Screening And Identification Of NF001 Against Fusarium Graminearum And Its Biological Control Mechanism

Fusarium graminearum is a major pathogenic fungus that causes Fusarium head blight,which infects wheat ears and secretes mycotoxins such as deoxynivalenol（DON）and zearalenone（ZEN）.It coused the production and quality of wheat are reduced.At present,the main means of controlling Fusarium head blight are to strengthen field management,cultivate resistant varieties,use chemical pesticides and use biological agents for biological control.However,due to the long period required for cultivating resistant varieties,chemical pesticides are likely to cause resistance,and increasing the amount will increase the pollution of the environment.Therefore,the research and application of biological prevention and control diseases are more and more popular.In this experiment,a strain of a high-efficiency antagonistic Fusarium graminearum was screened from wheat grains grown in a certain place in hubei province.The strain was identified as Burkholderia gladioli（NF001）by morphological and molecular biological identification.NF001 inhibited all 12 plant pathogenic fungi,among which the inhibition rate of fusarium gramineae was as high as 64.52%.In the NF001 antagonizing Fusarium graminearum condition optimization experiment,the inhibition rate of NF001 against Fusarium graminearum was determined under different conditions such as p H,temperature,distance between NF001 and Fusarium graminearum,and shake flask culture method.The results show that:at p H 5,the inhibition rate is the highest,reaching 74.3%;within the temperature of 26℃～29℃,the highest inhibitory activity;When the distance between NF001and Fusarium graminearum is 1.5 cm,the antibacterial activity reaches the maximum,reaching 62%;It was found that the antibacterial activity of the 20%fermentation supernatant obtained by shaking the flask for 2 d then standing for 3 d was about12.1%higher than the fermentation broth obtained by shaking the flask for 5 d.Study on the effect of NF001 on the inhibition of mycelial growth,spore germination and DON production of Fusarium graminearum.It was found that after NF001 treated Fusarium graminearum mycelium will twist and deform the mycelium of Fusarium graminearum,protoplasts will coagulate,the mycelium will break and the cell wall will collapse,thus inhibiting the growth of Fusarium graminearum hyphae.When the bacterial concentration of NF001 is higher than 5×10⁵CFU m L^-1,NF001 can almost completely inhibit the germination of Fusarium graminearum spores,When the bacterial concentration of NF001 is 5×10⁴CFU m L^-1,NF001 can coexist with Fusarium graminearum.NF001 bacterial inhibits the growth of Fusarium graminearum in wheat grains,thereby inhibiting the content of DON in wheat grains.After treatment with NF001 bacterial for 15 days,the DON content in the wheat grains decreased by 58%.The mechanism of NF001 antagonizing Fusarium graminearum was studied.By using solvent extraction,silica gel column chromatography,thin layer chromatography,high performance liquid chromatography and LC-MS,nuclear magnetic resonance and other methods,the NF001 antagonistic substance of Fusarium graminearum was isolated,purified and identified.After LC-MS analysis,the purified sample mainly contains dipeptide His-His,tetrapeptide Ala-Phe-His-Lys,Hydroxypromazine,toxoflavin,cyclic dipeptide pro-leu,C16 Sphinganine Hexadecasphinganine.Among them,the purified toxoflavin has a good bacteriostatic effect.Dipeptide His-His and cyclic dipeptide pro-leu can not inhibit Fusarium graminearum,but can promote the antifungal activity of toxoflavin against Fusarium graminearum.The minimum inhibitory concentration of toxoflavin against Fusarium graminearum was higher than 256 ug/m L.In the experiment of optimizing the condition of NF001 to produce toxoflavin,it was found that co-cultivation of Fusarium graminearum and NF001 would result in the reduction of NF001’s antibacterial activity and toxoflavin.When NF001 was cultured in a shake the flask for 2 d then standing for 3 d,the amount of biofilm produced increased,and the amount of toxoflavin was also significantly increased.Studing the application of NF001 in the control of Fusarium graminearum in winter wheat.The experiment showed that in the pot experiment of wheat,after spraying NF001,the content of DON in wheat grains decreased by 1155 ug/m L,the reduction rate reached 84%,and the disease index decreased by 25.1%;The plant height,100-kernel weight,ear length,and ear weight of wheat have also increased.In the field experiment of wheat,after spraying NF001,compared with blank control and negative control,the DON content of wheat decreased by 1324.9 ug/m L and 168.8ug/m L,and the disease index decreased by 36.14%,13.81%,respectively.Compared spraying NF001 group with non-fertilizer group,the number of tillers,plant height,yield per mu,ear length,ear weight,fresh weight,and effective ear number of wheat were also increased.The experimental results show that in the field environment,NF001 has a good control effect on Fusarium head blight caused by Fusarium graminearum,and has the potential as a biological control strain.