Studies On The Pathogen Of Soybean Fusarium Root Rot And Multiplex PCR Detection

Soybean Fusarium Root Rot is one of the most important diseases in the soybean production process.Once the soybean plant becomes diseased,it will cause serious economic losses.When the disease is serious,the particles may not be harvested.In order to clarify the types of pathogens that cause soybean Fusarium root rot in Liaoning region and provide a theoretical basis for the prevention and control of Fusarium pathogens,the laboratory carried out soybean root rot diseases in soybean production areas in China in 2017 Investigate and conduct pathogen isolation.In this experiment,the morphological identification and molecular biological identification were used to identify pathogens isolated from the roots of soybean Fusarium root rot plants.The biological characteristics of the pathogen were studied,and the differences in resistance to different soybean varieties to Fusarium acuminatum were evaluated.In addition,a rapid PCR multiple detection technology for Fusarium was developed.The results are as follows: 1.The colony morphology,conidiophore,conidia morphology,and pathogenicity verification were observed.The amplified ITS,translation elongation factor sequence and phylogenetic analysis were identified as Fusarium acuminatum.2.Through the study of the effects of mycelial growth,spore production and spore germination on Fusarium under different environmental conditions,it was observed that the fungus can achieve the best culture under the dark conditions of 25 ° C and p H 8 as well as with sucrose as carbon source and sodium nitrate as nitrogen source.3.According to the pathogenicity research of the Fusarium on different varieties of soybeans,it was found that one disease-resistant soybean variety was Tiefeng 29,wild soybean and William82 soybean belonged to high-sensitivity varieties,and Liao 15 soybean and Shen soybean were mediumsensitivity varieties.4.In addition,hyphae growth and spore germination methods were used to evaluate inhibition of different fungicides.The toxicity of 16 common fungicides against the pathogen was determined.The results show that 98% flupronil has the strongest toxicity,98% fluazinam,95% chlorothalonil,95% acetazol,95% prochloraz,97.2% triazodone,and 98% Carbendazim,98% azoxystrobin,96% metalaxyl,98% oxime mycolipids,97.2% azoxystrobin,98% azoxystrobin,99% azoxystrobin,98% carbendazim virulence,90.1% enazolol and 99% Pythium had poor toxicity.5.In addition,based on the research of O'Donnellet and others in 2015,using the EF-1? gene sequence to identify Fusarium.By comparing gene sequences,selecting different interspecific fragments,designing specific primers for each Fusarium and One shared reverse primer,and established multiple PCR reaction system and reaction program.Multiplex PCR program: pre-denaturation at 95 ° C for 3 min,denaturation at 95 ° C for 30 s;annealing at 54 ° C for 30 s;extension at 72 ° C for 50 s;30 cycles;extension at 72 ° C for 10 min.This method can distinguish between Fusarium acuminatum,Fusarium oxysporum,Fusarium solani and Fusarium graminearu by the size of the amplified fragments.Sensitivity tests showed that the minimum detection genomic DNA concentration was 1 × 10-4ng/?l;experiments with simulated infection samples showed that using the mixed sample of Fusarium and soybean genome as a template,the target band could still be accurately detected.In summary,the multiplex PCR technology established with the translation elongation factor sequence as the target can detect four Fusarium species sensitively and specifically.