Department of Biology, Federal University of Lavras, CEP 37200-000 Lavras, Minas Gerais, Brazil.
Microb Ecol. 2012 Feb;63(2):405-17. doi: 10.1007/s00248-011-9919-3. Epub 2011 Aug 12.
This study used a multiphasic approach, characterized by the simultaneous use of culture-dependent and culture-independent methods, to investigate endophytic bacterial communities in strawberry (Fragaria ananassa) fruit. A total of 92 bacterial endophytes were isolated and initially grouped by their repetitive extragenic palindromic (rep)-PCR banding pattern and biochemical features. Phylogenetic analysis of the 16S rRNA gene sequences of 45 representatives showed that the isolates belonged to the species Bacillus subtilis (eight isolates), Bacillus sp. (seven isolates), Enterobacter sp. (seven isolates), Enterobacter ludwigii (six isolates), Lactobacillus plantarum (six isolates), Pseudomonas sp. (five isolates), Pantoea punctata (three isolates), and Curtobacterium citreum (three isolates). Nucleic acids were extracted from the strawberry fruit and subjected to 16S rRNA gene directed polymerase chain reaction denaturing gradient gel electrophoresis (16S rRNA PCR-DGGE). The species B. subtilis, Enterobacter sp., and Pseudomonas sp. were detected both by isolation and DGGE. The DGGE fingerprints of total bacterial DNA did not exhibit bands corresponding to several of the representative species isolated in the extinction dilution (L. plantarum, C. citreum, and P. punctata). In contrast, bands in the DGGE profile that were identified as relatives of Arthrobacter sp. and one uncultivable Erythrobacter sp. were not recovered by cultivation techniques. After isolation, the nitrogen fixation ability and the in vitro production of indole-3-acetic acid (IAA) equivalents and siderophores were evaluated. A high percentage of isolates were found to possess the ability to produce siderophores and IAA equivalents; however, only a few isolates belonging to the genera Pseudomonas and Enterobacter showed the ability to fix nitrogen. Plant growth promotion was evaluated under greenhouse conditions and revealed the ability of the Bacillus strains to enhance the number of leaves, shoot length, root dry weight, and shoot dry weight. The activity of the bacterial isolate identified as B. subtilis NA-108 exerted the greatest influence on strawberry growth and showed a 42.8% increase in number of leaves, 15.26% for high shoot, 43.5% increase in root dry weight, and a 77% increase in shoot dry weight when compared with untreated controls.
本研究采用多相方法，即同时使用依赖培养和不依赖培养的方法，来研究草莓（ Fragaria ananassa ）果实中的内生细菌群落。共分离出 92 株内生细菌，并根据其重复外回文（rep）-PCR 带型和生化特征初步分组。45 个代表物的 16S rRNA 基因序列的系统发育分析表明，这些分离株属于枯草芽孢杆菌（8 株）、芽孢杆菌属（7 株）、肠杆菌属（7 株）、路德维希肠杆菌（6 株）、植物乳杆菌（6 株）、假单胞菌属（5 株）、斑点泛菌（3 株）和柠檬色短小杆菌（3 株）。从草莓果实中提取核酸，进行 16S rRNA 基因定向聚合酶链反应变性梯度凝胶电泳（16S rRNA PCR-DGGE）。通过分离和 DGGE 检测到芽孢杆菌属 B. subtilis 、肠杆菌属和假单胞菌属。总细菌 DNA 的 DGGE 指纹图谱没有显示从灭绝稀释中分离出的几个代表种（植物乳杆菌、柠檬色短小杆菌和斑点泛菌）的带。相比之下，通过培养技术无法回收在 DGGE 图谱中鉴定为节杆菌属和一种不可培养的红杆菌属亲缘关系的条带。分离后，评估了固氮能力和体外产生吲哚-3-乙酸（IAA）当量和铁载体的能力。发现很大比例的分离株具有产生铁载体和 IAA 当量的能力；然而，只有少数属于假单胞菌属和肠杆菌属的分离株具有固氮能力。在温室条件下评估了植物生长促进作用，结果表明芽孢杆菌菌株能够增加叶片数量、茎长、根干重和茎干重。鉴定为枯草芽孢杆菌 NA-108 的细菌分离株的活性对草莓生长的影响最大，与未处理对照相比，叶片数增加了 42.8%，高茎增加了 15.26%，根干重增加了 43.5%，茎干重增加了 77%。
