Screening And Identification Of MEJ05 Against Fusarium Graminearum And And Its Effect On The Degradation Of ZEN

Fusarium graminearum（Fg）causes a variety of plant diseases,which not only leads to economic losses,but also produces toxins such as Deoxynivalanol（DON）and Zearalenone（ZEN）.These toxins will be harmful to the health of humans and animals.In the control of Fg,due to the lack of disease-resistant wheat varieties and the problems of environmental pollution of chemical fungicides such as carbendazim and chlorothalonil,biological fungicides have gradually become the most promising alternative methods,which have significant effects on inhibiting the growth and development of Fg,inhibiting the production of fungal toxins and detoxification.Microbial diversity analysis of normal and diseased wheat grains on diseased wheat ears infested with Fg showed that Bacillus was present on the normal grains that could resist the infestation.Normal wheat grains collected were cultured against Fg to isolate bacteria around the grains that could antagonize Fg,and six strains of Bacillus were obtained.MEJ05 showed the highest inhibition rate of 41.57%against Fg when using the agar disk diffusion method.After morphological observation,16S r DNA sequence determination and phylogenetic tree analysis,MEJ05 was identified as Bacillus velezensis.Subsequently,MEJ05 was subjected to agar disk diffusion experiments with six pathogenic fungi.The MEJ05 solution was found to be antagonistic to Alternaria alternata,Fusarium graminearum,Rhizoctonia cerealis,Trichoderma viride,Pestalotiopsis neglecta and Sclerotinia sclerotiorum.MEJ05 inhibited both Fg and Aa on PDA plates at p H 5,6,7,8,9 and 10,and it showed the greatest inhibition at p H 7 with 41.57%and 46.27%,respectively.In the process of culturing MEJ05,it was found that it could form biofilm,so the characteristics of MEJ05 forming biofilm were investigated.Firstly,MEJ05 were inoculated on PDB,LB and KMB semi-solid plates,and it was found to grow all over the culture dish in24 h with good motility.Next,the effects of different culture mediums and p H values on the biofilm formation of MEJ05 were explored,and it was found that MEJ05 could form biofilm in PDB,LB,KMB and NB culture mediums for 2 days with the thickest and more wrinkled biofilm generated in KMB and PDB.MEJ05 could form biofilm in LB culture medium at p H5,6,7,8 and 9 for 2 days with the resting culture biofilm,p H≦4 or≧10 were no bacterial growth and no biofilm generation.The biofilms formed by MEJ05 on wheat ears and grains were observed by scanning electron microscopy,and it could be seen that the bacteria were surrounded by a membrane-like structure connected to the surfaces of wheat ears and grains,and the bacteria showed the phenomenon of adhesion and aggregation.The uneven structure of the wheat grain surface was favorable to the movement and colonization of MEJ05,so the biofilm was thicker and more extensive,and even some MEJ05 bacteria were embedded under the biofilm.These indicate that MEJ05 has the characteristics of motility and biofilm formation,which will facilitate its growth and reproduction in different environments and play the role of antagonistic bacteria.The effects of different factors on the growth of MEJ05 were next explored.When a single factor in the basal medium was changed and the OD600 of MEJ05 reached the maximum after 2 days of growth,the optimal carbon source was 10 g/L glucose,nitrogen source was 10 g/L peptone,Fe SO₄·7H₂O was 0.02 g/L,Mn SO₄·H₂O was 0.04 g/L,Cu SO4was 0.02 g/L,Mg SO₄·7H₂O was 3 g/L,Na₂HPO₄was 1 g/L,KH₂PO₄was 0.5 g/L,p H was 7.In order to investigate the mechanism of MEJ05 antagonizing Fg,this experiment was conducted to isolate,purify and identify the antagonistic Fg substance.The uninfested part of MEJ05 confronted with Fg plate was cut off,extracted twice with n-butanol solution,filtered and spin-dried,and then extracted twice with an equal volume of deionized water to verify the inhibitory effect of crude extract on Fg.Then more crude extracts were collected and spin-dried,the concentrated solution was obtained by adding ethyl acetate to re-solvate.The concentrates of the antagonistic Fg substances were subjected to HPLC-MS,and the substances in the 4:4 fraction were identified as fengycin.Meanwhile,experiments on the effect of MEJ05 antagonizing Fg were conducted.Firstly,the minimum inhibitory concentration of MEJ05 against Fg was obtained as 5×10⁷CFU/m L.Then MEJ05 was co-cultured with Fg,and it was found that the addition of MEJ05 produced an inhibitory effect on Fg mycelium,and the number and length of mycelium were significantly reduced;the inhibition rate of MEJ05 with a concentration of 2?10⁹CFU/m L on 10⁵spores/m L of Fg reached 40%at the fourth hour.Moreover,the mixture of MEJ05 and Fg sprayed on wheat grains could effectively inhibit the growth of Fg on wheat grains,and the inhibitory effect was similar to that of pesticide carbendazim.In addition,using HPLC-MS,it was found that the addition of MEJ05 reduced the DON and ZEN produced by Fg-infested wheat grains by 60%and 96%,respectively.Finally,the degradation effect of MEJ05 on ZEN was investigated.In this experiment,two degradation conditions were investigated.First,MEJ05 with a final concentration of 10⁸CFU/m L was added to LB culture solution containing standard ZEN with a final concentration of 2.83μg/m L.The content of ZEN was measured by HPLC-MS after 7 days of shaking culture.The degradation rate of standard ZEN by MEJ05 was 97%.Secondly,the Fg-infested wheat grains were dried and crushed,and the sterilized crushed wheat grains were added with the final concentration of 10⁸CFU/g of MEJ05 in the LB culture medium.The ZEN content was detected by HPLC-MS after 7 days of shaking culture.The degradation rate of MEJ05 on ZEN produced by Fg on wheat grains was 76%.It can be seen that MEJ05 has a very good degradation effect on ZEN.