Establishment Of A Rapid System For Detection Of Wheat Soil-Borne Fungi Diseases And Soil Microbial Diversity In Wheat Crown Rot Nursery

Wheat sharp eyespot,wheat common root rot,and wheat crown rot are three devastating soil-borne fungal diseases which greatly diminish wheat quality and yield.These symptoms may be confounding in the early stages of wheat growth,thus making them difficult to diagnose.On the one hand,this study aimed to realize the rapid detection of three wheat soilborne diseases.Specific primers were designed for the pathogens of these diseases.A multiplex PCR system for simultaneous detection of these three soil-borne diseases was established.The system was performed on wheat seedlings collected from filed.On the other hand,we analyzed the structure and diversity of soil microbial community in wheat fields infected with Fusarium crown rot by high-throughput sequencing to explain the relationship between fertility and microbial community of soil and wheat crown rot.Follows are the mian conclusions: 1.Establishment and application of a multiple PCR system(1)Primer design and PCR amplification.Specific primers were designed on the basis of the sequences of following genes: internal transcribed spacer(ITS)of R.cerealis,ITS of B.sorokiniana,and translation elongation factor(TEF1-?)of Fusarium spp.,generating amplicons of 174,418,and 600 bp,respectively.(2)Optimization of multiple PCR reaction system.To obtain three clear target bands,the annealing temperature of mPCR was optimized and the mPCR system was optimized through an orthogonal design(three levels of five factors: three sets of primers,Taq DNA polymerase,and dNTPs).The results showed that the optimal annealing temperature is 54.9 ?.The optimal reaction system included primers WKF-S18/WKR-S8,0.24 ?mol/L;primers RBFS2/RBR-S1,0.48 ?mol/L;primers EF-SF9/EF-SR8,0.24 ?mol/L;Taq DNA polymerase,1.5 U;dNTPs,0.15 mmol/L.(3)Specificity of the mPCR assay.DNA templates of other fungal strains were amplified under optimized reaction conditions.The results showed that no nonspecific products were obtained upon amplification of nontarget species of the fungi,indicating high specificity for the primers.(4)Sensitivity analysis of single and multiplex PCR.R.cerealis,B.sorokiniana,Fusarium spp.genomic DNA and mixed DNA were initially diluted to 10 ng and then serially diluted 10-fold in distilled water(10 ng to 10 fg).Single and multiplex PCR was performed under optimized conditions.The results showed that single PCR detected 10 pg,10 pg,and 100 pg DNA for R.cerealis,B.sorokiniana,and Fusarium spp.,respectively,whereas the limits of detection of the mPCR assay were 100 pg,100 pg,and 100 pg for R.cerealis,B.sorokiniana,and Fusarium spp.,respectively.(5)Application of mPCR assay for wheat samples collected from Shandong Province.In March 2018,wheat samples were collected from fields in Xiajin and Linyi counties in Dezhou,Dongchangfu and Donge counties in Liaochng,suburb in Taian and Hanting counties in Weifang in Shandong Province.Total genomic DNA was extracted from each sample and analyzed via mPCR under the optimized conditions.In May 2018,symptoms of diseases of wheat from these areas were investigated via visual diagnosis to validate the mPCR assay.The results showed that all of the target pathogens were detected in wheat samples from Dezhou and the dominant pathogen was Fusarium spp..R.cerealis and Fusarium spp.were detected in Liaocheng.R.cerealis was the only pathogen detected in samples from Weifang.R.cerealis and Fusarium spp.were detected in wheat samples from Taian.The results of the mPCR assay and subsequent symptom identification were highly consistent,indicating that the results of mPCR upon wheat seedling samples could provide reference for the early diagnosis of wheat soil-borne fungal diseases.2.Soil microbial diversity in wheat crown rot nurseryRhizosphere soils were collected from wheat severely infected with crown rot in Xinshengdian town and wheat lightly infected with crown rot in Mazhuang town.Physical and chemical properties of soils were analyzed.The diversity analysis was performed by the Illumina Miseq high-throughput sequencing platform based on the variation of 16 S rRNA V4 area of bacteria and ITS1 of fungus.The results showed that soil fertility and diversity of bacteria and fungi were higher in the lightly infected soils in Mazhuang.The dominant bacteria were Arthrobacter,Bacillus,and Mizugakiibacter and the dominant fungi were Penicillium,Trichoderma,and Trechispora;Soil fertility and diversity of bacteria and fungi were lower in the severely affected soils in Xinshengdian.The dominant bacteria were Nocardioides and Iamia and the dominant fungi were Fusarium,Mortierella,and Pyrenochaetopsis.These results indicated that the occurrence of wheat crown rot may be related to the decrease of soil fertility,microbial diversity and beneficial genes.