红三叶根际溶磷菌的筛选与培养基优化
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摘要
为从岷山红三叶根际土壤中筛选出大量高效溶磷菌株,本研究采用Pikovaskaia’s、PKOC1和PKOC2三种溶磷培养基进行溶磷菌株的初步筛选和优良溶磷菌株16SrRNA基因序列鉴定,并对分离筛选效果较好的PKOC2培养基进行响应面优化。结果表明:采用Pikovaskaia’s、PKOC1和PKOC2三种溶磷培养基,从红三叶根际分离出26株溶磷菌株;经16SrRNA基因序列对7株优良溶磷菌株进行初步比对鉴定,初步鉴定结果为:菌株MHS4为蜡样芽胞杆菌(Bacillus cereus);菌株MHS7和MHS19为枯草芽胞杆菌(Bacillus subtilis);菌株MHS27为苏云金杆菌(Bacillus thuringiensis);菌株MHS30为荧光假单胞菌(Pseudomonas fluorescens);菌株MHS31为盖氏假单胞菌(Pseudomonas gessardii);菌株MHS49为产酸克雷伯菌(Klebsiella oxytoca)。PKOC2溶磷培养基优化配方为:葡萄糖18.19g·L~(-1),Ca_3(PO_4)25g·L~(-1),MgCl2·6H_2O 3.21g·L~(-1),MgSO_4·7H_2O 0.25g·L~(-1),KCl 0.2g·L~(-1),(NH_4)_2SO_40.08g·L~(-1)。红三叶根际溶磷菌资源丰富,优化后的溶磷培养基为高效溶磷菌资源筛选提供资源支持。
In order to optimize isolation and screening of phosphate solubilizing bacteria from the rhizosphere soil of Trifolium pratense(Cv.Minshan),apreliminary extraction using Pikovaskaia's,PKOC1 and PKOC2phosphate solubilizing media was carried out,and the 16 SrRNA gene sequence was determined for the bacterial strains with excellent phosphate solubilizing ability.The separation and screening effect of PKOC2 was better than that of PKOC1 and Pikovaskaia's.The composition of the culture medium was optimized using a response surface experiment design.The optimal composition so identified for the PKOC2 medium was:glucose 18.19g·L~(-1),Ca_3(PO_4)25g·L~(-1),MgCl2·6H_2O 3.21g·L~(-1),MgSO_4·7H_2O 0.25g·L~(-1),KCl 0.2g·L~(-1),(NH_4)_2SO_40.08g·L~(-1).It was found that 26 strains of phosphate solubilizing bacteria were isolated from T.pratense rhizosphere using these techniques.A preliminary identification of 7isolated bacterial strains with excellent phosphate solubilizing ability using the strains 16 SrRNA gene sequence indicated that a strain designated MHS4 was Bacillus cereus,strain MHS7 and MHS19were Bacillus subtilis,strain MHS27 was Bacillus thuringiensis,strain MHS30 was Pseudomonas fluorescens,strain MHS31 was Pseudomonas gessardii,and strain MHS49 was Klebsiella oxytoca.To summarise,phosphate-solubilizing bacteria were abundant in the rhizosphere of T.pretense were abundant,and the optimized phosphate-solubilizing medium provided support for the screening of bacterial strains with excellent phosphate-solubilizing ability.
In order to optimize isolation and screening of phosphate solubilizing bacteria from the rhizosphere soil of Trifolium pratense(Cv.Minshan),apreliminary extraction using Pikovaskaia's,PKOC1 and PKOC2phosphate solubilizing media was carried out,and the 16 SrRNA gene sequence was determined for the bacterial strains with excellent phosphate solubilizing ability.The separation and screening effect of PKOC2 was better than that of PKOC1 and Pikovaskaia's.The composition of the culture medium was optimized using a response surface experiment design.The optimal composition so identified for the PKOC2 medium was:glucose 18.19g·L~(-1),Ca_3(PO_4)25g·L~(-1),MgCl2·6H_2O 3.21g·L~(-1),MgSO_4·7H_2O 0.25g·L~(-1),KCl 0.2g·L~(-1),(NH_4)_2SO_40.08g·L~(-1).It was found that 26 strains of phosphate solubilizing bacteria were isolated from T.pratense rhizosphere using these techniques.A preliminary identification of 7isolated bacterial strains with excellent phosphate solubilizing ability using the strains 16 SrRNA gene sequence indicated that a strain designated MHS4 was Bacillus cereus,strain MHS7 and MHS19were Bacillus subtilis,strain MHS27 was Bacillus thuringiensis,strain MHS30 was Pseudomonas fluorescens,strain MHS31 was Pseudomonas gessardii,and strain MHS49 was Klebsiella oxytoca.To summarise,phosphate-solubilizing bacteria were abundant in the rhizosphere of T.pretense were abundant,and the optimized phosphate-solubilizing medium provided support for the screening of bacterial strains with excellent phosphate-solubilizing ability.
引文
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[9] Meng X J,Yu L P,Cheng W D,et al.Influence of Rhizobia inoculation and nitrogen fertilizer application on isoflavonoides content of Minshan red clover.Pratacultural Science,2010,27(5):97-100.孟祥君,俞联平,程文定,等.接种根瘤菌与施肥对岷山红三叶异黄酮含量的影响.草业科学,2010,27(5):97-100.
[10] Han T H,Cheng W D,Yu L P,et al.Current situation and strategies to the development of local characterized grass industry in high and cold regions of southern Dingxi.Pratacultural Science,2007,24(11):6-10.韩天虎,程文定,俞联平,等.定西南部高寒阴湿地区特色草产业发展现状及对策探讨.草业科学,2007,24(11):6-10.
[11] Yue Y.Studies on the effects of different enviroments and fertilizing rates on hay yield and isoflavone content of Trifolium pratense cv.Minshan.Lanzhou:Gansu Agricultural University,2007.岳燕.不同生境和施肥量对岷山红三叶草产量及异黄酮含量影响的研究.兰州:甘肃农业大学,2007.
[12] Ma W B,Yao T,Wang G J,et al.Assessment of rhizobacteria strains for Vicia sativa.Scientia Agricultura Sinica,2014,23(5):241-248.马文彬,姚拓,王国基,等.根际促生菌筛选及其接种剂对箭筈豌豆生长影响的研究.草业学报,2014,23(5):241-248.
[13] Fan B Q,Jin J Y,Ge C.Isolation of penicillium oxalicum and its effect on solubilization of insoluble phosphate under different conditions.Scientia Agricultura Sinica,2002,35(5):525-530.范丙全,金继运,葛诚.溶磷草酸青霉菌筛选及其溶磷效果的初步研究.中国农业科学,2002,35(5):525-530.
[14] Gupta R,Singal R,Shankar A,et al.A modified plate assay for screening phosphate solubilizing microorganisms.The Journal of General and Applied Microbiology,1994,40(3):255-260.
[15] Lin Q M,Zhao H Y,Zhao X R.The characteristics of solubilizing rock phosphate by four isolates of bacteria and fungi.Microbiology China,2002,29(6):24-28.林启美,赵海英,赵小蓉.4株溶磷细菌和真菌溶解磷矿粉的特性.微生物学通报,2002,29(6):24-28.
[16] Qi W J,Long R J,Zhou W H,et al.Study on the phosphate solubilization ability of phosphate solubilizing bacteria in five different mediums.Grassland and Turf,2007,124:37-41.齐文娟,龙瑞军,周万海,等.溶磷菌在5种不同培养基中溶解磷矿粉的性能比较.草原与草坪,2007,124:37-41.
[17] Rong L Y.Beneficial characteristics of PGPR fromTrifolium pretense L.cv.Minshan.Lanzhou:Gansu Agricultural University,2015.荣良燕.岷山红三叶根际促生菌及有益特性研究.兰州:甘肃农业大学,2015.
[18] Tian H,Yao T,Zhang D G.Primary study on isolation and ability of phosphorus-solubilizing bacteria in rhizosphere of turfgrass in Lanzhou.Grassland and Turf,2004,4:39-41.田宏,姚拓,张德罡.兰州地区草坪草根际溶磷菌分离及溶磷能力初步测定.草原与草坪,2004,4:39-41.
[19] Chen J,Lu J K,Kang L H,et al.Primary identification,cvapability of phosphatesolubilization and optimization of medium of some microorganism from mangrove.Microbiology China,2009,36(8):1183-1188.陈俊,陆俊棍,康丽华,等.红树林溶磷菌的初步鉴定、溶磷能力测定及其优化培养.微生物学通报,2009,36(8):1183-1188.
[20] Ma R Y,Zhang A M,Hui X S,et al.Screening,identification and sporulation conditions optimization of NX-11strain having the ability of solubilizing phosphorus and potassium.Acta Agriculturae Boreali-Sinica,2013,28(2):202-208.麻瑞阳,张爱民,惠小双,等.高效解磷解钾菌NX-11菌株的分离筛选、鉴定及最佳培养条件的确定.华北农学报,2013,28(2):202-208.
[21] Huang D M,Li Q,Guan G Q,et al.Selection,identification and medium optimization of a phosphate solubilizing bacterium.Biotechnology Bulletin,2015,31(2):173-178.黄达明,李倩,管国强,等.一株解磷细菌的筛选、鉴定及其溶磷培养条件的优化.生物技术通报,2015,31(2):173-178.
[22] Liu Y H,Wu Y X,Yang S C,et al.Screening of phosphorus-solubilizing strain Burkholderia cenocepacia and optimizing of phosphate-dissolving culture condition.Journal of South China Agricultural University,2015,36(3):78-82.刘云华,吴毅歆,杨绍聪,等.洋葱伯克霍尔德溶磷菌的筛选和溶磷培养条件优化.华南农业大学学报,2015,36(3):78-82.
[23] Shekhar Nautiyal C.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms.Federation of European Microbiological Societies,1999,170:265-270.
[24] Gao X,Qiao S,Lu W.Determination of an economical medium for growth of Lactobacillus fermentumusing response surface methodology.Letters in Applied Microbiology,2009,49:556-561.
[25] Lotfy W A,Ghanem K M,El-Helow E R.Citric acid production by a novel Aspergillus niger isolate:II.Optimization of process parameters through statistical experimental designs.Bioresource Technology,2007,98:3470-3477.
[26] Leyval C,Berthelin J.Interactions between Laccaria laccata,Agrobacterium radiobacter and beech roots:Influence on P,K,Mg,and Fe mobilization from minerals and plant growth.Plant and Soil,1989,117:103-110.
[27] Chen Y,Rekha P,Arun A,et al.Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities.Applied Soil Ecology,2006,34(1):33-41.
[2] Liu J L,Fang F,Shi X H,et al.Isolation and characterization of PGPR from the rhizosphere of the Avena sativain saline-alkali soil.Acta Prataculturae Sinica,2013,22(2):132-139.刘佳莉,方芳,史煦涵,等.2株盐碱地燕麦根际促生菌的筛选及其促生作用研究.草业学报,2013,22(2):132-139.
[3] Aslantas R,Cakmakci R,sahin F.Effect of plant growth promoting rhizobacteria on young apple tree growth and fruit yield under orchard conditions.Scientia Horticulturae,2007,111:371-377.
[4] Yao T,Pu X P,Zhang D G,et al.Associative nitrogen-fixing bacteria in the rhizosphere of Avena sativain an alpine regionⅢEffect on Avena sativa growth and quantification of nitrogen fixed.Acta Prataculturae Sinica,2004,13(5):101-105.姚拓,蒲小鹏,张德罡,等.高寒地区燕麦根际联合固氮菌研究Ⅲ固氮菌对燕麦生长的影响及其固氮量测定.草业学报,2004,13(5):101-105.
[5] Rong L Y,Chai Q,Yao T,et al.Partial replacement of chemical fertilizer by compound microbial inoculant and potential for promoting growth of intercropped Zea mays and Pisum sativum.Acta Prataculturae Sinica,2015,24(2):22-30.荣良燕,柴强,姚拓,等.复合微生物接种剂替代部分化肥对豌豆间作玉米的促生效应.草业学报,2015,24(2):22-30.
[6] Yanni Y G,Rizk R Y,El-Fattah F K,et al.The beneficial plant growth-promoting association of Rhizobium leguminosarum bv.trifolii with rice roots.Functional Plant Biology,2001,28:845-870.
[7] Turan M,Gulluce M,Karadayi M,et al.Role of soil enzymes produced by PGPR strains in wheat growth and nutrient uptake parameters in the filed conditions.Current Opinion in Biotechnology,2011,22:133-140.
[8] Udayashankar A,Chandra N S,Reddy M,et al.Plant growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv.Oryzae.Biological Control,2011,59:114-122.
[9] Meng X J,Yu L P,Cheng W D,et al.Influence of Rhizobia inoculation and nitrogen fertilizer application on isoflavonoides content of Minshan red clover.Pratacultural Science,2010,27(5):97-100.孟祥君,俞联平,程文定,等.接种根瘤菌与施肥对岷山红三叶异黄酮含量的影响.草业科学,2010,27(5):97-100.
[10] Han T H,Cheng W D,Yu L P,et al.Current situation and strategies to the development of local characterized grass industry in high and cold regions of southern Dingxi.Pratacultural Science,2007,24(11):6-10.韩天虎,程文定,俞联平,等.定西南部高寒阴湿地区特色草产业发展现状及对策探讨.草业科学,2007,24(11):6-10.
[11] Yue Y.Studies on the effects of different enviroments and fertilizing rates on hay yield and isoflavone content of Trifolium pratense cv.Minshan.Lanzhou:Gansu Agricultural University,2007.岳燕.不同生境和施肥量对岷山红三叶草产量及异黄酮含量影响的研究.兰州:甘肃农业大学,2007.
[12] Ma W B,Yao T,Wang G J,et al.Assessment of rhizobacteria strains for Vicia sativa.Scientia Agricultura Sinica,2014,23(5):241-248.马文彬,姚拓,王国基,等.根际促生菌筛选及其接种剂对箭筈豌豆生长影响的研究.草业学报,2014,23(5):241-248.
[13] Fan B Q,Jin J Y,Ge C.Isolation of penicillium oxalicum and its effect on solubilization of insoluble phosphate under different conditions.Scientia Agricultura Sinica,2002,35(5):525-530.范丙全,金继运,葛诚.溶磷草酸青霉菌筛选及其溶磷效果的初步研究.中国农业科学,2002,35(5):525-530.
[14] Gupta R,Singal R,Shankar A,et al.A modified plate assay for screening phosphate solubilizing microorganisms.The Journal of General and Applied Microbiology,1994,40(3):255-260.
[15] Lin Q M,Zhao H Y,Zhao X R.The characteristics of solubilizing rock phosphate by four isolates of bacteria and fungi.Microbiology China,2002,29(6):24-28.林启美,赵海英,赵小蓉.4株溶磷细菌和真菌溶解磷矿粉的特性.微生物学通报,2002,29(6):24-28.
[16] Qi W J,Long R J,Zhou W H,et al.Study on the phosphate solubilization ability of phosphate solubilizing bacteria in five different mediums.Grassland and Turf,2007,124:37-41.齐文娟,龙瑞军,周万海,等.溶磷菌在5种不同培养基中溶解磷矿粉的性能比较.草原与草坪,2007,124:37-41.
[17] Rong L Y.Beneficial characteristics of PGPR fromTrifolium pretense L.cv.Minshan.Lanzhou:Gansu Agricultural University,2015.荣良燕.岷山红三叶根际促生菌及有益特性研究.兰州:甘肃农业大学,2015.
[18] Tian H,Yao T,Zhang D G.Primary study on isolation and ability of phosphorus-solubilizing bacteria in rhizosphere of turfgrass in Lanzhou.Grassland and Turf,2004,4:39-41.田宏,姚拓,张德罡.兰州地区草坪草根际溶磷菌分离及溶磷能力初步测定.草原与草坪,2004,4:39-41.
[19] Chen J,Lu J K,Kang L H,et al.Primary identification,cvapability of phosphatesolubilization and optimization of medium of some microorganism from mangrove.Microbiology China,2009,36(8):1183-1188.陈俊,陆俊棍,康丽华,等.红树林溶磷菌的初步鉴定、溶磷能力测定及其优化培养.微生物学通报,2009,36(8):1183-1188.
[20] Ma R Y,Zhang A M,Hui X S,et al.Screening,identification and sporulation conditions optimization of NX-11strain having the ability of solubilizing phosphorus and potassium.Acta Agriculturae Boreali-Sinica,2013,28(2):202-208.麻瑞阳,张爱民,惠小双,等.高效解磷解钾菌NX-11菌株的分离筛选、鉴定及最佳培养条件的确定.华北农学报,2013,28(2):202-208.
[21] Huang D M,Li Q,Guan G Q,et al.Selection,identification and medium optimization of a phosphate solubilizing bacterium.Biotechnology Bulletin,2015,31(2):173-178.黄达明,李倩,管国强,等.一株解磷细菌的筛选、鉴定及其溶磷培养条件的优化.生物技术通报,2015,31(2):173-178.
[22] Liu Y H,Wu Y X,Yang S C,et al.Screening of phosphorus-solubilizing strain Burkholderia cenocepacia and optimizing of phosphate-dissolving culture condition.Journal of South China Agricultural University,2015,36(3):78-82.刘云华,吴毅歆,杨绍聪,等.洋葱伯克霍尔德溶磷菌的筛选和溶磷培养条件优化.华南农业大学学报,2015,36(3):78-82.
[23] Shekhar Nautiyal C.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms.Federation of European Microbiological Societies,1999,170:265-270.
[24] Gao X,Qiao S,Lu W.Determination of an economical medium for growth of Lactobacillus fermentumusing response surface methodology.Letters in Applied Microbiology,2009,49:556-561.
[25] Lotfy W A,Ghanem K M,El-Helow E R.Citric acid production by a novel Aspergillus niger isolate:II.Optimization of process parameters through statistical experimental designs.Bioresource Technology,2007,98:3470-3477.
[26] Leyval C,Berthelin J.Interactions between Laccaria laccata,Agrobacterium radiobacter and beech roots:Influence on P,K,Mg,and Fe mobilization from minerals and plant growth.Plant and Soil,1989,117:103-110.
[27] Chen Y,Rekha P,Arun A,et al.Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities.Applied Soil Ecology,2006,34(1):33-41.