一株兼具秸秆腐解能力玉米促生菌的筛选、鉴定及发酵优化

Isolation and identification of a maize growth-promoting bacterial strain with straw-decomposing capacity and optimization of fermentation conditions

摘要:

【背景】砂姜黑土地区存在秸秆腐解缓慢、秸秆还田后作物幼苗生长不良等问题。【目的】从砂姜黑土区农田筛选一株兼具秸秆腐解能力的玉米促生菌MC29，以促进秸秆腐解和玉米作物生长。【方法】通过16S rRNA基因序列分析对该菌株进行鉴定；采用液态摇瓶及盆栽试验验证菌株实际促腐、促生能力及土壤养分的提升效果，并且探究菌株的最佳生长及产吲哚乙酸(indole-3-acetic acid, IAA)条件；采用电击转化法将绿色荧光蛋白(green fluorescent protein, GFP)基因导入菌株细胞，并通过PCR琼脂糖凝胶电泳进行验证。【结果】分离筛选的玉米促生菌鉴定为纤维化纤维微细菌(Cellulosimicrobium cellulans)。该菌株MC29羧甲基纤维素(carboxymethyl cellulose, CMC)酶活可达13.32 U/mL，产IAA量为8.63 mg/L。与对照相比，施用菌株MC29后秸秆腐解率显著提高24.8%；玉米soil and plant analyzer development (SPAD)值、植株总重、根表面积和根长分别提高7.6%、21.3%、30.9%和18.3%；土壤碱解氮含量显著提高68.1%，土壤速效磷及土壤速效钾含量分别提高5.8%及6.0%。菌株MC29最佳生长条件为pH 7.0、装液量为25/250 mL、碳源为麦芽糖、氮源为酵母粉；最佳产IAA条件为pH 7.0、装液量为50/250 mL、碳源为果糖、氮源为硝酸钾；成功构建荧光标记菌株MC29-GFP，并据此追踪到其接入砂姜黑土15 d后定殖量为2.8×10⁵-9.5×10⁵ copies/g。【结论】所筛选的纤维化纤维微细菌MC29对于指导砂姜黑土区多功能秸秆促腐菌剂、微生物菌肥的研制及提升作物产量有一定的积极意义，并为探究其在砂姜黑土中的实际应用奠定基础。

关键词:纤维化纤维微细菌;玉米促生;秸秆腐解;土壤改良;发酵优化;绿色荧光蛋白

Abstract:

[Background] Shajiang black soil is characterized by heavy texture, poor structure, and low fertility. The long-term straw returning in the area with Shajiang black soil leads to stunted growing of crops and slow straw decomposition. [Objective] To accelerate straw decomposition and increase crop yield, we screened a strain producing both carboxymethyl cellulase (CMCase) and indoleacetic acid (IAA) from Shajiang black soil. [Methods] The strain was identified based on physiological and biochemical properties and 16S rRNA gene sequence analysis. Shake flask of straw decomposition and maize pot experiments were carried out to verify the straw-decomposing, growth-promoting, and soil-improving effects of the strain. Single factor tests were then carried out to optimize the conditions for strain growth and IAA production. Green fluorescent protein (GFP) gene was introduced into the strain cells by electroporation and verified by PCR combined with agarose gel electrophoresis. The colonization rate was determined by fluorescence quantitative experiment. [Results] The straw-decomposing and maize growth-promoting strain was screened out and named as MC29, which belonged to Cellulosimicrobium cellulans. It presented the CMCase activity of 13.32 U/mL and the IAA production of 8.63 mg/L. Compared with the control, the application of MC29 increased the straw decomposition rate by 24.8% and the soil and plant analyzer development (SPAD) value, total plant weight, root surface area, and root length of maize by 7.6%, 21.3%, 30.9%, and 18.3%, respectively. Furthermore, the strain increased the soil available nitrogen, available phosphorus, and available potassium by 68.1%, 5.8%, and 6.0%, respectively. The optimum growth conditions of strain MC29 were pH 7.0, liquid-loading volume of 25/250 mL, maltose as carbon source, and yeast powder as nitrogen source, while the optimum conditions for IAA production were pH 7.0, liquid-loading volume 50/250 mL, fructose as carbon source, and potassium nitrate as nitrogen source. The strain MC29-GFP was successfully constructed, which showed the colonization amount of 2.8×10⁵ to 9.5×10⁵ copies/g 15 days after inoculation in the Shajiang black soil, demonstrating good colonization ability. [Conclusion] C. cellulans MC29 has the ability of decomposing straw and promoting maize growth, which plays a role in the development of multifunctional straw-decomposing agent and microbial fertilizer and the yield improvement of maize. The good and stable colonization ability lays a foundation for the practical application of strain MC29 in Shajiang black soil.

Key words:Cellulosimicrobium cellulans;maize growth-promoting effect;straw decomposing;soil amelioration;optimization of fermentation conditions;green fluorescent protein