玉米根际溶磷菌Bucillus megaterium XS2的筛选鉴定与溶磷特性分析
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摘要
为了充分挖掘土壤"磷库"资源,减少化学磷肥的施用量,减轻磷肥施用导致的农业面源污染问题,以PVK培养基为分离培养基从玉米根际土壤中分离到1株溶磷菌XS2,结合菌落形态特征、生理生化试验和16S r DNA方法对其进行分类鉴定;利用L25(53)正交设计试验,从碳源、氮源及p H对菌株XS2溶磷培养条件进行优化;利用优化后的PVK培养基测定了菌株XS2对多种难溶性磷源的溶解特点;采用高效液相色谱法分析了菌株XS2发酵液中有机酸的变化,对其溶磷机制进行初步分析。结果表明:溶磷菌株XS2为巨大芽孢杆菌(Bucillus megaterium)。正交试验结果表明:在以葡萄糖为碳源,硫酸铵为氮源,p H 5条件下溶磷量最高,为401.33 mg/L;菌株XS2对Ca3(PO4)2、Al3(PO4)2、Fe PO4和磷矿粉均有较好的溶解能力;发酵液中含有草酸、苹果酸、柠檬酸、乳酸、乙酸、琥珀酸和几种未知酸,其中有机酸的种类和含量随培养时间的变化而不同,柠檬酸、乳酸、乙酸和草酸可能是影响菌株溶磷的重要有机酸,试验获得了1株溶解难溶性磷的巨大芽孢杆菌XS2,它能够将多种难溶性磷转化为植物可利用的有效磷,从而增加土壤中的有效磷含量,提高作物产量,为从源头上解决磷肥施用导致的农业面源污染问题提供材料。
In order to fully mobilize phosphorus resources of soil,reduce the application amount of chemical phosphorus fertilizer,and then reduce agricultural non-point source pollution caused by the application of phosphate fertilizer,one phosphate-solubilizing bacterium named XS2 was isolated fromrhizosphere soil of maize by PVK medium and identified by colony morphology characteristics,physiological and biochemical tests and 16 S rD NA sequence analysis. The phosphate-solubilizing conditions were optimized in 3 aspects of carbon source,nitrogen source and p H value by using L25( 53)orthogonal tests,the dissolution characteristics of strain XS2 were determined for a variety of insoluble phosphate sources,the changes of organic acids in the fermentation broth of strain XS2 were analyzed by HPLC and the phosphate-dissolving mechanism was primarily analyzed. The results show that strain XS2 belonged to Bucillus megaterium,and the phosphate-solubilizing capacity reached the highest level of 401. 33 mg/L under the conditions of glucose as carbon source,ammonium sulfate as nitrogen source and p H 5. The strain had better ability to dissolve Ca3( PO4)2,Al3( PO4)2,Fe PO4 and phosphate powder. Oxalic acid,malic acid,citric acid,lactic acid,acetic acid,acetic anhydride and several unknown acids were determined by HPLC and the kinds and contents of organic acids in culture medium were different with the change of culture time. Citric acid,lactic acid,acetic acid and oxalic acid seemed to be the major organic acids which affect the dissolution of phosphorus. A strain of Bacillus megaterium XS2 was obtained and identified as P. decumbens. It can convert insoluble phosphorus into available phosphorus for plants,increasing content of effective phosphorus in soil and crop yield and providing data and theoretical basis for the solution of widespread pollution caused by the overuse of phosphate fertilizer from the source.
In order to fully mobilize phosphorus resources of soil,reduce the application amount of chemical phosphorus fertilizer,and then reduce agricultural non-point source pollution caused by the application of phosphate fertilizer,one phosphate-solubilizing bacterium named XS2 was isolated fromrhizosphere soil of maize by PVK medium and identified by colony morphology characteristics,physiological and biochemical tests and 16 S rD NA sequence analysis. The phosphate-solubilizing conditions were optimized in 3 aspects of carbon source,nitrogen source and p H value by using L25( 53)orthogonal tests,the dissolution characteristics of strain XS2 were determined for a variety of insoluble phosphate sources,the changes of organic acids in the fermentation broth of strain XS2 were analyzed by HPLC and the phosphate-dissolving mechanism was primarily analyzed. The results show that strain XS2 belonged to Bucillus megaterium,and the phosphate-solubilizing capacity reached the highest level of 401. 33 mg/L under the conditions of glucose as carbon source,ammonium sulfate as nitrogen source and p H 5. The strain had better ability to dissolve Ca3( PO4)2,Al3( PO4)2,Fe PO4 and phosphate powder. Oxalic acid,malic acid,citric acid,lactic acid,acetic acid,acetic anhydride and several unknown acids were determined by HPLC and the kinds and contents of organic acids in culture medium were different with the change of culture time. Citric acid,lactic acid,acetic acid and oxalic acid seemed to be the major organic acids which affect the dissolution of phosphorus. A strain of Bacillus megaterium XS2 was obtained and identified as P. decumbens. It can convert insoluble phosphorus into available phosphorus for plants,increasing content of effective phosphorus in soil and crop yield and providing data and theoretical basis for the solution of widespread pollution caused by the overuse of phosphate fertilizer from the source.
引文
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[3]尹秀玲,张璐,贾丽,等.玉米秸秆生物炭对暗棕壤性质和氮磷吸附特性的影响[J].吉林农业大学学报,2016,38(4):439-445.
[4]Teymouri A,Akhtari J,Karkhane M,et al.Assessment of phosphate solubilization activity of Rhizobacteria in mangrove forest[J].Biocatalysis and Agricultural Biotechnology,2016,5(1):168-172
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[6]Matilla M A,Pizarro-Tobias P,Roca A,et al.Complete genome of the plant growth-promoting rhizobacterium Pseudomonas putida BIRD-1[J].Journal Bactetial,2011,193(5):1290.
[7]Jiang Chun-yu,Sheng Xia-fang,Qian Meng,et al.Isolation and characterization of a heavy metal-resistant Burkholderia sp.from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil[J].Chemosphere,2008,72:157-164
[8]钟传青,黄为一.磷细菌P17对不同来源磷矿粉的溶磷作用及机制[J].土壤学报,2004,41(6):931-937.
[9]Chen Y P,Rekha P D,Arun A B,et al.Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities[J].Applied Soil Ecology,2006,34(1):33-41.
[10]Yi Y,Huang W,Ge Y.Exopolysaccharide:A novel important factor in the microbial dissolution of tricalcium phosphate[J].World Journal of Microbiology and Biotechnology,2008,24(7):1059-1065
[11]吴海燕,全荣德,范作伟,等.解磷巨大芽孢杆菌(Bacillus megaterium)的溶磷机理探讨[J].吉林农业大学学报,2014,36(2):171-175.
[12]张建峰,苗天瑶,张洁婧,等.盐碱地中溶磷真菌的筛选及培养条件优化[J].吉林农业大学学报,2016,38(2):169-174.
[13]史发超,殷中伟,江红梅,等.一株溶磷真菌筛选鉴定及其溶磷促生效果[J].微生物学报,2014,54(11):1333-1343.
[14]刘文干,何园球,张坤,等.一株红壤溶磷菌的分离、鉴定及溶磷特性[J].微生物学报,2012,52(3):326-333.
[15]中国科学院微生物研究所.伯杰氏细菌鉴定手册[M].9版.北京:科学出版社,1994:3-112.
[16]黄静,盛下放,何琳燕.具溶磷能力的植物内生促生细菌的分离筛选及其生物多样性[J].微生物学报,2010,50(6):710-716.
[17]Hamdali H,Bouizgarne B,Hafidi M,et al.Screening for rock phosphate solubilizing Actinomycetes from Moroccan phosphate mines[J].Applied Soil Ecology,2008,38:12-19.
[18]刘文干,曹慧,樊建波,等.一株红壤花生根际溶磷真菌的分离、鉴定及溶磷能力的研究[J].土壤学报,2012,49(5):988-995.
[19]卫星,徐鲁荣,张丹,等.一株耐硝酸盐的巨大芽孢杆菌溶磷特性研究[J].环境科学学报,2015,35(7):2052-2058.
[20]朱颖,姚拓,李玉娥,等.红三叶根际溶磷菌分离及其溶磷机制初探[J].草地学报,2009,17(2):259-263.
[21]Ghosh R,Barman S,Mukherjee R,et al.Role of phosphate solubilizing Burkholderia spp.for successful colonization and growth promotion of Lycopodiun cernuum L.(Lycopodiaceae)in lateritic belt of Birbhum district of West Bengal,India[J].Microbiological Research,2016,183:80-91.
[22]Perez E,Sulbaran M,Ball M M,et al.Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region[J].Soil Biol Biochem,2007,39:2905-2914.
[23]Gothwal R K,Nigam V K,Mohan M K,et al.Phosphate solubilization by rhizospheric bacterial isolatesfrom economically important desert plants[J].Indian Journal of Microbiology,2006,46(4):355-361.
[24]Rashid M,Khalil S,Ayub N,et al.Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms(PSM)under in vitro conditions[J].Pakistan Journal of Biological Sciences,2004,7:187-196.
[25]Yadav H,Gothwal P K,Nigam V K,et al.Optimization of culture conditions for phosphate solubilizationby a thermo-tolerant phosphate-solubilizing bacterium Brevibacillus sp.BISR-HY65 isolated from phosphate mines[J].Biocatalysis and Agricultural Biotechnology,2013,2(3):217-225.
[26]Montenegro A,Zapata F.Rape genotypic differences in P uptake and utilization from phosphate rocks in an Andisol of Chile[J].Nutrient Cycling in Agroecosystems,2002,63(1):27-33.
[27]Reyes I,Bernier L,Simard R R,et al.Effect of nitrogen source on the solubilization of different inorganic phosphates by an isolate of Penicillium rugulosum and two UV-induced mutants[J].FEMS Microbiology Ecology,1999,28(3):281-290.
[28]贺梦醒,高毅,胡正雪,等.解磷菌株B25的筛选、鉴定及其解磷能力[J].应用生态学报,2012,23(1):235-239.
[29]冯瑞章,姚拓,周万海,等.不同生存环境和磷酸盐对4株溶磷菌溶磷能力的影响[J].应用与环境生物学报,2009,15(6):856-860.
[30]Vora M S,Shelath H N.Impact of addition of different carbon and nitrogen sources on solubization of rock phosphate by phosphate-solubilizing micro-organisms[J].Journal of Pineal Research,1998,68(6):292-294.
[31]林启美,赵小蓉,孙众鑫,等.四种不同生态系统的土壤中解磷细菌的数量及种群分布[J].土壤与环境,2000,9(1):34-37.
[32]王同,孔令雅,焦加国,等.红壤溶磷菌的筛选及溶磷机制[J].土壤学报,2014,51(2):373-380.
[33]杨慧,范丙全,龚明波,等.一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究[J].微生物学报,2008,48(1):51-56.
[34]王光华,周克琴,赵英.黑土区高效溶磷真菌筛选及其溶解磷矿粉效果的研究[J].中国农业生态学报,2003,12(3):144-145.
[35]赵小蓉,林启美,李保国.溶磷菌对4种难溶性磷酸盐溶解能力的初步研究[J].微生物学报,2002,42(2):236-241.
[36]Narsian V,Ahmed ASSM,Patel H H.Rock phosphate dissolution by specific yeast[J].Indian Journal of Microbiology,2010,50(1):57-62.
[37]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[J].微生物学通报,2002,29(6):24-28.
[38]杨芳,何园球,李成亮.不同施肥条件下红壤旱地磷素形态及有效性分析[J].土壤学报,2006,43(5):794-799.
[2]王光华,周克琴,金剑,等.不同碳源对三种溶磷真菌解磷矿粉能力的影响[J].生态学杂志,2004,23(2):32-36.
[3]尹秀玲,张璐,贾丽,等.玉米秸秆生物炭对暗棕壤性质和氮磷吸附特性的影响[J].吉林农业大学学报,2016,38(4):439-445.
[4]Teymouri A,Akhtari J,Karkhane M,et al.Assessment of phosphate solubilization activity of Rhizobacteria in mangrove forest[J].Biocatalysis and Agricultural Biotechnology,2016,5(1):168-172
[5]Kaplan D,Maymon M,Agapakis C,et al.A survey of the microbial community in the rhizosphere of two dominant shrubs of the Negev Desert highlands Zygophyllum dumosum(Zygophyllaceae)and Atriplex halimus(Amaranthaceae),using cultivation-dependent and cultivation-independent methods[J].American Journal of Botany,2013,100(100):1713-1725
[6]Matilla M A,Pizarro-Tobias P,Roca A,et al.Complete genome of the plant growth-promoting rhizobacterium Pseudomonas putida BIRD-1[J].Journal Bactetial,2011,193(5):1290.
[7]Jiang Chun-yu,Sheng Xia-fang,Qian Meng,et al.Isolation and characterization of a heavy metal-resistant Burkholderia sp.from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil[J].Chemosphere,2008,72:157-164
[8]钟传青,黄为一.磷细菌P17对不同来源磷矿粉的溶磷作用及机制[J].土壤学报,2004,41(6):931-937.
[9]Chen Y P,Rekha P D,Arun A B,et al.Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities[J].Applied Soil Ecology,2006,34(1):33-41.
[10]Yi Y,Huang W,Ge Y.Exopolysaccharide:A novel important factor in the microbial dissolution of tricalcium phosphate[J].World Journal of Microbiology and Biotechnology,2008,24(7):1059-1065
[11]吴海燕,全荣德,范作伟,等.解磷巨大芽孢杆菌(Bacillus megaterium)的溶磷机理探讨[J].吉林农业大学学报,2014,36(2):171-175.
[12]张建峰,苗天瑶,张洁婧,等.盐碱地中溶磷真菌的筛选及培养条件优化[J].吉林农业大学学报,2016,38(2):169-174.
[13]史发超,殷中伟,江红梅,等.一株溶磷真菌筛选鉴定及其溶磷促生效果[J].微生物学报,2014,54(11):1333-1343.
[14]刘文干,何园球,张坤,等.一株红壤溶磷菌的分离、鉴定及溶磷特性[J].微生物学报,2012,52(3):326-333.
[15]中国科学院微生物研究所.伯杰氏细菌鉴定手册[M].9版.北京:科学出版社,1994:3-112.
[16]黄静,盛下放,何琳燕.具溶磷能力的植物内生促生细菌的分离筛选及其生物多样性[J].微生物学报,2010,50(6):710-716.
[17]Hamdali H,Bouizgarne B,Hafidi M,et al.Screening for rock phosphate solubilizing Actinomycetes from Moroccan phosphate mines[J].Applied Soil Ecology,2008,38:12-19.
[18]刘文干,曹慧,樊建波,等.一株红壤花生根际溶磷真菌的分离、鉴定及溶磷能力的研究[J].土壤学报,2012,49(5):988-995.
[19]卫星,徐鲁荣,张丹,等.一株耐硝酸盐的巨大芽孢杆菌溶磷特性研究[J].环境科学学报,2015,35(7):2052-2058.
[20]朱颖,姚拓,李玉娥,等.红三叶根际溶磷菌分离及其溶磷机制初探[J].草地学报,2009,17(2):259-263.
[21]Ghosh R,Barman S,Mukherjee R,et al.Role of phosphate solubilizing Burkholderia spp.for successful colonization and growth promotion of Lycopodiun cernuum L.(Lycopodiaceae)in lateritic belt of Birbhum district of West Bengal,India[J].Microbiological Research,2016,183:80-91.
[22]Perez E,Sulbaran M,Ball M M,et al.Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region[J].Soil Biol Biochem,2007,39:2905-2914.
[23]Gothwal R K,Nigam V K,Mohan M K,et al.Phosphate solubilization by rhizospheric bacterial isolatesfrom economically important desert plants[J].Indian Journal of Microbiology,2006,46(4):355-361.
[24]Rashid M,Khalil S,Ayub N,et al.Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms(PSM)under in vitro conditions[J].Pakistan Journal of Biological Sciences,2004,7:187-196.
[25]Yadav H,Gothwal P K,Nigam V K,et al.Optimization of culture conditions for phosphate solubilizationby a thermo-tolerant phosphate-solubilizing bacterium Brevibacillus sp.BISR-HY65 isolated from phosphate mines[J].Biocatalysis and Agricultural Biotechnology,2013,2(3):217-225.
[26]Montenegro A,Zapata F.Rape genotypic differences in P uptake and utilization from phosphate rocks in an Andisol of Chile[J].Nutrient Cycling in Agroecosystems,2002,63(1):27-33.
[27]Reyes I,Bernier L,Simard R R,et al.Effect of nitrogen source on the solubilization of different inorganic phosphates by an isolate of Penicillium rugulosum and two UV-induced mutants[J].FEMS Microbiology Ecology,1999,28(3):281-290.
[28]贺梦醒,高毅,胡正雪,等.解磷菌株B25的筛选、鉴定及其解磷能力[J].应用生态学报,2012,23(1):235-239.
[29]冯瑞章,姚拓,周万海,等.不同生存环境和磷酸盐对4株溶磷菌溶磷能力的影响[J].应用与环境生物学报,2009,15(6):856-860.
[30]Vora M S,Shelath H N.Impact of addition of different carbon and nitrogen sources on solubization of rock phosphate by phosphate-solubilizing micro-organisms[J].Journal of Pineal Research,1998,68(6):292-294.
[31]林启美,赵小蓉,孙众鑫,等.四种不同生态系统的土壤中解磷细菌的数量及种群分布[J].土壤与环境,2000,9(1):34-37.
[32]王同,孔令雅,焦加国,等.红壤溶磷菌的筛选及溶磷机制[J].土壤学报,2014,51(2):373-380.
[33]杨慧,范丙全,龚明波,等.一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究[J].微生物学报,2008,48(1):51-56.
[34]王光华,周克琴,赵英.黑土区高效溶磷真菌筛选及其溶解磷矿粉效果的研究[J].中国农业生态学报,2003,12(3):144-145.
[35]赵小蓉,林启美,李保国.溶磷菌对4种难溶性磷酸盐溶解能力的初步研究[J].微生物学报,2002,42(2):236-241.
[36]Narsian V,Ahmed ASSM,Patel H H.Rock phosphate dissolution by specific yeast[J].Indian Journal of Microbiology,2010,50(1):57-62.
[37]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[J].微生物学通报,2002,29(6):24-28.
[38]杨芳,何园球,李成亮.不同施肥条件下红壤旱地磷素形态及有效性分析[J].土壤学报,2006,43(5):794-799.