毛竹根部可培养细菌种群多样性及其促生功能
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摘要
本文以我国特色植物毛竹为研究材料,采用传统的分离培养方法,对毛竹根部内生细菌的多样性进行了研究。利用LB和KB两种培养基对温室毛竹野生株和温室毛竹航天诱变株根部的内生细菌进行分离和计数,发现毛竹野生株内生细菌菌落数在两种培养基上分别为1.68×106 cfu/g FW和1.95×106 cfu/g FW,毛竹诱变株内生细菌菌落数分别为9.7×106 cfu/g FW和1.355×107 cfu/g FW,诱变株内生细菌菌落数大于野生株内生细菌菌落数。根据细菌菌落形态特征,共得到126株细菌,通过ARDRA及16S rDNA序列系统发育分析表明,毛竹野生株的66株内生细菌分属于6大类群,15个菌属;毛竹诱变株的60株内生细菌分属于5大类群,8个菌属。其中,Alphaproteobacteria、Gammaproteobacteria、Firmicutes、Actinobacteria、Bacteroidetes是共同存在的5大类群,毛竹野生株和诱变株内生细菌中优势类群均为Alphaproteobacteria,最优势种群均为根瘤菌属(Rhizobium)。另外,在毛竹诱变株根内得到了1株Betaproteobacteria类群的细菌,而毛竹野生株根内未分离到此类群的细菌。
本文发现一株Parapedobacter属的细菌,通过生物学特性和系统发育上的研究确定为一个新种并命名为Parapedobacter phyllostachys sp. nov.。
此外,本文对387株毛竹根部细菌解磷、产IAA及分泌铁载体的功能进行了初步测定,结果表明具有解磷活性的内生细菌为36株,菌株BK24的解磷活性最高,为369.06 mg/L;产IAA的菌株为58株,菌株AP 5-2产IAA的活性最高,为4.08μg/mL;分泌铁载体的菌株为78株,菌株H87的分泌能力最强,达到了+++++的极高量水平。
The diversity of culturable endophytic bacteria in the roots of greenhouse moso bamboo (Phyllostachys edulis) plants was investigated by culture-dependent methods. The endophytic bacterial strains isolated from the roots of wild plants are less than that of the space mutant plants. 126 strains with different colony characteristics were isolated with Luria-Bertani and King′s B media. Amplified ribosomal DNA restriction analysis (ARDRA) and partial sequence analysis of 16S rDNA gene indicated that the population diversity of culturable endophytic bacteria was abundant in the roots of greenhouse moso bamboo plants. 66 strains isolated from wild plants were grouped into six groups and fifteen genera, while 60 strains isolated form mutant plants were clustered into five groups and eight genera. The members of Alphaproteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were found both in the roots of wild and mutant plants. The majority of root endophytic bacteria were the members of Alphaproteobacteria, while the most dominant genera was Rhizobium. The members of Betaproteobacteria were only found in the roots of space mutant plants. Several potential novel strains were found at the same time.
A novel endophytic strain AK43 6.1T was studied on the basis of morphological, phylogenetic and chemotaxonomic data, for which the name Parapedobacter phyllostachys sp. nov. is proposed.
Plant-growth promotion fuctions of 387 bacterial strains isolated from the roots of moso bamboo plants were analyzed. Results showed that there were 36 strains with phosphate-solubilizing activity, 58 strains with IAA producing activity, and 78 strains with siderophore-producing activity. The strain with highest phosphate-solubilizing activity was BK24, 369.06 mg/L; the strain with maximal IAA concentration was AP5-2, 4.08μg/mL; the strain with highest siderophore producing activity was H87.
本文发现一株Parapedobacter属的细菌,通过生物学特性和系统发育上的研究确定为一个新种并命名为Parapedobacter phyllostachys sp. nov.。
此外,本文对387株毛竹根部细菌解磷、产IAA及分泌铁载体的功能进行了初步测定,结果表明具有解磷活性的内生细菌为36株,菌株BK24的解磷活性最高,为369.06 mg/L;产IAA的菌株为58株,菌株AP 5-2产IAA的活性最高,为4.08μg/mL;分泌铁载体的菌株为78株,菌株H87的分泌能力最强,达到了+++++的极高量水平。
The diversity of culturable endophytic bacteria in the roots of greenhouse moso bamboo (Phyllostachys edulis) plants was investigated by culture-dependent methods. The endophytic bacterial strains isolated from the roots of wild plants are less than that of the space mutant plants. 126 strains with different colony characteristics were isolated with Luria-Bertani and King′s B media. Amplified ribosomal DNA restriction analysis (ARDRA) and partial sequence analysis of 16S rDNA gene indicated that the population diversity of culturable endophytic bacteria was abundant in the roots of greenhouse moso bamboo plants. 66 strains isolated from wild plants were grouped into six groups and fifteen genera, while 60 strains isolated form mutant plants were clustered into five groups and eight genera. The members of Alphaproteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were found both in the roots of wild and mutant plants. The majority of root endophytic bacteria were the members of Alphaproteobacteria, while the most dominant genera was Rhizobium. The members of Betaproteobacteria were only found in the roots of space mutant plants. Several potential novel strains were found at the same time.
A novel endophytic strain AK43 6.1T was studied on the basis of morphological, phylogenetic and chemotaxonomic data, for which the name Parapedobacter phyllostachys sp. nov. is proposed.
Plant-growth promotion fuctions of 387 bacterial strains isolated from the roots of moso bamboo plants were analyzed. Results showed that there were 36 strains with phosphate-solubilizing activity, 58 strains with IAA producing activity, and 78 strains with siderophore-producing activity. The strain with highest phosphate-solubilizing activity was BK24, 369.06 mg/L; the strain with maximal IAA concentration was AP5-2, 4.08μg/mL; the strain with highest siderophore producing activity was H87.
引文
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[15] Popavath R N, Gurusamy R, Kannan B N, et al. Assessment of genetic and functional diversity of phosphate solubilizing Fluorescent pseudomonads isolated from rhizospheric soil[J]. BMC microbiology, 2008, 8: 230-243.
[16] Masalha J, Kosegarten H, Elmaci O, et al. The central role of microbial activity for iron acquisition in maize and sunflower[J]. Biology and fertility of soils, 2000, 30: 433-439.
[17] Buyer J S, Kratzke M G, Sikora L J. A method for detection of pseudobactin, the siderophore produced by a plant-growth-promoting Pseudomonas strains, in the barley rhizosphere[J]. Applied and environmental microbiology, 1993, 59, 677-681.
[18]余贤美.海南岛橡胶根际嗜铁细菌B.subtilis CAS15筛选及嗜铁素基因dhbC克隆、表达与功能分析[D].泰安:山东农业大学, 2002.
[19] David C, Herve C, Nicolas F, et al. The crystal structure of the pyoverdine outer membrance FpyA from Pseudomonas aeruginosa at 3.6。A resolution[J]. Journal of molecular, 2005, 347: 121-134.
[20] Duffy B K, Defago G. Environmental factors modulating antibiotic and siderophore by Pseudomonas fluorescens biocontrol strains[J]. Applied and environmental microbiology, 1999, 65: 2429-2438.
[21] Neilands J B, Nakamura K. Detectiom, determination, isolation, characterization and of nicrobial iron chelates[M]. In G. Winkelmann (Eds.) CRC handbook of microbial iron chelates. London: CRC Press, 1991.
[22] Ryu C M, Farag M A, Hu C H, et al. Bacterial volatiles induce systemic resistance in Arabidopsis[J]. Plant physiology, 2004, 134: 1017-1026.
[23] Whipps J M. Microbial interactions and biocontrol in the rhizosphere[J]. Journal of experimental biology, 2001, 52: 487-511.
[24] Honyou Z, Hailei W, Xili L, et al. Improving biocontrol activity of Pseudomonas fluorscens through chromosomal integration of 2,4-diacetyl-phloroglucinol biosynthesis genes[J]. Chinese science bulletin, 2005, 50: 775-781.
[25] Ongena M, Daayf F, Jacques P, et al. Protection of cucumber against pythium root rot byPseudomonas fluorescent: predominant role of induced resistance over siderophores and antibiosis [J]. Plant pathology, 1999, 48: 66-76.
[26] Voisard C, Keel C, Haas D, et al. Cyanide production by Pseudomonas fluorscens helps black root rot of tobacco under gnotobiotic conditions[J]. EMBO Journal, 1989, 8: 351-358.
[27] Walsh U F, Morrissey J P, O’Gara F, et al. Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation[J]. Current opinion in biotechnology, 2001, 12: 289-295.
[28] Tanaka H, Watanabe T. Glucanases and chitinases of bacillus circulans WL-12[J]. Journal of industrial microbiology, 1995, 114: 478-438.
[29] Kisore G K, Pande S, Rao J N, et al. Pseudomonas aeruginosa inhibits the plant cell wall degrading enzymes of Sclerotium rolfsii and reduces the severity of groundnut stem rot[J]. European journal of plant pathology, 2005.
[30] Pieterse C M J, Van P J A, Van W S C M, et al. Rhizobacteria-nediated induced systenic resistance: triggering, signaling and expression[J]. European journal of plant pathology, 2001, 107: 51-61.
[31] Kobayashi D Y, Palumbo J D. Bacterial endophytes and their effects on plants and uses in agriculture [M]. Microbial endophytes. New York: Marcel Dekker, Inc, 2000, 199-233.
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