高秆野生稻内生固氮细菌多样性

doi:10.17520/biods.2019366

生物多样性 ›› 2020, Vol. 28 ›› Issue (8): 1018-1025. DOI: 10.17520/biods.2019366

高秆野生稻内生固氮细菌多样性

刘丽平¹, 宋瑞凤¹, 张馥¹^,³, 张秀香¹, 彭桂香²^,^*(), 谭志远¹^,^*()

^1. 华南农业大学农学院, 广州 510642
^2. 华南农业大学资源环境学院, 广州 510642
^3. 河北省保定市乐凯中学, 河北保定 071051

收稿日期:2019-11-12 接受日期:2020-03-03 出版日期:2020-08-20 发布日期:2020-06-12
通讯作者: 彭桂香,谭志远
作者简介:zytan@scau.edu.cn
*. E-mail: gxpeng@scau.edu.cn;
基金资助:
国家自然科学基金(31970001);广东省科技计划项目(2018A050506075);广东省科技创新战略专项资金(2018B020205003);广东省科技创新战略专项资金(2018B020206001);广东省植物分子育种重点实验室开放基金

Diversity of endophytic diazotrophs isolated from Oryza alta

Liping Liu¹, Ruifeng Song¹, Fu Zhang¹^,³, Xiuxiang Zhang¹, Guixiang Peng²^,^*(), Zhiyuan Tan¹^,^*()

1 College of Agriculture, South China Agricultural University, Guangzhou 510642
2 College of Resource and Environment, South China Agricultural University, Guangzhou 510642
3 Baoding Lucky Middle School, Hebei Province, Baoding, Hebei 071051

Received:2019-11-12 Accepted:2020-03-03 Online:2020-08-20 Published:2020-06-12
Contact: Guixiang Peng,Zhiyuan Tan

摘要/Abstract

摘要：

高秆野生稻(Oryza alta)是一种重要的种质资源, 其组织内也蕴藏着非常宝贵的功能微生物资源。本实验采用无氮培养基, 从高秆野生稻中分离到43株内生固氮菌, 结合乙炔还原法测定其固氮酶活性。经固氮酶基因(nifH)的PCR扩增检测, 43株内生固氮菌代表菌株均能扩增出固氮酶基因片段。利用IS-PCR DNA指纹图谱和SDS-PAGE全细胞蛋白电泳图谱将获得的菌株聚类为6个类群(I、II、III、IV、V、VI)。对各个类群的代表菌株(ZF3, ZF8, ZF13, ZF15, ZF24, ZF43)进行16S rRNA基因序列测定, 结果表明, 类群I属于土生拉乌尔菌(Raoultella terrigena), 类群II属于类肺炎克雷伯氏菌类肺炎亚种(Klebsiella quasipnmoniae subsp. quasipneumoniae), 类群III属于越南伯克氏菌(Burkholderia vietnamiensis), 类群IV的菌株代表肠秆菌属的一个类群(Enterobacter sp.), 类群V的菌株属于门多萨假单胞菌(Pseudomonas mendocina), 类群VI归属于固氮植物菌(Phytobacter diazotrophicus)。Biolog聚类结果与IS-PCR指纹图谱类型及SDS-PAGE全细胞蛋白聚类结果一致。Biolog板测定结果显示, 来自不同类群的代表菌株对碳源的利用差异显著, 说明野生稻内多样的内生固氮菌从环境中获取碳源和氮素的适应能力较强。

关键词: 内生固氮菌, 多样性, 全细胞蛋白电泳, 指纹图谱, 聚类分析

Abstract

Forty-three strains of endophytic nitrogen-fixing bacteria were obtained from wild rice Oryza alta, and grown on nitrogen-free medium. Their potential nitrogen-fixing ability was determined with the acetylene reduction method and a PCR amplification of the nifH gene fragment. The obtained isolates were grouped into six groups (I, II, III, IV, V, VI) based on their DNA patterns of IS-PCR and protein patterns of SDS-PAGE. The 16S rRNA gene sequence analysis identified representative strains, suggesting that group I belonged to Raoultella terrigena, group II to Klebsiella quasipneumoniae subsp. quasipneumoniae, group III to Burkholderia vietnamiensis, group IV to genus Enterobacter, group V to Pseudomonas mendocina, and group VI to Phytobacter diazotrophicus. A Biolog plate test showed strains from differing groups had significant differences in carbon source utilization, often clustering consistently within IS-PCR and SDS-PAGE patterns. The diversity of endophytic nitrogen-fixing bacteria in wild rice indicates that endophytic bacteria is strongly adaptable in obtaining both carbon and nitrogen from varying environments.

Key words: endophytic diazotrophs, diversity, eletrophoresis of whole cell protein patterns, fingerprinting patterns, clustering analysis

刘丽平, 宋瑞凤, 张馥, 张秀香, 彭桂香, 谭志远 (2020) 高秆野生稻内生固氮细菌多样性. 生物多样性, 28, 1018-1025. DOI: 10.17520/biods.2019366.

Liping Liu, Ruifeng Song, Fu Zhang, Xiuxiang Zhang, Guixiang Peng, Zhiyuan Tan (2020) Diversity of endophytic diazotrophs isolated from Oryza alta. Biodiversity Science, 28, 1018-1025. DOI: 10.17520/biods.2019366.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2019366

https://www.biodiversity-science.net/CN/Y2020/V28/I8/1018

图/表 5

参考文献 28

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菌株编号 Strain no.	部位 Organ	固氮酶活性 Nitrogenase activity (nmol C₂H₂/mL·h)	菌株编号 Strain no.	部位 Organ	固氮酶活性 Nitrogenase activity (nmol C₂H₂/mL·h)
ZF1	根 Root	10.82 ± 0.15	ZF23	叶 Leave	72.63 ± 0.93
ZF2	叶 Leave	26.19 ± 0.19	ZF24	叶 Leave	57.67 ± 0.45
ZF3	茎 Stem	15.76 ± 0.31	ZF25	茎 Stem	59.60 ± 0.41
ZF4	茎 Stem	10.72 ± 0.24	ZF26	茎 Stem	13.18 ± 0.18
ZF5	茎 Stem	15.39 ± 0.28	ZF27	茎 Stem	184.15 ± 1.05
ZF6	茎 Stem	20.22 ± 0.39	ZF28	叶 Leave	67.17 ± 0.79
ZF7	叶 Leave	76.00 ± 0.45	ZF29	根 Root	10.91 ± 0.22
ZF8	根 Root	254.31 ± 4.51	ZF30	根 Root	5.44 ± 0.15
ZF9	根 Root	26.09 ± 0.29	ZF31	茎 Stem	257.42 ± 1.57
ZF10	茎 Stem	506.46 ± 3.63	ZF32	叶 Leave	29.41 ± 0.33
ZF11	茎 Stem	1.23 ± 0.11	ZF33	茎 Stem	1.82 ± 0.17
ZF12	根 Root	69.44 ± 0.65	ZF34	茎 Stem	43.62 ± 0.43
ZF13	茎 Stem	17.20 ± 0.16	ZF35	茎 Stem	87.18 ± 0.55
ZF14	叶 Leave	41.82 ± 0.39	ZF36	茎 Stem	132.52 ± 0.66
ZF15	茎 Stem	59.84 ± 0.48	ZF37	根 Root	90.59 ± 0.48
ZF16	根 Root	94.03 ± 0.57	ZF38	茎 Stem	33.36 ± 0.34
ZF17	根 Root	107.85 ± 1.11	ZF39	茎 Stem	9.67 ± 0.19
ZF18	茎 Stem	33.02 ± 0.43	ZF40	根 Root	98.96 ± 0.51
ZF19	茎 Stem	11.74 ± 0.21	ZF41	叶 Leave	35.50 ± 0.43
ZF20	茎 Stem	108.38 ± 0.67	ZF42	茎 Stem	38.05 ± 0.42
ZF21	根 Root	64.74 ± 0.71	ZF43	茎 Stem	80.49 ± 0.59
ZF22	茎 Stem	20.50 ± 0.23

菌株编号 Strain no.	部位 Organ	固氮酶活性 Nitrogenase activity (nmol C₂H₂/mL·h)	菌株编号 Strain no.	部位 Organ	固氮酶活性 Nitrogenase activity (nmol C₂H₂/mL·h)
ZF1	根 Root	10.82 ± 0.15	ZF23	叶 Leave	72.63 ± 0.93
ZF2	叶 Leave	26.19 ± 0.19	ZF24	叶 Leave	57.67 ± 0.45
ZF3	茎 Stem	15.76 ± 0.31	ZF25	茎 Stem	59.60 ± 0.41
ZF4	茎 Stem	10.72 ± 0.24	ZF26	茎 Stem	13.18 ± 0.18
ZF5	茎 Stem	15.39 ± 0.28	ZF27	茎 Stem	184.15 ± 1.05
ZF6	茎 Stem	20.22 ± 0.39	ZF28	叶 Leave	67.17 ± 0.79
ZF7	叶 Leave	76.00 ± 0.45	ZF29	根 Root	10.91 ± 0.22
ZF8	根 Root	254.31 ± 4.51	ZF30	根 Root	5.44 ± 0.15
ZF9	根 Root	26.09 ± 0.29	ZF31	茎 Stem	257.42 ± 1.57
ZF10	茎 Stem	506.46 ± 3.63	ZF32	叶 Leave	29.41 ± 0.33
ZF11	茎 Stem	1.23 ± 0.11	ZF33	茎 Stem	1.82 ± 0.17
ZF12	根 Root	69.44 ± 0.65	ZF34	茎 Stem	43.62 ± 0.43
ZF13	茎 Stem	17.20 ± 0.16	ZF35	茎 Stem	87.18 ± 0.55
ZF14	叶 Leave	41.82 ± 0.39	ZF36	茎 Stem	132.52 ± 0.66
ZF15	茎 Stem	59.84 ± 0.48	ZF37	根 Root	90.59 ± 0.48
ZF16	根 Root	94.03 ± 0.57	ZF38	茎 Stem	33.36 ± 0.34
ZF17	根 Root	107.85 ± 1.11	ZF39	茎 Stem	9.67 ± 0.19
ZF18	茎 Stem	33.02 ± 0.43	ZF40	根 Root	98.96 ± 0.51
ZF19	茎 Stem	11.74 ± 0.21	ZF41	叶 Leave	35.50 ± 0.43
ZF20	茎 Stem	108.38 ± 0.67	ZF42	茎 Stem	38.05 ± 0.42
ZF21	根 Root	64.74 ± 0.71	ZF43	茎 Stem	80.49 ± 0.59
ZF22	茎 Stem	20.50 ± 0.23

类群 Groups	代表菌株 Representative strain	最相似菌株名称(GenBank登录号) Closely related strain (GenBank accession number)	相似性 Similarity (%)
I	ZF3	土生乌拉尔菌 Raoultella terrigena ATCC33257^T(NR_114503)	99.79
II	ZF8	类肺炎克雷伯氏菌肺炎亚种 Klebsiella quasipneumoniae subsp. quasipneumoniae 01A030^T (HG933296)	99.86
III	ZF13	越南伯克霍尔德氏菌 Burkholderia vietnamiensis LMG10929^T (AF097534)	99.93
IV	ZF15	肠杆菌 Enterobacter sp. R4-368 (CP005991_s)	99.59
V	ZF24	门多萨假单胞菌 Pseudomonas mendocina NBRC14162^T (BBQC01000018)	99.03
VI	ZF43	固氮植物菌 Phytobacter diazotrophicus LS8^T (DQ821583.2)	99.37

类群 Groups	代表菌株 Representative strain	最相似菌株名称(GenBank登录号) Closely related strain (GenBank accession number)	相似性 Similarity (%)
I	ZF3	土生乌拉尔菌 Raoultella terrigena ATCC33257^T(NR_114503)	99.79
II	ZF8	类肺炎克雷伯氏菌肺炎亚种 Klebsiella quasipneumoniae subsp. quasipneumoniae 01A030^T (HG933296)	99.86
III	ZF13	越南伯克霍尔德氏菌 Burkholderia vietnamiensis LMG10929^T (AF097534)	99.93
IV	ZF15	肠杆菌 Enterobacter sp. R4-368 (CP005991_s)	99.59
V	ZF24	门多萨假单胞菌 Pseudomonas mendocina NBRC14162^T (BBQC01000018)	99.03
VI	ZF43	固氮植物菌 Phytobacter diazotrophicus LS8^T (DQ821583.2)	99.37

高秆野生稻内生固氮细菌多样性

Diversity of endophytic diazotrophs isolated from Oryza alta

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