| Wheat crown rot occurs in all wheat producing areas of the world,such as Asia,Africa,North America,South America and Oceania,and is a worldwide disease.Wheat crown rot is a soil facsomum disease caused by a variety of Fusarium pseudograminearum,Fusarium graminearum,Fusarium verticillioides and Fusarium proliferatum,which in recent years has been due to changes in straw return and agricultural farming methods.In China,especially in the Huang-Huai-Hai wheat area,the incidence area of wheat crown rot has gradually increased,and the incidence of the disease has increased year by year,which has a major impact on the production and safety of wheat.In this study,using Real-time PCR technology,a pair of specific detection primers for Fusarium were designed,and the biomass of Fusarium in the soil was quantitatively detected in the wheat-maize rotation field in Liutuan Town,Changyi City,Weifang District,Shandong Province,and Mazhuang Town,Daiyue District,and the incidence of stem base rot in wheat at different growth stages was investigated,and combined with local temperature and rainfall,the digestion dynamics of wheat crown rot bacteria in wheat-maize rotation field soil were preliminarily analyzed.The main findings are as follows:1.Design primers and verify their specificity.Based on the conserved nucleotide sequence of Fusarium 28 Sr DNA-ITS region,the specific primers of Fusarium ITS-F/ITS-R were designed,and the DNA of Fusarium pseudoflora,Fusarium graminearum,Fusarium rotiferii,Fusarium fusarium,Fusarium layered out and other pathogenic bacteria was extracted by CTAB method,and conventional PCR amplification was carried out as a template to obtain a target fragment of about 427 bp.The detection of pathogenic bacteria from other rhizome diseases in wheat without the presence of the same band indicates that the primer is specific for a variety of Fusarium species.2.Optimization of reaction conditions and detection of primer sensitivity of conventional PCR and Real-time PCR.The SYBR Green I dye method of conventional PCR and Real-time PCR is optimized for optimal temperature between 50℃ and 64℃,and the experimental results show that conventional PCR and Real-time PCR are the best at 60℃.The DNA of the extracted Fusarium pseudograves were diluted in a 10-fold gradient and tested by conventional PCR and Real-time PCR,and the results showed that the minimum concentration of conventional PCR detection was 1.0×10-1 ng/μL,and the minimum concentration of Real-time PCR detection was1.0×10-3 ng/μL.3.Establishment of a standard curve for quantitative detection of various Fusarium Real-time PCR.The DNA of Fusarium pseudograces,Fusarium graminearum,Fusarium rotundifolia,and Fusarium layered on the same conditions was subjected to fluorescence quantitative PCR under the same conditions,and the Ct values were the same at the same concentration.Therefore,with Fusarium pseudogracea as the representative bacterium,a standard curve between the Ct value of Real-time PCR and the log value of DNA concentration of Fusarium pseudograces is plotted.When the standard curve was drawn,the Ct value obtained by the gradient concentration was between 10-35,the coefficient of determination was0.9996,the linear range could reach 6 orders of magnitude,the dissolution peak was single,and no primer dimer was generated,indicating that the standard curve could be used for quantitative detection of Fusarium in the soil.4.Annual dynamic detection of Fusarium biomass in wheat-maize rotation field soil.From March 2021 to February 2022,every 15-30 days between the Tai’an City and Liutuan Town wheat-corn rotation field soil fusarium content was detected,in the detection cycle of fusarium in the soil of the two places have two rapid growth periods and three peaks,in the wheat growth period there are two rapid growth periods and peaks,there is a small peak during the corn growth period,Mazhuang Town The first rapid growth period of Fusarium in the soil from March 8 to June 8 reaches the first peak,The second period of rapid growth began on October2 and reached a third peak on November 3.The first rapid growth period of Fusarium in the soil began from March 18 to June 12 to reach the first peak,and the second rapid growth period reached the third peak from October 4 to November 5,through the analysis of the growth trend of Fusarium content in the soil of the two places,Shandong Province Winter wheat sowing,the selection of effective seed coating mixing and wheat before and after the timely spraying of effective fungicides,can effectively inhibit the infection and transfer of Fusarium in the soil to the base of the wheat stem.5.Relationship between Fusarium biomass in soil,disease status of wheat crown rot and climatic conditions.Based on the comprehensive analysis of the detection of Fusarium biomass in wheat-maize rotation field soil and the incidence of disease at different growth stages of wheat and the meteorological data monitored by local meteorological stations,it can be seen that the content of Fusarium in soil has a certain correlation with the effective accumulated temperature and rainfall,such as the 15-day cumulative temperature and rainfall during the first and third peak testing periods,which are 349.5℃ and 7.4 mm,and 190.5℃ and 8.4 mm,respectively.When the amount of Fusarium in the soil is low,the cumulative temperature and rainfall of 348℃ and 219.4 mm in 15 days,and 0℃ and 0.6 mm and 415℃ and 41.5 mm,respectively,show that the temperature is too high or too low,and when the rainfall is too large,the biomass of Fusarium in the soil is low.The results of the simultaneous investigation of the incidence and disease index of wheat crown rot at the Tai’an test site showed that the rapid growth period of Fusarium in the soil was significantly earlier than the rapid growth period of wheat crown rot incidence and disease index.6.Greenhouse study of the effects of temperature,humidity and straw return on the change of Fusarium biomass in soil.In the greenhouse environment,the growth rate of Fusarium and the change of Fusarium biomass in soil were observed and detected by controlling the three conditions of single variable temperature,humidity and straw presence,and the experimental results showed that Fusarium growth rate was the fastest at a temperature of about 25℃ and about 40%of the soil relative humidity,and the biomass of Fusarium in the soil was the highest.In soils where stalks were artificially added,the growth rate,spread range and biomass of Fusarium fusarium in soils were significantly higher than those in soils without straw,which was consistent with the results of dynamic monitoring of Fusarium in the field. |
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