三株酚酸降解菌的筛选与鉴定及其生物活性

Three phenolic acid-degrading bacterial strains:screening,identification,and biological activities

【背景】常见作物连续种植后会导致根系分泌酚酸类自毒物质,该类物质是引起土传病害和连作障碍频发的主要因素。【目的】探讨利用有益微生物降解作物根系分泌物苯甲酸,缓解作物土传病害发生,并储备有益菌种资源。【方法】通过以苯甲酸为唯一碳源的无机盐培养基筛选无量山菌株资源,结合高效液相色谱仪测定酚酸降解菌株对苯甲酸的降解率;利用平板对峙试验,并基于缓解苯甲酸抑制番茄种子萌发试验及番茄幼苗促生长和对番茄枯萎病防效试验验证菌株的生物活性;结合形态学、生理生化和16S rRNA基因序列对菌株进行分类学鉴定。【结果】从无量山筛选得到4株放线菌和3株细菌具有酚酸降解能力,酚酸降解菌株在摇瓶培养72 h时对苯甲酸降解率分别为100%、99.83%、99.89%、99.87%、64.91%、56.92%和49.33%;其中2株放线菌YNK-FS0018、YNK-FS0019和一株细菌YNK-FB0022能较好地利用苯甲酸。番茄种子萌发试验结果表明菌株YNK-FS0018、YNK-FS0019和YNK-FB0022均可以缓解苯甲酸对种子萌发的抑制,促进种子萌发,萌发率分别为73%、73%和97%,并促进种子根生长,平均根长为13.31、13.04和14.56 cm;平板对峙试验结果表明菌株YNK-FS0018、YNK-FS0019和YNK-FB0022均对病原菌有广谱抗性;抗生素基因检测结果为菌株YNK-FS0018、YNK-FS0019具有PKSII、NRPS基因,菌株YNK-FB0022具有fenD、bioA、yndJ、ysnE、ituC、sboA和srfAB基因;盆栽试验结果显示菌株YNK-FS0018对番茄株高、地上鲜重、地上干重、根重、根长和茎粗有促生作用,各项分别增加53.40%、67.50%、57.50%、73.50%、77.28%和9.40%;菌株YNK-FS0019处理的株高、地上鲜重、地上干重、根重、根长和茎粗分别增加23%、40.90%、14.00%、57.00%、26.30%和16.80%;菌株YNK-FB0022处理的株高、地上鲜重、地上干重、根重、根长和茎粗分别增加24.50%、44.70%、26.40%、75.10%、98.00%和9.00%;3株菌对番茄枯萎病的防效分别为87.10%、74.18%和80.65%;结合形态学和分子生物学鉴定,菌株YNK-FS0018为稻瘟霉素链霉菌(Streptomyces blastmyceticus,GenBank登录号为OR523286),YNK-FS0019为白黄链霉菌(Streptomyces alboflavus,GenBank登录号为OR523289),YNK-FB0022为耐盐芽孢杆菌(Bacillus halotolerans,GenBank登录号为OR523290);此外,菌株均有解有机磷、溶锌、分泌铁载体、水解淀粉酶和水解蛋白酶的能力。【结论】三株酚酸降解菌具有解有机磷、溶锌、分泌铁载体、水解淀粉酶和水解蛋白酶的能力,对番茄幼苗株高、茎粗、根长、根重、地上部分鲜重和干重等农艺性状具有显著促进作用,对番茄枯萎病防效较好,可为土传病害的生物防治上提供微生物资源。

[Background]Continuous planting of common crops can lead to the secretion of phenolic acid autotoxic substances in roots,which are the main factors causing soil-borne diseases and continuous cropping obstacles.[Objective]To mine the microorganisms capable of degrading benzoic acid secreted by crop roots,reduce the occurrence of soil-borne diseases,and reserve beneficial microbial resources.[Methods]We used benzoic acid as the sole carbon source in the inorganic salt medium to screen the strain resources of Wuliang Mountain and determined the degradation rate of benzoic acid by high-performance liquid chromatography.The plate confrontation test,tomato seed germination experiment,tomato seedling growth experiment,and pathogen(Fusarium wilt)inoculation experiment with tomato seedlings were carried out to examine the biological activities of the strains.The strains were identified based on morphological,physiological,and biochemical characteristics and 16S rRNA gene sequences.[Results]Four strains of Actinomycetes and three strains of bacteria with phenolic acid-degrading ability were screened from Wuliang Mountain.After culture in shake flasks for 72 h,the strains showed the benzoic acid-degrading rate of 100%,99.83%,99.89%,99.87%,64.91%,56.92%,and 49.33%,respectively.Two Actinomycetes strains(YNK-FS0018 and YNK-FS0019)and one bacterial strain(YNK-FB0022)could make good use of benzoic acid.The treatments with the three strains alleviated the inhibitory effect of benzoic acid on tomato seed germination,with the seed germination rates of 73%,73%,and 97%,the root length of 13.31,13.04,and 14.56 cm,respectively.Furthermore,the three strains had broad-spectrum resistance to pathogens.YNK-FS0018 and YNK-FS0019 carried PKSII and NRPS,and YNK-FB0022 carried fenD,bioA,yndJ,ysnE,ituC,sboA,and srfAB.The results of pot experiments showed that strain YNK-FS0018 increased the plant height,above ground fresh weight,above ground dry weight,root weight,root length,and stem diameter by 53.40%,67.50%,57.50%,73.50%,77.28%,and 9.40%,respectively.The increases in the above indicators by YNK-FS0019 were 23.00%,40.90%,14.00%,57.00%,26.30%,and 16.80%,respectively.The increases by YNK-FB0022 were 24.50%,44.70%,26.40%,75.10%,98.00%,and 9.00%,respectively.The control effects of three strains on tomato Fusarium wilt were 87.10%,74.18%,and 80.65%,respectively.YNK-FS0018,YNK-FS0019,and YNK-FB0022 were identified as Streptomyces blastmyceticus(GenBank accession number:OR523286),Streptomyces alboflavus(GenBank accession number:OR523289),and Bacillus halotolerans(GenBank accession number:OR523290),respectively.All the strains had the abilities of solubilizing organic phosphorus and zinc,secreting siderophores,and producing amylase and protease.[Conclusion]The three phenolic acid-degrading bacterial strains have the abilities of solubilizing organic phosphorus and zinc,secreting siderophores,and producing amylase and protease.They can significantly increase the plant height,stem thickness,root length,root weight,above ground fresh weight and above ground dry weight of tomato seedlings.Moreover,they demonstrate strong control effects on tomato Fusarium wilt,serving as the microbial resources for the biocontrol of soil-borne diseases.