水稻内生细菌新资源分离培养方案探究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0961

生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 201-211.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0961

水稻内生细菌新资源分离培养方案探究

李怡君¹(), 吴晨晨¹, 李睿¹, 王喆², 何山文³, 韦善君¹(), 张晓霞²()

1.中央民族大学生命与环境科学学院，北京 100081
2.中国农业科学院农业资源与农业区划研究所农业农村部农业微生物资源收集与保藏重点实验室，北京 100081
3.上海市园林科学规划研究院，上海 200232

收稿日期:2022-08-08 出版日期:2023-04-26 发布日期:2023-05-16
通讯作者: 韦善君，女，博士，副教授，研究方向：农业资源微生物学；E-mail: wei.s.j@163.com；
张晓霞，女，博士，研究员，研究方向：农业资源微生物学；E-mail: zhangxiaoxia@caas.cn
作者简介:李怡君，女，硕士研究生，研究方向：微生物学；E -mail：1624968646@qq.com
第一联系人：
吴晨晨为本文共同第一作者
基金资助:
国家自然科学基金项目(31970007);中国农业科学院科技创新工程(CAAS-ZDRW202201);北京市乡村振兴科技项目(BJXCZX2022);中央民族大学大学生创新训练计划项目(URTP2021110092)

Exploring Cultivation Approaches for New Endophytic Bacterial Resource in Oryza sativa

LI Yi-jun¹(), WU Chen-chen¹, LI Rui¹, WANG Zhe², HE Shan-wen³, WEI Shan-jun¹(), ZHANG Xiao-xia²()

1. College of Life and Environmental Science, Minzu University of China, Beijing 100081
2. Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture and Rural Affairs, Beijing 100081
3. Shanghai Academy of Landscape Architecture Science and Planning, Shanghai 200232

Received:2022-08-08 Published:2023-04-26 Online:2023-05-16

摘要/Abstract

摘要：

为了探究适用于分离水稻内生细菌的方案以获得水稻内细菌新资源，本研究基于培养组的方法，以水稻3个品种种子及幼苗为材料，从材料表面消毒、菌悬液的制备和培养基配比3个方面进行探究，并对分离获得代表性纯培养物的16S rRNA基因进行测序鉴定。结果表明，75%乙醇和含2%有效氯的次氯酸钠溶液组合对种子先后处理90 s和12 min，对植株组织先后处理90 s和5 min即可获得良好的消毒效果。水稻种子内生细菌量为10⁵-10⁶CFU/g，其中90%-95%分布于颖壳内，不同品种或同一品种的不同生境来源的种子之间内生细菌群落组成有明显差异；在制备种子菌悬液时需注意控制研磨程度以获得种类丰富的菌落，剥去颖壳有利于分离培养糙米中的内生菌。对于根、茎、叶内生细菌的分离，需要根据材料不同控制接种菌悬液的浓度以获得其中微量的内生细菌；在培养基中添加组织浸提液和延长培养时间至两周以上，可促进根、茎、叶内生细菌的分离培养。采用调整后的方案，本研究对3个水稻品种的种子和它们的植株根、茎、叶内生细菌进行分离培养，分别完成了71、14、10和2个代表性纯培养物的16S rRNA基因测序鉴定，其中有8株与已知类群模式菌株同源序列相似度在97.22%-98.74%，新种获得率为8.25%。研究结果为分离培养水稻内生细菌新资源提供了方法参考。

关键词: 水稻, 内生细菌, 培养基, 组织浸提液, 表面消毒, 菌悬液制备

Abstract:

In order to explore approaches of isolating new endophytic bacteria in rice and obtain novel bacterial resources in rice, 3 aspects of material surface sterilization, preparing bacterial suspension, and the formulation of cultural medium were explored while using seeds and seedlings of three rice varieties as materials. The representative pure isolates were identified based on the sequence of the 16S rRNA gene. The results indicated that promising surface sterilization could be achieved by treating seeds with 75% ethanol for 90 s plus NaClO solution（containing 2% available chlorine）for 12 min, or by treating plant tissues with 75% ethanol for 90 s plus NaClO solution for 5 min. In rice seeds, the amount of endophytic bacteria was up to 10⁵-10⁶ CFU per gram，of which 90%-95% were harbored in glumes, and the bacterial community varied according to varieties and localities of growth. The grinding degree should be considered during the preparation of bacterial suspension so as to obtain colonies as diverse as possible, and stripping off glumes was beneficial to isolating species distributed mainly in the brown rice. For the separation of endophytic bacteria in rice root, stem and leave, it was necessary to control the concentration of bacterial suspension according to the material characteristics so as to obtain the trace species harbored inside. The isolation and culture of some endophytic bacteria in rice root, stem and leave could be facilitated by adding tissue extract to the culture medium and by prolonging the cultivation time to over two weeks. Using the modified protocol, we separated and cultivated the endophytic bacteria in seeds as well as roots, stems and leaves of three rice varieties, and identified 71, 14, 10 and 2 pure isolates respectively, by 16S rRNA gene sequencing. A total of 8 isolates, accounting for 8.25% of the total identified isolates, showed a similarity of 97.22%-98.74% to the model strains recorded in the Ezbiocloud database and were putative new species. The results of our study may provide a referenced method to isolate and cultivate new resources of endophytic bacteria in rice.

Key words: Oryza sativa, endophytic bacteria, culture medium, tissue extract, surface sterilization, bacterial suspension preparation

李怡君, 吴晨晨, 李睿, 王喆, 何山文, 韦善君, 张晓霞. 水稻内生细菌新资源分离培养方案探究[J]. 生物技术通报, 2023, 39(4): 201-211.

LI Yi-jun, WU Chen-chen, LI Rui, WANG Zhe, HE Shan-wen, WEI Shan-jun, ZHANG Xiao-xia. Exploring Cultivation Approaches for New Endophytic Bacterial Resource in Oryza sativa[J]. Biotechnology Bulletin, 2023, 39(4): 201-211.

图/表 14

参考文献 36

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材料 Materials	处理时间 Treatment time/min
材料 Materials	75%乙醇 75% ethanol	NaClO溶液 NaClO solution
种子、植株组织Seeds, and plant tissues	1.5	12
种子 Seeds	1.5	15
种子 Seeds	3	12
种子 Seeds	3	15
植株组织 Plant tissues	1.5	5

材料 Materials	处理时间 Treatment time/min
材料 Materials	75%乙醇 75% ethanol	NaClO溶液 NaClO solution
种子、植株组织Seeds, and plant tissues	1.5	12
种子 Seeds	1.5	15
种子 Seeds	3	12
种子 Seeds	3	15
植株组织 Plant tissues	1.5	5

菌落类型 Colony type	形状和色泽 Shape and color	直径 Diameter/mm
I	黄色，圆形Yellow and round	3-5
Ⅱ	黄色，圆形Yellow and round	1-3
Ⅲ	中间黄色，边缘浅白Yellow in the middle, and light white on the edge	3-5
Ⅳ	白色，圆形White and round	3-5
Ⅴ	白色，圆形White and round	1-3
Ⅵ	粉红色，圆形Pink and round	1-3
Ⅶ	橙色，圆形Orange and round	3-5

菌落类型 Colony type	形状和色泽 Shape and color	直径 Diameter/mm
I	黄色，圆形Yellow and round	3-5
Ⅱ	黄色，圆形Yellow and round	1-3
Ⅲ	中间黄色，边缘浅白Yellow in the middle, and light white on the edge	3-5
Ⅳ	白色，圆形White and round	3-5
Ⅴ	白色，圆形White and round	1-3
Ⅵ	粉红色，圆形Pink and round	1-3
Ⅶ	橙色，圆形Orange and round	3-5

品种 Cultivar	种子部位 Seed part	稀释度 Dilution	菌落数/皿* CFU/ dish *								内生细菌总数 Total number of endophytic bacteria/（CFU·g^-1）
品种 Cultivar	种子部位 Seed part	稀释度 Dilution	I	Ⅱ	Ⅲ	Ⅳ	Ⅴ	Ⅵ	Ⅶ	总数 Total	内生细菌总数 Total number of endophytic bacteria/（CFU·g^-1）
日本晴 Nipponbare	完整种子 Intact seeds	10^-2	26	69	31	9	227	83	0	445	3.2×10⁵
	完整种子 Intact seeds	10^-3	10	8	14	1	7	23	0	63	3.2×10⁵
	糙米 Brown rice	10^-1	78	0	0	0	4	178	0	260	1.3×10⁴
	糙米 Brown rice	10^-2	24	6	0	0	1	63	0	94	1.3×10⁴
日本晴-LF Nipponbare-LF	完整种子 Intact seeds	10^-2	24	0	0	9	260	1	0	293	1.5×10⁵
	完整种子 Intact seeds	10^-3	9	0	0	0	68	0	0	77	1.5×10⁵
	糙米 Brown rice	10^-1	1	2	0	1	19	139	6	168	8.5×10³
	糙米 Brown rice	10^-2	6	0	0	9	0	35	0	50	8.5×10³
培矮64 Peiai 64	完整种子 Intact seeds	10^-2	220	40	0	0	376	2	0	638	1.6×10⁶
	完整种子 Intact seeds	10^-3	119	35	0	0	147	13	0	314	1.6×10⁶
	糙米 Brown rice	10^-1	240	40	0	10	64	0	0	354	1.2×10⁵
	糙米 Brown rice	10^-2	143	37	0	30	21	1	0	232	1.2×10⁵
Kashlath	完整种子 Intact seeds	10^-2	44	144	0	43	653	0	0	884	8.0×10⁵
	完整种子 Intact seeds	10^-3	24	5	0	8	121	0	0	158	8.0×10⁵
	糙米 Brown rice	10^-1	530	9	0	0	0	25	0	564	9.0×10³
	糙米 Brown rice	10^-2	120	10	0	0	0	60	0	180	9.0×10³

水稻内生细菌新资源分离培养方案探究

Exploring Cultivation Approaches for New Endophytic Bacterial Resource in Oryza sativa

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品种 Cultivar	部位 Seed part	成功鉴定菌株数量 Number of identified strains	种的数量 Number of species	部位共有种数量 Number of site-common species	部位特有种数量 Number of site-specific species
日本晴 Nipponbare	完整种子 Intact seed	24	6	1	5
日本晴 Nipponbare	糙米 Brown rice	2	2	1	1
日本晴-LF Nipponbare-LF	完整种子 Intact seed	7	4	1	3
日本晴-LF Nipponbare-LF	糙米 Brown rice	2	1	1	0
培矮64 Peiai 64	完整种子 Intact seed	19	6	3	3
培矮64 Peiai 64	糙米 Brown rice	6	4	3	1
Kashlath	完整种子 Intact seed	6	3	1	2
Kashlath	糙米 Brown rice	5	3	1	2

植物组织Plant tissue	培养基Culture medium	菌悬液浓度Concentration of bacterial suspension	菌落数/皿CFU/dish
根Root	基础培养基Basic culture medium	10^-1	0-5
	基础培养基Basic culture medium	5×10^-1	20-50
	组织浸提液Tissue extract	10^-1	0-5
	组织浸提液Tissue extract	5×10^-1	20-50
	基础培养基+组织浸提液 Basic culture medium + tissue extract	10^-1	5-10
	基础培养基+组织浸提液 Basic culture medium + tissue extract	5×10^-1	20-50
茎Stem	基础培养基Basic culture medium	10^-1	0
	基础培养基Basic culture medium	5×10^-1	5-20
	组织浸提液Tissue extract	10^-1	0-5
	组织浸提液Tissue extract	5×10^-1	5-20
	基础培养基+组织浸提液 Basic culture medium + tissue extract	10^-1	5-10
	基础培养基+组织浸提液 Basic culture medium + tissue extract	5×10^-1	20-50
叶Leaf	基础培养基Basic culture medium	10^-1	0
		5×10^-1	0
		原浆 Protoplasm	0-2
	组织浸提液Tissue extract	10^-1	0
	组织浸提液Tissue extract	5×10^-1	0
	基础培养基+组织浸提液 Basic culture medium + tissue extract	10^-1	0-5
	基础培养基+组织浸提液 Basic culture medium + tissue extract	5×10^-1	5-10

菌落类型 Colony type	形状和色泽 Shape and color	直径 Diameter/mm
A	橙色，圆形 Orange and round	1-3
B	白色，圆形 White and round	1-3
C	青色，圆形 Cyan and round	1-3
D	黄棕色，圆形 Yellowish brown and round	3-5
E	透明，圆形 Transparent and round	1-3
F	白色，圆形，表面干燥 White, round, and dry surface	1-3
G	黄色，圆形 Yellow and round	1-3
H	中央黄色，边缘透明，圆形 Yellow center, transparent edge, and round	>5

分离物编号 Isolate ID	来源 Source	最高匹配种 Top-hit taxon	模式菌株编号 Top-hit strain	相似率 Similarity/%	菌落类型 Colony type
SD-7	根、茎 Roots and stems	Chitinophaga ginsengihumi	SR18	98.18	B
SD-9	根、茎 Roots and stems	Sphingomonas Canadensis	FWC47（T）	97.35	G
SD-10	茎 Stems	Diaminobutyricibacter tongyongensis	KIS66-7（T）	98.74	G
SD-11	茎Stems	Hephaestia caeni	DSM 25527（T）	97.59	B
SD-22	根Roots	Phycicoccus endophyticus	IP6SC6	98.41	B
SD-26	茎Stems	Sphingomonas astaxanthinifaciens	DSM 22298	97.56	A

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