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

Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) is a destructive wheat disease worldwide. Over-summering and over-wintering are two important phases of the pathogen in its life cycle. The common key issue of the disease forecast is to timely and accurately estimate and quantify the infection levels in overwintering seedlings and oversummering volunteer seedlings that may serve as sources of initial inoculum of epidemics. However, under unfavorable conditions, the infected leaves are at latency phase which may last for a long period of time before symptoms appear. Culturing and incubating plants to promote symptom appearation is the troduction method to determine the latent infection levels. Obviously, this method is time consuming and labor intensitve.In this study, we designed the Bgt-specific primers (Bgt-F/Bgt-R) and wheat-specific primers (Wheat-F1/Wheat-R1), tested their specificities by using template DNA from wheat and different speciries of wheat pathogens, determined the sensitivities of these primers and optimaized the PCR conditions. Based on above achievements, a real-time PCR method was established to quantify the latent infection level in wheat leaves. The standard curve established with 10-fold serial dilutions of Bgt DNA and wheat leaves DNA by using the corresponding primer pairs were established. We defined the quantity of the fungal DNA /quantity of the plant DNA (μg/mg) as the molecular-detected disease index (MDX).Artificial inoculation experiments were conducted in which different concentrations of inoculum were used. Real-time PCR can detecte the latent infection leaves at minimum inoculation density of 64 spores /cm2 on the first day after inoculate. The linear regressions of MDX and disease index DX were obtained for different days after inoculation.The real-time PCR assay was also used to quantify the latent infection level of naturally and artificially infected leaves collected from wheat fields at different ecological locations in China. Results showed that there were the significant linear relationships between MDX and DX in the field for most cases. The field distribution of MDX and DX anlysised by using the computersoftware SURFER (Golden Software, Surfer 8.0) showed that the distribution patterns of MDX matched well with those of DX in the most fields. This study provided a useful tool that can be used to timely, rapidly and accurately monitor and quantify the latent infections and efficiently estimate the initial inoculum potential of wheat powdery mildew epidemics in the fields.