The Extraction, Analysis And Antibacterial Mechanism Study From Endophytic Bacteria From Dendrobium Nobile Of Guizhou

Plant bacterial diseases seriously threat global agricultural production.Two important rice bacterial diseases,rice bacterial blight（BLB）and rice bacterial leaf streak（BLS）caused by Xanthomonas oryzae pv.oryzae（Xoo）and Xanthomonas oryzae pv.oryzicola（Xoc）,respectively,seriously affect the yield and quality of rice.Long-term use of traditional bactericide such as bismerthiazol,Zinc thiazole and thiodiazole copper will cause pathogens to develop resistance to drugs and have certain side effects on the safety of the environment and plants.Antimicrobial agents derived from natural products usually show more advantages than synthetic chemicals,including low mammalian toxicity,easy decomposition,environment,friendliness.So,microbial pesticide has been a hot research topic in recent years.In this work,the multiple active metabolites were extracted from Paenibacillus polymyxa Y-1（P.polymyxa Y-1）isolated from Dendrobium nobile（D.nobile）,their structural were elucidated through ¹H NMR,¹³C NMR and high-resolution mass spectrometry.The multiple active metabolites were found to anti-Xoo and anti-Xoc activities for the first time in vitro.In addition,the multiple active metabolites were also found to possesse outstanding protective activity against BLB and BLS for the first time in vivo.The antiviral mechanisms indicated that polymyxins B₁could activate phenylpropane biosynthesis pathway to induce plant to express defense related proteins.Meanwhile,it also could increase defense enzyme activity and promote lignin synthesis to enhance rice resistance to pathogens.These results indicate that biogenic pesticides have broad application prospects as a potential antibacterial agent.This study attempts to explore the activity of active metabolites against plant bacterial diseases.The specific process and content of the study are as follows:1.Single factors such as carbon source,nitrogen source,concentration,temperature, pH value and time were selected to study their effects on the bacteriostatic ability of P.polymyxa Y-1 fermentation broth.Using 10 g/L peptone,0.4 g/L Mg SO₄,and 2 g/L KH₂PO₄as basal medium（pH=7）,30 g/L lactose,glucose,maltose,fructose,glycerol,and starch carbon sources were respectively added to prepare different carbon sources culture medium（200m L）,and then P.polymyxa Y-1 strain（10 m L）was inoculated.Using 20 g/L glycerol,0.4 g/L Mg SO₄,and 2 g/L KH₂PO₄as basal medium（pH=7）,10 g/L peptone,tryptone,beef extract,yeast powder,and urea nitrogen sources were respectively added to prepare different nitrogen sources culture medium（200 m L）,and then P.polymyxa Y-1 strain（10 m L）was inoculated.Using 0.4 g/L Mg SO₄and 2g/L KH₂PO₄as the basal medium（pH=7）,carbon source and nitrogen source were respectively added to prepare concentrations of 50 g/L,40 g/L,30 g/L,20 g/L,and 10g/L nutrient medium（200 m L）,and then P.polymyxa Y-1 strain（10 m L）was inoculated.The effect of pH value showed the pH value was adjusted to 5.0,5.5,6.0,6.5,7.0,7.5,and 8.0 with 1 mol/L HCl and Na OH in the basal medium（200m L）,then P.polymyxa Y-1 strain（10 m L）was inoculated.The temperature effect showed that P.polymyxa Y-1 was cultured on a shaking table at 25°C,26°C,27°C,28°C,29°C,and 30°C,respectively.The time effect showed that P.polymyxa Y-1 was incubated on a shaker for 48,72,96,120,144,168,192,and 216 hours,respectively.The paper disk method was used to detect the antibacterial activity of the P.polymyxa Y-1fermentation broth to Xoo and Xoc.The experiments were all performed three times and in triplicate.2.The metabolites were separated from the P.Polymyxa Y-1 fermentation broth by biological activity tracking separation.The fermentation broth was centrifuged to take the supernatant,which was placed on an Amberlite XAD-16 column and eluted with water-methanol,the eluent polarity was gradually reduced,the ratios of pure water to methanol were 100:0（A）,75:25（B）,50:50（C）,20:80（D）,0:100（E）in turn.The same polarity sections were combined and concentrated into a yellow solid.Then,10metabolites were obtained by Amberlite XAD-16 column,SKP-10-1800reverse-phase resin,Sephadex LH-20 column,Phenomenex Gemini C18（00G-4435-N0）.Including 2,4-di-tert-butylphenol（Y1）,3-（2-aminoethyl）indole（Y2）,polymyxin B₁（Y3）,polymyxin E₂（Y4）,N-acetyl-5-Methoxytryptamine（Y5）,2,4-dihydroxy-5-methylpyrimidine（Y6）,glutathione（Y7）,mannitol（Y8）,p-hydroxybenzoic acid（Y9）,serine（Y10）.The structures of 10 compounds were identified by ¹H NMR,¹³C NMR and high-resolution mass spectrometry.3.The activity of active metabolites was detected by turbidity method,leaf clipping method and acupuncture method.The results are as follows:（1）At a concentration of 200μg/m L,polymyxin B₁and E₂had remarkable in vitro inhibitory activities to Xoo and Xoc with the EC₅₀values of 0.19μg/m L and 0.21μg/m L against Xoo,and 0.32μg/m L and 0.41μg/m L against Xoc,respectively,which were better than those of Zhongshengmycin（0.31μg/m L and 0.73μg/m L,respectively）and Bismerthiazol（77.48μg/m L and 85.30μg/m L,respectively）.Polymyxins B₁and E2 had good protective and curative activities against rice bacterial leaf blight（BLB）and rice bacterial leaf streak（BLS）in vivo.The protective and curative activities of polymyxins B₁（45.8%and 35.8%,respectively）and E₂（41.2%and 37.0%,respectively）to BLB were superior to those of Zhongshengmycin（34.8%and 29.8%,respectively）and Bismerthiazol（38.0%and 33.5%,respectively）.Meanwhile,the protective and curative activities of polymyxins B₁（44.8%and 39.8%,respectively）and E₂（42.9%and 39.9%,respectively）to BLS were also superior to those of Zhongshengmycin（39.7%and 32.0%,respectively）and Bismerthiazol（41.5%and 34.3%respectively）.（2）At a concentration of 200μg/m L,the bioassay results showed that 2,4-di-tert-butylphenol,N-acetyl-5-methoxytryptamine,P-hydroxybenzoic acid had good antibacterial activity against Xoo and Xoc,with the 50%effective concentration values of 49.45μg/m L,64.22μg/m L,and 16.32μg/m L to Xoo,and 34.33μg/m L,71.17μg/m L,and 15.58μg/m L to Xoc,respectively,compared with Zhongshengmycin（0.42 and 0.82μg/m L,respectively）and Bismerthiazol（85.64 and92.49μg/m L,respectively）.In vivo experiments found that 2,4-di-tert-butylphenol（35.9%and 35.4%,respectively）,N-acetyl-5-methoxytryptamine（42.9%and 36.7%,respectively）,and P-hydroxybenzoic acid（40.6%and 36.8%,respectively）demonstrated excellent protective and curative activity against rice bacterial leaf blight,which were better than that of Zhongshengmycin（38.4%and 34.4%,respectively）.4.Based on the good protective activity of polymyxin B₁against BLB.Fengyouxiangzhan rice was cultivated for 8 weeks,and 200μg/m L active metabolite solutions were uniformly sprayed onto rice leaves of the treatment group until dripping down,whereas Tween was used as control groups.One day after spraying,Xoo in the logarithmic phase was inoculated onto rice leaves with scissors method.Then,the rice plants were cultivated in greenhouse at 28°C and 90%RH.Five days after inoculation,rice leaves were extracted and enzymatically digested for LC-MS/MS.DEPs were analyzed using the gene ontology（GO）annotation（http://www.geneontology.org/）,and the kyoto encyclopedia of genes and genomes（KEGG）annotation（http://www.genome.jp/Pathway）.And then,the databases were searched using the Uniprot software（http://www.uniprot.org/）.The results showed that a total of 12 pathways were enriched,including the phenylpropanoid biosynthesis,MAPK signaling pathway,fatty acid degradation,tryptophan metabolism,lysine degradation,and others.Among them,16 DEPs was enriched in the phenylpropane biosynthetic pathway,including trans-cinnamate-4-monooxygenase（CYP73A）,β-glucosidase（GLU）,CAD and POD.There were 9 up-regulated proteins（A0A0N7KI36,A0A0P0XR31,Q5JMS4,Q94DM2,A2YPX2,Q6ZCC2,Q6K4J4,Q7XSU7,and Q7XSU8）and 2 down-regulated ones（A0A0P0V2C2,Q9AS12）involved in the regulation of POD.Meanwhile,CAD（Q8H859,Q6ERW9,and Q6ERW7）and GLU（Q60DX8）proteins were up-regulated,but CYP73A（A2Y375）was down-regulated.It is well-known that POD and CAD are able to regulate the formation of lignin,which can provide structural support,prevent pathogen invasion through a physical barrier and act as defensive components in plants.Up-regulation of CAD and POD can increase the resistance of plants to invaded pathogens and reduce the infection incidence.The results indicated that polymyxin B₁could enhance the resistance of rice to Xoo pathogen through up-regulating CAD and POD in rice.Parallel Reaction Monitoring was performed to confirm the potential relationship of16 DEPs to the phenylpropanoid biosynthesis pathway.As shown in Table 4 and Figures 10,expression levels of 16 DEPs were consistent with proteomic analysis results.Three POD proteins were significantly up-regulated,including Q94DM2,Q6ZCC2,and Q7XSU7.Notably,the expression level of Q7XSU7 increased17.64-fold after Xoo+PB treatment than that of Xoo group（PB/CK）.Meanwhile,the PB/CK ratio of Q94DM2 and Q6ZCC2 protein was 9.89 and 8.34,respectively.Expression levels of Q8H859 and Q6ERW9 of CAD up-regulated 2.34-fold and2.19-fold versus CK groups.Among DEPs of phenylpropanoid biosynthesis pathway,A0A0P0V2C2,Q9AS12,and A2Y375 were down-regulated.PRM results demonstrated that polymyxin B₁could significantly affect the expression level of POD and then regulate the phenylpropanoid biosynthesis pathway,which were consistent with DEPs analysis.