted in which artificial inoculations of wheat seedlings were implemented by using different inoculum densities. Leaf samples were collected on different days after inoculation and samples of inoculated leaves with latent infections were processed to obtain the pathogen DNA. The regular and nested PCR approaches were used to detect the infections and compared in sensitivity for detection of latent infection. When the inoculum concentration of 300 spores/cm of leaf area was used, the pathogen DNA in infected leaves was detected three days after inoculation with the regular PCR assay, while only one day after inoculation with the nested PCR assay. Generally, the nested PCR could detect latent infection 2-3 days earlier than did the regular PCR. The nested PCR shows a higher advantage over regular PCR in detection of latent infection especially when low inoculum concentration was used at early infection stage.The nested PCR assay was also used to detect latent infection level of naturally and artificially infected leaves collected from wheat fields in Beijing region and Shandong Province. A half mount of these leaf samples were incubated under controlled condition to determine latent infection rate and the other half amount of the leaf samples were processed with the regular and the nested PCR assays to determine the infection rates, too. Regression analyses showed that there was a significant correlation between incidence of latent infection determined with the regular PCR assay and that with leaf incubation method at P=0.05 level but not at P=0.01 level (r~2=0.49). Among 11 sample sites, the regular PCR did not detect infections in 6 sample sites. However, regression between latent infection rate determined with the nested PCR assay and that determined with conventional leaf incubation method was significant at P=0.0023 (r~2=0.66). The results demonstrated a high sensitivity and predictability of the nested PCR compared with the regular PCR in estimation of latent infection level of wheat seedlings infected by B. graminis f. sp. tritici. Moreover, the nested PCR consistently overestimated the latent infection level determined with conventional method, implying a high sensitivity in detection latent infection. This study provided useful tool that can be used to timely, rapidly and accurately monitor latent infection of wheat powdery mildew in seedlings that benefits the disease prediction.