| Wheat crown rot（WCR）is a soil-borne fungal disease caused by a variety of pathogens,which seriously affects the yield and quality of wheat,and poses a serious threat to the safe production of wheat in China.Fusarium pseudograminearum is a dominant pathogen of WCR in Huang-Huai-Hai River Plain of China.Biological control is gaining more attention due to their safe,efficient and environmentally friendly properties.However,few study was focused on biological control of WCR.It is urgent to screen new efficient biocontrol agentsand clarify their interaction mechanism,which would provide theoretical basis for the green control of WCR.Based on this,the following studies are carried out:1.Isolation and screening of WCR biocontrol bacteria.Ten wheat rhizosphere soil samples were collected from diseased fields of WCR in different areas of Henan Province,and 98 strains of bacteria were isolated by dilution coating method.The results of the preliminary screening showed that 27 of the isolated bacteria showed strong antagonistic activity to F.pseudograminearum in confrontation culture.The results of the greenhouse experiment showed that 14 strains were effective against F.pseudograminearum.What’s more,YB-161 had the best control efficiency against F.pseudograminearum（68.15%）and showed a good effect on promoting growth of wheat plants.The results of the inhibition spectrum showed that strain YB-161 had good antagonistic effects against five plant pathogenic fungi,including Colletotrichum acutatum and Phytophthora parasitica.According to the above analysis,YB-161 was used for the following studies.2.Identification and inhibition activity determination of the biocontrol bacterium YB-161.The surface of strain YB-161 colonies was rough,with irregular edges and a milky white opaque appearance.Electron microscopy showed a rod-like morphology with bluntly rounded ends.Gram staining was positive,and the budding spores were mesogenic to subterminal.The results of the Biolog automated microbial identification system showed that strain YB-161 had the highest similarity value to Bacillus amyloliquefaciens in the database.gyrA sequence analysis also showed that strain YB161 had the highest sequence homology with B.amyloliquefaciens.The strain YB-161 was identified as B.amyloliquefaciens based on the above results.The F.pseudograminearum colonies showed shrinkage,yellowing and edge deformation under the fermentation filtrate of YB-161of.In addition,the 5× and 10× dilutions of YB-161 fermentation filtrate inhibited F.pseudograminearum colonies by 66.67%and 44.33%,respectively.Microscopic observation also showed that the mycelium of F.pseudograminearum exhibited expansion and breakage under the fermentation filtrate of strain YB-161.The results indicated that YB-161 had strong antagonistic activity on the growth of F.pseudograminearum.3.Efficacy of the biocontrol bacterium YB-161 against WCR in field conditions.The results of the field survey showed that the disease index of YB-161 treatment was 6.27 and 8.47 on 2 March and 4 May 2019,respectively,and the control effect of YB161 on wheat stem rot reached 58.33%and 52.35%respectively,which was close to that of the chemical treatment（4.8%difenoconazole·fludioxonil）.The yield of wheat treated by YB-161 was 9046.37 kg/hm2,increased by 6.63%.The results indicated that YB-161 have the ability to control WCR effectively and have great potential for farther developed.4.Transcriptomic response of F.pseudograminearum to the stress of bacterium YB-161.The transcriptome sequencing results of F.pseudograminearum under coculture with YB-161 showed that:（1）2394 genes were significantly differentially expressed in the 4 h sample,of which 1524 genes were significantly up-regulated and 870 genes were significantly down-regulated;4174 genes were significantly differentially expressed in the 16 h sample,of which 2130 genes were significantly upregulated and 2044 genes were significantly down-regulated.The expression of 4174 genes in the 16-h samples was significantly different,of which 2130 genes were significantly up-regulated and 2044 genes were significantly down-regulated.（2）GO functional annotation showed that the differentially expressed genes in the 4 h and 16 h samples were mainly concentrated in metabolic processes,cellular processes and cell membranes.（3）The functional annotation of KEGG showed that the up-regulated genes in the 4 h samples were mainly concentrated in the pathways of aminyl biosynthesis and steroid biosynthesis,while the down-regulated genes in the 4 h samples were mainly concentrated in the pathways of ribosome and citric acid cycle pyruvate metabolism,etc.The up-regulated genes in the 16 h samples were mainly concentrated in the pathways of ribosome synthesis and aminyl biosynthesis,while the down-regulated genes in 16 h samples were mainly expressed in the citric acid cycle,glycerol ester metabolism and other pathways.The results showed that F.pseudograminearum was significantly differentially expressed under the stress of YB161,which laid the foundation for the mechanism of action of the microorganism on F.pseudograminearum.5.Expression analysis of apoptosis-related gene FpTatD4 and its biological function under the stress of biocontrol bacterium YB-161.The results of transcriptome sequencing and RT-qPCR showed that FPSE05373 gene was significantly upregulated after 4 h and 16 h of treatment with YB-161.The gene was identified as FpTatD4,which was homologous to the apoptosis-related gene TatD4.Compared with the wild-type and the complementary strains,there were no significant changes in the growth of Δfptatd4 colonies,tolerance to various stresses,pathogenicity and susceptibility to the growth control bacterium YB-161,but conidial production of wasΔfptatd4 significantly reduced.The results suggested that FpTatD4 is associated with the formation of F.pseudograminearum conidia.This study enriched the biological control resources of WCR,and explored the response mechanism of F.pseudograminearum to B.amyloliquefaciens stress,and preliminarily analyzed the expression pattern and biological function of the apoptosisrelated gene FpTatD4 of F.pseudograminearum under the stress conditions of YB-161,which providing a theoretical basis for the green control of WCR. |
