新疆木碱蓬(Suaeda dendroides)根际耐盐促生细菌的筛选及鉴定
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摘要
【背景】新疆是全国最大的盐渍化土壤分布区,土壤盐渍化严重影响作物生长,耐盐促生菌可以有效改善土壤肥力,提高作物抗逆性,促进植物生长,提高土壤利用率。【目的】分离筛选获得木碱蓬(Suaeda dendroides)根际土壤中耐盐促生菌菌株,对优良促生作用的菌株进行分子鉴定,挖掘微生物资源,为微生物菌剂的研制奠定基础。【方法】采用传统的分离方法筛选获得木碱蓬根际耐盐微生物,采用"三级筛选体系"筛选获得耐盐促生菌菌株,用CTAB法提取菌株DNA,对菌株16S rRNA基因测序进行系统发育分析,确定耐盐促生菌菌株的分类地位。【结果】从木碱蓬根际共分离获得耐盐微生物58株,8株具有固氮活性,解磷活性菌株12株,具有解钾活性菌株15株,产IAA活性的菌株3株,具有较强产氨活性的菌株2株。经过促生平板筛选,菌株GTZW50-5和MH-F促进了拟南芥生长,表现出较好的促生效果,通过小麦盆栽试验,菌株GTZW50-5对小麦的根长以及株高具有显著的促生作用,在一定程度上提高了植物体内的叶绿素含量。MH-F菌株对小麦的根系具有较显著的促生作用,且对小麦的叶绿素及脯氨酸含量在不同盐浓度下都有所提高。经过系统发育分析,GTZW50-5与Bacillus vallismortis (AY603658)序列相似度达到99.43%,鉴定属于Bacillus vallismortis,MH-F与Enterobacterludwigii(JTLO01000001)序列相似度为98.34%,鉴定属于Enterobacter属。【结论】菌株GTZW50-5与MH-F均具有较好的促生效果,这为耐盐微生物资源的开发和利用提供了理论依据。
[Background] Xinjiang is the largest salinized soil distribution area in China. Soil salinization inhibits plant growth seriously. Salt-tolerant promoting bacteria can improve soil fertility, crop stress resistance and soil utilization effectively, thus it promotes plant growth. [Objective] We isolated and screened salt-tolerant strains from rhizosphere soil of Suaeda dendroides. The strains with excellent growth-promoting effect were identified, and the microbial resources were excavated. [Methods] The traditional method of separation was used to screen the salt-tolerant bacteria in the rhizosphere of S. dendroides. The strains of salt-tolerant promoting bacteria were obtained through the three-stage screening system. DNA of the strains was extracted by CTAB method, and the 16 S r RNA gene sequencing was performed for phylogenetic analysis to determine the classification status of the salt-tolerant bacteria. [Results] A total of 58 salt-tolerant bacteria were isolated from the rhizosphere of S. dendroides. There were 8 strains with nitrogen fixation activity, 12 strains with phosphate solubilizing activity, and 15 strains with potassium solubilizing activity, 3 strains with IAA activity, and 2 strains with strong ammonia producing ability. Strains named GTZW50-5 and MH-F promoted the growth of Arabidopsis thaliana significantly. The results of wheat pot experiment showed strain GTZW50-5 had significant growth promotion effect on root length and plant height of wheat, and to some extent, the chlorophyll content in plants was improved. Strain MH-F had a significant promoting effect on the roots of wheat, and the chlorophyll content and proline content of wheat were increased at different salt concentrations. According to phylogenetic analysis, the similarity between GTZW50-5 and Bacillus vallismortis(AY603658) was 99.43%, GTZW50-5 was identified as Bacillus vallismortis. The similarity between MH-F and Enterobacter ludwigii(JTLO01000001) sequence was 98.34%, which was identified as Enterobacter. [Conclusion] Strains GTZW50-5 and MH-F had a good effect of promoting growth, providing a theoretical basis for the development and utilization of salt-tolerant microbial resources.
[Background] Xinjiang is the largest salinized soil distribution area in China. Soil salinization inhibits plant growth seriously. Salt-tolerant promoting bacteria can improve soil fertility, crop stress resistance and soil utilization effectively, thus it promotes plant growth. [Objective] We isolated and screened salt-tolerant strains from rhizosphere soil of Suaeda dendroides. The strains with excellent growth-promoting effect were identified, and the microbial resources were excavated. [Methods] The traditional method of separation was used to screen the salt-tolerant bacteria in the rhizosphere of S. dendroides. The strains of salt-tolerant promoting bacteria were obtained through the three-stage screening system. DNA of the strains was extracted by CTAB method, and the 16 S r RNA gene sequencing was performed for phylogenetic analysis to determine the classification status of the salt-tolerant bacteria. [Results] A total of 58 salt-tolerant bacteria were isolated from the rhizosphere of S. dendroides. There were 8 strains with nitrogen fixation activity, 12 strains with phosphate solubilizing activity, and 15 strains with potassium solubilizing activity, 3 strains with IAA activity, and 2 strains with strong ammonia producing ability. Strains named GTZW50-5 and MH-F promoted the growth of Arabidopsis thaliana significantly. The results of wheat pot experiment showed strain GTZW50-5 had significant growth promotion effect on root length and plant height of wheat, and to some extent, the chlorophyll content in plants was improved. Strain MH-F had a significant promoting effect on the roots of wheat, and the chlorophyll content and proline content of wheat were increased at different salt concentrations. According to phylogenetic analysis, the similarity between GTZW50-5 and Bacillus vallismortis(AY603658) was 99.43%, GTZW50-5 was identified as Bacillus vallismortis. The similarity between MH-F and Enterobacter ludwigii(JTLO01000001) sequence was 98.34%, which was identified as Enterobacter. [Conclusion] Strains GTZW50-5 and MH-F had a good effect of promoting growth, providing a theoretical basis for the development and utilization of salt-tolerant microbial resources.
引文
[1] Lin J, Anwar M, Dilbar S. Investigation of the spatial variability of soil salts in saline soil in Xinjiang[J]. Research of Soil and Water Conservation, 2007, 14(6):189-192(in Chinese)蔺娟,艾尼瓦尔·买买提,地里拜尔·苏力坦.新疆盐渍化区土壤盐分离子的空间变异特征[J].水土保持研究, 2007, 14(6):189-192
[2] Liu XR. Overview of salinization of cultivated land in Xinjiang[J]. Chinese Science&Technology, 2011(11):345,251(in Chinese)刘湘茹.概述新疆的盐碱化耕地整治[J].中国科技纵横,2011(11):345,251
[3] Jiazila AS, Wang XG, Yao WY. Preliminary study on soil salinization treatment technology in Xinjiang[J]. Water Saving Irrigation, 2011(11):50-52(in Chinese)加孜拉·阿山,王修贵,姚宛艳.新疆土壤盐碱化治理技术初步研究[J].节水灌溉, 2011(11):50-52
[4] Zhang AQ, Pang QY, Yan XF. Advances in salt-tolerance mechanisms of Suaeda plants[J]. Acta Ecologica Sinica, 2013,33(12):3575-3583(in Chinese)张爱琴,庞秋颖,阎秀峰.碱蓬属植物耐盐机理研究进展[J].生态学报, 2013, 33(12):3575-3583
[5] Zhang LB, Xu HL, Zhao GX. Salt tolerance of Suaeda salsa and its soil ameliorating effect on coastal saline soil[J]. Soils, 2007,39(2):310-313(in Chinese)张立宾,徐化凌,赵庚星.碱蓬的耐盐能力及其对滨海盐渍土的改良效果[J].土壤, 2007, 39(2):310-313
[6] Wu X, Gan BC, Huang ZQ, et al. Screening and identification of a IAA biosynthesis strain and optimization of its culture conditions[J]. Journal of Sichuan Agricultural University, 2014,32(4):432-435(in Chinese)吴翔,甘炳成,黄忠乾,等.一株产IAA菌株的筛选、鉴定及培养条件优化[J].四川农业大学学报, 2014, 32(4):432-435
[7] Liao JW, Wen YC, Yang M. Effects of different nitrogen fixing bacteria on growth index of Eucalyptus[J]. South China Agriculture, 2016, 10(27):78-79(in Chinese)廖静文,闻玉昌,杨蒙.不同固氮菌对桉树生长指标的影响[J].南方农业, 2016, 10(27):78-79
[8] Jiang Y, Wang H, Wang SD, et al. Application potential of microorganism fertilizer in agricultural production[J]. Soybean Science&Technology, 2010(5):25-27(in Chinese)姜妍,王浩,王绍东,等.微生物菌肥在农业生产中的应用潜力[J].大豆科技, 2010(5):25-27
[9] Wu XW. Improvement of saline land by microorganism fertilization in Weibei Area[D]. Xi’an:Master’s Thesis of Northwest University, 2015(in Chinese)吴晓卫.微生物菌肥改良渭北地区盐碱化土壤作用及效果研究[D].西安:西北大学硕士学位论文, 2015
[10] Wang Q, Li WT, Zhang PD, et al. Isolation and characterization of nitrogen-fixing bacteria in the rhizosphere of Zostera marina and optimization of its culture conditions[J]. Journal of Fishery Sciences of China, 2017, 24(4):791-801(in Chinese)王琦,李文涛,张沛东,等.鳗草根际固氮菌的分离鉴定及培养条件的筛选[J].中国水产科学, 2017, 24(4):791-801
[11] Lv R, Jia FA, Liu C, et al. Isolation, identification and phosphate-dissolving ability of inorganic phosphorus degrading strains[J]. Jiangsu Agricultural Sciences, 2017, 45(20):295-298(in Chinese)吕睿,贾凤安,刘晨,等.无机磷降解菌株的分离、鉴定及解磷能力[J].江苏农业科学, 2017, 45(20):295-298
[12] Zhang MY, Chen YF, Zhou DB, et al. Isolation, identification and fermentation conditions optimization of potassium bacteria in castor rhizosphere soil[J]. Chinese Journal of Tropical Crops,2016, 37(12):2268-2275(in Chinese)张妙宜,陈宇丰,周登博,等.蓖麻根际土壤解钾菌的筛选鉴定及发酵条件的优化[J].热带作物学报, 2016, 37(12):2268-2275
[13] Yang J, Sun HY, Zhao YZ, et al. Influence of alanine stress on culture in vitro of Arabidopsis thaliana and Nicotiana tabacum Linn[J]. Biotechnology, 2009, 19(1):81-84(in Chinese)杨靖,孙海燕,赵元增,等.丙氨酸胁迫对拟南芥和烟草离体培养的影响[J].生物技术, 2009, 19(1):81-84
[14] Pang FH, Huang SL, Wang T, et al. Isolation and identification of endogenous Bacillus megaterium and its biocontrol effect on wheat leaf rust[A]//Papers Collection of the Annual Meeting of the Chinese Society of Plant Pathology in 2015[C]. Haikou:Chinese Society for Plant Pathology, 2015:1(in Chinese)庞发虎,黄思良,王坦,等.内生性巨大芽孢杆菌的分离、鉴定及其对小麦叶锈病的生防效果[A]//中国植物病理学会2015年学术年会论文集[C].海口:中国植物病理学会, 2015:1
[15] Zheng WB, Shen F, Yan XM, et al. Isolation and identification of the IAA-producing and phosphate-dissolving bacteria and its promoting effects on red soil[J]. Soil, 2015, 47(2):361-368(in Chinese)郑文波,申飞,闫小梅,等.红壤中产吲哚乙酸并具解磷作用的促生菌筛选鉴定及促生效果研究[J].土壤, 2015, 47(2):361-368
[16] Wang KP, Zheng Y, Chu GY, et al. Screening of bacterial strains for phosphate solubilization, nitrogen fixation and IAA production and their promotive effects on plant growth[J]. Jiangsu Journal of Agricultural Sciences, 2013, 29(6):1352-1359(in Chinese)王奎萍,郑颖,褚光耀,等.解磷、固氮、产吲哚乙酸微生物菌株的筛选及其对植物的促生效果[J].江苏农业学报, 2013,29(6):1352-1359
[17] Taguchi F, Suzuki T, Inagaki Y, et al. The siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection[J]. Journal of Bacteriology, 2010,192(1):117-126
[18] Lalitha Kumari B, Lalitha R, Sudhakar P. Studies on isolation,purification and molecular identification of pectinase producing bacteria[J]. International Journal of Advanced Research, 2013,1(5):204-212
[19] Chen LY, Zhang GX, Huang CP, et al. Isolation, identification and enzymatic characteristics of cellulose-producing strains with high cellulase activity[J]. Microbiology China, 2011, 38(4):531-538(in Chinese)陈丽燕,张光祥,黄春萍,等.两株高产纤维素酶细菌的筛选、鉴定及酶学特性[J].微生物学通报, 2011, 38(4):531-538
[20] Park JW, Balaraju K, Kim JW, et al. Systemic resistance and growth promotion of chili pepper induced by an antibiotic producing Bacillus vallismortis strain BS07[J]. Biological Control, 2013, 65(2):246-257
[21] Noh SW, Seo R, Park JK, et al. Cyclic dipeptides from Bacillus vallismortis BS07 require key components of plant immunity to induce disease resistance in Arabidopsis against Pseudomonas infection[J]. The Plant Pathology Journal, 2017, 33(4):402-409
[22] Probanza A, Lucas Garc?a?JA, Palomino MR, et al. Pinus pinea L.seedling growth and bacterial rhizosphere structure after inoculation with PGPR Bacillus(B. licheniformis CECT 5106 and B. pumilus CECT 5105)[J]. Applied Soil Ecology, 2002, 20(2):75-84
[23] Cao JJ, Yang HY, Xie XS, et al. Control effect of Bacillus amyloliquefaciens on wheat yellow mosaic disease[J]. Journal of Plant Protection, 2016, 43(4):588-593(in Chinese)曹晶晶,杨海艳,谢咸升,等.解淀粉芽胞杆菌对小麦黄花叶病的生物防治[J].植物保护学报, 2016, 43(4):588-593
[24] Xiong WD, Liu Y, Wang GW, et al. Effects of plant growth-promoting rhizobacteria agents application on wheat in sandy soil[J]. Journal of Triticeae Crops, 2016, 36(7):945-950(in Chinese)熊伟东,刘晔,王国文,等.促生菌剂在砂质潮土麦田的应用效果[J].麦类作物学报, 2016, 36(7):945-950
[25] Allaoua H, Silini A, Yahiaoui B, et al. Phylogenetic and plant-growth-promoting characteristics of Bacillus isolated from the wheat rhizosphere[J]. Annals of Microbiology, 2016, 66(3):1087-1097
[26] Shoebitz M, Ribaudo CM, Pardo MA, et al. Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere[J]. Soil Biology and Biochemistry, 2009, 41(9):1768-1774
[27] Yousaf S, Afzal M, Reichenauer TG, et al. Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains[J].Environmental Pollution, 2011, 159(10):2675-2683
[28] Sarron E, Clément N, Pawlicki-Jullian N, et al. Stimulating effects of two plant growth-promoting bacteria, Enterobacter ludwigii Ez-185-17 and Raoultella terrigena Ez-555-6, on flax culture[J]. AIP Conference Proceedings, 2018:1954(1):020003
[2] Liu XR. Overview of salinization of cultivated land in Xinjiang[J]. Chinese Science&Technology, 2011(11):345,251(in Chinese)刘湘茹.概述新疆的盐碱化耕地整治[J].中国科技纵横,2011(11):345,251
[3] Jiazila AS, Wang XG, Yao WY. Preliminary study on soil salinization treatment technology in Xinjiang[J]. Water Saving Irrigation, 2011(11):50-52(in Chinese)加孜拉·阿山,王修贵,姚宛艳.新疆土壤盐碱化治理技术初步研究[J].节水灌溉, 2011(11):50-52
[4] Zhang AQ, Pang QY, Yan XF. Advances in salt-tolerance mechanisms of Suaeda plants[J]. Acta Ecologica Sinica, 2013,33(12):3575-3583(in Chinese)张爱琴,庞秋颖,阎秀峰.碱蓬属植物耐盐机理研究进展[J].生态学报, 2013, 33(12):3575-3583
[5] Zhang LB, Xu HL, Zhao GX. Salt tolerance of Suaeda salsa and its soil ameliorating effect on coastal saline soil[J]. Soils, 2007,39(2):310-313(in Chinese)张立宾,徐化凌,赵庚星.碱蓬的耐盐能力及其对滨海盐渍土的改良效果[J].土壤, 2007, 39(2):310-313
[6] Wu X, Gan BC, Huang ZQ, et al. Screening and identification of a IAA biosynthesis strain and optimization of its culture conditions[J]. Journal of Sichuan Agricultural University, 2014,32(4):432-435(in Chinese)吴翔,甘炳成,黄忠乾,等.一株产IAA菌株的筛选、鉴定及培养条件优化[J].四川农业大学学报, 2014, 32(4):432-435
[7] Liao JW, Wen YC, Yang M. Effects of different nitrogen fixing bacteria on growth index of Eucalyptus[J]. South China Agriculture, 2016, 10(27):78-79(in Chinese)廖静文,闻玉昌,杨蒙.不同固氮菌对桉树生长指标的影响[J].南方农业, 2016, 10(27):78-79
[8] Jiang Y, Wang H, Wang SD, et al. Application potential of microorganism fertilizer in agricultural production[J]. Soybean Science&Technology, 2010(5):25-27(in Chinese)姜妍,王浩,王绍东,等.微生物菌肥在农业生产中的应用潜力[J].大豆科技, 2010(5):25-27
[9] Wu XW. Improvement of saline land by microorganism fertilization in Weibei Area[D]. Xi’an:Master’s Thesis of Northwest University, 2015(in Chinese)吴晓卫.微生物菌肥改良渭北地区盐碱化土壤作用及效果研究[D].西安:西北大学硕士学位论文, 2015
[10] Wang Q, Li WT, Zhang PD, et al. Isolation and characterization of nitrogen-fixing bacteria in the rhizosphere of Zostera marina and optimization of its culture conditions[J]. Journal of Fishery Sciences of China, 2017, 24(4):791-801(in Chinese)王琦,李文涛,张沛东,等.鳗草根际固氮菌的分离鉴定及培养条件的筛选[J].中国水产科学, 2017, 24(4):791-801
[11] Lv R, Jia FA, Liu C, et al. Isolation, identification and phosphate-dissolving ability of inorganic phosphorus degrading strains[J]. Jiangsu Agricultural Sciences, 2017, 45(20):295-298(in Chinese)吕睿,贾凤安,刘晨,等.无机磷降解菌株的分离、鉴定及解磷能力[J].江苏农业科学, 2017, 45(20):295-298
[12] Zhang MY, Chen YF, Zhou DB, et al. Isolation, identification and fermentation conditions optimization of potassium bacteria in castor rhizosphere soil[J]. Chinese Journal of Tropical Crops,2016, 37(12):2268-2275(in Chinese)张妙宜,陈宇丰,周登博,等.蓖麻根际土壤解钾菌的筛选鉴定及发酵条件的优化[J].热带作物学报, 2016, 37(12):2268-2275
[13] Yang J, Sun HY, Zhao YZ, et al. Influence of alanine stress on culture in vitro of Arabidopsis thaliana and Nicotiana tabacum Linn[J]. Biotechnology, 2009, 19(1):81-84(in Chinese)杨靖,孙海燕,赵元增,等.丙氨酸胁迫对拟南芥和烟草离体培养的影响[J].生物技术, 2009, 19(1):81-84
[14] Pang FH, Huang SL, Wang T, et al. Isolation and identification of endogenous Bacillus megaterium and its biocontrol effect on wheat leaf rust[A]//Papers Collection of the Annual Meeting of the Chinese Society of Plant Pathology in 2015[C]. Haikou:Chinese Society for Plant Pathology, 2015:1(in Chinese)庞发虎,黄思良,王坦,等.内生性巨大芽孢杆菌的分离、鉴定及其对小麦叶锈病的生防效果[A]//中国植物病理学会2015年学术年会论文集[C].海口:中国植物病理学会, 2015:1
[15] Zheng WB, Shen F, Yan XM, et al. Isolation and identification of the IAA-producing and phosphate-dissolving bacteria and its promoting effects on red soil[J]. Soil, 2015, 47(2):361-368(in Chinese)郑文波,申飞,闫小梅,等.红壤中产吲哚乙酸并具解磷作用的促生菌筛选鉴定及促生效果研究[J].土壤, 2015, 47(2):361-368
[16] Wang KP, Zheng Y, Chu GY, et al. Screening of bacterial strains for phosphate solubilization, nitrogen fixation and IAA production and their promotive effects on plant growth[J]. Jiangsu Journal of Agricultural Sciences, 2013, 29(6):1352-1359(in Chinese)王奎萍,郑颖,褚光耀,等.解磷、固氮、产吲哚乙酸微生物菌株的筛选及其对植物的促生效果[J].江苏农业学报, 2013,29(6):1352-1359
[17] Taguchi F, Suzuki T, Inagaki Y, et al. The siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection[J]. Journal of Bacteriology, 2010,192(1):117-126
[18] Lalitha Kumari B, Lalitha R, Sudhakar P. Studies on isolation,purification and molecular identification of pectinase producing bacteria[J]. International Journal of Advanced Research, 2013,1(5):204-212
[19] Chen LY, Zhang GX, Huang CP, et al. Isolation, identification and enzymatic characteristics of cellulose-producing strains with high cellulase activity[J]. Microbiology China, 2011, 38(4):531-538(in Chinese)陈丽燕,张光祥,黄春萍,等.两株高产纤维素酶细菌的筛选、鉴定及酶学特性[J].微生物学通报, 2011, 38(4):531-538
[20] Park JW, Balaraju K, Kim JW, et al. Systemic resistance and growth promotion of chili pepper induced by an antibiotic producing Bacillus vallismortis strain BS07[J]. Biological Control, 2013, 65(2):246-257
[21] Noh SW, Seo R, Park JK, et al. Cyclic dipeptides from Bacillus vallismortis BS07 require key components of plant immunity to induce disease resistance in Arabidopsis against Pseudomonas infection[J]. The Plant Pathology Journal, 2017, 33(4):402-409
[22] Probanza A, Lucas Garc?a?JA, Palomino MR, et al. Pinus pinea L.seedling growth and bacterial rhizosphere structure after inoculation with PGPR Bacillus(B. licheniformis CECT 5106 and B. pumilus CECT 5105)[J]. Applied Soil Ecology, 2002, 20(2):75-84
[23] Cao JJ, Yang HY, Xie XS, et al. Control effect of Bacillus amyloliquefaciens on wheat yellow mosaic disease[J]. Journal of Plant Protection, 2016, 43(4):588-593(in Chinese)曹晶晶,杨海艳,谢咸升,等.解淀粉芽胞杆菌对小麦黄花叶病的生物防治[J].植物保护学报, 2016, 43(4):588-593
[24] Xiong WD, Liu Y, Wang GW, et al. Effects of plant growth-promoting rhizobacteria agents application on wheat in sandy soil[J]. Journal of Triticeae Crops, 2016, 36(7):945-950(in Chinese)熊伟东,刘晔,王国文,等.促生菌剂在砂质潮土麦田的应用效果[J].麦类作物学报, 2016, 36(7):945-950
[25] Allaoua H, Silini A, Yahiaoui B, et al. Phylogenetic and plant-growth-promoting characteristics of Bacillus isolated from the wheat rhizosphere[J]. Annals of Microbiology, 2016, 66(3):1087-1097
[26] Shoebitz M, Ribaudo CM, Pardo MA, et al. Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere[J]. Soil Biology and Biochemistry, 2009, 41(9):1768-1774
[27] Yousaf S, Afzal M, Reichenauer TG, et al. Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains[J].Environmental Pollution, 2011, 159(10):2675-2683
[28] Sarron E, Clément N, Pawlicki-Jullian N, et al. Stimulating effects of two plant growth-promoting bacteria, Enterobacter ludwigii Ez-185-17 and Raoultella terrigena Ez-555-6, on flax culture[J]. AIP Conference Proceedings, 2018:1954(1):020003
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