| Wheat（Triticum aestivum L.）is an important cereal crop which widely planted.Fusarium graminearum is the main cause of wheat scab in China,which seriously damages wheat production.The most serious infection time of Fusarium graminearum on wheat is the flowering stage.After infection,Fusarium graminearum rapidly proliferated in the ear and produced a variety of toxins in the wheat grain.At present,there is a lack of disease resistant germplasm resources and the progress of disease resistant breeding is slow.It is of great significance for the control of wheat scab and breeding of disease resistance to screen the biocontrol bacteria against wheat scab,reveal the mechanism of disease resistance,screen the antimicrobial substances and clone the gene resistant to Fusarium graminearum.In this study,we used F0609 strain as the target bacteria to study the control effect of endophytic bacteria Bacillus amylolyticus C3 and 41B-1 on F0609 and the preliminary mechanism of inhibition.Endophytic bacterium C3 is a highly efficient mutant of 41B-1 treated by Co irradiation.The cell morphology is similar,and the colony folds become less and thinner.Strains C3 and 41B-1 could significantly inhibit the growth of Fusarium graminearum mycelium.C3 had obvious antagonistic effect on the growth of hyphae of different races of Fusarium graminearum in the plate test,which indicated that C3 had broad-spectrum resistance to Fusarium graminearum.Field experiments showed that 41B-1 strains could reduce the incidence rate of wheat scab of different wheat varieties and had good biological control effect.The disease index of 41B-1 in 201 treatment group was 0.9167,the control effect was 86.36%,and the disease index of 208 treatment group was 0.6250,the control effect was 86.67%.Strains 41B-1 and C3 can induce the apoptosis of Fusarium graminearum mycelium cells,and the morphology of mycelium is abnormal,and the organelles are disordered.In particular,the cell wall of F0609 was thickened abnormally.The antagonistic effect of C3 on the chitin of cell wall may be due to the antagonistic effect of C3.The crude protein of C3 mutant was obtained by ammonium sulfate precipitation salting out method with a saturation of 80%after 48 hours of culture.The concentrated crude protein samples were obtained by desalting in dialysis bags and freeze drying.After trypsin digestion,the digested liquid was detected by high sensitive sodium rising liquid phase and high-resolution mass spectrometry（LTQ-Orbitrap）technology.The data obtained were compared,retrieved and evaluated with information base of Bacillus amyloliquefaciens and Bacillus subtilis in UniProt website,and 72 kinds of proteins were obtained.The function of C3 secreted crude protein was analyzed by Panther classification system under gene ontology.Strain C3 is a variant strain of 41B-1 and compared with the crude protein of 41B-1,there are 43 different proteins.According to UniProt website,these proteins include 16 hydrolases,5 glycosidases,2 lyases,1 decarboxylase,4 oxidoreductases,4 aminopeptidases,1 proteinase and 18 unknown ones.Among the 72 proteins obtained by the above search,TasA protein has inhibitory effect on a variety of pathogenic bacteria,including mulberry blight,citrus canker and cucumber grey mould.Therefore,this antibacterial protein is selected for further study.Based on the results of sequence alignment,primers were designed to clone the TasA protein gene from the genomic DNA of cotton endophyte strain C3.The sequencing results showed that the tasA gene was 660bp in size.Through the amino acid sequence analysis of the tasA gene sequence translated by ExPASY software,it was found that there were 232 amino acids in the primary structure of the TasA protein.The protein encoded by the TasA protein was rich in lysine 26（11.2%）,alanine 23（9.9%）,isoleucine 18（7.8%）,aspartate 17（7.7%）,and It is a stable protein.The tasA gene was cloned from strain C3 and connected to the transgenic expression vector pBI121 which was digested by Xbal and SacI.The recombinant vector pBI121-tasA was transformed into E.coli DH5a,and the recombinant plasmid was extracted.After sequencing,the recombinant plasmid was transformed into the sensitive Agrobacterium EHA105,the positive bacterial solution was screened by PCR,and the Arabidopsis plants with tasA gene were obtained.Strains 41B-1 and C3 have a good inhibitory effect on wheat scab.C3 crude protein can induce the apoptosis of mycelia cells of Fusarium graminearum.The tasA gene cloned from C3 has the potential to be used as a disease resistant gene. |
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