棉花黄萎病拮抗细菌产铁载体测定及其对抑菌活性的影响
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摘要
【背景】棉花是新疆农业的支柱产业,棉花黄萎病严重影响棉花产量和纤维品质,有效的防治途径是目前解决的首要任务。【目的】探究BacillusvelezensisBHZ-29、Bacillusatrophaeus SHZ-24、Bacillus subtilis SHT-15、Bacillus vanillea SMT-24菌株产铁载体能力及其对抑菌活性的影响。【方法】MKB-CAS双层平板检测铁载体,Arnow法与高氯酸铁法鉴定铁载体类型,CAS染液定量铁载体,琼脂扩散法测定低铁条件下铁载体对抑菌物质的影响。【结果】MKB-CAS固体平板菌体周围出现橙紫色显色反应,Arnow实验4株拮抗菌反应液颜色为红色,高氯酸铁实验反应液颜色为黄色;BHZ-29、SHZ-24、SHT-15、SMT-24菌株铁载体定量活性单位最大值分别为8.27%±0.61%、31.80%±2.06%、16.06%±3.61%、15.53%±0.51%,吸光度值As/Ar比值最小分别为0.917±0.092、0.682±0.021、0.845±0.005、0.846±0.008。BHZ-29、SHZ-24、SMT-24菌株含铁离子浓度0、10、20μmol/L 3组处理发酵无菌滤液均未出现抑菌圈,SHT-15菌株3组处理出现抑菌圈,抑菌圈直径大小无差异性。【结论】4株拮抗菌株均产铁载体,铁载体类型为儿茶酚型,4株拮抗菌株整体产铁载体能力较低,最高达31.80%±2.06%,产铁能力As/Ar比值高于0.682时,BHZ-29、SHZ-24、SMT-24菌株含铁离子浓度0、10、20μmol/L 3组处理发酵无菌滤液对抑菌活性无影响,SHT-15菌株3组不同铁离子浓度处理对抑菌物质影响差异不显著。
[Background] Cotton plays a key role in Xinjiang's agriculture, but cotton production and fiber quality are seriously affected by Verticillium dahliae, effective prevention and control approaches are the primary tasks to be solved. [Objective] To study the ability of Bacillus velezensis BHZ-29, Bacillus atrophaeus SHZ-24, Bacillus subtilis SHT-15 and Bacillus vanillea SMT-24 to produce siderophore and siderophore's effects on antibacterial activity. [Methods] MKB-CAS double-layer plate was used to detect siderophore, Arnow method and iron perchlorate method were used to identify the type of siderophore,CAS dye liquid quantitative siderophore and agar diffusion method was used to determine the effect of siderophore on antibacterial substances under low iron conditions. [Results] An orange-purple color appeared around the bacteria on the MKB-CAS solid plate, Solutions color of the four antagonistic antibiotics in the Arnow experiment was red and perchlorate reaction solutions was yellow. The maximum values of quantitative activity units of siderophore of BHZ-29, SHZ-24, SHT-15 and SMT-24 strains were 8.27%±0.61%, 31.80%±2.06%, 16.06%±3.61%, and 15.53%±0.51%, respectively, the absorbance values of As/Ar the smallest ratios were 0.917±0.092, 0.682±0.021, 0.845±0.005 and0.846±0.008, respectively. No inhibitory zone appeared in the 0, 10 and 20 μmol/L iron ion treatment groups of BHZ-29, SHZ-24 and SMT-24 strains, in three treatment groups of SHT-15 strains had inhibition zones, but there was no difference in the diameter of the inhibition zone. [Conclusion] The four strains of antagonistic bacteria were all siderophore-producing, the type of siderophore was catechol, and the four antagonistic strains had low siderophore-producing ability, with a maximum of31.80%±2.06%, when the siderophore-producting capacity of As/Ar ratio was higher than 0.682,BHZ-29, SHZ-24 and SMT-24 fermentation sterile filtrates with 0, 10 and 20 μmol/L iron ions, had no effect on the antibacterial activity, SHT-15 fermentation sterile filtrates with 0, 10 and 20 μmol/L had no significant difference in the effect of antibacterial substances.
[Background] Cotton plays a key role in Xinjiang's agriculture, but cotton production and fiber quality are seriously affected by Verticillium dahliae, effective prevention and control approaches are the primary tasks to be solved. [Objective] To study the ability of Bacillus velezensis BHZ-29, Bacillus atrophaeus SHZ-24, Bacillus subtilis SHT-15 and Bacillus vanillea SMT-24 to produce siderophore and siderophore's effects on antibacterial activity. [Methods] MKB-CAS double-layer plate was used to detect siderophore, Arnow method and iron perchlorate method were used to identify the type of siderophore,CAS dye liquid quantitative siderophore and agar diffusion method was used to determine the effect of siderophore on antibacterial substances under low iron conditions. [Results] An orange-purple color appeared around the bacteria on the MKB-CAS solid plate, Solutions color of the four antagonistic antibiotics in the Arnow experiment was red and perchlorate reaction solutions was yellow. The maximum values of quantitative activity units of siderophore of BHZ-29, SHZ-24, SHT-15 and SMT-24 strains were 8.27%±0.61%, 31.80%±2.06%, 16.06%±3.61%, and 15.53%±0.51%, respectively, the absorbance values of As/Ar the smallest ratios were 0.917±0.092, 0.682±0.021, 0.845±0.005 and0.846±0.008, respectively. No inhibitory zone appeared in the 0, 10 and 20 μmol/L iron ion treatment groups of BHZ-29, SHZ-24 and SMT-24 strains, in three treatment groups of SHT-15 strains had inhibition zones, but there was no difference in the diameter of the inhibition zone. [Conclusion] The four strains of antagonistic bacteria were all siderophore-producing, the type of siderophore was catechol, and the four antagonistic strains had low siderophore-producing ability, with a maximum of31.80%±2.06%, when the siderophore-producting capacity of As/Ar ratio was higher than 0.682,BHZ-29, SHZ-24 and SMT-24 fermentation sterile filtrates with 0, 10 and 20 μmol/L iron ions, had no effect on the antibacterial activity, SHT-15 fermentation sterile filtrates with 0, 10 and 20 μmol/L had no significant difference in the effect of antibacterial substances.
引文
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[15]Long YC,Chen X,Zhou SQ.Isolation,identification and assessment on selenium biofortification of siderophore-producing rhizobacteria[J].Current Biotechnology,2017,7(5):402-408(in Chinese)龙云川,陈轩,周少奇.高产铁载体根际菌的筛选鉴定及硒活化特性评价[J].生物技术进展,2017,7(5):402-408
[16]Yang CE,Lu YL,Ni HC,et al.Screening of siderophoreproducing vibrio vulnificus and optimization of induction conditions using response surface methodology[J].Science and Technology of Food Industry,2017,38(3):159-165(in Chinese)杨常娥,鲁艳莉,倪捍成,等.创伤弧菌产铁载体菌株的筛选及其诱导条件的响应面优化[J].食品工业科技,2017,38(3):159-165
[17]Rong LY,Yao T,Zhao GQ,et al.Screening of siderophoreproducing PGPR bacteria and their antagonism against the pathogens[J].Plant Protection,2011,37(1):59-64(in Chinese)荣良燕,姚拓,赵桂琴,等.产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J].植物保护,2011,37(1):59-64
[18]Sun M.ARTP selection of high siderophore productions trains and preliminary analysis on mechanism of the siderophore improving[D].Wuxi:Master’s Thesis of Jiangnan University,2017(in Chinese)孙萌.铁载体高产菌株的ARTP选育及其铁载体产量提高机理的初步分析[D].无锡:江南大学硕士学位论文,2017
[19]Shenker M,Oliver I,Helmann M,et al.Utilization by tomatoes of iron mediated by a siderophore produced by Rhizopus arrhizus[J].Journal of Plant Nutrition,1992,15(10):2173-2182
[20]Schwyn B,Neilands JB.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56
[21]Yoshida T,Hayashi KI,Ohmoto H.Dissolution of iron hydroxides by marine bacterial siderophore[J].Chemical Geology,2002,184(1/2):1-9
[22]Payne SM.Detection,isolation,and characterization of siderophores[J].Methods in Enzymology,1994,235:329-344
[23]Chen SX.Detection methods on siderophores from bacteria[J].Journal of Honghe University,2005,3(6):16-18(in Chinese)陈绍兴.细菌铁载体检测方法[J].红河学院学报,2005,3(6):16-18
[24]Chen W,Wang XL,Fu W,et al.Screening,identification and antagonistic against the pathogens of a siderophore-producing bacteria HMGY6B from rhizosphere of ryegrass[J].Microbiology China,2016,43(10):2207-2215(in Chinese)陈伟,王小利,付薇,等.黑麦草根际产铁载体细菌HMGY6B的筛选鉴定及对病原菌的拮抗作用[J].微生物学通报,2016,43(10):2207-2215
[25]Han S,Zhang SC,Huang XY,et al.Antagonism of a siderophore-producing entophytic Bacteria on Fusarium oxysporum[J].Acta Botanica Boreali-Occidentalia Sinica,2011,31(5):1039-1044(in Chinese)韩松,张守村,黄晓艳,等.一株产铁载体内生细菌对尖孢镰刀菌的拮抗作用[J].西北植物学报,2011,31(5):1039-1044
[2]Deng LW,Zhang LY,Ma JT,et al.Research progress on the iron uptake of strategy I plant and iron sensors to iron deficiency[J].Heilongjiang Agricultural Sciences,2015(9):140-146(in Chinese)邓凌韦,张丽艳,马军韬,等.机理I植物根系铁吸收及缺铁感应信号研究进展[J].黑龙江农业科学,2015(9):140-146
[3]Saha M,Sarkar S,Sarkar B,et al.Microbial siderophores and their potential applications:a review[J].Environmental Science and Pollution Research,2016,23(5):3984-3999
[4]Sasirekha B,Srividya S.Siderophore production by Pseudomonas aeruginosa FP6,a biocontrol strain for Rhizoctonia solani and Colletotrichum gloeosporioides causing diseases in chilli[J].Agriculture and Natural Resources,2016,50(4):250-256
[5]Farina R,Beneduzi A,Ambrosini A,et al.Diversity of plant growth-promoting rhizobacteria communities associated with the stages of canola growth[J].Applied Soil Ecology,2012,55:44-52
[6]Rana A,Joshi M,Prasanna R,et al.Biofortification of wheat through inoculation of plant growth promoting rhizobacteria and cyanobacteria[J].European Journal of Soil Biology,2012,50:118-126
[7]Lin TX,Tang M,Huang MY,et al.Screening and identification of a high yield sideropho-res-producing bacteria SS05 isolated from cotton soil[J].Microbiology China,2012,39(5):668-676(in Chinese)林天兴,唐梅,黄明远,等.高产铁载体棉田土壤细菌SS05的筛选与鉴定[J].微生物学通报,2012,39(5):668-676
[8]Couillerot O,Prigent-Combaret C,Caballero-Mellado J,et al.Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens[J].Letters in Applied Microbiology,2009,48(5):505-512
[9]Chaiharn M,Chunhaleuchanon S,Lumyong S.Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in thailand[J].World Journal of Microbiology and Biotechnology,2009,25(11):1919-1928
[10]Sayyed RZ,Chincholkar SB.Siderophore-producing Alcaligenes feacalis exhibited more biocontrol potential Vis-à-Vis chemical fungicide[J].Current Microbiology,2009,58(1):47-51
[11]Zeng JH,Li YY,Ruan DS,et al.Phytoremediation of heavy metal contaminated soils by plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungi[J].Microbiology China,2017,44(5):1214-1221(in Chinese)曾加会,李元媛,阮迪申,等.植物根际促生菌及丛枝菌根真菌协助植物修复重金属污染土壤的机制[J].微生物学通报,2017,44(5):1214-1221
[12]Liu X,Fu JW,Da Silva E,et al.Microbial siderophores and root exudates enhanced goethite dissolution and Fe/As uptake by As-hyperaccumulator Pteris vittata[J].Environmental Pollution,2017,223:230-237
[13]Lamb AL.Breaking a pathogen’s iron will:inhibiting siderophore production as an antimicrobial strategy[J].Biochimica et Biophysica Acta(BBA)-Proteins and Proteomics,2015,1854(8):1054-1070
[14]Pan XX,Peng JH,Liu XB,et al.Isolation and identification of a strain of endogenous antagonist bacteria from banana[J].Tropical Agricultural Engineering,2009,33(4):4-8(in Chinese)潘羡心,彭建华,刘先宝,等.一株产铁载体香蕉拮抗内生细菌的分离及鉴定[J].热带农业工程,2009,33(4):4-8
[15]Long YC,Chen X,Zhou SQ.Isolation,identification and assessment on selenium biofortification of siderophore-producing rhizobacteria[J].Current Biotechnology,2017,7(5):402-408(in Chinese)龙云川,陈轩,周少奇.高产铁载体根际菌的筛选鉴定及硒活化特性评价[J].生物技术进展,2017,7(5):402-408
[16]Yang CE,Lu YL,Ni HC,et al.Screening of siderophoreproducing vibrio vulnificus and optimization of induction conditions using response surface methodology[J].Science and Technology of Food Industry,2017,38(3):159-165(in Chinese)杨常娥,鲁艳莉,倪捍成,等.创伤弧菌产铁载体菌株的筛选及其诱导条件的响应面优化[J].食品工业科技,2017,38(3):159-165
[17]Rong LY,Yao T,Zhao GQ,et al.Screening of siderophoreproducing PGPR bacteria and their antagonism against the pathogens[J].Plant Protection,2011,37(1):59-64(in Chinese)荣良燕,姚拓,赵桂琴,等.产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J].植物保护,2011,37(1):59-64
[18]Sun M.ARTP selection of high siderophore productions trains and preliminary analysis on mechanism of the siderophore improving[D].Wuxi:Master’s Thesis of Jiangnan University,2017(in Chinese)孙萌.铁载体高产菌株的ARTP选育及其铁载体产量提高机理的初步分析[D].无锡:江南大学硕士学位论文,2017
[19]Shenker M,Oliver I,Helmann M,et al.Utilization by tomatoes of iron mediated by a siderophore produced by Rhizopus arrhizus[J].Journal of Plant Nutrition,1992,15(10):2173-2182
[20]Schwyn B,Neilands JB.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56
[21]Yoshida T,Hayashi KI,Ohmoto H.Dissolution of iron hydroxides by marine bacterial siderophore[J].Chemical Geology,2002,184(1/2):1-9
[22]Payne SM.Detection,isolation,and characterization of siderophores[J].Methods in Enzymology,1994,235:329-344
[23]Chen SX.Detection methods on siderophores from bacteria[J].Journal of Honghe University,2005,3(6):16-18(in Chinese)陈绍兴.细菌铁载体检测方法[J].红河学院学报,2005,3(6):16-18
[24]Chen W,Wang XL,Fu W,et al.Screening,identification and antagonistic against the pathogens of a siderophore-producing bacteria HMGY6B from rhizosphere of ryegrass[J].Microbiology China,2016,43(10):2207-2215(in Chinese)陈伟,王小利,付薇,等.黑麦草根际产铁载体细菌HMGY6B的筛选鉴定及对病原菌的拮抗作用[J].微生物学通报,2016,43(10):2207-2215
[25]Han S,Zhang SC,Huang XY,et al.Antagonism of a siderophore-producing entophytic Bacteria on Fusarium oxysporum[J].Acta Botanica Boreali-Occidentalia Sinica,2011,31(5):1039-1044(in Chinese)韩松,张守村,黄晓艳,等.一株产铁载体内生细菌对尖孢镰刀菌的拮抗作用[J].西北植物学报,2011,31(5):1039-1044
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