Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop
Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop
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DOI：
10.1007/s11104-013-1828-4
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发表时间：
2013-07
期刊：
影响因子：
4.9
通讯作者：
S. S. Videira-S.;M. D. C. Pereira E Silva;Péricles Souza Galisa;A. C. F. Dias;R. Nissinen;Vera Lúcia Baldani Divan-Vera-Lúcia-B
中科院分区：
文献类型：
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作者：
S. S. Videira-S.;M. D. C. Pereira E Silva;Péricles Souza Galisa;A. C. F. Dias;R. Nissinen;Vera Lúcia Baldani Divan-Vera-Lúcia-B
目的以往的研究表明，象草被固氮细菌定植；然而，这些结果是基于培养依赖的方法，这种方法由于对微生物群落的评估不完整而引入了偏差。在这项研究中，我们采用不依赖培养的方法调查了5种用于生物能源生产的象草基因型的内生菌和植物相关细菌群落的多样性。方法从农学Embrapa Agrobiologia试验区收获5个基因型的象草植株，将其分为茎和根组织。从植物组织中提取总DNA和RNA，利用DGGE和16S rRNA和nifh基因克隆文库在cDNA和DNA水平上对细菌群落进行分析。结果总体而言，基于DNA和rna衍生的dgge谱的模式不同，特别是在组织样本中。基于dna的DGGE表明，与根（根面+内生菌）相关的总细菌和重氮营养群落与茎（内生菌）相关的细菌和重氮营养群落明显不同。这些结果通过rna衍生的16S rRNA序列的系统发育分析得到了证实（细菌总数为586条），但不包括fornifH（186条）。事实上，稀薄分析显示，与茎相关的重氮营养生物的多样性高于与根相关的重氮营养生物。基于16S rRNA序列，克隆文库中以细链菌属（leptotrix）的附属序列为主（12.8%），其次是伯克霍尔德菌属（9%）和慢生根瘤菌属（6.5%），而大多数h克隆与慢生根瘤菌属（26%）密切相关。结论我们的研究结果揭示了具有代谢活性的细菌的多样性，为研究主要与象草相关的细菌种类提供了新的见解。此外，这些结果可以为开发新的策略来选择潜在的细菌，有效地促进生物固氮和提高象草作为生物能源作物的可持续生产提供有益的帮助。
AimsPrevious studies have shown that elephant grass is colonized by nitrogen-fixing bacterial species; however, these results were based on culture-dependent methods, an approach that introduces bias due to an incomplete assessment of the microbial community. In this study, we used culture-independent methods to survey the diversity of endophytes and plant-associated bacterial communities in five elephant grass genotypes used in bioenergy production.MethodsThe plants of five genotypes of elephant grass were harvested from the experimental area of Embrapa Agrobiologia and divided into stem and root tissues. Total DNA and RNA were extracted from plant tissues and the bacterial communities were analyzed by DGGE and clone library of the 16S rRNA andnifH genes at both the cDNA and DNA levels.ResultsOverall, the patterns based on DNA- and RNA-derived DGGE-profiles differed, especially within tissue samples. DNA-based DGGE indicated that both total bacterial and diazotrophic communities associated with roots (rhizoplane + endophytes) differed clearly from those obtained from stems (endophytes). These results were confirmed by the phylogenetic analyses of RNA-derived sequences of 16S rRNA (total bacteria; 586 sequences), but not fornifH (186). In fact, rarefaction analyses showed a higher diversity of diazotrophic organisms associated with stems than roots. Based on 16S rRNA sequences, the clone libraries were dominated by sequences affiliated to members ofLeptotrix(12.8 %) followed byBurkholderia(9 %) andBradyrhizobium(6.5 %), while most of thenifH clones were closely related to the genusBradyrhizobium(26 %).ConclusionsOur results revealed an unexpectedly large diversity of metabolically active bacteria, providing new insights into the bacterial species predominantly found in association with elephant grass. Furthermore, these results can be very useful for the development of new strategies for selection of potential bacteria that effectively contribute to biological nitrogen fixation and enhance the sustainable production of elephant grass as bioenergy crop.