| In the process of agricultural production,plant diseases are the main cause of crop yield loss.With the negative impact of traditional pesticides and fertilizers on agro-ecosystem becoming more and more serious,part or all of the use of new biocontrol agents to replace the use of chemical pesticides and chemical fertilizers.It plays a positive role in solving the problems caused by excessive application of traditional pesticides and fertilizers.In recent years,the studies on biocontrol agents,especially protein elicitors,are mostly limited to model plants,such as arabidopsis,tomato and tobacco,and lack of comprehensive and systematic studies on biocontrol bacteria promoting growth and disease resistance of maize.In this study,the biocontrol,growth-promoting and resistance-inducing effect and mechanism of Paenibacillus terrae on maize were studied comprehensively and systematically.A new type of protein elicitor-ATi En was excavated on the basis of genome sequencing by means of bioinformatics and molecular biology,and the mechanism of induced disease resistance of maize and tobacco was analyzed systematically and in details.It broadens the train of thought for the research and development of biocontrol agents.The specific results are as follows:(1)In order to determine the bacteriostatic activity of Paenibacillus terrae NK3-4 against Exserohilum turcicum,the bacteriostatic effect was studied by plate confrontation method.It was found that the bacteriostatic effect of Paenibacillus terrae NK3-4 was favorable,and width of bacteriostatic zone is 1.97 cm,the control effect is 68%.Different components had a certain inhibitory effect on spore germination and cell growth of Exserohilum turcicum.The best inhibition effect of fermentation broth,inhibition ratio was 75.71% The best control effect was sprayed 8 hours in advance with the concentration of 1×107CFU/m L.The best combination of culture medium for the production of bacteriostatic substances by response surface analysis was sucrose 1.7%,peptone 1.6%,Mn Cl2 0.01%.The bacteriostatic activity of the active substance produced by neutral culture under the condition of one fifth as the volume of liquid,2%inoculum and neutral culture at 28? for 72 h was the best.(2)Based on the analysis of antibacterial activity,this study carried out the study on the stability of bacteriostatic substances from Paenibacillus terrae NK3-4.The results showed that the strain had good storage stability,thermal stability,acid-base stability,light stability,UVstability and genetic stability.This strain can destroy the mycelial structure of Exserohilum turcicum,inhibit the formation of mycelium and spore germination.The alkaline phosphatase activity,protophosphorus metabolism level,electrical conductivity and total sugar concentration of the pathogen were determined.The results showed that the fermentation broth could destroy the cell wall structure,cell membrane metabolism,cell membrane permeability and integrity.At the same time,it can reduce the pathogenicity of HT toxin and melanin content.It is suggested that Paenibacillus terrae NK3-4 can reduce the pathogenicity of pathogens and play a role in disease prevention by destroying the morphology and structure of pathogens and inhibiting the production of pathogenic factors.(3)The pot experiment and field experiment method were used to analyze the growth-promoting effect of Paenibacillus terrae on on maize.The pot experiment showed that compared with the control group,the germination rate of Paenibacillus terrae treatment group increased by 6%,the length of radicle and germ increased by 32% and 65%,respectively,and the increment of plant height was 24%.The content of chlorophyll increased by 25%,and the dry matter accumulation of aboveground and underground parts increased by 79% and 93%,respectively.The results showed that the strain could promote the growth and development of maize,accelerate the accumulation of dry matter,enhance the photosynthesis of maize and promote the growth of maize.Field experiments showed that in different growth stages of maize,Paenibacillus terrae could enhance the SPAD value of maize in different degrees,increased by9% to 15%.The growth of maize could be promoted,and the plant height could be increased by7% to 16%.The determination of soil nutrient content and enzyme activity showed that the strain could improve the soil nutrient content and enzyme activity of maize rhizosphere,and could effectively control NCLB,and the field control effect was 50.38%.And increase corn yield by10%.The results showed that the yield-increasing effect of Paenibacillus NK3-4 was not played by a single factor,but was formed by the function of promoting growth,disease prevention and inducing resistance of the strain.Each function complemented each other and promoted each other to increase the yield of maize.(4)In this study,on the basis of confirming the growth-promoting and biocontrol effect of Paenibacillus terrae NK3-4,the physiological index determination and real-time quantitative PCR method were used to study the mechanism of NCLB resistance induced by Paenibacillusterrae NK3-4.The results showed that Paenibacillus terrae could induce disease resistance of maize by inducing the outbreak of reactive oxygen species,enhancing the activity of protective enzymes,reducing the accumulation of harmful substances and increasing the activity of rate-limiting enzyme PAL in phenylpropane metabolism pathway.The mechanism of disease resistance induced by Paenibacillus terrae NK3-4 was analyzed by real-time quantitative method from the point of view of molecular biology.The results showed that Paenibacillus terrae NK3-4 could induce the expression of disease-related protein coding genes PR5 and PR10,and can induce the expression of PAL and CAT genes,indicating that it could enhance the disease resistance of maize by regulating physiological level,phenylpropane metabolism pathway and initiating the accumulation of disease-related proteins.It shows that Paenibacillus terrae can enhance the resistance of maize to NCLB.(5)In order to explore a new type of small protein elicitor,on the basis of genome sequencing,the target gene-ATi En,which has the potential of protein elicitor,was selected to analyze the physical and chemical properties of the protein elicitor by bioinformatics method.The gene encodes 93 amino acids,belongs to non-secretory hydrophilic protein and does not contain transmembrane region.Protein structure analysis showed that it contained the typical functional domain of antitoxin gene.The results of secondary structure study showed that the content of ? helix was high,which was 83.87%,indicating that the protein was stable.The ATi En gene was expressed in E.coli BL21 by prokaryotic expression method,the recombinant protein was induced by IPTG induction method,and the target protein was purified by nickel strain.The biological activity of the protein was determined by tobacco injection method in vitro.It was found that the protein could induce typical HR reaction,had the function of inducing plant systemic disease resistance,and had a certain thermal stability and acid-base stability.The lowest concentration of allergic reaction was 2 ?mol.(6)In order to further analyze the induced resistance and mechanism of recombinant protein,the induction of resistance to plant disease by recombinant protein was studied by challenge inoculation method.It was found that the recombinant protein could induce the resistance of maize to NCLB and the resistance of tobacco to Alternaria alternata,TMV virus and Verticillium dahlia,indicating that the resistance of the protein was broad-spectrum.Physiological,biochemical and molecular biological methods were used to explain themechanism of tobacco disease resistance induced by recombinant protein.The results showed that the treatment of recombinant protein could induce the accumulation of ROS,the accumulation of lignin,the alkalinization of extracellular medium and enhance the activity of defense enzymes in tobacco leaves.The results of defense gene detection showed that the recombinant protein could participate in defense response and improve plant disease resistance by activating SA and JA/ET signal pathways and activating the expression of PAL,a key gene in phenylpropane metabolism pathway. |
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