Identification And Molecular Detection Of Pathogen Of Soilborne Fungal Diseases In Wheat Of Shandong Region

Wheat Sharp Eyespot, Wheat Common Rot and Wheat Take-all are main fungal diseases in wheat roots in Shandong region, the three fungal diseases seriously affected the wheat production.This three diseases are soilborne fungal diseases, their pathogenic fungi are all soil inhabitant microbes and they can be accumulated for years, they can infect the wheats at seeding to adult stage. The symptom of the wheat roots are not easy to distinguish, so it’s difficult to make specific and effective prevention measures. So it is necessary to know the composition of the pathogens in wheat roots in Shandong region and develop a repid detection technology. In this paper, we collected the infected wheat plants by the three main fungal diseases in most areas of Shandong province, Then we isolated and identified the pathogenics from the infected wheat plants to initially know the composition of pathogenic of the three main fungal diseases; On this basis,we designed specific primers to detect the three main fungal diseases. The main results are summarized as follows:1. We collected the infected wheat plants more than140copies in the main areas of wheat production of Shandong in2012and2013year. Using conventional isolation method, we get357isolates from the wheat samples in most areas of Shandong pronince. Among the357isolates, we identified that136isolates of the pathogenics are Rhizoctonia cerealis,169isolates of the pathogenics are Bipolaris sorokiniana,52isolates of the pathogenics are Fusarium spp. by using morphology and ITS sequences and EF-la sequences analysis, the Fusarium including Fusarium oxysporum, Fusarium proliferatum and Fusarium culmorum, and the Fusarium culmorum has the highest proportion in Fusarium pathogenics.2. Used the methods of soil inoculation for pathogenicity identification,29isolates of Bipolaris sorokiniana,2isolates of Fusarium oxysporum,5isolates of Fusarium proliferatum and9isolates of Fusarium culmorum were test in the Seedling stage of wheat. Result indicate that, the pathogenicity of all the Bipolaris sorokiniana and2isolates of Fusarium oxysporum (WF42and WF51) and4isolates of Fusarium culmorum (WF5, WF19, WF24and WF25) were the best strong, the average disease index was about30.67to46.67, the average disease index of the WF10(Fusarium culmorum) and WF38(Fusarium proliferatum) is18.00and16.67, the two isolates have weak pathogenicity, the average disease index of the other4isolates of Fusarium proliferatum is24.00to25.33, the average disease index of the other4isolates of Fusarium culmorum is19.33to28.67, the8isolates has moderate virulence. The result of the pathogenicity identification proved once again, the Bipolaris sorokiniana is the main pathogen of the Wheat Common Rot.3. A portion of the infected wheat plants collected in2013were used to isolate and identify the pathogenics, the others were used to extract DNA of the infected wheat roots using CTAB method, then we used the primers for different pathogenics designed by others to amplificate the DNA, the result showed that, we detected the Rhizoctonia cerealis in all the wheat samples presenting symptom of Wheat Sharp Eyespot; we also detected the Rhizoctonia cerealis in all the wheat samples presenting symptom of Wheat Common Rot; we detected the Bipolaris sorokiniana in the samples presenting symptom of Wheat Common Rot of13regions, but we can’t detect the Bipolaris sorokiniana in some samples presenting symptom of Wheat Common Rot. We detected the Gaeumannomyces graminis var. tritici in the samples presenting symptom of Wheat Take-all from Jining, also we detected the Gaeumannomyces graminis var. tritici in the samples unpresented symptom of Wheat Take-all from Yucheng and Juye.4. We designed specific primer RBF-2/RBR-2for Bipolaris sorokiniana using Primer Premier5.0according to the5.8SrDNA-ITS sequence, it is specific to Bipolaris sorokiniana, the product is about417bp. Thus, primer EF-1H/EF-2T were used to the amplification of Fusarium, the amplification of wheat take-all pathogenics used the primer for Gaeumannomyces graminis var. tritici that were designed by Qiongxian Lu, the amplification of Rhizoctonia cerealis used the primer WKF-8/WKR-8designed by Nana Xu. We designed a Multiplex PCR amplification to detect Bipolaris sorokiniana, Fusarium and Gaeumannomyces graminis var. tritici in the same time, the Rhizoctonia cerealis should be detected alone. so we can detect the4different pathogenics using the PCR method.5. We collected wheat samples in the Seedling stage of wheat in14areas of Shandong pronince, then we extracted DNA of the wheat samples using CTAB method and using the Multiplex PCR to detect the DNA samples. The results show that, we detected the Rhizoctonia cerealis in all the28wheat samples, we detected the Bipolaris sorokiniana in21wheat samples, the detection rate is75%, The Fusarium is detected in the wheat sample from Mazhuang. The above result show that, Wheat Sharp Eyespot is a commonly disease in Shandong province, the Wheat Common Rot and Wheat Sharp Eyespot occurs mixed usually.