| Rhizoctonia cerealis E.P. van der Hoeven (teleomorph of Ceratobasidium cereale D.I. Murray&Burpee), which caused sharp eyespot in wheat, is a major soilborne fungal pathogen. This disease affects winter wheat production regions of China and causes severely impairs yield and quality. Analysis the population genetic structure of R. cerealis and understanding the distribution and survival of pathogen in soil are very important to study the population biology and molecular epidemiology of R. cerealis, and is helpful to detect and control of this fungus in nature.In this study, genetic structure of4R. cerealis poplations were analyzed using simple sequence repeat (SSR), and designed6pair SSR primers based on the transcriptome of R. cerealisR0301. Populations were collected from different province, including Bengbu city in Anhui province, Linyi city in Shangdong province, Jiangyan city in Jiangsu province and Shangqiu city in Henan province. In the results, Six of the microsatellite loci were detected to be polymorphic and they are considered to be effective markers for analysis of genetic diversity and relationship among R. cerealis populations. The results of Popgene analysis showed that the number of alleles observed for4populations was44with average of7.3alleles, allele frequencies for each locus were not evenly distributed. The expected heterozygosity of four populations ranged from0.514to0.533with average of0.522, which showing rich genetic diversity in populations. Hardy-Weinberg equilibrium tests show that there were tow loci in all populations under HWE, but other loci were out of the equilibrium significantly, the absence of heterozygote was prominent in most of populations, which suggested inbreeding at the different degree might existed in populations. The analysis of F-statistics shows that genetic divergence was found in and between populations. Fst was0.042, which showing that the divergence between populations was low,4.2%of divergence was present among populations and95.2%was detected within populations. Gene flow among populations was frequnently and Nm value was5.75. Genetic distance was low and genetic identity was high, genetic structure among populations was similar. It is difficult to identify and quantify R. cerealis in soil based on conventional methods. Therefore, a rapid and reliable method would be advantageous for the detection and quantification of this pathogen. In this study, a SYBR Green-based quantitative real-time polymerase chain reaction assay was developed for specific and sensitive detection and quantification of R. cerealis in soil samples. A specific primer pair was designed based on the β-tubulin (tubB) gene of the fungal DNA sequence. Using the real-time PCR assays, we were able to quantify R. cerealis in artificially infested soil samples and naturally soil samples in China.The specific detection of R. cerealis was successful with quantities as low as100fg in purified pathogen DNA. A separate standard curve was generated to correlate the Ct values with the CFU of R. cerealis, and the amounts of R. cerealis quantified in nature soil samples were9.3to73.1cfu, the corresponding amounts of DNA was20.3to133pg. |
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