Dalton David A, Kramer Sasha, Azios Nico, Fusaro Suzanne, Cahill Elizabeth, Kennedy Christina
Biology Department, Reed College, Portland, OR 97202-8199, USA.
FEMS Microbiol Ecol. 2004 Sep 1;49(3):469-79. doi: 10.1016/j.femsec.2004.04.010.
Several tropical grasses harbor symbiotic nitrogen-fixing bacteria within their stem and rhizome tissue that may contribute to the nitrogen nutrition of the host plant. We present evidence here that sand dune grasses (Ammophila arenaria and Elymus mollis) from Oregon also contain nitrogen-fixing bacteria. Surface-sterilized stem and rhizome tissue from these species possess acetylene reduction (nitrogen fixation) activity and large populations (10(5) to 10(6) cfu/g fresh weight) of bacteria. These bacteria were cultured on N-free media and identified by sequencing of 16S rRNA genes or by GC-FAME. Random sequencing of numerous colonies from the initial isolation plates of mixed isolates showed that pseudomonads (Stenotrophomonas and Pseudomonas) were by far the most common microorganism. One isolate -Burkholderia sp. strain Aa1 - reduced acetylene in culture with maximum activity at an O(2) concentration of 2% (v/v) in liquid media or 10% on solid media. PCR screening of all the isolates with nifH and nifD primers was positive only for this species. Immunolocalization studies with antibodies to nitrogenase resulted in labeling within plant cell walls of stems and rhizomes. Evidence for a similar nitrogen-fixing association was also detected in Uniola paniculata (sea oats) and Ammophila brevigulata (American beachgrass). We conclude that these grasses, and probably other dune grasses from temperate climates, contain endophytic, diazotrophic bacteria that may contribute to the phenomenal success of these grasses on nutrient-poor sand.
几种热带禾本科植物在其茎和根茎组织中含有共生固氮细菌，这可能有助于宿主植物的氮素营养。我们在此提供证据表明，来自俄勒冈州的沙丘禾本科植物（沙茅草和软穗披碱草）也含有固氮细菌。这些物种经表面消毒的茎和根茎组织具有乙炔还原（固氮）活性，且含有大量细菌（10⁵至10⁶ cfu/g鲜重）。这些细菌在无氮培养基上培养，并通过16S rRNA基因测序或气相色谱 - 脂肪酸甲酯分析（GC - FAME）进行鉴定。对混合分离物初始分离平板上的众多菌落进行随机测序表明，假单胞菌（嗜麦芽窄食单胞菌和假单胞菌）是迄今为止最常见的微生物。一个分离株——伯克霍尔德氏菌Aa1菌株——在液体培养基中O₂浓度为2%（v/v）或固体培养基中为10%时，培养物中乙炔还原活性最高。用nifH和nifD引物对所有分离物进行PCR筛选，仅该物种呈阳性。用针对固氮酶的抗体进行免疫定位研究，结果显示在茎和根茎的植物细胞壁内有标记。在圆锥单胞草（海燕麦）和短柄沙茅草（美洲海滩草）中也检测到了类似的固氮关联证据。我们得出结论，这些禾本科植物，可能还有其他来自温带气候的沙丘禾本科植物，含有内生的、能进行固氮的细菌，这可能是这些禾本科植物在营养贫瘠的沙地上取得显著成功的原因。
