Breeding of Highly Efficient Growth-promoting Mutant Strain of Pseudomonas fluorescens CLW17 by Atmospheric and Room Temperature Plasma

doi:10.11924/j.issn.1000-6850.casb2025-0016

Abstract

Abstract:

The purpose of this study is to select and breed highly efficient and excellent growth-promoting mutant strains of Pseudomonas fluorescent CLW17, to provide high-quality bacterial strain resources for the production of microbial fertilizer. The wild strain CLW17 with the phosphate-solubilizing and siderophore-producing abilities was used as the starting strain, and the strain was improved by ARTP mutagenesis technology. Through the three-stage screening system of 'qualitative primary screening-quantitative re-screening-molecular identification', the high-efficiency growth-promoting mutant strain was obtained and the mutagenesis conditions were optimized. The results showed that the optimal mutagenesis for CLW17 was as follows: output power of 100 W, irradiation distance of 2 mm, and treatment time of 240 s. Under these conditions, the mortality rate of the strain was 91.67%, and the positive mutation rate was 11.27%. After ARTP mutagenesis, two mutants with high efficiency and excellent growth-promoting characteristics (CLW17-240-2-1 and CLW17-240-3-2) were finally screened out. Compared to the original strain, the phosphate-solubilizing capacity of CLW17-240-2-1 and CLW17-240-3-2 was enhanced by 21.89% and 30.40% respectively, and their siderophore-producing ability was enhanced by 21.27% and 40.29%. 16S rDNA molecular identification confirmed that the two mutants were genetic mutants of CLW17 without exogenous pollution. In this study, the synergistic improvement of the dual growth-promoting traits of CLW17 strain 'phosphate-solubilizing and siderophore-producing' was successfully achieved through ARTP mutagenesis, which provided a technical reference for the directional breeding of plant growth-promoting rhizobacteria (PGPR), and also laid a strain foundation for the research and development of microbial fertilizer for trees such as Taxus chinensis var.mairei.

Key words: Pseudomonas fluorescens CLW17, atmospheric and room temperature plasma mutagenesis, phosphorus-solubilizing, siderophore-producing, growth-promoting

CHEN Yanbin, ZHI Mengtao, BAI Bianxia, JIA Rui, REN Jiahong. Breeding of Highly Efficient Growth-promoting Mutant Strain of Pseudomonas fluorescens CLW17 by Atmospheric and Room Temperature Plasma[J]. Chinese Agricultural Science Bulletin, 2025, 41(33): 131-138.

Figures/Tables 8

References 25

[1]	JIANG H, LI S, WANG T, et al. Interaction between halotolerant phosphate-solubilizing bacteria (Providencia rettgeri Strain TPM23) and rock phosphate improves soil biochemical properties and peanut growth in saline soil[J]. Frontiers in microbiology, 2021,12:777351.
[2]	FU W, PAN Y, SHI Y, et al. Root morphogenesis of Arabidopsis thaliana tuned by plant growth-promoting Streptomyces isolated from root-associated soil of Artemisia annua[J]. Frontiers in plant science, 2022,12:802737.
[3]	白洁, 姚拓, 雷杨, 等. 欧李多功能PGPR菌株筛选,鉴定及促生防病特性研究[J]. 草原与草坪, 2023, 43(1):20-28.
[4]	杨晓玫, 冯起, 吕丹彤, 等. 植物根际促生菌Bacillus mycoides Gnyt1菌株生物学特性比较研究[J]. 草地学报, 2022, 30(3):553-559. doi: 10.11733/j.issn.1007-0435.2022.03.006
[5]	HASSEN W, NEIFAR M, CHERIF H, et al. Pseudomonas rhizophila S211, a new plant growth-promoting rhizobacterium with potential in pesticide-bioremediation[J]. Frontiers in microbiology, 2018,9:34.
[6]	沈德龙, 李俊, 姜昕. 我国微生物肥料产业现状及发展方向[J]. 微生物学杂志, 2013, 33(3):1-4.
[7]	李琬, 刘淼, 张必弦, 等. 植物根际促生菌的研究进展及其应用现状[J]. 中国农学通报, 2014, 30(24):1-5.
[8]	BAHADUR A, SINGH J, SINGH K P. Effect of Organic amendments and biofertilizers on growth, yield and quality attributes of Chinese cabbage (Brassica pekinensis)[J]. Indian journal of agricultural sciences, 2016, 76(10):596-598.
[9]	凌思雨, 王洲, 张会敏, 等. 常压室温等离子体诱变与微生物微液滴培养选育谷胱甘肽高产菌株[J]. 食品科学, 2023, 44(4):200-208.
[10]	陆欢, 沈玲, 尚晓冬, 等. 常压室温等离子体技术在微生物诱变育种中的研究进展[J]. 生物学杂志, 2023, 40(4):92-97.
[11]	祁田甜, 张婵, 胡济美, 等. 常压室温等离子体诱变技术选育高产Monacolin K紫色红曲霉突变株[J]. 食品科学, 2015, 36(9):66-70. doi: 10.7506/spkx1002-6630-201509013
[12]	张名帆, 张立飞, 马婷, 等. 基于常压室温等离子体诱变技术选育耐高温马克斯克鲁维酵母菌株及其代谢机制解析[J]. 食品与发酵工业, 2024, 50(22):90-97. doi: 10.13995/j.cnki.11-1802/ts.037948
[13]	TONG X, FAN S, LI X, et al. Improving agar degradation activity of Vibrio natriegens WPAGA4 via atmospheric and room temperature plasma (ARTP)[J]. Journal of marine science & engineering, 2024, 12(7):1154.
[14]	任嘉红, 刘辉, 吴晓蕙, 等. 南方红豆杉根际溶无机磷细菌的筛选、鉴定及其促生效果[J]. 微生物学报, 2012, 52(3):295-303.
[15]	BAI B X, YANG X, ZHAO Q S, et al. Inoculations with Pseudomonas fluorescens and Bacillus cereus affect the soil enzyme activity, growth and rhizosphere microbial diversity of Taxus chinensis var. mairei[J]. Plant and soil, 2020, 455(1):45-52.
[16]	吴娟丽. 嗜铁素产生菌的筛选鉴定及其对供试植物促生作用研究[D]. 兰州交通大学,2020:1-70.
[17]	韩坤, 田曾元, 刘珂, 等. 具有ACC脱氨酶活性的海滨锦葵(Kosteletzkya pentacarpos)内生细菌对小麦耐盐性的影响[J]. 植物生理学报, 2015, 51(2):212-220.
[18]	DONG T T, GONG J S, GU B C, et al. Significantly enhanced substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma and cell immobilization[J]. Bioresource technology, 2017, 244:1104-1110. doi: 10.1016/j.biortech.2017.08.039 URL
[19]	JIANG M, WAN Q, LIU R M, et al. Succinic acid production from corn stalk hydrolysate in an E.coli mutant generated by atmospheric and room-temperature plasmas and metabolic evolution strategies[J]. Journal of industrial microbiology and biotechnology, 2014, 41(1):115-123. doi: 10.1007/s10295-013-1346-7 URL
[20]	薛应钰, 叶巍, 张树武, 等. 紫外诱变选育木霉高效解磷菌株[J]. 核农学报, 2015, 29(8):1509-1516. doi: 10.11869/j.issn.100-8551.2015.08.1509
[21]	ZHANG X, ZHANG X F, LI H P, et al. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool[J]. Applied microbiology and biotechnology, 2014, 98(12):5387-5396. doi: 10.1007/s00253-014-5755-y pmid: 24769904
[22]	ZHANG X, ZHANG C, ZHOU Q Q, et al. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis[J]. Applied microbiology and biotechnology, 2015, 99(13):5639-5646. doi: 10.1007/s00253-015-6678-y pmid: 26025015
[23]	张朝正, 李意, 赵华. ARTP诱变法提高蜡状芽孢杆菌中壳聚糖酶的产量[J]. 食品工业科技, 2022, 43(1):141-146.
[24]	闵勇, 王颖, 刘晓艳, 等. 绿针假单胞菌ARTP诱变及高产菌株的选育[J]. 农业科学, 2020,11:89-90.
[25]	梁英, 辛红翠, 闫译允, 等. 小新月菱形藻的常压室温等离子体诱变及高产岩藻黄素藻株的筛选[J]. 中国海洋大学学报(自然科学版), 2022, 52(7):39-48.

菌株	透明圈直径/mm	菌落大小/mm	解磷量（水解圈D/ 菌落直径d）
野生株	8.64	5.18	1.668
210-2-4	8.06	4.56	1.768
210-3-5	8.87	4.95	1.792
240-1-1	11.23	1.28	8.773
240-1-2	12.62	1.99	6.342
240-1-3	7.48	1.5	4.987
240-1-4	11.49	1.96	5.862
240-1-5	11.49	1.95	5.892
240-2-1	14.50	1.9	7.632
240-3-1	17.40	3.87	4.496
240-3-2	14.19	1.77	8.017
270-1-7	9.57	5.65	1.694
270-2-1	11.61	2.29	5.070
300-1-1	8.58	4.23	2.028
300-1-2	15.63	5.28	2.960
300-2-1	9.57	5.07	1.888
300-2-3	10.16	5.64	1.801
300-2-4	10.22	5.83	1.753
300-2-5	9.57	5.58	1.715

菌株	透明圈直径/mm	菌落大小/mm	解磷量（水解圈D/ 菌落直径d）
野生株	8.64	5.18	1.668
210-2-4	8.06	4.56	1.768
210-3-5	8.87	4.95	1.792
240-1-1	11.23	1.28	8.773
240-1-2	12.62	1.99	6.342
240-1-3	7.48	1.5	4.987
240-1-4	11.49	1.96	5.862
240-1-5	11.49	1.95	5.892
240-2-1	14.50	1.9	7.632
240-3-1	17.40	3.87	4.496
240-3-2	14.19	1.77	8.017
270-1-7	9.57	5.65	1.694
270-2-1	11.61	2.29	5.070
300-1-1	8.58	4.23	2.028
300-1-2	15.63	5.28	2.960
300-2-1	9.57	5.07	1.888
300-2-3	10.16	5.64	1.801
300-2-4	10.22	5.83	1.753
300-2-5	9.57	5.58	1.715

诱变时间/s	纯化获得菌株数/个	正突变菌株数/个	正突变率/%
210	89	2	2.25
240	71	8	11.27
270	57	2	3.51
300	26	6	23.08
总计	243	18	7.41

诱变时间/s	纯化获得菌株数/个	正突变菌株数/个	正突变率/%
210	89	2	2.25
240	71	8	11.27
270	57	2	3.51
300	26	6	23.08
总计	243	18	7.41