设施农业土壤中聚磷菌的筛选及其生物学特性分析
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摘要
为在设施农业土壤中筛选获得兼具高效除磷能力和良好环境适应性的聚磷菌(Phosphorus accumulating organisms,PAOs),从设施大棚采集40 cm以上土样,梯度稀释后经PAOs平板初筛、异染粒染色复筛聚磷菌,结合形态学和16S rRNA测序鉴定种属。采用钼锑抗分光光度计法测定聚磷菌的除磷率和菌体磷含量,PCR验证聚磷作用关键基因ppk。采用活菌计数法分析聚磷菌的耐盐和耐高温率,对峙培养法分析对病原菌的抑菌率。本研究共筛选得到7株聚磷菌,经鉴定分别属于节杆菌(Arthrobacter spp.)、微杆菌(Microbacterium spp.)、红球菌(Rhodococcus spp.)和芽孢杆菌(Bacillus spp.)4个菌属。好氧条件下,所有菌株均具有除磷作用,其中A. ureafaciens Fp64的除磷率最高(70.52%),R. rhodochrous Fp31的菌体含磷量最高(18.74%)。在除磷率大于50%的4株聚磷菌中,均可扩增获得ppk基因。所筛选聚磷菌兼具多种其他生物学特性,其中A. ureafaciens Fp64和A.keyseri Fp38具有病原菌拮抗和耐盐性,M. esteraromaticum Np20具有病原菌拮抗和耐高温性,R. rhodochrous Fp31具有极其显著的耐盐性。本研究所筛选的菌株具有较高的除磷能力和菌体含磷量,兼具拮抗病原菌和良好环境适应性等特点,有望用于控制土壤渗漏液磷流失,为进一步研发针对设施农业土壤环境的高效生物除磷技术提供了优良菌种。
The objective of this study was to isolate efficient phosphorus accumulating organisms(PAOs)with good environment adaptability. Soil samples(0～40 cm depth)were collected from within greenhouses. The PAOs were isolated and screened by serial dilution, PAOplate culturing, and metachromatic granule staining. Taxonomic identification of PAO isolates was based on morphological characters and16 S rRNA sequencing. Phosphorus removal and phosphorus contents of these PAOs were determined by the Mo-Sb colorimetric method.Gene ppk, the key gene in phosphorus-accumulation, was amplified using PCR. Salt and high temperature tolerances were analyzed by counting the number of surviving bacteria following exposure. Antagonistic activity against pathogens was determined by a confrontation test.Seven bacterial PAOs belonging to 4 genera, Arthrobacter, Microbacterium, Rhodococcus, and Bacillus were screened.All strains showed phosphorus-removal effects, of which A. ureafaciens Fp64 showed the largest removal effect(70.52%), and R. rhodochrous Fp31 had the highest phosphorus content(18.74%). The ppk gene was detected in 4 strains with phosphorus removal rate >50%. The PAO strains also showed variable biological characters. A. ureafaciens Fp64 and A. keyseri Fp38 were salt tolerant and antagonistic to pathogens. M. esteraromaticum Np20 showed antagonistic activities and high temperature tolerance. R. rhodochrous Fp31 exhibited prominent salt tolerance. The PAO strains screened in this study had high phosphorus contents and phosphorus removal capacities. Furthermore, they exhibited variable features, including antagonistic activity and environmental adaptability. They are expected to be used to reduce phosphorus loss from soil leaching and act as good agents for biological phosphorus removal technology in the greenhouse industry.
The objective of this study was to isolate efficient phosphorus accumulating organisms(PAOs)with good environment adaptability. Soil samples(0～40 cm depth)were collected from within greenhouses. The PAOs were isolated and screened by serial dilution, PAOplate culturing, and metachromatic granule staining. Taxonomic identification of PAO isolates was based on morphological characters and16 S rRNA sequencing. Phosphorus removal and phosphorus contents of these PAOs were determined by the Mo-Sb colorimetric method.Gene ppk, the key gene in phosphorus-accumulation, was amplified using PCR. Salt and high temperature tolerances were analyzed by counting the number of surviving bacteria following exposure. Antagonistic activity against pathogens was determined by a confrontation test.Seven bacterial PAOs belonging to 4 genera, Arthrobacter, Microbacterium, Rhodococcus, and Bacillus were screened.All strains showed phosphorus-removal effects, of which A. ureafaciens Fp64 showed the largest removal effect(70.52%), and R. rhodochrous Fp31 had the highest phosphorus content(18.74%). The ppk gene was detected in 4 strains with phosphorus removal rate >50%. The PAO strains also showed variable biological characters. A. ureafaciens Fp64 and A. keyseri Fp38 were salt tolerant and antagonistic to pathogens. M. esteraromaticum Np20 showed antagonistic activities and high temperature tolerance. R. rhodochrous Fp31 exhibited prominent salt tolerance. The PAO strains screened in this study had high phosphorus contents and phosphorus removal capacities. Furthermore, they exhibited variable features, including antagonistic activity and environmental adaptability. They are expected to be used to reduce phosphorus loss from soil leaching and act as good agents for biological phosphorus removal technology in the greenhouse industry.
引文
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[2]唐珧,李丽君,白光洁,等.山西南部设施蔬菜施肥及土壤氮磷累积现状调查分析[J].山西农业科学, 2017, 45(5):773-776, 785.TANG Yao, LI Li-jun, BAI Guang-jie, et al. Investigation and analysis on the current status of fertilization and soil nitrogen and phosphorus accumulation in greenhouse vegetable in southern of Shanxi[J]. Journal of Shanxi Agriculture Sciences, 2017, 45(5):773-776, 785.
[3]魏玉奎,李新平,刘瑞丰,等.设施农业土壤磷素富集的动态变化[J].干旱地区农业研究, 2011, 29(1):126-132.WEI Yu-kui, LI Xin-ping, LIU Rui-feng, et al. Dynamic changes of phosphorus enrichment in facility agricultural soil[J]. Agricultural Research in the Arid Areas, 2011, 29(1):126-132.
[4]张作新,刘建玲,廖文华,等.磷肥和有机肥对不同磷水平土壤磷渗漏影响研究[J].农业环境科学学报, 2009, 28(4):729-735.ZHANG Zuo-xin, LIU Jian-ling, LIAO Wen-hua, et al. The effect of phosphate fertilizer and manure on phosphorus leaching in different phosphorus levels soil[J]. Journal of Agro-Environment Science, 2009,28(4):729-735.
[5]金晶,高扬,王洋,等.土-水界面磷的稳定性与生物有效性对水体富营养化的影响[J].绿色科技, 2018, 4:60-65.JIN Jing, GAO Yang, WANG Yang, et al. Phosphorus stability, transformation in soil-water layer and associated impact on water eutrophication[J]. Journal of Green Science&Technology, 2018, 4:60-65.
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[7]杨栋,陈航,华玉妹,等.不同材料对河岸带土壤吸附除磷的强化能力及机制[J].环境工程, 2018, 36(2):74-77, 119.YANG Dong, CHEN Hang, HUA Yu-mei, et al. Improvement of three materials on phosphorus removal by riparian soil and the mechanism[J]. Environmental Engineering, 2018, 36(2):74-77, 119.
[8] Yang Y, Shi X, Ballent W, et al. Biological phosphorus recovery:review of current progress and future needs[J]. Water environment research:A Research Publication of the Water Environment Federation,2017, 89(12):2122-2135.
[9]刘菡.土壤中聚磷菌的聚磷特性研究[D].长春：吉林农业大学,2013.LIU Han. Study on phosphorus accumulation characteristics of polyphosphorus bacteria in soil[D]. Changchun:Jilin Agricultural University, 2013.
[10]南亚萍,周国标,袁林江.多聚磷酸盐激酶基因在污水生物除磷中的功能[J].环境科学, 2017, 38(4):1529-1535.NAN Ya-ping, ZHOU Guo-biao, YUAN Lin-jiang. Function of polyphosphate kinase gene in biological phosphate removal during the wastewater treatment process[J]. Environmental Science, 2017, 38(4):1529-1535.
[11]邵闯,袁淑博,陈晓通,等.一株高效聚磷菌的筛选鉴定及基因组分析[J].湖北大学学报（自然科学版）, 2018, 40(5):452-456.SHAO Chuang, YUAN Shu-bo, CHEN Xiao-tong, et al. Screening and identification and genomic analysis of a high-efficient polyphosphate accumulating organisms[J]. Journal of Hubei University(Natural Science Edition), 2018, 40(5):452-456.
[12]王呈玉,萧龙珍,都郁馨.兼具康抗病促生活性的溶磷聚磷菌XF1的分离、鉴定与特性分析[J].吉林农大大学学报, 2018, 40(3):290-298.WANG Cheng-yu, XIAO Long-zhen, DU Yu-xin. Isolation, identification and characteristic analysis of a phosphorus solubilizing and phosphorus accumulating bacterium XF1 with disease resistance and growth promoting activity[J]. Journal of Jilin Agricultural University,2018, 40(3):290-298.
[13]孙明辉,孙会忠,王小东,等.一株兼有β-甘露聚糖酶活性聚磷菌的分离和鉴定[J].中国烟草科学, 2016, 37(2):23-28.SUN Ming-hui, SUN Hui-zhong, WANG Xiao-dong, et al. Isolation and identification of a phosphate accumulating strain withβ-mannanase activity[J]. Chinese Tobacco Science, 2016, 37(2):23-28.
[14]邱旭.聚磷菌对矿物和黑土中不同形态磷素转化的作用研究[D].长春：吉林农业大学, 2016.QIU Xu. Study PAOs of different forms of phosphorus transformation in the role of minerals and black soil[D]. Changchun:Jilin Agricultural University, 2016.
[15] Suresh N, Warburg R, Timmerman M. New strategies for the isolation of microorganisms responsible for phosphate accumulation[J]. Waterence&Technology, 2011, 17(11):99-111.
[16]李海峰,李志建,屈建航.高效聚磷鞘氨醇杆菌XF-5的分离与鉴定[J].河南农业科学, 2012, 41(9):68-72.LI Hai-feng, LI Zhi-jian, QU Jian-hang. Isolation and identification of a high-efficient phosphate accumulating Novosphingobium sp.Strain XF-5[J]. Journal of Henan Agricultural Sciences, 2012, 41(9):68-72.
[17]蔡天明,管莉菠,崔中利,等.高效聚磷菌株GM1的分离和聚磷特性研究[J].土壤学报, 2005, 42(4):635-641.CAI Tian-ming, GUAN Li-bo, CUI Zhong-li, et al. Isolation and characterization of strain GM1 with high capability of accumulating Poly-P[J]. Acta Pedologica Sinica, 2005, 42(4):635-641.
[18] Moreno C, Romero J, Espejo R T. Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio[J]. Microbiology, 2002, 148(4):1233-1239.
[19] Li X, Yuan H, Yang J, et al. Genome sequence of the polyphosphateaccumulating organism Arthrobacter sp. strain PAO19 isolated from maize rhizosphere soil[J]. Genome Announc, 2013, 1(4):e00566-13.
[20]闫海啸,崔俊涛,田立生,等.农田黑土中聚磷菌的筛选及其聚磷特性研究[C]//中国水土保持学会2012学术年会, 2012:281-283.YAN Hai-xiao, CUI Jun-tao, TIAN Li-sheng, et al. Screening of phosphorus accumulating organisms from black soil and their characteristics of phosphorus accumulation[C]//Chinese Society of Soil and Water Conservation(2012), 2012:281-283.
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