| Bacillus is a kind of microorganisms with strong biocontrol potential and is widely used in biological control.Two strains of biocontrol bacteria HN-1 and HN-2 were isolated in the preliminary laboratory.This study classifies HN-1 and HN-2 The status,antibacterial active ingredients,the function of antibacterial related genes,the effect and mechanism of promoting plant growth and preventing plant virus infection have been studied.The results of the study are as follows:1 The biocontrol bacteria HN-1 and HN-2 were identified by morphological observation combined with modern molecular biology.Both strains were identified as Bacillus velezensis,and the n-butanol extraction of the fermentation supernatant of the two strains was found.The size of the zone of inhibition for Colletotrichum gloeosporioides Penz was 30.29 ± 0.8 mm and 30.44 ± 0.03 mm,the zone of inhibition for Xanthomonas oryzae pv.oryzae was 34.93 ± 0.08 mm and 38.01 ± 0.23 mm;it was found in the hemolysis experiment The size of the hemolysis circle of the crude n-butanol extracts of the two strains was 15.16 ± 0.13 mm and 16.47 ± 0.02 mm.Use MALDI-TOF-MS to analyze the active substances in the crude n-butanol extracts of the two strains,and the results showed that the main active components in the n-butanol extracts of the two strains were lipopeptides,including: surfactin,iturins and fengycin etc.2 In order to explore the antibacterial molecular mechanism of Bacillus velezensis group of biocontrol bacteria.Our research group transformed the temperature-sensitive plasmid vector p Mar A carrying the transposon Tn YLB-1 into the Bacillus velezensis,HN-1and HN-2 strains by chemical transformation.The results show that only HN-1 is currently available.The strain was successfully transferred into the p Mar A plasmid.Through high temperature induction,a total of 1800 single clones of transposon insertion mutations were screened,and a mutant library of Bacillus velezensis HN-1 strain was constructed.The transposition efficiency reached 8.01×10-3 cfu/μg,p Mar A plasmid The elimination rate is99.7%.A mutant strain HN-1-△682 with loss of hemolytic activity and antibacterial effect was screened.3 Reverse PCR technology was used to identify the Tn YLB insertion position gene of the mutant.This gene has 99% homology with the Bacillus velezensis FZB42 arsenic resistance protein(RBAM-550730)gene,and is shown in the phylogenetic tree.The gene in Bacillus velezensis FZB42 belongs to the same branch.The gene was named Arp gene,and the translated amino acid sequence was subjected to BLAST comparison.The results showed that the amino acid homology with arsenic resistance protein was as high as 99.13%.The structure and function prediction results of the protein showed that the protein is a transport protein and its function is encoding.arsenic resistance protein.4 The site-directed knockouts HN-1△Cm Arp and HN-1△Tn YLBArp of HN-1 were constructed,and the fermentation broth and bacterial lipopeptides were extracted with nbutanol for testing.Firstly,it was found that the hemolytic activity of the fermentation broth extract of the knockout strain was only 59.69% and 41.29% of HN-1;the inhibitory activity against Colletotrichum gloeosporioides Penz was only 83.49% and 60.31% of HN-1;against rice bacterial blight The inhibitory activity of bacteria inhibition is only 74.09% and 80.58%of HN-1.The n-butanol extract of the designated knockout strain showed hemolytic activity,while the HN-1 bacterial extract did not show hemolytic activity;the inhibitory activity against Colletotrichum gloeosporioides Penz increased to 122.77% and 174.73% of HN-1.The inhibitory activity of Xanthomonas oryzae pv.oryzae was increased to 155.21% and236.73% of HN-1.Secondly,time-of-flight mass spectrometry showed that the amount of lipopeptides in the fermentation broth of site-specific knockouts was lower than HN-1,and the amount of lipopeptides in bacterial cells was higher than HN-1 at the same concentration.Finally,combined with the antibacterial activity,we found that the knockout body knocked out the Arp gene,so that the cell lost the ability to actively transport lipopeptides,and lipopeptides diffused from high concentration to low concentration,intracellular and extracellular The amount of lipopeptides is almost the same,while the wild-type strain has the Arp gene for active transport,which makes the activity and content of extracellular lipopeptides far higher than those of the extracellular.The above results indicate that the Arp gene may be involved in lipopeptides.The transport of substances is a key protein for the transport of lipopeptides to the outside of the cell.5 Preliminary experiments found that HN-1 and HN-2 strains can promote the growth of Capsicum chinense,and can effectively delay the infection of Capsicum chinense by PVMV.After comparing the growth-promoting effect of the HN-2 strain on the Capsicum chinense and the delayed PVMV infection on the Capsicum chinense,the effect of PVMV on the Capsicum chinense is better than that of HN-1,and the genetic relationship between the two strains is very similar.And the lipopeptide compound Surfactin to explore its prevention and treatment mechanism of PVMV.After tobacco is treated with HN-2 and the lipopeptide compound Surfactin,the up-regulated expression of RBOH gene and NPR1 gene in tobacco leaves,and the increase in the content of active oxygen scavenging enzymes(CAT,POD),proves that the plant has initiated a defense response;at the same time The upregulated expression of JA gene in the JA pathway means that it is activated under the treatment of bacteria and drugs(ISR);however,the content of salicylic acid hormone in the leaves has not increased,and the expression of PR1 gene has not changed significantly,indicating that SAR The pathway has not been activated.In summary,HN-2 and the lipopeptide compound Surfactin can effectively induce plant ISR response to activate plant immunity.6 After the tobacco plants are inoculated with PVMV,the tobacco plants pretreated with HN-2 and the lipopeptide compound Surfactin have the effect of delaying the infection of PVMV.This plant strain’s defense method through the ISR pathway is very effective in resisting PVMV virus infection in the future.The HN-2 strain and its lipopeptide compound component Surfactin reduces PVMV by activating the antioxidant pathway in the plant and increasing active oxygen scavenging enzymes.Oxidative damage caused by infestation.Subsequently,the synthesis of plant hormones JA and SA is promoted,leading to the upregulation of disease resistance-related genes PR1 and NPR1,and ultimately enhancing the plant’s immunity to viruses.It can be concluded that Bacillus velezensis HN-2 and its lipopeptide compound Surfactin can delay the effect of PVMV on the plant growth,the fluorescence signal under ultraviolet is reduced or even no fluorescence signal,and the GFP protein content cannot be detected by Western blot.Infection of Tobacco.Combined with the rhizosphere colonization experiment,it can be concluded that HN-2 can effectively colonize the roots of plants as a rhizosphere growth-promoting bacteria,and enter the plant after a period of time to exert its superior biocontrol bacteria effect.7 By constructing antibiotic marker strains,combined with rhizosphere colonization experiments,we found that the biocontrol bacteria HN-2 can colonize plant roots effectively and lastingly,and form a good rhizosphere flora in plant roots,and promote plant growth and development.It continuously stimulates plants to produce ISR response and exerts its superior biological control effect.8 At present,the HN-2 strain and its metabolites have applied for a number of patents and registered the "Refengbao" trademark.In order to further study the stability of its field test,we proceeded to compare the persistence of the HN-2 strain and its metabolites.The activity of several commercially available agents was evaluated and the results showed that after 180 days of application,Capsicum chinense treated with the HN-2 strain and the lipopeptide compound Surfactin can effectively delay the infection of PVMV.In addition,the HN-2 strain and its metabolites have a better control effect than the currently marketed antiviral agents benzothiadiazole and fluorophos in the prevention and treatment of PVMV virus,and the effect is longer. |
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