Exploring Cultivation Approaches for New Endophytic Bacterial Resource in Oryza sativa

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

Abstract

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

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.

Figures/Tables 14

References 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