辽宁海域细菌多样性及刺参养殖水域细菌组成研究
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摘要
通过构建大连湾(DL17)和辽东湾(ZD-LDW017)两个站点夏季海水的细菌16S rRNA文库及辽东湾和海参养殖池塘秋季海水的细菌16S rRNA文库,利用16S rRNA序列比对法,对四个站点海水细菌多样性进行了分析,为海水微生物资源调查和开发及海参的水产养殖提供有益的信息。结果表明这4个水域的细菌主要为非培养细菌(Uncultured bacterium)。每个海区分别随机选取了200个克隆进行酶切谱型分析和序列测定,共得到180个不同的DNA序列,其中大连湾51个序列,分别属于13种(属);辽东湾(夏季)53个序列,分别属于13种(属);辽东湾(秋季)37个序列,分别属于10种(属);海参池塘海水39个序列,分别属于12种(属)。这4个水域各自有不同的优势菌,在大连湾和辽东湾中没有检测到致病菌,研究结果表明大连湾和辽东湾海水细菌具有丰富的多样性;海参池塘海水中检测到3种与水产养殖相关的致病菌,分别为溶藻弧菌(Vibrio.alginolyticus)、副凝聚短状杆菌( Brachybacterium qaraconglomeratum)、巨大芽孢杆菌(Bacillus megaterium)和涅斯捷连科氏菌(Nesterenkonia菌),因此控制刺参养殖环境和减少致病菌,对预防和保证刺参养殖生产具有重要作用。
自2005年-2006年,连续3次从室内不同养殖时期的刺参附着基上分离优势异养细菌,以期探索刺参附着环境优势异养细菌结构与刺参发病的相关性。每次均分离出6株优势异养细菌,通过进一步对这6种优势菌株群体、个体培养特征观察、生理性状试验和16SrRNA基因序列同源性及系统发育分析表明,菌株0506-1与溶藻弧菌(Vibrio.alginolyticus)具有较高的相似性,菌株0506-2与副凝聚短状杆菌(Brachybacterium qaraconglomeratum)具有较高的相似性,菌株0506-3和0506-4具有相近的亲缘关系,两者均与Nesterenkonia sp.具有较高的相似性,菌株0506-5与巨大芽孢杆菌(Bacillus megaterium)具有较高的相似性,菌株0506-6与涅斯捷连科氏菌(Nesterenkonia菌)的相似性为99%。经人工感染试验证明,这6株菌对刺参具有不同程度的致病性。药物敏感性试验结果表明,呋南妥因、新霉素和先锋孟多对这6种优势菌株均显示高度敏感性。
Content:The diversity of bacteria in Summer sea water of Dalian Bay and Liaodong Bay and the Autumn sea water of Liaodong Bay and aquaculture pond of sea cucumber were analyzed by constructing 16S rRNA libraries of bacteria samples collected from four Survey Stations, this will provide helpful information for investigating and empoldering microbial resource and sea cucumber cultured. The results showed that there were mainly uncultured bacteria in the four Stations. In Dalian Bay there were 51 unique sequences belonging to 13 bacteria species (genera), in Liaodong Bay (summer) there were 53 unique sequences belonging to 13 bacteria species (genera), in Liaodong Bay (autumn) there were 37 unique sequences belonging to 10 bacteria species (genera) and in aquaculture pond there were 39 unique sequences belonging to 12 bacteria species (genera). Different preponderant bacteria existed in the different stations, no pathogenic bacterial species were found in Dalian Bay and Liaodong Bay stations. This indicated that the bacterial diversity exists in the two stations. We found Vibrio.alginolyticus, Brachybacterium qaraconglomeratum,Bacillus megaterium and Nesterenkonia sp. in the aquaculture pond, so we should control it and reduce baleful bacteria, this will provide helpful information for the cultured of sea cucumber.
The strains were studies using a polyphasic approach, which included bacillus culture character observation, biochemical characterization and phylogenetic analyses by direct sequencing of 16S rRNA genes, et al. The associated with dominance heterotrophic bacillus composing on accrete media of different periods in cultured Apostichopus japonicus and disease revealed that the dominance strains which were isolated continuously three times from the year 2005 to 2006 were identical. And the dominance strains mainly included Vibrio.alginolyticus, Brachybacterium qaraconglomeratum, Nesterenkonia sp., Bacillus megaterium. Manual infection tests showed that bacillus could cause different degree disease. Drugs sensitivity tests showed that these dominance bacillus were sensitive to Nitrofurantoin、Neomycinum and Cefamandde.
自2005年-2006年,连续3次从室内不同养殖时期的刺参附着基上分离优势异养细菌,以期探索刺参附着环境优势异养细菌结构与刺参发病的相关性。每次均分离出6株优势异养细菌,通过进一步对这6种优势菌株群体、个体培养特征观察、生理性状试验和16SrRNA基因序列同源性及系统发育分析表明,菌株0506-1与溶藻弧菌(Vibrio.alginolyticus)具有较高的相似性,菌株0506-2与副凝聚短状杆菌(Brachybacterium qaraconglomeratum)具有较高的相似性,菌株0506-3和0506-4具有相近的亲缘关系,两者均与Nesterenkonia sp.具有较高的相似性,菌株0506-5与巨大芽孢杆菌(Bacillus megaterium)具有较高的相似性,菌株0506-6与涅斯捷连科氏菌(Nesterenkonia菌)的相似性为99%。经人工感染试验证明,这6株菌对刺参具有不同程度的致病性。药物敏感性试验结果表明,呋南妥因、新霉素和先锋孟多对这6种优势菌株均显示高度敏感性。
Content:The diversity of bacteria in Summer sea water of Dalian Bay and Liaodong Bay and the Autumn sea water of Liaodong Bay and aquaculture pond of sea cucumber were analyzed by constructing 16S rRNA libraries of bacteria samples collected from four Survey Stations, this will provide helpful information for investigating and empoldering microbial resource and sea cucumber cultured. The results showed that there were mainly uncultured bacteria in the four Stations. In Dalian Bay there were 51 unique sequences belonging to 13 bacteria species (genera), in Liaodong Bay (summer) there were 53 unique sequences belonging to 13 bacteria species (genera), in Liaodong Bay (autumn) there were 37 unique sequences belonging to 10 bacteria species (genera) and in aquaculture pond there were 39 unique sequences belonging to 12 bacteria species (genera). Different preponderant bacteria existed in the different stations, no pathogenic bacterial species were found in Dalian Bay and Liaodong Bay stations. This indicated that the bacterial diversity exists in the two stations. We found Vibrio.alginolyticus, Brachybacterium qaraconglomeratum,Bacillus megaterium and Nesterenkonia sp. in the aquaculture pond, so we should control it and reduce baleful bacteria, this will provide helpful information for the cultured of sea cucumber.
The strains were studies using a polyphasic approach, which included bacillus culture character observation, biochemical characterization and phylogenetic analyses by direct sequencing of 16S rRNA genes, et al. The associated with dominance heterotrophic bacillus composing on accrete media of different periods in cultured Apostichopus japonicus and disease revealed that the dominance strains which were isolated continuously three times from the year 2005 to 2006 were identical. And the dominance strains mainly included Vibrio.alginolyticus, Brachybacterium qaraconglomeratum, Nesterenkonia sp., Bacillus megaterium. Manual infection tests showed that bacillus could cause different degree disease. Drugs sensitivity tests showed that these dominance bacillus were sensitive to Nitrofurantoin、Neomycinum and Cefamandde.
引文
[1] ZoBell CE. Marine Microbiology. A Monograph on Hydrobacteriology Massachusetts[M]. Chronica Botantea company,1946.
[2] Ferguson R L,Buckley E N, Palumbo AV.Response of marine bacterioplankton to differential filtration and confinement[J]. Appl Environ Microbioi, 1984,47:49～55.
[3] Hugenholtz P,Goebel B M,Pace N R.Impact of culture—independent studies on the emerslng phylogenetic view of bacterial diversity[J].J Bacteriol,1998,180:4765～4774.
[4]张春云,等.国内外海参自然资源与养殖现状[J].海洋水产研究,2004,25(3):89-93.
[5]王印庚,荣小军,张春云,孙素凤.养殖海参主要疾病及防治技术[J].海洋科学,2005,29(3):1～7.
[6]邓欢,隋锡林.仿刺参育苗期常见疾病[M].水产科学,2004,23(3):40.
[7]王印庚,方波,张春云,等.养殖刺参保苗期重大疾病“腐皮综合征”病原及其感染源分析[J].中国水产科学,2006,13(4):610～616.
[8]马悦欣,徐高蓉,张恩鹏,等.仿刺参幼参急性口围肿胀症的细菌性病原[J].水产学报,2006,30(3):377～382.
[9] Becker P,Gillan D,Lanterbecq D,et al. The Skin Ulceration Disease in Cultivatted Juveniles of Holothuria scabra(Holothuroidea, Echinodermata)[J].Aquaulture,2004,242:13～30.
[10]董颖,邓欢,隋锡林,宋伦.养殖仿刺参溃烂病病因初探[J].水产科学, 2005, 24(3):4～6.
[11]赵伟.渤海辽东湾环境状况及污染防治对策[J].辽宁城乡环境科技,19(3):4～6.
[12]刘金屏,曹文伟,张楚佳,等.大连湾石油微生物的数量分布及分类鉴定[J].辽宁师范大学学报,1986,2,38～43.
[13]徐辉,王日东.大连湾海域赤潮状况及预测模式研究[J].资源与环境,2007,87.
[14]邱春霞,邵秘华,李玉娜,等.大连湾海域生态环境现状的评估[J].大连海事大学学报,2005,31(1):77～80.
[15]于大江.近海资源保护与可持续利用[M].北京:海洋出版社,2001,78～96.
[16]李震,刘景泰.大连湾海域水体富营养状况分析[J].中国环境监测, 2000,16(1):38～41.
[17]王惠卿,杜广玉.大连市近岸海域赤潮状况、预测及防治对策[J].中国环境监测, 2000,16 (6):42～46.
[18]郑建平,王芳,华祖林.辽东湾北部河口区生态环境问题及对策[J].东北水利水电,2005,10,47～50.
[19]李建军,冯慕华,喻龙.辽东湾浅水区水环境质量现状评价[J].海洋环境科学,2001,20(3):42～45.
[20]宛立,田继辉,马志强,等.辽东湾北部海域表层水体夏季油类的污染状况[J].水产科学,2007,26(9):515～517.
[21]廖玉麟.中国动物志(第1卷)[M].北京:科学出版社,1997,61.
[22]崔清晨.海洋资源[M].北京:商务印书馆,1981,294.
[23]常亚青,丁军,宋坚等.海参、海胆生物学研究与养殖[M].海洋出版社, 2004,113～192.
[24]孙素凤.养殖刺参(Apostichopus japonicus)苗期细菌性疾病及其病原学初步研究[J].中国学术期刊,2006,13～14.
[25]王印庚,荣小军,张春云,等.养殖海参主要疾病及防治技术[J].海洋科学,2005(3):1～7.
[26]李琳,李槿年,余为一.细菌分类鉴定方法的研究概况[J].安徽农业科学,2004,32(3):549～551.
[27]阎东辉,许淑珍,马纪平,等. VITEK- AMS在临床细菌鉴定中的应用[J].中华实用医学,2000,(10):9～10.
[28]姜远良.化学分类法在细菌和真菌系统分类中的应用和发展[J].辽宁师范大学学报(自然科学版),1995,(2):152～155.
[29]乐毅全,顾国维.16S rRNA基因技术在环境科学领域中的应用[J].四川环境,2003,22(6),1～4.
[30] GrayM. W, et al. On the evo lutionary desent of o rganelles: a global phylogeny based on a h igly conserved structural co re in small subunit ribo somal RNA[J]. N ucleic. Acid. R es. ,1984,12(14):5 837～5851.
[31] Rashidian E Q ,Payne G. Ward A G1 A Phylogenetic analysis of the Strep2 tomyces cyaneus species-group[A]1 11th International Symposium on the Biology of Actinomycetes Abstracts[C]. Heraklion, GRT,1999,164.
[32]徐平,张利平,余利岩.应用16S rRNA基因V-2高变区序列进行链霉菌分子分类[J].生物多样性,2001,9(2):129～137.
[33]东秀珠,沈德龙,辛玉华. 16S rRNA同源性所揭示的双歧杆菌与有关细菌的亲缘关系[J].生物多样性,2000,8(2):146～152.
[34]赵庆新,谭远德.利用肠道菌群的分布和16S DNA序列研究鲤科肠道菌系统演化关系[J].遗传,2002,24(4):447～454.
[35]李颖,白雨,陈文新.沙坡头地区根瘤菌DNA同源性及16S rRNA全序列[J].微生物学报,1999,39(2):95～99.
[36]汪恩涛,曲春枫,等. 16S rRNA基因测序确定根瘤菌的系统发育[J].高技术通讯,1995,5(11):55～57.
[37]彭桂香,陈文新.16S rRNA-RFLP分析新疆快生大豆根瘤菌的分类地位[J].微生物学通报,2000,27(4):237～241.
[38] Vaneechoutte M,R Roseau,et al.Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA)[J].FEMS Microbial.Lett.,1992,93(2):227～234.
[39] Weidner S,W Arnold,et al.Diversity of Uncultured Microorganisms Associated with the Seagrass Halophila stipulacea estimated by Restriction Fragment length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes[J]. Appl1Environ. Microbiol., 1996,62(3):766～771.
[40]刘忠,关少慧,等.应用ARDRA技术研究Frankia菌多样性[J].微生物学杂志,2001,21(1):20～23.
[41]刘志培,杨惠芳.微生物分子生态学进展[J].应用与环境生物学报,1995(5):43～48.
[42]李琳,李槿年,余为一.细菌分类鉴定方法的研究概况[J].安徽农业科学, 2004,32(3):549～551.
[43] Ostein B White RL, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. Am J Hum Genet,1980,32:314～331.
[44]王志林等.分子标记技术及其进展[J].生命的化学,2002,2:4～5.
[45]常弘,柯亚永.分子标记及其在鸟类分子生态学研究中的应用[J].动物学杂志,2002,37(1):79～84.
[46] [美]C.W.迪芬巴赫,G.S.德维克斯勒著,黄培堂,俞炜源,陈添弥等译.PCR技术实验指南[M].北京:科学出版社,2003,7,12～14.
[47] Polz,M.F.,and Cavanaugh,C.M. Bias in template-to-product ratios in multitemplate PCR[J].App Environ Microbiol,1998,64:3724～3730.
[48]焦念志等著.海洋微型生物生态学[M].科学出版社,2006,12:453～454.
[49]周进.分子生物学技术在水产养殖中的应用[J].河北渔业,2003,(6):21～24.
[50]孔祥会,王桂忠,李少菁.现代生物技术在水产动物健康养殖中的应用[J].河南师范大学学报,2003,31(3):66～71.
[51]萨姆布鲁克,弗里奇,曼尼阿蒂斯.分子克隆实验指南(第二版)[M].北京:科学出版社,1996.
[52]卢圣栋.现代分子生物学实验技术[M].北京:高等教育出版社,1993.
[53]戴欣,周惠,蔡创华,等.海洋沉积物中特有细菌类群的初步探讨[J].中山大学学报,2001,40(6):51～54.
[54]曾润颖,赵晶.深海细菌的分子鉴定分类[J].微生物学通报,2002,29(6):12～16.
[55]萨姆布鲁克J,拉塞尔DW,黄培堂,等.译.分子克隆实验指南[M].3版.北京:科学出版社,2002.
[56] Devereux R, He S H , Doyle C L , et al.Diversity and origin of desulfovibrio species: phylogenetic definition of a family[J].J Bacteriol, 1990, 172: 3609～3619.
[57] Fry N K, Warwick S, Saunders N A, et al. The use of 16S ribosomalRNA analyses to investigate the phylogeny of the family Legionellacea[J]. J Gen Microbiol,1991,137:1215～1222.
[58] Patel JB. 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn, 2001, 6: 313～321.
[59] PetersRP, Van SgtmaelMA, Danner JA, et al. New development in the diagnosis of bloodstream infections. Lancet InfectDis,2004, 4:751～760.
[60] Distel D L,DeLong E F,Waterbury J B.Phylogenetic characterization and in situ loc alization of tIle bacterial symbiont of shipworms (Teredinidae:Bivalvia) by using 16S rRNA sequence analysis and Oligo deoxynucleotide probe hybridization[J].Appl Environ Microblol,1991,57:2376～2382.
[61] Polz F M,Distel D L,Zlda B,et a1. Phylogenetic analysis of a highly specificassociation between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode[J].Appl Environ Microbiof,1994,60:4461～4467.
[62] Distel D L,DeLong E F,Waterbury J B.Phylogenetic characterization and in situ loc alization of tIle bacterial symbiont of shipworms (Teredinidae:Bivalvia) by using 16S rRNA sequence analysis and Oligo deoxynucleotide probe hybridization[J].Appl Environ Microblol,1991,57:2376～2382.
[63] Polz F M,Distel D L,Zlda B,et a1. Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode[J].Appl Environ Microbiof,1994,60:4461～4467.
[64]方再光,黄惠琴,蔡海宝,等.海绵(Pachychalina sp.)体内细菌多样性的研究[J].微生物学报,2004,8(44): 427～430.
[65] Darby A C , Birkle L M, Turner S L , et al. FEMS Microbiol Ecol,2001,36:43～501.
[66] Zhang T, Fang H H P1 Biotechnol Lett,2000,22:399～4051.
[67] Sekiguchi Y, Kamagata Y, Syutsubo K, et al. Microbiol ,1998 ,144 : 2655～26651.
[68] Zhang T, Fang H H P1 Biotechnol Lett, 2000, 22:399～4051.
[69] Zhang T, Fang H H P1 Appl Microbiol Biotechnol, 2001,57:437～4401.
[70]叶应妍,王毓上.全国临床检验操作规程(第2版)[M].南京:东南大学出版社,1997,556～562.
[71]常亚青,丁君,宋坚.海胆、海参生物学和增养殖技术[M].北京:海洋出版社2004.
[72]李筠,生菊,纪伟尚,徐怀恕.海湾扇贝(Argopecten irradians)附着基异养细菌区系初探[J].青岛海洋大学学报,2001,31(1):69～74.
[73]郑国兴,李何,黄宁宇,等.文蛤病原菌(溶藻胶弧菌)的分离、性状及文蛤的电镜观察[J].水产学报,1991,15(2):85～95.
[74]朱传华,何建国,黄志坚.网箱养殖石斑鱼暴发性溃疡病病原菌分离、鉴定及致病性研究[J].中山大学学报,自然科学版,2000,39(增刊):278～282.
[75]周永灿,张本,陈雪芬,等.养殖对虾细菌性红体病的初步研究[J].海洋科学,2003,27(5):61～65.
[76]刘广锋,周世宁,徐力文,等.杂色鲍幼苗“急性死亡脱落症”病原菌分析[J].中国水产科学,2006,13(4):655～661.
[77]张伟妮,周丽,邢静,战文斌.养殖大菱鲆腹水症病原菌SR1的分离及鉴定[J].中国水产科学,2006,13(4):603～609.
[78] Rikelme, C., A. E. Toranzo, J. L. Barja, N. Vergara, and R. Araya. Association of Aeromonas hydrophila and Vibrio alginolyticus with larval mortalities of scallop (Argopecten purpuratus)[J]. J. Invert. Pathol. 1996,67:213～218.
[79] Lee, K. K. Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus Bloch et Schneider[J]. Microb. Pathog,1995,19:39～48.
[80]马悦欣,徐高蓉,张恩鹏,等.仿刺参幼参急性口围肿胀症的细菌性病原[J].水产学报,2006,30(3):377～382.
[2] Ferguson R L,Buckley E N, Palumbo AV.Response of marine bacterioplankton to differential filtration and confinement[J]. Appl Environ Microbioi, 1984,47:49～55.
[3] Hugenholtz P,Goebel B M,Pace N R.Impact of culture—independent studies on the emerslng phylogenetic view of bacterial diversity[J].J Bacteriol,1998,180:4765～4774.
[4]张春云,等.国内外海参自然资源与养殖现状[J].海洋水产研究,2004,25(3):89-93.
[5]王印庚,荣小军,张春云,孙素凤.养殖海参主要疾病及防治技术[J].海洋科学,2005,29(3):1～7.
[6]邓欢,隋锡林.仿刺参育苗期常见疾病[M].水产科学,2004,23(3):40.
[7]王印庚,方波,张春云,等.养殖刺参保苗期重大疾病“腐皮综合征”病原及其感染源分析[J].中国水产科学,2006,13(4):610～616.
[8]马悦欣,徐高蓉,张恩鹏,等.仿刺参幼参急性口围肿胀症的细菌性病原[J].水产学报,2006,30(3):377～382.
[9] Becker P,Gillan D,Lanterbecq D,et al. The Skin Ulceration Disease in Cultivatted Juveniles of Holothuria scabra(Holothuroidea, Echinodermata)[J].Aquaulture,2004,242:13～30.
[10]董颖,邓欢,隋锡林,宋伦.养殖仿刺参溃烂病病因初探[J].水产科学, 2005, 24(3):4～6.
[11]赵伟.渤海辽东湾环境状况及污染防治对策[J].辽宁城乡环境科技,19(3):4～6.
[12]刘金屏,曹文伟,张楚佳,等.大连湾石油微生物的数量分布及分类鉴定[J].辽宁师范大学学报,1986,2,38～43.
[13]徐辉,王日东.大连湾海域赤潮状况及预测模式研究[J].资源与环境,2007,87.
[14]邱春霞,邵秘华,李玉娜,等.大连湾海域生态环境现状的评估[J].大连海事大学学报,2005,31(1):77～80.
[15]于大江.近海资源保护与可持续利用[M].北京:海洋出版社,2001,78～96.
[16]李震,刘景泰.大连湾海域水体富营养状况分析[J].中国环境监测, 2000,16(1):38～41.
[17]王惠卿,杜广玉.大连市近岸海域赤潮状况、预测及防治对策[J].中国环境监测, 2000,16 (6):42～46.
[18]郑建平,王芳,华祖林.辽东湾北部河口区生态环境问题及对策[J].东北水利水电,2005,10,47～50.
[19]李建军,冯慕华,喻龙.辽东湾浅水区水环境质量现状评价[J].海洋环境科学,2001,20(3):42～45.
[20]宛立,田继辉,马志强,等.辽东湾北部海域表层水体夏季油类的污染状况[J].水产科学,2007,26(9):515～517.
[21]廖玉麟.中国动物志(第1卷)[M].北京:科学出版社,1997,61.
[22]崔清晨.海洋资源[M].北京:商务印书馆,1981,294.
[23]常亚青,丁军,宋坚等.海参、海胆生物学研究与养殖[M].海洋出版社, 2004,113～192.
[24]孙素凤.养殖刺参(Apostichopus japonicus)苗期细菌性疾病及其病原学初步研究[J].中国学术期刊,2006,13～14.
[25]王印庚,荣小军,张春云,等.养殖海参主要疾病及防治技术[J].海洋科学,2005(3):1～7.
[26]李琳,李槿年,余为一.细菌分类鉴定方法的研究概况[J].安徽农业科学,2004,32(3):549～551.
[27]阎东辉,许淑珍,马纪平,等. VITEK- AMS在临床细菌鉴定中的应用[J].中华实用医学,2000,(10):9～10.
[28]姜远良.化学分类法在细菌和真菌系统分类中的应用和发展[J].辽宁师范大学学报(自然科学版),1995,(2):152～155.
[29]乐毅全,顾国维.16S rRNA基因技术在环境科学领域中的应用[J].四川环境,2003,22(6),1～4.
[30] GrayM. W, et al. On the evo lutionary desent of o rganelles: a global phylogeny based on a h igly conserved structural co re in small subunit ribo somal RNA[J]. N ucleic. Acid. R es. ,1984,12(14):5 837～5851.
[31] Rashidian E Q ,Payne G. Ward A G1 A Phylogenetic analysis of the Strep2 tomyces cyaneus species-group[A]1 11th International Symposium on the Biology of Actinomycetes Abstracts[C]. Heraklion, GRT,1999,164.
[32]徐平,张利平,余利岩.应用16S rRNA基因V-2高变区序列进行链霉菌分子分类[J].生物多样性,2001,9(2):129～137.
[33]东秀珠,沈德龙,辛玉华. 16S rRNA同源性所揭示的双歧杆菌与有关细菌的亲缘关系[J].生物多样性,2000,8(2):146～152.
[34]赵庆新,谭远德.利用肠道菌群的分布和16S DNA序列研究鲤科肠道菌系统演化关系[J].遗传,2002,24(4):447～454.
[35]李颖,白雨,陈文新.沙坡头地区根瘤菌DNA同源性及16S rRNA全序列[J].微生物学报,1999,39(2):95～99.
[36]汪恩涛,曲春枫,等. 16S rRNA基因测序确定根瘤菌的系统发育[J].高技术通讯,1995,5(11):55～57.
[37]彭桂香,陈文新.16S rRNA-RFLP分析新疆快生大豆根瘤菌的分类地位[J].微生物学通报,2000,27(4):237～241.
[38] Vaneechoutte M,R Roseau,et al.Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA)[J].FEMS Microbial.Lett.,1992,93(2):227～234.
[39] Weidner S,W Arnold,et al.Diversity of Uncultured Microorganisms Associated with the Seagrass Halophila stipulacea estimated by Restriction Fragment length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes[J]. Appl1Environ. Microbiol., 1996,62(3):766～771.
[40]刘忠,关少慧,等.应用ARDRA技术研究Frankia菌多样性[J].微生物学杂志,2001,21(1):20～23.
[41]刘志培,杨惠芳.微生物分子生态学进展[J].应用与环境生物学报,1995(5):43～48.
[42]李琳,李槿年,余为一.细菌分类鉴定方法的研究概况[J].安徽农业科学, 2004,32(3):549～551.
[43] Ostein B White RL, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. Am J Hum Genet,1980,32:314～331.
[44]王志林等.分子标记技术及其进展[J].生命的化学,2002,2:4～5.
[45]常弘,柯亚永.分子标记及其在鸟类分子生态学研究中的应用[J].动物学杂志,2002,37(1):79～84.
[46] [美]C.W.迪芬巴赫,G.S.德维克斯勒著,黄培堂,俞炜源,陈添弥等译.PCR技术实验指南[M].北京:科学出版社,2003,7,12～14.
[47] Polz,M.F.,and Cavanaugh,C.M. Bias in template-to-product ratios in multitemplate PCR[J].App Environ Microbiol,1998,64:3724～3730.
[48]焦念志等著.海洋微型生物生态学[M].科学出版社,2006,12:453～454.
[49]周进.分子生物学技术在水产养殖中的应用[J].河北渔业,2003,(6):21～24.
[50]孔祥会,王桂忠,李少菁.现代生物技术在水产动物健康养殖中的应用[J].河南师范大学学报,2003,31(3):66～71.
[51]萨姆布鲁克,弗里奇,曼尼阿蒂斯.分子克隆实验指南(第二版)[M].北京:科学出版社,1996.
[52]卢圣栋.现代分子生物学实验技术[M].北京:高等教育出版社,1993.
[53]戴欣,周惠,蔡创华,等.海洋沉积物中特有细菌类群的初步探讨[J].中山大学学报,2001,40(6):51～54.
[54]曾润颖,赵晶.深海细菌的分子鉴定分类[J].微生物学通报,2002,29(6):12～16.
[55]萨姆布鲁克J,拉塞尔DW,黄培堂,等.译.分子克隆实验指南[M].3版.北京:科学出版社,2002.
[56] Devereux R, He S H , Doyle C L , et al.Diversity and origin of desulfovibrio species: phylogenetic definition of a family[J].J Bacteriol, 1990, 172: 3609～3619.
[57] Fry N K, Warwick S, Saunders N A, et al. The use of 16S ribosomalRNA analyses to investigate the phylogeny of the family Legionellacea[J]. J Gen Microbiol,1991,137:1215～1222.
[58] Patel JB. 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn, 2001, 6: 313～321.
[59] PetersRP, Van SgtmaelMA, Danner JA, et al. New development in the diagnosis of bloodstream infections. Lancet InfectDis,2004, 4:751～760.
[60] Distel D L,DeLong E F,Waterbury J B.Phylogenetic characterization and in situ loc alization of tIle bacterial symbiont of shipworms (Teredinidae:Bivalvia) by using 16S rRNA sequence analysis and Oligo deoxynucleotide probe hybridization[J].Appl Environ Microblol,1991,57:2376～2382.
[61] Polz F M,Distel D L,Zlda B,et a1. Phylogenetic analysis of a highly specificassociation between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode[J].Appl Environ Microbiof,1994,60:4461～4467.
[62] Distel D L,DeLong E F,Waterbury J B.Phylogenetic characterization and in situ loc alization of tIle bacterial symbiont of shipworms (Teredinidae:Bivalvia) by using 16S rRNA sequence analysis and Oligo deoxynucleotide probe hybridization[J].Appl Environ Microblol,1991,57:2376～2382.
[63] Polz F M,Distel D L,Zlda B,et a1. Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode[J].Appl Environ Microbiof,1994,60:4461～4467.
[64]方再光,黄惠琴,蔡海宝,等.海绵(Pachychalina sp.)体内细菌多样性的研究[J].微生物学报,2004,8(44): 427～430.
[65] Darby A C , Birkle L M, Turner S L , et al. FEMS Microbiol Ecol,2001,36:43～501.
[66] Zhang T, Fang H H P1 Biotechnol Lett,2000,22:399～4051.
[67] Sekiguchi Y, Kamagata Y, Syutsubo K, et al. Microbiol ,1998 ,144 : 2655～26651.
[68] Zhang T, Fang H H P1 Biotechnol Lett, 2000, 22:399～4051.
[69] Zhang T, Fang H H P1 Appl Microbiol Biotechnol, 2001,57:437～4401.
[70]叶应妍,王毓上.全国临床检验操作规程(第2版)[M].南京:东南大学出版社,1997,556～562.
[71]常亚青,丁君,宋坚.海胆、海参生物学和增养殖技术[M].北京:海洋出版社2004.
[72]李筠,生菊,纪伟尚,徐怀恕.海湾扇贝(Argopecten irradians)附着基异养细菌区系初探[J].青岛海洋大学学报,2001,31(1):69～74.
[73]郑国兴,李何,黄宁宇,等.文蛤病原菌(溶藻胶弧菌)的分离、性状及文蛤的电镜观察[J].水产学报,1991,15(2):85～95.
[74]朱传华,何建国,黄志坚.网箱养殖石斑鱼暴发性溃疡病病原菌分离、鉴定及致病性研究[J].中山大学学报,自然科学版,2000,39(增刊):278～282.
[75]周永灿,张本,陈雪芬,等.养殖对虾细菌性红体病的初步研究[J].海洋科学,2003,27(5):61～65.
[76]刘广锋,周世宁,徐力文,等.杂色鲍幼苗“急性死亡脱落症”病原菌分析[J].中国水产科学,2006,13(4):655～661.
[77]张伟妮,周丽,邢静,战文斌.养殖大菱鲆腹水症病原菌SR1的分离及鉴定[J].中国水产科学,2006,13(4):603～609.
[78] Rikelme, C., A. E. Toranzo, J. L. Barja, N. Vergara, and R. Araya. Association of Aeromonas hydrophila and Vibrio alginolyticus with larval mortalities of scallop (Argopecten purpuratus)[J]. J. Invert. Pathol. 1996,67:213～218.
[79] Lee, K. K. Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus Bloch et Schneider[J]. Microb. Pathog,1995,19:39～48.
[80]马悦欣,徐高蓉,张恩鹏,等.仿刺参幼参急性口围肿胀症的细菌性病原[J].水产学报,2006,30(3):377～382.