Construction of Mixed Fermentation System of Four PGPR Strains and Evaluation of Its Promoting Effect

doi:10.13560/j.cnki.biotech.bull.1985.2020-0989

Abstract

Abstract:

This objective of this work is to construct a mixed fermentation system from 4 plant growth-promoting rhizobacteria（PGPR）strains and to inoculate pepper for laying a foundation for the research and development of microbial compound agents. The medium and fermentation conditions of the mixed fermentation system were optimized by single factor experiment,orthogonal experiment and response surface method. And experiment of planting pepper in pot was conducted to evaluate the growth-promoting effect of compound broth. In mixed fermentation system,the optimal carbon source was sucrose,the optimal nitrogen source was yeast extract,and the optimal carbon-nitrogen ratio was 5∶1. The optimal fermentation conditions were as follows,temperature 29.7℃,pH 7.05,inoculation proportion 3.19%,and the medium volume 50 mL/250 mL. Under these culture conditions,the viable bacterial count in the mixed fermentation culture was 2.94×10¹⁴CFU/mL,which was about 5.13 times compared with single strain fermentation. The growth and physiological indexes of pepper seedlings were significantly improved after irrigating with the mixed fermentation broth,i.e.,the plant height,root length,fresh weight,and chlorophyll content increased by 8.01%,50.22%,43.66% and 6.94% compared with the single strain fermentation compound. In Conclusion,the compound agents prepared by the mixed fermentation system of 4 PGPR strains may have higher viable microbial counts,and play better growth-promoting effects on pepper,and have obvious application potential in the development of PGPR compound agent.

Key words: mixed fermentation, fermentation optimization, compound agent, growth-promoting effect of pepper

ZHOU Jing, HUANG Wen-mao, QIN Li-jun, HAN Li-zhen. Construction of Mixed Fermentation System of Four PGPR Strains and Evaluation of Its Promoting Effect[J]. Biotechnology Bulletin, 2021, 37(4): 116-126.

Figures/Tables 13

References 28

[1]	Reddy PP . Plant Growth-Promoting Rhizobacteria(PGPR)[M]// Recent advances in crop protection. New Delhi:Springer, 2013.
[2]	万水霞, 王静, 李帆, 等. 玉米根际高效溶磷菌的筛选、鉴定及促生效应研究[J]. 生物技术通报, 2020,36(5):98-103.
	Wan SX, Wang J, Li F, et al. Screening and identification of phosphate solubilizing bacteria from maize rhizosphere soil and its growth promoting effect[J]. Biotechnology Bulletin, 2020,36(5):98-103.
[3]	周璇, 李玉明, 丛聪, 等. 外源腐解微生物的物种组合对土壤微生物群落结构及代谢活性的影响[J]. 中国生态农业学报, 2018,26(7):1056-1066.
	Zhou X, Li YM, Cong C, et al. Effects of species-combined exogenous decomposing micro-organisms on soil microbial community structure and metablic activity[J]. Chinese Journal of Eco-Agriculture, 2018,26(7):1056-1066.
[4]	张英, 姚拓, 朱颖. 复合接种剂对三叶草生长特性和品质的影响[J]. 植物营养与肥料学报, 2012,18(5):1277-1285.
	Zhang Y, Yao T, Zhu Y. Effect of compound inoculum on growth characteristics and quality of Trifolium[J]. Plant Nutrition and Fertilizer Science, 2012,18(5):1277-1285.
[5]	谢雨歆, 曾庆宾, 杨军伟, 等. 植物根际促生细菌在烤烟提质增产中的作用[J]. 烟草科技, 2017,50(7):14-21, 30.
	Xie YX, Zeng QB, Yang JW, et al. Effects of growth-promoting rhizobacteria on quality and yield of flue-cured tobacco[J]. Tobacco Science and Technology, 2017,50(7):14-21, 30.
[6]	王继雯, 岳丹丹, 赵俊杰, 等. 两株芽胞孢杆菌混菌发酵产芽胞孢条件的优化[J]. 中国酿造, 2017,36(5):95-99.
	Wang JW, Yue DD, Zhao JJ, et al. Optimization of spore-producing conditions of two Bacillus spp. mixed fermentation[J]. China Brewing, 2017,36(5):95-99.
[7]	陈庆彩, 李敬龙, 胡晓珂. 基于高活菌数的瑞士乳杆菌和鼠李糖乳杆菌混合发酵工艺优化[J]. 中国乳品工业, 2016,44(6):12-15, 19.
	Chen QC, Li JL, Hu XK. Optimization of mixed fermentation technology of Lactobacillus helveticus and Lactobacillus rhamnosus based on high viable count[J]. China Dairy Industry, 2016,44(6):12-15, 19.
[8]	崔美兰, 刘金凤, 黄忠刚, 等. 9株益生菌混合发酵条件的优化[J]. 大连工业大学学报, 2015(6):417-421.
	Cui ML, Liu JF, Huang ZG, et al. Optimization of the mixed fermentation conditions of nine strains of probiotics[J]. Journal of Dalian Polytechnic University, 2015(6):417-421.
[9]	韩丽珍, 林佳静, 郑欢, 等. 一株溶磷菌的抗逆促生特性及对种子萌发的研究[J]. 种子, 2019,38(10):34-40.
	Han LZ, Lin JJ, Zhen H, et al. Study on the stress resistance and growth-promoting characteristics of a phosphate-solubilizing bacteria strain and its effect on seed germination[J]. Seed, 2019,38(10):34-40.
[10]	韩丽珍, 周静, 王欢. 两株对花生促生的芽胞孢杆菌的鉴定及溶磷特性研究[J]. 基因组学与应用生物学, 2019,38(9):4066-4076.
	Han LZ, Zhou J, Wang H. Identification and phosphorus-dissolving characteristics of two Bacillus strains promoted on peanut growth[J]. Genomics and Applied Biology, 2019,38(9):4066-4076.
[11]	花玉鹏, 文才艺, 刘伟成, 等. 响应曲面法优化利迪链霉菌A02发酵培养基[J]. 中国生物防治学报, 2011,27(4):520-527.
	Hua YP, Wen CY, Liu WC, et al. Optimization of medium components for production of Natamycin by Streptomyces lydicus A02 with response surface methodology[J]. Chinese Journal of Biological Control, 2011,27(4):520-527.
[12]	杨继业, 崔冠慧, 习彦花, 等. 产黄纤维单胞菌CR-14菌株诱变选育及发酵条件优化[J]. 农业资源与环境学报, 2016,33(3):262-267.
	Yang JY, Cui GH, Xi YH, et al. Screening and mutation breeding of the Celloulonmonas flavigena CR-14 and optimization of its fermentation conditions[J]. Journal of Agricultural Resources and Environment, 2016,33(3):262-267.
[13]	金丽娜, 闻建平. 4-羟基环孢素高产菌株诱变筛选及培养基优化[J]. 中国科学院大学学报, 2017,34(5):558-566.
	Jin LN, Wen JP. Mutagenesis, screening, and medium optimization of high[(4’-OH)MeLeu] ⁴-CsA producing strain-Nonomuraea dietziae [J]. Journal of University of Chinese Academy of Sciences, 2017,34(5):558-566.
[14]	梁剑光, 祝金山, 赵明霞, 等. 一株高产春雷霉素菌株生长特性及其培养基优化[J]. 中国抗生素杂志, 2019,44(8):920-923.
	Liang JG, Zhu JS, Zhao MX, et al. Growth characteristics and optimization of fermentation medium for a high-yield kasugamycin producing Streptomyces microaureus[J]. Chinese Journal of Antibiotics, 2019,44(8):920-923.
[15]	李静舒, 赵佳. 生防细菌ML-3抗菌蛋白发酵条件的优化及其防治应用[J]. 生物技术通报, 2020,36(6):83-92.
	Li JS, Zhao J. Optimization of fermentation conditions of biocontrol bacterium ML-3 antifungal protein and its application[J]. Biotechnology Bulletin, 2020,36(6):83-92.
[16]	Gopal K, Wang RH, Hemant P, et al. Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties[J]. Process Biochemistry, 2007,42(1):65-70. doi: 10.1016/j.procbio.2006.07.011 URL
[17]	袁辉林, 康丽华, 王胜坤. 红树林植物促生菌SZ7-1菌株的培养基优化[J]. 微生物学通报, 2011,38(3):333-340.
	Yuan HL, Kang LH, Wang SK. Optimization of growth medium for a PGPR strain(SZ7-1)from mangrove rhizosphere[J]. Microbiology China, 2011,38(3):333-340.
[18]	吴翔, 谢丽源, 谭昊, 等. 一株烟草根际促生菌的鉴定及响应面法优化其发酵条件[J]. 农业生物技术学报, 2019,27(12):2248-2257.
	Wu X, Xie LY, Tan H, et al. Identification of a tobacco(Nicotiana tobacum)PGPR strain and optimization for its fermentation conditions by response surface analysis method[J]. Journal of Agricultural Biotechnology, 2019,27(12):2248-2257.
[19]	李雅华, 张启航, 王姣, 等. 产IAA菌株的UV和DES诱变筛选及培养条件优化[J]. 核农学报, 2020,34(9):1873-1880.
	Li YH, Zhang QH, Wang J, et al. Screening of IAA-producing strains by UV and DES mutagenesis and optimization of culture conditions[J]. Journal of Nuclear Agricultural Sciences, 2020,34(9):1873-1880.
[20]	王呈玉, 温芳悦, 萧龙珍, 等. 溶磷菌剂发酵工艺条件的优化及其促生效果[J]. 吉林农业大学学报, 2019,41(4):408-413.
	Wang CY, Wen FY, Cui LZ, et al. Optimization of fermentation conditions for phosphate-solubilizing microbial inoculants and their growth promoting effect[J]. Journal of Jilin Agricultural University, 2019,41(4):408-413.
[21]	曹恩珲, 侯宪文, 李光义, 等. 复合菌剂对盆栽番茄土壤理化性质及微生物活性的影响[J]. 生态环境学报, 2011,20(5):875-880.
	Cao EH, Hou XW, Li GY, et al. Effect of combination on soil physicochemical properties and soil microbiol activity by pot tomato experiments[J]. Ecology and Environment, 2011,20(5):875-880.
[22]	邓振山, 党军龙, 张海洲, 等. 植物根际促生菌的筛选及其对玉米的促生效应[J]. 微生物学通报, 2012,39(7):980-988.
	Deng ZS, Dang JL, Zhang HZ, et al. Screening of plant growth-promoting rhizobacteria and their promoting effects on maize[J]. Microbiology China, 2012,39(7):980-988.
[23]	刘丽, 马鸣超, 姜昕, 等. 根瘤菌与促生菌双接种对大豆生长和土壤酶活的影响[J]. 植物营养与肥料学报, 2015,21(3):644-654.
	Liu L, Ma MC, Jiang X, et al. Effect of Rhizobia and PGPR co-inoculant on soybean characteristics and soil enzyme activities[J]. Plant Nutrition and Fertilizer Science, 2015,21(3):644-654.
[24]	Korir H, Mungai NW, Thuita M, et al. Co-inoculation effect of Rhizobia and plant growth Rhizobacteria on common bean growth in a low phosphorus soil[J]. Frontiers in Plant Science, 2017(8):141.
[25]	Egamberdieva D, Wirth S, Jabborova D, et al. Coordination between Bradyrhizobium and Pseudomonas alleviates salt stress in soybean through altering root system architecture[J]. Journal of Plant Interactions, 2017,12(1):100-107. doi: 10.1080/17429145.2017.1294212 URL
[26]	Madhaiyan M, Poonguzhali S, Kang BG, et al. Effect of co-inoculation of methylotrophic Methylobacterium oryzae with Azospirillum brasilense and Burkholderia pyrrocinia on the growth and nutrient uptake of tomato, red pepper and rice[J]. Plant and Soil, 2010,328(1/2):71-82. doi: 10.1007/s11104-009-0083-1 URL
[27]	赵思崎, 王敬敬, 杨宗政, 等. 微生物复合菌剂的制备[J]. 微生物学通报, 2020,47(5):1492-1502.
	Zhao SQ, Wang JJ, Yang ZZ, et al. Preparation of microbial compound agents[J]. Microbiology China, 2020,47(5):1492-1502.
[28]	韩梅, 李丽娜, 魏冉, 等. 混合培养提高菌株解磷解钾能力的探讨[J]. 微生物学杂志, 2010,30(5):74-77.
	Han M, Li LN, Wei R, et al. Investigation of capacity increment to dissolve phosphate and potassium by blended culture[J]. Journal of Microbiology, 2010,30(5):74-77.

碳源 Carbon source	碳源添加量Dosage/（g·L^-1）	氮源 Nitrogen source	氮源添加量Dosage/（g·L^-1）
果糖 Fructose	20.0	牛肉膏 Beef extract	12.08
蔗糖 Sucrose	19.0	酵母浸出物 Yeast extract	12.53
乳糖 Lactose	19.0	硫酸铵 Ammonium sulfate	6.84
麦芽糖 Maltose	19.0	硝酸钠 Sodium nitrate	8.79

碳源 Carbon source	碳源添加量Dosage/（g·L^-1）	氮源 Nitrogen source	氮源添加量Dosage/（g·L^-1）
果糖 Fructose	20.0	牛肉膏 Beef extract	12.08
蔗糖 Sucrose	19.0	酵母浸出物 Yeast extract	12.53
乳糖 Lactose	19.0	硫酸铵 Ammonium sulfate	6.84
麦芽糖 Maltose	19.0	硝酸钠 Sodium nitrate	8.79

水平 Level	因素Factors
水平 Level	A碳源 Carbon source	B氮源Nitrogen source	C碳氮比 Carbon/Nitrogen
1	葡萄糖Glucose	蛋白胨Peptone	1∶1
2	蔗糖Sucrose	牛肉膏 Beef extract	5∶1
3	乳糖Lactose	酵母浸出物Yeast extract	10∶1
4	麦芽糖Maltose	硫酸铵Ammonium sulfate	15∶1

水平 Level	因素Factors
水平 Level	A碳源 Carbon source	B氮源Nitrogen source	C碳氮比 Carbon/Nitrogen
1	葡萄糖Glucose	蛋白胨Peptone	1∶1
2	蔗糖Sucrose	牛肉膏 Beef extract	5∶1
3	乳糖Lactose	酵母浸出物Yeast extract	10∶1
4	麦芽糖Maltose	硫酸铵Ammonium sulfate	15∶1

试验号 Experiment No	A碳源 Carbon source	B氮源 Nitrogen source	C碳氮比 Carbon-Nitrogen ratio	D空白列 Blank	OD₆₀₀值 OD₆₀₀ value
1	1（葡萄糖Glucose）	1（蛋白胨Peptone）	1（1∶1）	1	0.741
2	1	2（牛肉膏Beef extract）	3（10∶1）	2	1.022
3	1	3（酵母浸出物Yeast extract）	4（15∶1）	3	1.747
4	1	4（硫酸铵Ammonium sulfate）	2（5∶1）	4	0.428
5	2（蔗糖Surcose）	1	2	4	1.428
6	2	2	4	3	1.619
7	2	3	3	2	1.805
8	2	4	1	1	0.228
9	3（乳糖Lactose）	1	3	2	1.463
10	3	2	1	1	1.509
11	3	3	2	4	1.628
12	3	4	4	3	0.154
13	4（麦芽糖Maltose）	1	4	3	1.313
14	4	2	2	4	1.590
15	4	3	1	1	1.607
16	4	4	3	2	0.250
K1	3.938	4.946	4.085	4.085
K2	5.080	5.740	5.075	4.540
K3	4.755	6.787	4.540	4.833
K4	4.760	1.060	4.833	5.075
k1	0.985	1.237	1.021	1.021
k2	1.270	1.435	1.269	1.135
k3	1.189	1.697	1.135	1.208
k4	1.190	0.265	1.208	1.269
R	1.142	5.727	0.990	0.990
因素主次Order of factors	B﹥A﹥C
最优组合Optimal combination	A₂B₃C₂