Isolation and Identification of Endophytic Bacteria from Saposhnikovia divaricata and Analysis of Its Growth-promoting Characteristics

doi:10.13560/j.cnki.biotech.bull.1985.2025-0070

Abstract

Abstract:

Objective The growth-promoting strains were isolated and screened from the healthy roots of Saposhnikovia divaricata, and their growth-promoting effects on S. divaricata were investigated. The potential of endophytic bacteria for development and utilization was mined. Method Endophytic bacteria were isolated from healthy S. divaricata roots by dilution coating method and growth-promoting bacteria with nitrogen fixation, phosphorus solubilization, IAA production and siderophore production were screened by selective medium. The growth-promoting strains were classified by morphological, physiological and biochemical identification and 16S rRNA analysis. The colonization ability of endophytic bacteria in soil and its effect on the growth of S. divaricata were investigated based on antibiotic labeling method and pot experiment. Result A total of 202 endophytic bacteria were isolated from the roots of S. divaricata, 26 strains had the ability of nitrogen fixation, 21 strains had the ability to dissolve phosphorus, 24 strains had the ability to produce IAA, and 27 strains had the ability to produce siderophores. Two strains of endophytic bacteria MX-56 and MX-31 had three or more different growth-promoting functions, were identified as Klebsiella michiganensis and Pseudomonas koreensis. Two rifampicin-resistant strains stably colonized in the rhizosphere soil of S. divaricata. In the pot experiment, compared with CK, MX-56 had the most significant effect on plant height, root length, root diameter, aboveground fresh weight, root fresh weight and root dry weight of S. divaricata, increased 13.47%, 43.04%, 42.75%, 31.43%, 63.21% and 77.12% (P<0.05). Compared to CK, the total contents of two chromones in S. divaricata plants treated with strain MX-31 and MX-56 respectively increased by 33.72% and 30.23%. Conclusion In this study, two strains of P. koreaensis MX-31 and K. michiganensis MX-56 with good growth-promoting effect are isolated and screened from healthy S. divaricata, it may significantly increase the biomass and the content of effective components of S. divaricata.

Key words: Saposhnikovia divaricata, endophytic bacteria, Klebsiella michiganensis, Pseudomonas koreensis, growth promotion

SUN Meng-xue, ZHANG Yi-ying, Xu Peng, SUN Zhuo, WANG Yun-he, HAN Zhong-ming. Isolation and Identification of Endophytic Bacteria from Saposhnikovia divaricata and Analysis of Its Growth-promoting Characteristics[J]. Biotechnology Bulletin, 2025, 41(7): 299-311.

Figures/Tables 17

Fig. 1 Detection of nitrogen-fixing effect of endophytic bacteria in S. divaricata

Fig. 2 Nitrogenase activity of nitrogen-fixing bacteria in S. divaricataDifferent letters in the figure indicate significant differences (P<0.01), the same below

Fig. 3 Effect of endophytic bacteria in S. divaricata on inorganic phosphorus-dissolving

Fig. 4 Determination of endophytic bacteria on inorganic phosphorus-dissolving ability in S. divaricata

Fig. 5 Effect of endophytic bacteria on organophosphorus-dissolving in S. divaricata

Fig. 6 Determination of endophytic bacteria on organophosphorus-dissolving ability in S. divaricata

Fig. 7 Qualitative staining effect of IAA-producing strain

Fig. 8 Determination of endophytic strain on IAA-producing capacity in S. divaricata

Fig. 9 Effect of endophytic bacteria on siderophore-producing of S. divaricata

Fig. 10 Determination of endophytic bacteria on siderophore-producing ability in S. divaricata

Fig. 11 Morphological and Gram staining observations of endophytic bacterial strainsA: Colony morphology of MX-56 in NA medium. B: Microscopic observation of MX-56 Gram staining. C: Colony morphology of MX-31 in in NA medium. D: Microscopic observation of MX-31 Gram staining; The microscopic image was taken with a Leica DM 2500 100X lens and magnified 35% by LAS X software without changing the resolution

Fig. 12 Phylogenetic tree of strain MX-31 and MX-56 based on 16S rRNA gene sequences

Fig. 13 Growth morphology of S. divaricata

Table 1 Results of physiological and biochemical identification of endophytic bacteria

Item	MX-56	MX-31
木糖	+	+
葡萄糖分解	+	-
L-阿拉伯糖	+	+
D-甘露醇	-	+
甲基红试验	-	-
V-P测试	+	-
OF试验	氧化型	氧化型
硫化氢产生	-	-
明胶水解	-	-
淀粉水解	-	-
氧化酶	-	-
硝酸盐还原	+	-

Table 2 Amount of colonizing bacteria of marked strains in soil

标记菌株 Strain	定殖数量 Colonization quantity （1×10⁴ CFU/g）
标记菌株 Strain	0 d	4 d	7 d	10 d	15 d	20 d	30 d
MX-31	1 000	343.33±14.53b	172.67±4.63c	20±0.58e	513.33±12.02a	118.33±4.41d	45±2.89e
MX-56	1 000	245±2.89b	20.33±0.33e	510±11.55a	224.3±4.48c	108.6±1.45d	26.7±0.57e

Table 3 Effect of different bacterial treatments on the morphological indexes of S. divaricata

菌株

Strain

株高

Plant height （cm）

根长

Root length （cm）

根粗

Root diameter （mm）

地上鲜重

Shoot fresh weight （g）

根鲜重

Root fresh weight （g）

根干重

Root dry weight （g）

Table 4 Effects of different treatments on the contents of prim-O-glucosylcimifugin and 5-O-methylvisaminide

处理

Treatments

升麻素苷含量

Content of prim-O-glucosylcimifugin （%）

5-O-甲基维斯阿米醇苷含量

Content of 5-O-methylvisaminide （%）

药典规定总含量

Total content specified in pharmacopoeia （%）

References 37

[1]	张亚琴, 邓秋林, 文秋姝, 等. 浅谈科学施肥在中药材生态种植中的作用与措施 [J]. 中国中药杂志, 2020, 45(20): 4846-4852.
	Zhang YQ, Deng QL, Wen QS, et al. Brief discussion about role and measures of scientific fertilization in ecological cultivation of Chinese medicinal materials [J]. Chinese Journal Of Chinese Materia Medicine, 2020, 45(20): 4846-4852.
[2]	许明双. 番茄和水稻种子可培养内生细菌的多样性分析及促生菌功能研究 [D]. 北京: 中国农业大学, 2014.
	Xu MS. Culturable bacterial community compositions from seeds of tomato and rice and function of plant growth promoting endophytic bacteria [D]. Beijing: China Agricultural University, 2014.
[3]	Hallmann J, Quadt-Hallmann A, Mahaffee WF, et al. Bacterial endophytes in agricultural crops [J]. Can J Microbiol, 1997, 43(10): 895-914.
[4]	Vurukonda SSKP, Giovanardi D, Stefani E. Plant growth promoting and biocontrol activity of Streptomyces spp. as endophytes [J]. Int J Mol Sci, 2018, 19(4): 952.
[5]	Cueva-Yesquén LG, Goulart MC, Attili de Angelis D, et al. Multiple plant growth-promotion traits in endophytic bacteria retrieved in the vegetative stage from passionflower [J]. Front Plant Sci, 2021, 11: 621740.
[6]	Nimaichand S, Devi AM, Li WJ. Direct plant growth-promoting ability of Actinobacteria in grain legumes [M]. Plant Growth Promoting Actinobacteria. Singapore: Springer Singapore, 2016: 1-16.
[7]	Tian QL, Gong YR, Liu S, et al. Endophytic bacterial communities in wild rice (Oryza officinalis) and their plant growth-promoting effects on perennial rice [J]. Front Plant Sci, 2023, 14: 1184489.
[8]	杜佳慧, 徐伟芳, 杨晓冬, 等. 多花黄精产吲哚乙酸内生菌的分离筛选及其对黄精种子萌发的影响 [J]. 生物技术通报, 2022, 38(12): 223-232.
	Du JH, Xu WF, Yang XD, et al. Isolation and screening of endophytes producing indole acetic acid from Polygonatum cyrtonema Hua. and its effect on seed germination of polygonatum [J]. Biotechnol Bull, 2022, 38(12): 223-232.
[9]	黄淑芬, 常华瑜, 顾李成, 等. 九层塔内生细菌系统发育分析及生物学功能 [J]. 应用与环境生物学报, 2020, 26(1): 96-105.
	Huang SF, Chang HY, Gu LC, et al. Diversity of endophyte isolated from Ocimum basilicum L. and its biological function [J]. Chin J Appl Environ Biol, 2020, 26(1): 96-105.
[10]	田义新, 王志清, 王英平. 药用植物栽培学 [M]. 4版. 北京: 中国农业出版社, 2023.
	Tian YX, Wang ZQ, Wang YP. Cultivation of medicinal plants [M]. 4th ed. Beijing: China Agriculture Press, 2023.
[11]	Gao JW, Zhan Y, Wang YH, et al. Advances in phytochemistry and modern pharmacology of Saposhnikovia divaricata (Turcz.) schischk [J]. Chin J Integr Med, 2023, 29(11): 1033-1044.
[12]	李阳, 王旭, 李壮壮, 等. 防风化学成分分离鉴定 [J]. 中国实验方剂学杂志, 2017, 23(15): 60-64.
	Li Y, Wang X, Li ZZ, et al. Isolate and identify chemical constituents of methylene chloride and ethyl acetate extract of Saposhnikovia Radix [J]. Chinese Journal Of Experimental Traditional Medical Formulae, 2017, 23(15): 60-64.
[13]	李琪. 吉林省西部地区栽培防风施肥策略研究 [D]. 长春: 吉林农业大学, 2024.
	Li Q. Study on planting Saposhnikovia divaricata (Turcz.) Schischk fertilization strategy in western Jilin [D]. Changchun: Jilin Agricultural University, 2024.
[14]	Wang YM, Zhang YJ, Cong H, et al. Cultivable endophyte resources in medicinal plants and effects on hosts [J]. Life, 2023, 13(8): 1695.
[15]	戚昱琦, 陈爱明, 蔡灏漾, 等. 防风内生生防细菌FFR3的筛选、鉴定及其抑菌物质特性研究[J]. 微生物前沿, 2020, 9(4): 171-184.
	Qi YQ, Chen AM, Cai HY, et al. Screening, identification of endophytic bacterium FFR3 from Saposhnikovia divaricata (Trucz.) Schischk. and study on characteristics of its antibacterial substances[J]. Advances in Microbiology, 2020, 9(4): 171-184.
[16]	赵银. 白及优质种苗繁育技术及其促生内生菌的筛选和鉴定 [D]. 杭州: 浙江大学, 2021.
	Zhao Y. The high-quality propagation of Bletilla striata seedlings and screening and identification of growth-promoting endophytes [D]. Hangzhou: Zhejiang University, 2021.
[17]	王春艳, 腊贵晓, 苏秀红, 等. 地黄不同时期内生促生细菌的筛选及其促生特性分析 [J]. 生物技术通报, 2022, 38(4): 242-252.
	Wang CY, La GX, Su XH, et al. Screening of endophytic bacteria from Rehmannia glutinosa at different stages and analysis of their growth-promoting characteristics [J]. Biotechnol Bull, 2022, 38(4): 242-252.
[18]	江美彦, 周杨, 刘仁浪, 等. 白芷根际促生菌的筛选及其促生效果研究 [J]. 生物技术通报, 2022, 38(8): 167-178.
	Jiang MY, Zhou Y, Liu RL, et al. Screening and plant growth promoting of grow-promoting bacteria in rhizosphere bacteria of Angelica dahurica var. formosana [J]. Biotechnol Bull, 2022, 38(8): 167-178.
[19]	杨凯元, 韩玲娟, 梁银萍, 等. 达乌里胡枝子产铁载体根瘤内生细菌的鉴定及其促生抗逆效应 [J]. 微生物学通报, 2023, 50(10): 4413-4432.
	Yang KY, Han LJ, Liang YP, et al. Siderophore-producing endophytic bacteria in the root nodules of Lespedeza daurica: identification and examination of growth-promoting effect and stress resistance [J]. China Ind Econ, 2023, 50(10): 4413-4432.
[20]	李振东, 陈秀蓉, 李鹏, 等. 珠芽蓼内生菌Z5产IAA和抑菌能力测定及其鉴定 [J]. 草业学报, 2010, 19(2): 61-68.
	Li ZD, Chen XR, Li P, et al. Identification of Polygonum viviparum endophytic bacteria Z5 and determination of the capacity to secrete IAA and antagonistic capacity towards pathogenic fungi [J]. Acta Prataculturae Sin, 2010, 19(2): 61-68.
[21]	布坎南. 伯杰细菌鉴定手册 [M]. 8版. 北京: 科学出版社, 1984.
	Buchanan RE. Bergey’s manual of determinative bacteriology [M]. 8th ed. Beijing: Science Press, 1984.
[22]	孙卓. 人参病害生防细菌的筛选及其防病作用研究 [D]. 长春: 吉林农业大学, 2014.
	Sun Z. Study on screening of bacteria for ginseng pathogenic fungical disease and effect of ginseng disease control [D]. Changchun: Jilin Agricultural University, 2014.
[23]	马丙如, 崔静轩, 王雨懿, 等. 防风药材质量的多指标综合评价 [J]. 浙江农林大学学报, 2024, 41(4): 715-723.
	Ma BR, Cui JX, Wang YY, et al. Comprehensive quality evaluation of Saposhnikoviae Radix based on multi-component contents determination [J]. J Zhejiang A F Univ, 2024, 41(4): 715-723.
[24]	国家药典委员会. 中华人民共和国药典-一部: 2020年版 [M]. 北京: 中国医药科技出版社, 2020: 156.
	National Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China (Part I) [M]. Beijing: China Medical Science Press, 2020: 156.
[25]	马静, 田义新, 王志清, 等. 防风内生拮抗细菌的分离与鉴定 [J]. 吉林农业大学学报, 2022, 44(3): 323-328.
	Ma J, Tian YX, Wang ZQ, et al. Isolation and identification of endophytic antagonistic bacteria from Saposhnikovia divaricata [J]. J Jilin Agric Univ, 2022, 44(3): 323-328.
[26]	褚衍亮, 刘宇佳, 姚婷, 等. 一株产蛋白酶植物内生菌的分离、鉴定及产酶条件研究 [J]. 江苏科技大学学报: 自然科学版, 2019, 33(1): 93-99, 120.
	Chu YL, Liu YJ, Yao T, et al. Isolation, identification of a protease-producing endophytic bacteria and its culture conditions [J]. J Jiangsu Univ Sci Technol Nat Sci Ed, 2019, 33(1): 93-99, 120.
[27]	孟彤彤, 朱宁, 崔兴帅, 等. 党参内生细菌荧光假单胞菌RHBA17生物学特性及其对宿主的促生作用 [J]. 甘肃农业大学学报, 2023, 58(4): 146-155.
	Meng TT, Zhu N, Cui XS, et al. Growth-promoting properties of Pseudomonas fluorescens RHBA17 and its effect on Codonopsis pilosula [J]. J Gansu Agric Univ, 2023, 58(4): 146-155.
[28]	罗书岚, 刘丽, 李肖莉, 等. 不同时期白花前胡内生菌的多样性和差异性 [J]. 中国野生植物资源, 2022, 41(10): 25-29.
	Luo SL, Liu L, Li XL, et al. Diversity and difference of endophytes in Peucedanum praeruptorum Dunn at different stages [J]. Chinese Wild Plant Resources, 2022, 41(10): 25-29.
[29]	刘书铭. 外加功能菌/菌群-芒草联合修复土壤重金属镉污染 [D]. 长沙: 湖南农业大学, 2021.
	Liu SM. Remediation of Cd contaminated soil by combined remediation approaches of addition of functional bacteria/microflora and Miscanthus [D]. Changsha: Hunan Agricultural University, 2021.
[30]	Lin BS, Song ZZ, Jia YL, et al. Biological characteristics and genome-wide sequence analysis of endophytic nitrogen-fixing bacteria Klebsiella variicola GN02 [J]. Biotechnol Biotechnol Equip, 2019, 33(1): 108-117.
[31]	程亮. 青藏高原矮火绒草内生细菌多样性及生物功能分析 [J]. 南方农业学报, 2019, 50(10): 2222-2233.
	Cheng L. Diversity and biological function of endophytic bacteria in Leontopodium nanum of Qinghai-Tibetan Plateau [J]. J South Agric, 2019, 50(10): 2222-2233.
[32]	李慧萍, 甘雅楠, 韩庆庆, 等. 祁连山云杉林土壤溶磷细菌的分离及对白三叶的促生效应 [J]. 草地学报, 2022, 30(4): 879-888.
	Li HP, Gan YN, Han QQ, et al. Isolation of phosphate solubilizing bacteria from soil of spruce forest in the qilian mountains and their growth promotion effects in white clover [J]. Acta Agrestia Sinica, 2022, 30(4): 879-888.
[33]	韩梅, 罗培宇, 肖亦农, 等. 玉米内生固氮菌的分离鉴定及其促生长作用研究 [J]. 沈阳农业大学学报, 2010, 41(1): 94-97.
	Han M, Luo PY, Xiao YN, et al. Isolation of endophytic N₂-fixing bacteria of corn rice and their function for promoting growth [J]. J Shenyang Agric Univ, 2010, 41(1): 94-97.
[34]	吕泽勋, 宋未. 产酸克雷伯氏菌SG-11生物合成吲哚-3-乙酸的检测 [J]. 分析测试学报, 1999(5): 42-44.
	Lv ZX, Song W. Determination of indole-3-acetic acid biosynthesized by Klebsiella Oxytoca SG-11 [J]. Journal Of Instrumental Analysis, 1999(5): 42-44.
[35]	孙真, 郑亮, 邱浩斌. 植物根际促生细菌定殖研究进展 [J]. 生物技术通报, 2017, 33(2): 8-15.
	Sun Z, Zheng L, Qiu HB. Research advances on colonization of plant growth-promoting rhizobacteria [J]. Biotechnol Bull, 2017, 33(2): 8-15.
[36]	张昊, 赵欣雨, 刘晓娜, 等. 促生菌对怀地黄苗生长及其根表、内生细菌群落的影响 [J/OL]. 微生物学通报, 2025. DOI: 10.13344/j.microbiol.China.241097 .
	Zhang H, Zhao XY, Liu XN, et al. Effects of plant growth-promoting bacteria on the growth and rhizoplane and endophytic bacterial communities of Rehmannia glutinosa seedlings [J/OL]. Microbiology China, 2025. DOI: 10.13344/j.microbiol.China.241097 .
[37]	Song XL, Wu H, Yin ZH, et al. Endophytic bacteria isolated from Panax ginseng improves ginsenoside accumulation in adventitious ginseng root culture [J]. Molecules, 2017, 22(6): 837.