Isolation and Screening of Endophytes Producing Indole Acetic Acid from Polygonatum cyrtonema Hua. and Its Effect on Seed Germination of Polygonatum

doi:10.13560/j.cnki.biotech.bull.1985.2022-0078

Abstract

Abstract:

The endophytic bacteria that can promote the seed germination of Polygonatum were isolated and screened from the healthy roots of Polygonatum cyrtonema Hua.，in order to lay a theoretical foundation for solving the shortage of Polygonatum resources. The endophytes were isolated and purified by the traditional tissue culture method，and the IAA-producing bacteria were screened by the Salkowski reaction（Salkowski method）. The taxonomic status of the target strains was determined by morphological observation，physiological and biochemical characterization，and 16S rDNA sequence analysis. The germination-promoting effect of the target strains on the seeds of Polygonatum was verified by greenhouse paper-bed experiments. The results showed that 34 endophytes were isolated and purified from Polygonatum cyrtonema Hua.，including 25 endophytic bacteria and 9 endophytic fungi. Two endophytic bacteria HNC2 and HPB1 with stable and high content of IAA were screened out，and their IAA production capacity was as high as 4.833 mg/L and 4.608 mg/L，respectively. The results of bacterial identification showed that strains HNC2 and HPB1 were Bacillus sp. and Staphylococcus sp.，respectively. The experimental results regarding the effect of the strains on seed germination showed that both of the growth-promoting bacteria Bacillus sp. HNC2 and Staphylococcus sp. HPB1 significantly increased the germination potential and germination rate of Polygonatum seeds compared to the sterile water and medium controls，and had different degrees of growth-promoting effects on the growth of seedlings. Polygonatum cyrtonema Hua. harbors abundant endophytic bacteria，of which Bacillus sp. HNC2 and Staphylococcus sp. HPB1 are expected to be further developed as microbial fertilizer to increase the germination rate of Polygonatum seeds.

Key words: Polygonatum cyrtonema Hua., endophytes, screening, indole acetic acid, plant-growth-promoting effect

DU Jia-hui, XU Wei-fang, YANG Xiao-dong, TAN Song, YIN Deng-ke, LIU Yuan-xu. Isolation and Screening of Endophytes Producing Indole Acetic Acid from Polygonatum cyrtonema Hua. and Its Effect on Seed Germination of Polygonatum[J]. Biotechnology Bulletin, 2022, 38(12): 223-232.

Figures/Tables 10

References 36

[1]	国家药典委员会. 中华人民共和国药典[M]. 北京: 中国医药科技出版社, 2015.
	National Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China[M]. Beijing: China Medical Science and Technology Press, 2015.
[2]	范佐旺, 柯晓燕, 陈靓雯, 等. 多花黄精的化学成分及药理研究进展[J]. 中医药信息, 2020, 37(5):119-126.
	Fan ZW, Ke XY, Chen LW, et al. Advances in chemical constituents and pharmacological acitivies of Polygonatum cyrtonema Hua[J]. Inf Tradit Chin Med, 2020, 37(5):119-126.
[3]	赵祺, 郭月琴, 鲍康德, 等. 黄精药材质量稳定性考察分析[J]. 人参研究, 2019, 31(6):10-15.
	Zhao Q, Guo YQ, Bao KD, et al. The investigation and analysis to quality retention of Polygonatum herb[J]. Ginseng Res, 2019, 31(6):10-15.
[4]	孟凡金. 多花黄精种苗繁育技术探讨[J]. 种子科技, 2019, 37(2):76, 78.
	Meng FJ. Discussion on seedling breeding technology of Polygonatum cyrtonema Hua[J]. Seed Sci Technol, 2019, 37(2):76, 78.
[5]	祝明珠, 俞年军, 史素影, 等. 多花黄精种子结构与休眠及萌发的关系研究[J]. 种子, 2020, 39(3):7-12, 19.
	Zhu MZ, Yu NJ, Shi SY, et al. Study on the relationship between seed structure and germination of Polygonatum cyrtonema Hua[J]. Seed, 2020, 39(3):7-12, 19.
[6]	陈怡, 杨赋祺, 陈松树, 等. 多花黄精种子萌发过程的内源激素含量变化研究[J]. 中药材, 2020, 43(3):523-527.
	Chen Y, Yang FQ, Chen SS, et al. Study on the changes of endogenous hormones contents in Polygonatum cyrtonema seed germination[J]. J Chin Med Mater, 2020, 43(3):523-527.
[7]	李立格, 张泽锐, 石艳, 等. 多花黄精生殖特性研究[J]. 中国中药杂志, 2021, 46(5):1079-1083.
	Li LG, Zhang ZR, Shi Y, et al. Investigation on reproductive characteristics of Polygonatum cyrtonema[J]. China J Chin Mater Med, 2021, 46(5):1079-1083.
[8]	罗敏, 章文伟, 邓才富, 等. 药用植物多花黄精研究进展[J]. 时珍国医国药, 2016, 27(6):1467-1469.
	Luo M, Zhang WW, Deng CF, et al. Advances in studies of medicinal crop Polygonatum cyrtonema Hua[J]. Lishizhen Med Mater Med Res, 2016, 27(6):1467-1469.
[9]	Dubey A, Malla MA, Kumar A, et al. Plants endophytes:unveiling hidden agenda for bioprospecting toward sustainable agriculture[J]. Crit Rev Biotechnol, 2020, 40(8):1210-1231. doi: 10.1080/07388551.2020.1808584 URL
[10]	王志伟, 纪燕玲, 陈永敢. 植物内生菌研究及其科学意义[J]. 微生物学通报, 2015, 42(2):349-363.
	Wang ZW, Ji YL, Chen YG. Studies and biological significances of plant endophytes[J]. Microbiol China, 2015, 42(2):349-363.
[11]	Jahn L, Hofmann U, Ludwig-Müller J. Indole-3-acetic acid is synthesized by the endophyte Cyanodermella asteris via a tryptophan-dependent and-independent way and mediates the interaction with a non-host plant[J]. Int J Mol Sci, 2021, 22(5):2651. doi: 10.3390/ijms22052651 URL
[12]	Turbat A, Rakk D, Vigneshwari A, et al. Characterization of the plant growth-promoting activities of endophytic fungi isolated from Sophora flavescens[J]. Microorganisms, 2020, 8(5):683. doi: 10.3390/microorganisms8050683 URL
[13]	丁绍武, 张鹏, 刘梦铭. 植物内生菌对植物生长的影响研究进展[J]. 现代农业科技, 2020(11):132-134.
	Ding SW, Zhang P, Liu MM. Research progress on the effects of plant endophytes on plant growth[J]. Mod Agric Sci Technol, 2020(11):132-134.
[14]	Ramanuj K, Shelat H. Plant growth promoting potential of bacterial endophytes from medicinal plants[J]. Adv Res, 2018, 13(6):1-15.
[15]	万兵兵, 刘晔, 吴越, 等. 一株玉米根际多功能促生菌的筛选鉴定及效应研究[J]. 生物技术通报, 2016, 32(8):169-176. doi: 10.13560/j.cnki.biotech.bull.1985.2016.08.025 URL
	Wan BB, Liu Y, Wu Y, et al. Screening and identification of maize growth-promoting rhizobacteria and its promoting effects on maize[J]. Biotechnol Bull, 2016, 32(8):169-176.
[16]	张东艳, 刘晔, 吴越, 等. 花生根际产IAA菌的筛选鉴定及其效应研究[J]. 中国油料作物学报, 2016, 38(1):104-110.
	Zhang DY, Liu Y, Wu Y, et al. Isolation and identification of IAA-producing strains from peanut rhizosphere and its promoting effects on peanut growth[J]. Chin J Oil Crop Sci, 2016, 38(1):104-110.
[17]	李培根, 要雅倩, 宋吉祥, 等. 马铃薯根际产IAA芽孢杆菌的分离鉴定及促生效果研究[J]. 生物技术通报, 2020, 36(9):109-116. doi: 10.13560/j.cnki.biotech.bull.1985.2019-1203 URL
	Li PG, Yao YQ, Song JX, et al. Isolation and identification of IAA-producing Bacillus sp on potato rhizosphere and its growth-promoting effect[J]. Biotechnol Bull, 2020, 36(9):109-116.
[18]	聂鑫, 王伟. 潜在促生菌Bacillus tequilensis U36的产IAA特性及其对辣椒的促生效果[J]. 广东农业科学, 2017, 44(1):36-42.
	Nie X, Wang W. IAA production characteristics and growth-promoting effects on pepper of potential plant grouth-promoting rhizobacteria(PGPR)Bacillus tequilensis U36[J]. Guangdong Agric Sci, 2017, 44(1):36-42.
[19]	谢洁, 任慧爽, 唐翠明, 等. 一株桑树内生细菌的鉴定和对桑椹核地杖菌的拮抗作用[J]. 蚕业科学, 2015, 41(5):815-824.
	Xie J, Ren HS, Tang CM, et al. Identification of a mulberry endophytic bacterium and its antagonistic activity on scleromitula shiraiana[J]. Sci Seric, 2015, 41(5):815-824.
[20]	徐婧, 邵锴, 李东芳, 等. 产IAA菌的筛选、鉴定及其培养基的优化[J]. 苏州科技大学学报:自然科学版, 2018, 35(2):45-54.
	Xu J, Shao K, Li DF, et al. Selection, identification of an IAA biosynthesis strain and the optimization of its culture medium[J]. J Suzhou Univ Sci Technol Nat Sci Ed, 2018, 35(2):45-54.
[21]	谭啸, 章熙东. 革兰氏染色法观察与区分细菌[J]. 生物学教学, 2019, 44(7):71-72.
	Tan X, Zhang XD. Gram staining method to observe and distinguish bacteria[J]. Biol Teach, 2019, 44(7):71-72.
[22]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
	Dong XZ, Cai MY. Manual of identification of common bacterial systems[M]. Beijing: Science Press, 2001.
[23]	王翌, 李淼, 李永锋, 等. 野猪粪便中乳酸菌的分离鉴定及特性研究[J]. 中国农业科学, 2020, 53(14):2964-2973.
	Wang Y, Li M, Li YF, et al. Identification and properties of lactic acid bacteria isolated from wild boar feces[J]. Sci Agric Sin, 2020, 53(14):2964-2973.
[24]	陈亚丽. 甜瓜嫁接亲和性连锁分子标记研究[D]. 南京: 南京农业大学, 2013.
	Chen YL. Studies of molecular marker linked to grafting compatibility in melon[D]. Nanjing: Nanjing Agricultural University, 2013.
[25]	萨娜, 王鹏摇, 鞠馥竹, 等. 芍药属SRAP反应体系优化与亲缘关系分析[J]. 北方园艺, 2019(10):93-101.
	Sa N, Wang PY, Ju FZ, et al. Optimization of SRAP reaction system and genetic relationship in Paeonia[J]. North Hortic, 2019(10):93-101.
[26]	周新华, 桂尚上, 肖智勇, 等. 温度和光照对多花黄精种子萌发的影响[J]. 南方林业科学, 2016, 44(6):1-4, 26.
	Zhou XH, Gui SS, Xiao ZY, et al. Influence of temperature and light on the seed germination of Polygonatum cyrtonema[J]. South China For Sci, 2016, 44(6):1-4, 26.
[27]	龙建吕, 陈松树, 王华磊, 等. 多花黄精植株及根际土水浸液对其种子萌发和种芽生长的影响[J]. 中药材, 2021(2):269-272.
	Long JL, Chen SS, Wang HL, et al. Effects of Polygonatum cyrtonema plants and rhizosphere soil water extract on seed germination and seed bud growth[J]. J Chin Med Mater, 2021(2):269-272.
[28]	陈龙, 梁子宁, 朱华. 植物内生菌研究进展[J]. 生物技术通报, 2015, 31(8):30-34. doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.005 URL
	Chen L, Liang ZN, Zhu H. Research advances in the studies of plant entophytic[J]. Biotechnol Bull, 2015, 31(8):30-34. doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.005 URL
[29]	Wang SS, Ji BY, Su XH, et al. Isolation of endophytic bacteria from Rehmannia glutinosa Libosch and their potential to promote plant growth[J]. J Gen Appl Microbiol, 2020, 66(5):279-288. doi: 10.2323/jgam.2019.12.001 URL
[30]	Petrini O, Fisher PJ. A comparative study of fungal endophytes in xylem and whole stem of Pinus sylvestris and Fagus sylvatica[J]. Trans Br Mycol Soc, 1988, 91(2):233-238. doi: 10.1016/S0007-1536(88)80210-9 URL
[31]	Xu WF, Wang F, Zhang M, et al. Diversity of cultivable endophytic bacteria in mulberry and their potential for antimicrobial and plant growth-promoting activities[J]. Microbiol Res, 2019, 229:126328. doi: 10.1016/j.micres.2019.126328 URL
[32]	Swain MR, Naskar SK, Ray RC. Indole-3-acetic acid production and effect on sprouting of yam(Dioscorea rotundata L.)minisetts by Bacillus subtilis isolated from culturable cowdung microflora[J]. Pol J Microbiol, 2007, 56(2):103-110.
[33]	康慧颖, 王伟, 刘佳莉, 等. 两株具促生作用的苜蓿内生菌的分离纯化与鉴定[J]. 微生物学通报, 2015, 42(2):280-288.
	Kang HY, Wang W, Liu JL, et al. Isolation and identification of two plant-growth promoting endophytes from alfalfa[J]. Microbiol China, 2015, 42(2):280-288.
[34]	阳湖荣, 陈飘雪, 戴传超. 白术内生细菌分离与促生活性[J]. 生态学杂志, 2019, 38(12):3680-3688.
	Yang HR, Chen PX, Dai CC. Isolation and plant-growth-promoting ability of endophytic bacteria in Atractylodes macrocephala Koidz[J]. Chin J Ecol, 2019, 38(12):3680-3688.
[35]	傅晓方, 韩红江, 郝勇锋, 等. 玉米内生固氮菌的分离鉴定及对小麦幼苗的促生效应[J]. 西北农业学报, 2012, 21(1):66-71.
	Fu XF, Han HJ, Hao YF, et al. Isolation and identification of endophytic diazotrophic bacteria from maize and their effect on wheat seedling growth[J]. Acta Agric Boreali Occidentalis Sin, 2012, 21(1):66-71.
[36]	迟惠荣, 张亚惠, 曾欣, 等. 多花黄精内生贝莱斯芽胞杆菌的分离鉴定及其抗菌与促生作用分析[J]. 植物保护, 2019, 45(4):122-131.
	Chi HR, Zhang YH, Zeng X, et al. Isolation and identification of antagonistic endophytic Bacillus velezensis from Polygonatum cyrtonema Hua and analysis of its antimicrobial and growth-promoting activity[J]. Plant Prot, 2019, 45(4):122-131.

培养基 Medium	内生细菌 Endophytic bacteria	内生真菌 Endophytic fungi
PDA培养基 PDA medium	10	7
LB培养基 LB medium	6	0
NA培养基NA medium	8	0
GA培养基GA medium	1	1
CZA培养基 CZA medium	0	1

培养基 Medium	内生细菌 Endophytic bacteria	内生真菌 Endophytic fungi
PDA培养基 PDA medium	10	7
LB培养基 LB medium	6	0
NA培养基NA medium	8	0
GA培养基GA medium	1	1
CZA培养基 CZA medium	0	1

菌株编号 Strain No.	产IAA强度 IAA production strength
IAA标准品 IAA standard substance（30 mg·L^-1）	+++
HNC2、HPB1	++
HPB3、HPC2、HPC4、HPA3、HLB3	+
HLB2、HNC1、HNB3、HGC1、HPB2、HNC3、HLA3、HPA2、HNA3、HPA1、HLA1、HLA4、HNB1、HNB2、HNA2、HPC3、HPC1、HLA2	-

菌株编号 Strain No.	产IAA强度 IAA production strength
IAA标准品 IAA standard substance（30 mg·L^-1）	+++
HNC2、HPB1	++
HPB3、HPC2、HPC4、HPA3、HLB3	+
HLB2、HNC1、HNB3、HGC1、HPB2、HNC3、HLA3、HPA2、HNA3、HPA1、HLA1、HLA4、HNB1、HNB2、HNA2、HPC3、HPC1、HLA2	-

生理生化测试项目 Test item	结果 Results
生理生化测试项目 Test item	HNC2	HPB1
葡萄糖 Glucose	+	-
乳糖 Lactose	-	-
麦芽糖 Maltose	+	-
甘露醇 Mannitol	-	-
蔗糖 Sucrose	+	-
靛基质试验 Indole test	+	+
甲基红试验Methyl Red（MR）	+	-
乙酰甲基甲醇试验 Voges-Proskaurer（VP）	+	+
西蒙氏柠檬酸盐 Simon’s citrate	+	+
硫化氢 Hydrogen sulphide	-	-
尿素 Urea	-	+
半固体琼脂 Semi-solid agar	+	+