一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

Characters identification and sugarcane growth promotion analysis of an endophytic nitrogen fixing bacteria GXS16

摘要:

[背景] 我国甘蔗生产中氮肥过量施用严重，导致生产成本居高不下，充分发挥甘蔗与内生固氮菌的联合固氮作用，减少氮肥施用量，对促进我国甘蔗产业可持续发展具有重要意义。[目的] 筛选优势甘蔗内生固氮菌，对其基本特性、联合固氮效率及促生长功能进行评价。[方法] 从甘蔗根系分离到一株内生固氮菌GXS16，利用乙炔还原法测定固氮酶活性，通过PCR扩增nifH基因确定菌株为固氮菌；通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类；通过接种盆栽甘蔗检测菌株的促生长作用，采用¹⁵N同位素稀释法检测菌株相对固氮效率。[结果] 菌株GXS16固氮酶活性为2.42μmol-C₂H₄/（h·mL），根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果，菌株GXS16属于伯克氏菌属（Burkholderia）；菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶（1-Aminocyclopropane-1-Carboxylate Deaminase，ACC）活性及合成生长素吲哚乙酸（Indole-3-acetic Acid，IAA）、降解无机磷的功能；接种GXS16处理甘蔗植株的株高比对照增长15%以上，干重增长20%以上，¹⁵N同位素测定显示甘蔗根、茎、叶从空气中获得氮的百分比分别为7.69%、15.64%和8.72%，效率显著优于模式菌株G.diazotrophicus PAL5。[结论] Burkholderia sp.GXS16是一株高效甘蔗内生固氮菌，具有良好应用前景。

Abstract:

[Background] The nitrogen fertilizer input for sugarcane crop production is very high in China, which result in the high planting costs and cause the field pollution. Sugarcane can obtain high levels of nitrogen via N₂-fixation by associated N₂-fixing bacteria. It is imperative to substantially reduce the nitrogen fertilizer inputs and to substitute nitrogen fertilizers with biological nitrogen fixation to develop a high benefit-cost ratio and environmentally benign sugarcane production in China. [Objective] The aim of this study was to systematically identify efficient endophytic nitrogen fixing bacteria of sugarcane, as well as demonstrate their potential for associative nitrogen fixation and plant growth promotion ability. [Methods] An endophytic diazotroph strain GXS16 was isolated from the roots of sugarcane, and its nitrogen fixation ability was tested by acetylene reduction assay and nifH gene sequence amplification. Strain GXS16 species classification was identified by its culture and the microscopic observation characters, Biolog detection and 16S rRNA gene sequence analysis together. Strain GXS16 was inoculated with micro propagated sugarcane seedlings to detect its growth promotion ability and associative nitrogen fixation activity with ¹⁵N isotope dilution method. [Results] Strain GXS16 showed a high acetylene reduction activity of 2.42 µmol-C₂H₄/(h·mL). Based on phenotypic characteristics, Biolog system test, 16S rRNA gene, nifH and acdS gene sequence analysis, GXS16 was classified into Burkholderia sp. Furthermore, GXS16 containing ACC deaminase activity, and other plant growth promoting traits as IAA production and phosphate solubilization ability. GXS16 significantly promoted the growth of sugarcane seedlings. Compared with the uninoculated controls, GXS16 increased plant height and dry weights more than 15% and 20%, respectively. ¹⁵N isotope dilution assays demonstrated that the associative nitrogen fixation rates of GXS16 in the sugarcane roots, stems and leaves were 7.69%, 15.64% and 8.72%, respectively, which were higher than that of model strain G. diazotrophicus PAL5. [Conclusion] Strain GXS16 has a great potential to efficiently fix N₂ in sugarcane and promote sugarcane growth.