广西甘蔗根际高效联合固氮菌的筛选及鉴定

Screening and identification of associative nitrogen fixation bacteria in rhizosphere of sugarcane in Guangxi

对广西主要甘蔗产区的根际联合固氮细菌进行了收集和评价,拟筛选获得对甘蔗具有潜在促生性能的联合固氮菌,为甘蔗生产节肥减耗提供依据。结合nifH基因扩增和固氮酶活性分析方法筛选获得36个固氮细菌菌株;进一步对所获得固氮菌株的固氮能力、溶磷性、分泌植物生长素IAA的特性等促进植物生长潜能进行评价,获得了5个同时具有较强固氮能力、降解无机磷和分泌植物生长激素IAA的功能菌株;通过Biolog鉴定系统和16S rRNA序列分析对5个具有较好应用潜力的固氮菌进行分类鉴定。结果表明这5个菌株分别属于Klebsiella sp.、Bacillus megaterium、Pseudomonas sp.、Pantoea sp.和Burkholderia sp.。本研究结果表明广西甘蔗根际联合固氮菌具有较大的开发利用潜力。

Sugarcane is one of the most important crops for sugar and energy production, and the Guangxi region is the major sugarcane and sugar producing area in China, producing about 60% of China＇s sugarcane and sugar. Nitrogen fertilizer input for sugarcane production in China is very high. It is essential to reduce nitrogen fertilizer input and thus cultivation cost while achieving a high yield for sustainable and environmentally friendly sugarcane production. Research on biological nitrogen fixation has increased significantly because of its potential importance to the economy and the environment. Sugarcane plants can obtain nitrogen from biological nitrogen fixation via diazotrophs, and 15N isotope assays have demonstrated that some Brazilian sugarcane varieties are able to obtain considerable nitrogen from biological nitrogen fixation. The aim of this work was to screen and identify nitrogen-fixing bacteria from the rhizosphere of sugarcane cultivated in the diverse geographical region of Guangxi, investigate their diversity and predominant affiliation, and to demonstrate their potential for associative nitrogen fixation with sugarcane as well as plant growth promotion. One hundred and forty eight bacterial isolates associated with sugarcane were isolated from 20 soil samples collected from 10 major production areas over the Guangxi region using Ashby nitrogen deficient minimal media. Thirty six of these isolates were found to have Na fixing ability as demonstrated by the acetylene reduction assay. All 36 isolates also yielded a polymerase chain reaction productwith primers targeting the n/fH gene, which encodes a key enzyme in the nitrogen fixation process. Further screening and selection indicated that five efficient isolates （GX0703, GX0798, GX07104, GX08125, and GX0797 ） were obtained which possessed multiple plant growth promoting traits such as higher N2 fixing, phosphate solubilization and IAA production, and all of the isolates with these attributes may well be valuable for agriculture. To demonstrate their potential for associative nitrogen fixation with sugarcane and growth promotion activities, the five isolates were inoculated into micropropagated sugarcane seedlings, and the seedlings were then incubated in a growth cabinet for 50 d at 28~C. The results indicated that all five isolates significantly promoted the growth of the micropropagated sugarcane seedlings. They also increased the dry weight and nitrogen contents by 29.15 to 57.61% and 29.35 to 45.04% , respectively, compared with the uninoculated controls. Based on phenotypic tests, biolog microplate tests and 16S rRNA gene sequence analysis, the five efficient isolates, GX0703, GX0798, GX07104, GX08125, and GX0797 were identified as KlebsieUa sp., Bacillus megaterium, Pseudomonas sp. , Pantoea sp. and Burkholderia sp. , respectively. These promising associative nitrogen fixation bacteria isolates from the rhizosphere of micropropagated sugarcane showed potential to promote sugarcane production via nitrogen fixation and other plant growth promoting functions. This will improve sugar production efficiency in China and reduce the environmental pollution that results from sugarcane production. The n/fl-I and 16S rDNA sequences of the N fixing bacterial isolates GX0703, GX0798, GX07104, GX08125 and GX0797 were deposited in the GenBank, and the accession numbers were FJ822998, FJ823001, EU938523, FJ822992 and EU938522 for n/fl-I, and FJ823041, FJ823003, FJ823047, FJ823045 and FJ823002 for 16S rDNA sequences, respectively.