Contaminated Microorganisms in Plant Tissue Culture: Isolation and Identification and Fungicides Screening

doi:10.11924/j.issn.1000-6850.casb2024-0298

Abstract

Abstract:

This research aims to identify the types of common contaminating bacteria during plant tissue culture, evaluate the antibacterial effect of bacteriostatic agents on contaminating bacteria, and screen out effective bacteriostatic agents. Taking six bacterial strains of common contaminants in tissue culture room as test materials, they were isolated and purified first, and then were classified and identified through 16S rDNA or ITS sequence homology analysis combined with phylogenetic tree analysis. The antibacterial effects of 5 antibacterial agents on 3 kinds of bacteria were evaluated by the inhibition zone method. The antibacterial effects of 5 antibacterial agents on 3 kinds of fungi were evaluated by the growth rate method. The results showed that Microbacterium paraoxydans, Acinetobacter radioresistens, and Sphingomonas yabuuchiaes were three contaminating bacteria, while Aspergillus versicolor, Cladosporium endophyticum, Aspergillus sydowii were three contaminating fungi. The bacteriostatic test results showed that 0.5 g/L 1,2-benzisothiazolin-3-one and 0.5 g/L enyl·mangano-zinc had good inhibitory effect on all the 6 tested bacteria; 0.5 mL/L isothiazolinone had good inhibitory effect on Acinetobacter radioresistens XJ-2, Sphingomonas yabuuchiaes XJ-3, Cladosporium endophyticum ZJ-2, Aspergillus sydowii ZJ-3, and Aspergillus versicolor ZJ-1. 0.05 g/L procymidone-thiram could effectively inhibit the growth of the three fungi, and 0.5 g/L procymidone-thiram had inhibitory effect on Microbacterium paraoxydans XJ-1 and Sphingomonas yabuuchiaes XJ-3. Bronopol had almost no inhibitory effect on bacterial growth, while 0.5 g/L bronopol had a certain inhibitory effect on fungi. To sum up, 1,2-benzisothiazolin-3-one, enoyl·manganese zinc and isothiazolinone show strong and broad-spectrum antibacterial effects, and have good application prospects as bacteriostatic agents in controlling plant tissue culture contamination.

Key words: bacteriostatic agent, bacteria, fungus, open tissue culture, tissue culture contamination, isolation and identification, 1,2-benzisothiazolin-3-one, enoyl·manganese zinc, phylogenetic tree analysis, 16S rDNA, ITS sequence, inhibition zone method

HAN Yao, YANG Xiaoyan, XIE Shuzhang. Contaminated Microorganisms in Plant Tissue Culture: Isolation and Identification and Fungicides Screening[J]. Chinese Agricultural Science Bulletin, 2025, 41(3): 131-138.

Figures/Tables 5

References 28

[1]	GUPTA N P, JAIN V, JOSEPH M, et al. A review on micropropagation culture method[J]. Asian journal of phar-maceutical research and development, 2020, 8(1):86-93.
[2]	MBAH E I, WAKIL S M. Elimination of bacteria from in vitro yam tissue cultures using antibiotics[J]. Journal of plant pathology, 2012, 94(1):53-58.
[3]	白一苇, 张世壮, 王雁楠, 等. 植物组织培养中抗污染培养基新配方的探索[J]. 中国农学通报, 2021, 37(6):89-96. doi: 10.11924/j.issn.1000-6850.casb2020-0093
[4]	田成津. 马铃薯组培中污染原因及解决对策[J]. 植物保护, 2012(1):35-36.
[5]	王秀英, 兰创业, 赵军良, 等. 大白菜小孢子培养中的污染问题及防治对策[J]. 上海蔬菜, 2018(6):72-74.
[6]	周颖怡, 王景飞, 钟建立, 等. 茶树组织培养技术与应用研究进展[J]. 分子植物育种, https://link.cnki.net/urlid/46.1068.S.20240221.1638.016
[7]	方丽, 王连平, 茹水江, 等. 植物组培过程中污染微生物种类及其季节性的变化[J]. 浙江农业学报, 2013, 25(2):284-287.
[8]	陈翠红, 苗春泽, 田年军, 等. 植物组织培养中常见污染类型及污染防控措施[J]. 现代园艺, 2023(24):155-159.
[9]	刘红坚, 李松, 何为中, 等. 甘蔗组培苗 2 种污染细菌的分离与鉴定[J]. 热带作物学报, 2021, 42(2):519-526. doi: 10.3969/j.issn.1000-2561.2021.02.030
[10]	FANG J Y, HSU Y R. Molecular identification and an-tibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures[J]. Plant cell tissue and organ culture, 2012, 110(1):53-62.
[11]	杨丽霞, 杨丽, 李永梅, 等. 4种食品防腐剂在铁皮石斛开放式组织培养中的抑菌效果[J]. 热带作物学报, 2017, 38(10):1921-1925.
[12]	韩宏伟, 廖晴, 玛尔哈巴·吾斯满, 等. 德国鸢尾组培快繁过程中内生菌的分离、鉴定及控制药剂筛选[J]. 新疆农业科学, 2017, 54(4):652-659.
[13]	王政, 王建格, 何松林, 等. 牡丹组织培养过程中抑菌剂的筛选及内生细菌的鉴定[J]. 分子植物育种, 2022, 20(8):2707-2712.
[14]	卢艳平, 陈玉军, 李玫, 等. 马占相思开放式组培体系的建立[J]. 西北农林科技大学学报(自然科学版), 2023, 51(7):107-114.
[15]	朱梦珠, 杨惠婷, 胡计红, 等. 切花菊‘白扇’开放式组培快繁体系的建立[J]. 热带作物学报, 2019, 40(8):1551-1558. doi: 10.3969/j.issn.1000-2561.2019.08.014
[16]	陈泽斌, 李冰, 高熹, 等. 抑菌剂在蓝莓开放组培中的应用试验研究[J]. 中国南方果树, 2017, 46(3):139-142.
[17]	陆春霞, 刘开莉, 梁贵秋, 等. 十一个桑黄菌株的形态学观察与分子鉴定[J]. 浙江农业学报, 2024, 36(4):800-810. doi: 10.3969/j.issn.1004-1524.20230417
[18]	刘绍雄, 王娟, 王金华, 等. 一株引起马来甜龙竹组培污染内生菌的分离与鉴定[J]. 微生物学通报, 2012, 39(8):1112-1119.
[19]	李伟杰, 朱孟乾, 曹广龙, 等. 水草组培中常见污染菌的初步鉴定分析[J]. 现代农业科技, 2023(2):69-74.
[20]	侯馨怡, 陈燕, 李彩侠, 等. 利用防腐剂控制植物组织培养中的微生物污染[J]. 应用与环境生物学报, 2023, 29(3):730-735.
[21]	杨钰泽, 宁楠楠, 马永强. 11种杀菌剂对马铃薯组培苗污染真菌室内毒力测定[J]. 青海大学学报, 2023, 41(2):56-65.
[22]	MUTSCHLERA J, GIMÉNEZ-ARNAUA E, FOERTSCH L. Mechanistic assessment of peptide reactivity assay to predict skin allergens with Kathon CG isothiazolinones[J]. Toxicology in vitro, 2009, 23(3):439-446. pmid: 19444925
[23]	陈晓君, 张饮江, 王芳, 等. 两种抑菌剂对马来眼子菜开放式组织培养的效应[J]. 浙江农业学报, 2015, 27(4):598-603.
[24]	牛艳, 吴燕, 杨静, 等. 烯酰吗啉在葡萄上残留及风险状况分析[J]. 浙江农业科学, 2021, 62(5):1022-1024. doi: 10.16178/j.issn.0528-9017.20210560
[25]	阎建辉, 朱政斌, 刘强, 等. 纳米Ag/TiO₂对烯酰吗啉的吸附与光催化降解[J]. 稀有金属, 2008, 32(1):78-83.
[26]	张艺, 张兴红, 王丰, 等. 代森锰锌对烟草靶斑病的防效及其叶际微生态的影响[J]. 江苏农业科学, 2023, 51(17):122-131.
[27]	赵建江, 张小风, 王文桥, 等. 氟硅唑与代森锰锌混配对梨黑星病菌的联合毒力及田间防效[J]. 植物保护, 2014, 40(3):95-198.
[28]	凌金锋, 周娟, 彭埃天, 等. 吡唑醚菌酯与代森锰锌对两种果树病原菌的联合毒力[J]. 植物保护, 2016, 42(5):246-250.

类型	菌株	最相似菌株	登录号	相似性/%
真菌	ZJ-1	Aspergillus versicolor isolate CA3	OR354725.1	99.64
	ZJ-2	Cladosporium endophyticum MFLUCC 17-0599	NR_158360.1	99.06
	ZJ-3	Aspergillus sydowii isolate H2Pb	KP994297.1	99.46
细菌	XJ-1	Microbacterium paraoxydans strain H396	MH669308.1	99.72
	XJ-2	Acinetobacter radioresistens strain LJ16106	KY767501.1	100
	XJ-3	Sphingomonas yabuuchiae strain OsEp_A&N_30A3	MT367848.1	97.67

类型	菌株	最相似菌株	登录号	相似性/%
真菌	ZJ-1	Aspergillus versicolor isolate CA3	OR354725.1	99.64
	ZJ-2	Cladosporium endophyticum MFLUCC 17-0599	NR_158360.1	99.06
	ZJ-3	Aspergillus sydowii isolate H2Pb	KP994297.1	99.46
细菌	XJ-1	Microbacterium paraoxydans strain H396	MH669308.1	99.72
	XJ-2	Acinetobacter radioresistens strain LJ16106	KY767501.1	100
	XJ-3	Sphingomonas yabuuchiae strain OsEp_A&N_30A3	MT367848.1	97.67

编号	抑菌剂	浓度	抑菌圈直径/mm
编号	抑菌剂	浓度	副氧化微杆菌XJ-1	抗辐射不动杆菌XJ-2	鞘氨醇单胞菌XJ-3
CK			0.00±0.00	0.00±0.00	0.00±0.00
Y1	烯酰·锰锌	0.1 g/L	13.88±0.38	9.40±0.61	7.17±0.25
		0.25 g/L	21.21±0.28	15.91±0.11	8.89±0.23
		0.5 g/L	22.93±0.08	18.49±0.14	9.44±0.37
Y6	腐霉·福美双	0.1 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.25 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.5 g/L	10.67±0.69	0.00±0.00	10.27±0.39
Y19	异噻唑啉酮	0.1 ml/L	0.00±0.00	5.11±0.35	7.33±0.37
		0.25 ml/L	0.00±0.00	11.11±0.22	14.39±0.25
		0.5 ml/L	6.72±0.21	17.06±0.70	17.84±0.33
Y22	1,2-苯并异噻唑啉-3-酮	0.1 g/L	13.60±0.18	15.50±0.39	7.38±0.00
		0.25 g/L	18.33±0.71	21.08±0.28	9.61±0.22
		0.5 g/L	27.56±0.87	27.44±0.28	18.67±0.32
Y32	溴硝醇	0.1 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.25 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.5 g/L	0.00±0.00	0.00±0.00	0.00±0.00

编号	抑菌剂	浓度	抑菌圈直径/mm
编号	抑菌剂	浓度	副氧化微杆菌XJ-1	抗辐射不动杆菌XJ-2	鞘氨醇单胞菌XJ-3
CK			0.00±0.00	0.00±0.00	0.00±0.00
Y1	烯酰·锰锌	0.1 g/L	13.88±0.38	9.40±0.61	7.17±0.25
		0.25 g/L	21.21±0.28	15.91±0.11	8.89±0.23
		0.5 g/L	22.93±0.08	18.49±0.14	9.44±0.37
Y6	腐霉·福美双	0.1 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.25 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.5 g/L	10.67±0.69	0.00±0.00	10.27±0.39
Y19	异噻唑啉酮	0.1 ml/L	0.00±0.00	5.11±0.35	7.33±0.37
		0.25 ml/L	0.00±0.00	11.11±0.22	14.39±0.25
		0.5 ml/L	6.72±0.21	17.06±0.70	17.84±0.33
Y22	1,2-苯并异噻唑啉-3-酮	0.1 g/L	13.60±0.18	15.50±0.39	7.38±0.00
		0.25 g/L	18.33±0.71	21.08±0.28	9.61±0.22
		0.5 g/L	27.56±0.87	27.44±0.28	18.67±0.32
Y32	溴硝醇	0.1 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.25 g/L	0.00±0.00	0.00±0.00	0.00±0.00
		0.5 g/L	0.00±0.00	0.00±0.00	0.00±0.00

编号	抑菌剂			浓度	杂色曲霉ZJ-1			枝孢内生菌ZJ-2		聚多曲霉ZJ-3
编号	抑菌剂			浓度	菌落直径/mm	抑菌率/%		菌落直径/mm	抑菌率/%	菌落直径/mm	抑菌率/%
CK					19.83			27.33		25.67
Y1	烯酰·锰锌			0.01 g/L	16.43	22.92		27.20	0.60	18.27	35.81
				0.05 g/L	12.97	46.29		22.07	23.58	15.90	47.26
				0.1 g/L	11.77	54.38		19.83	33.58	15.17	50.81
				0.25 g/L	9.67	68.54		14.07	59.40	13.17	60.48
				0.5 g/L	7.23	84.94		5.53	97.61	12.00	66.13
Y6	腐霉·福美双			0.01 g/L	7.33	84.27		25.67	7.46	5.00	100.00
				0.05 g/L	5.00	100.00		7.33	89.55	5.00	100.00
				0.1 g/L	5.00	100.00		5.67	97.01	5.00	100.00
				0.25 g/L	5.00	100.00		5.00	100.00	5.00	100.00
				0.5 g/L	5.00	100.00		5.00	100.00	5.00	100.00
Y19		异噻唑啉酮	0.01 mL/L		16.33		23.60	25.17	9.70	25.17		2.42
			0.05 mL/L		13.33		43.82	18.67	38.81	21.33		20.97
			0.1 mL/L		10.17		65.17	16.50	48.51	16.30		45.32
			0.25 mL/L		8.50		76.40	5.00	100.00	9.83		76.61
			0.5 mL/L		8.33		77.53	5.00	100.00	5.17		99.19
Y22		1,2-苯并异噻唑啉-3-酮	0.01 g/L		16.20		24.49	16.27	49.55	17.67		38.71
			0.05 g/L		13.17		44.94	10.17	76.87	14.83		52.42
			0.1 g/L		10.33		64.04	6.33	94.03	9.83		76.61
			0.25 g/L		8.67		75.28	5.67	97.01	8.83		81.45
			0.5 g/L		5.67		95.51	5.33	98.51	5.67		96.77
Y32		溴硝醇	0.01 g/L		18.83		6.74	26.33	4.48	22.17		16.94
			0.05 g/L		17.17		17.98	21.67	25.37	20.83		23.39
			0.1 g/L		16.27		24.04	21.60	25.67	16.07		46.45
			0.25 g/L		16.13		24.94	18.00	41.79	10.67		72.58
			0.5 g/L		12.33		50.56	13.67	61.19	7.33		88.71