一株高效甘蔗内生固氮菌NN08200的鉴定及其对甘蔗的促生长作用

Identification of an endophytic nitrogen-fixing bacterium NN08200 from sugarcane and its growth promotion of sugarcane

摘要:

【背景】生产上过高的氮肥投入是我国农业可持续发展的重要限制因子之一。利用生物固氮是减少氮肥施用量最为有效的途径，植物内生固氮菌资源的挖掘和利用对我国农业可持续发展具有重要实践意义。【目的】筛选高效甘蔗内生固氮菌，并对其联合固氮效率及促生长功能进行评价。【方法】从广西甘蔗茎基部组织分离筛选到一株内生固氮菌株NN08200，利用乙炔还原法测定固氮酶活性，通过菌落PCR扩增nifH基因确定菌株为固氮菌；通过菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16S rRNA基因序列分析确定该菌株的分类；采用盆栽接种测定菌株对甘蔗的实际促生长作用，并利用15N同位素稀释法测定其相对固氮效率。【结果】菌株NN08200的固氮酶活性达到2 445 nmol C2H4/(h·mL)，菌株的nifH基因长度为339 bp，与甘蔗内生固氮醋酸杆菌Gluconacetobacter diazotrophicus PAL5菌株的nifH相似性达99%；根据菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16S rRNA基因序列分析结果，菌株NN08200属于泛菌属(Pantoea sp.)细菌；盆栽接种菌株NN08200能显著提高甘蔗幼苗的株高和干重，15N同位素分析结果表明接种该菌株甘蔗植株的根、茎和叶从空气中获得氮素的百分率分别为7.49%、15.02%和10.79%，其联合固氮效率显著优于甘蔗内生固氮模式菌株G. diazotrophicus PAL5，利用后者接种的甘蔗根、茎和叶从空气中获得氮的百分率分别为3.53%、9.44%和4.87%。【结论】菌株Pantoea sp. NN08200是高效甘蔗内生固氮菌，其固氮促生长效果明显高于G. diazotrophicus PAL5菌株，可望研发成为优良固氮微生物肥料生产菌种，并可进一步用于甘蔗联合固氮菌作用机理的相关研究。

Abstract:

[Background] Nitrogen fertilizer input for production in China is very high, which is one of the most serious impediments of the sustainable development of agriculture. Research on biological nitrogen fixation (BNF) has increased significantly because of its potential importance to economy and the environment. [Objective] The aim of this work was to screen and identify efficient endophytic nitrogen-fixing bacteria of sugarcane, and to demonstrate their potential for associative nitrogen fixation with sugarcane as well as plant growth promotion. [Methods] An endophytic diazotroph strain NN08200 was isolated from the stem of sugarcane, and its nitrogen fixation was tested further by using the acetylene reduction assay and PCR amplification of the nifH gene encoding the iron protein of nitrogenase. Identification of the strain species according to its culture character and the microscopic observation, automatic bacteria identification system and by analyzing their 16S rRNA gene sequences. To demonstrate its potential for associative nitrogen fixation with sugarcane and growth promotion activities, strain NN08200 was inoculated into micropropagated sugarcane seedlings to investigate their plant growth-promoting and associative BNF activities using the 15N isotope dilution technique. [Results] The acetylene reduction activity of strain NN08200 was 2 445 nmol C2H4/(h·mL), and PCR amplification of nifH amplified approximately 339 bp fragments from the strain. Cloning and sequencing of the amplicon showed that strain NN08200 contained sequences that were the closest match to nifH. Based on phenotypic characteristics, Biolog system test, and 16S rRNA gene sequence analysis, strain NN08200 was identified as Pantoea sp. Compared with the uninoculated controls, strain NN08200 significantly promoted the growth of micropropagated sugarcane seedlings by increasing dry weight and plant height of sugarcane. 15N isotope dilution assays demonstrated that the associative nitrogen fixation rates of NN08200 in the sugarcane roots, stems and leaves were 7.49%, 15.02% and 10.79%, respectively, which were higher than that of Gluconacetobacter diazotrophicus strain PAL5, whose associative nitrogen fixation rates in the sugarcane roots, stems and leaves were 3.53%, 9.44% and 4.87%, respectively. [Conclusion] Strain NN08200 has the potential to efficiently fix N2 in sugarcane and promote sugarcane growth.