Characteristics and diversity of endophytic diazotrophs in three bamboo species

FENG Gelin; GAO Jing; YAN Shuxian; WANG Jing; LIANG Chenfei; QIN Hua; CHEN Junhui; XU Qiufang

doi:10.11833/j.issn.2095-0756.20190586

Volume 38 Issue 6

Dec. 2021

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FENG Gelin, GAO Jing, YAN Shuxian, WANG Jing, LIANG Chenfei, QIN Hua, CHEN Junhui, XU Qiufang. Characteristics and diversity of endophytic diazotrophs in three bamboo species[J]. Journal of Zhejiang A&F University, 2021, 38(6): 1203-1212. doi: 10.11833/j.issn.2095-0756.20190586

Citation:

FENG Gelin, GAO Jing, YAN Shuxian, WANG Jing, LIANG Chenfei, QIN Hua, CHEN Junhui, XU Qiufang. Characteristics and diversity of endophytic diazotrophs in three bamboo species[J]. Journal of Zhejiang A&F University, 2021, 38(6): 1203-1212. doi: 10.11833/j.issn.2095-0756.20190586

Characteristics and diversity of endophytic diazotrophs in three bamboo species

doi: 10.11833/j.issn.2095-0756.20190586

FENG Gelin^{1, 2
,},
GAO Jing^{1, 2},
YAN Shuxian^{1, 2},
WANG Jing^{1, 2},
LIANG Chenfei^{1, 2},
QIN Hua^{1, 2},
CHEN Junhui^{1, 2},
XU Qiufang^{1, 2
,
,}

1.
College of Environment and Resources, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
2.
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2020-10-10
Rev Recd Date: 2021-06-24

Available Online: 2021-08-09

Publish Date: 2021-12-08

Abstract

Objective The objective is to investigate the characteristics and biodiversity of endophytic diazotrophs in three bamboo species and in planting soil of root zones. Method Samples of leaves, roots and planting soil from Phyllostachys edulis, Bambusa multiplex and Pleioblastus chino were taken as the objects. The primers of nifH nitrogen fixation gene were designed. PCR-DGGE and random cloning technology were used to construct phylogenetic tree by cluster analysis and Shannon diversity index analysis. Result There were significant differences in community structure of diazotrophs among soils, between roots and leaves of the same bamboo species, between the same organs of different bamboo species, and between the soils of root zones and plants. The similarity of the same bamboo leaf or root was not high. More endophytic diazotrophs were detected in roots than in corresponding leaves. The similarity between diazotrophs in soil and those in roots was higher than that between soil and leaves. Significant differences of diazotrophs among plant species and plant tissues were observed by Shannon diversity index. The Shannon diversity index in leaf (2.73) and soil (2.70) of B. multiplex was higher than that in root (2.53) (P＜0.05), and that in roots of P. chino and Ph. edilus was higher than that in leaf and soil (P＜0.05). 83 strains of diazotrophs were obtained by random cloning and sequencing of plant samples, and only 12 strains were culturable, belonging to Bradyrhizobium, Dechlorosoma, Azospirillum, Enterobacter, Kosakonia, Gammaproteobacteria, Rubrivivax and Azohydromonas. The sequences of phylogenetic tree indicated that the endophytic diazotrophs in the three bamboo species differed greatly from those in other plants. Conclusion The structural characteristics and diversity of diazotrophs in bamboo are affected by soil properties, species and plant tissues. The diversity of endophytic diazotrophs in roots is significantly higher than that in leaves, and the corresponding taxa are distinct from those of Gramineae reported previously. [Ch, 4 fig. 4 tab. 30 ref.]
- bamboo,
- endophytic diazotroph,
- PCR-DGGE,
- cluster analysis,
- Shannon diversity index,
- random cloning,
- phylogenetic tree

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植物内生固氮菌是指定殖在健康植物体内、与宿主植物进行联合固氮活动的一类原核微生物，是植物微生态系统的天然组分^[1]。研究发现^[2]：因为存在固氮菌，某些亚热带地区稻田可以连续百年不施氮肥而土壤氮水平不下降。固氮微生物一般分为3类：自生固氮菌、与植物共生固氮的微生物、与植物联合固氮的微生物。共生固氮微生物常见于豆科Leguminosae植物中^[3]，其固氮机制、菌肥开发等研究已相当成熟。联合固氮微生物最早由DÖBEREINER ^[4]从热带禾木科Gramineae牧草雀裨Paspalum thunbergii根际中分离获得，这种被命名为雀裨固氮菌Azotobacter paspali的微生物被认为是存在于根际的自由生活的固氮菌。它们可以部分入侵植物表层组织，依靠根系分泌物生存繁殖，不与宿主形成特异分化结构，因而固氮能力不及共生固氮菌，但这类固氮菌与植物关系密切。BARRAQUIO等^[5]通过建立有效的水稻Oryza sativa内生固氮菌分离方法，测得水稻内生固氮菌数量为10⁵~10⁸ CFU·g⁻¹；袁梅等^[6]从湖南桂阳农田水稻分离出19株内生固氮菌，其中13株可抑制水稻苗期对镉的吸收；谭志远等^[7]从青香茅Cymbopogon caesius与五节芒Miscanthus floridulus中分离出66株内生固氮菌共10个类群；BALDANI等^[8]发现玉米Zea mays茎内存在内生固氮菌肺炎克雷伯氏菌Klebsiella pneumoniae，后者具有促进植物生长的作用。作为同属禾本科的木本植物，竹子内生固氮菌的报道较少。竹子是喜氮植物，对氮素要求较高；调查发现许多自然竹林没有氮肥补充但生长良好，提示竹子可能存在某种固氮菌，且后者具有较强的固氮潜能。顾小平等^[9]从浙江淡竹Phyllostachys glauca根际分离到3株活性较高的固氮菌，从毛竹Phyllostachys edulis根表面分离到36株固氮菌，分别为多黏芽孢杆菌Bacillus polymyxa和地衣芽孢杆菌Baclicus lincheniformis ^[10]；侯伟等^[11]从簕竹Bambusa blumeana中分离到40株高效固氮酶活性菌株，分别属固氮螺菌属Azosprillum、大肠埃希氏菌属Escherichias、绿脓杆菌属Pseudomonas和水螺旋菌属Aquaspirillum；同时从簕竹^[12]中分离到5株内生固氮菌，具有活性高、生长强势、可以通过自身代谢作用来适应环境酸碱性变化等特征。目前，国内关于竹子内生固氮菌的研究甚少。本研究以毛竹 (散生)、孝顺竹Bambusa multiplex (丛生)和狭叶青苦竹Pleioblastus chino (混生)为研究对象，利用分子生物技术研究不同生长型竹子根部及叶部的内生固氮菌、定殖土壤固氮菌的多样性和结构特征，探究不同竹种和不同器官间内生固氮菌多样性，探讨植株与定殖土壤内固氮菌多样性的关系，为竹子培育管理提供指导，也为开发有潜力的高效固氮菌，减少化学氮肥施用量、解决因化肥使用过度所造成的环境污染问题提供参考。

1. 材料与方法

1.1. 样品采集

狭叶青苦竹、孝顺竹栽植于浙江农林大学竹种园内，毛竹栽植于浙江农林大学后山。所有林相均为纯林，所有土壤均为红黄壤。采集竹叶、细根及不同竹种根部定植土壤。被选植株要求生长力旺盛，远离边缘地带，未受到病虫害感染。每种竹子选取3株间隔约15 m的不同植株作为重复。土壤样品采自所选竹子根周，深度为0~30 cm。所有样品放入4 ℃冰箱保存备用。

1.2. 土壤基本化学性质测定

经风干、磨细、过筛后，测定土样土壤化学性质。具体参考鲍士旦^[13]的方法，测定土壤含水量、pH、有机质、有效氮、速效磷、速效钾等指标。由表1可知：3种竹子定植土壤的pH、有机质、有效氮、速效钾、速效磷等指标均未表现出显著性差异(P＞0.05)，说明土壤样品的化学性质差异不影响固氮菌种类。

种类	pH	有机质/(g·kg⁻¹)	有效氮/(mg·kg⁻¹)	速效磷/(mg·kg⁻¹)	速效钾/(mg·kg⁻¹)
毛竹	4.32±0.24 a	33.01±7.04 a	58.20±16.46 a	5.40±0.85 a	79.00±8.80 a
孝顺竹	4.64±0.27 a	36.85±10.81 a	66.14±14.46 a	4.99±0.61 a	69.33±17.02 a
狭叶青苦竹	4.72±0.54 a	37.67±9.16 a	57.34±15.63 a	4.65±0.94 a	70.67±4.50 a
说明：同列相同字母表示差异不显著(P＞0.05)

Table 1. Basic chemical properties of three soil

1.3. 竹子内生微生物DNA提取

竹子叶与根的预处理参考谭致远等^[7]方法。叶与根灭菌消毒后，剪碎放入无菌研钵。用液氮充分研磨破碎植物样品，参考高志民等^[14]改良后的CTAB法提取植物内生微生物DNA，−40 ℃冰箱保存备用。

1.4. 土壤微生物DNA提取

土壤样品过20目筛，按Mobio土壤微生物DNA试剂盒(Powersoil kit，美国)提取土壤微生物DNA。

1.5. 固氮酶nifH基因扩增

固氮酶nifH基因采用聚合酶链式反应方法(PCR)扩增。第1轮正向引物FGPH19：5′-TACGGCAARGGTGGNATHG-3′，反向引物polR：5′-ATSGCATCATYTCRCCGGA-3′；第2轮正向引物AQER：5′-GACGATGTAGATYTCCTG-3′，反向引物polF-GC：5′-TGCGAYCCSAARGCBGACTC-3′。扩增程序参考DIALLO等^[15]。

1.6. 固氮酶nifH基因的变性梯度凝胶电泳(DGGE)

使用DCode^TM Universal Mutation Detection System (Bio-Rad，美国)对PCR产物进行变性梯度凝胶电泳，用GelDoc^TM EQ凝胶成像系统 (Bio-Rad，美国)拍照保存。

1.7. 固氮酶nifH基因的随机克隆

用polF(不含GC)与AQER引物扩增各竹种叶部、根部DNA，PCR产物用纯化试剂盒(Takara)纯化后与pEASY-T3 Vector(全金式生物技术公司，中国，北京)连接，转化到Trans1-T1感受态细胞中，涂在含有X-Gal、IPTG、氨苄青霉素的LB琼脂平板上进行蓝白斑筛选，用菌落PCR方法检测阳性克隆子。阳性克隆菌液送上海生工生物技术公司进行测序，序列通过NCBI进行Blast比对分析，获取相近典型菌株序列^[16]。

1.8. 数据分析

利用Quantity one软件对DGGE电泳图进行指纹图谱分析，根据条带对比结果，通过未加权算术平均法进行聚类分析；计算Shannon多样性指数，使用SPSS软件进行单因素方差分析与两两比较(Ducan法)；使用Mega 5.0，采用邻接法进行系统发育树分析。

3. 结论

不同竹种定植的土壤中存在着一定数量的同种固氮菌，但整体区系的相似度不高。同一竹种不同器官(根与叶)之间、不同竹种相同器官之间、土壤与植物之间的固氮菌群落结构差异显著；同种竹叶部或根部的重复样品相似度均不高。3种竹子根部内生固氮菌种类总体显著多于叶部，土壤固氮菌群落结构与根部内生固氮菌相似度高于叶部；不同竹种、不同器官之间固氮菌群落Shannon多样性指数差异显著，孝顺竹叶部与土壤大于根部，狭叶青苦竹和毛竹根部大于叶部和土壤。植物样品随机克隆测序BLAST对比得到83株固氮菌，其中12株为可培养固氮菌株，分别属于慢生根瘤菌属、Dechlorosoma、固氮螺菌属、肠杆菌属、Kosakonia、变形杆菌属、红长菌属、Azohydromonas等8个属，与已报道对竹子内生固氮菌研究相比，仅与侯伟等^[12]从簕竹中分离得到了相同的固氮螺菌，但未发现其他相同种属。系统发育树结果表明3种竹子内生固氮菌显著不同于其他植物中分离得到的内生固氮菌。

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种类	叶部	根部	土壤
狭叶青苦竹	2.43±0.30 Bb	2.89±0.12 ABa	2.36±0.09 Bb
毛竹	2.30±0.28 Bb	2.98±0.19 Aa	2.75±0.17 Aa
孝顺竹	2.73±0.10 Aa	2.53±0.26 Ba	2.70±0.15 Aa
说明：大写字母表示同列间差异显著(P＜0.05)，小写字母　　　　　表示同行间差异显著(P＜0.05)

样品来源	相似菌种基因
毛竹叶	HP-46 nitrogenase (nifH) gene、HP-76 nitrogenase (nifH) gene、nifH gene for nitrogenase iron protein、nitrogen- 　fixingbacteriumcloneb1 -HA3-1nitrogenase iron protein (nifH) gene、isolate DGGE gel band 5 nitrogenase iron protein (nifH) 　gene、J19I(16) nitrogenase (nifH) gene、HP-12 nitrogenase (nifH) gene、TNO_elv_99f12 dinitrogenase reductase (nifH) gene

毛竹根	nifH gene for nitrogenase iron protein、HP-27 nitrogenase (nifH) gene、HP-84 nitrogenase 　(nifH) gene、HP-84 nitrogenase (nifH) gene、CI(12) nitrogenase (nifH) gene、HP-14 nitrogenase (nifH) gene、TII(5) nitrogenase 　(nifH) gene、HP-48 nitrogenase (nifH) gene、HP-75 nitrogenase (nifH) gene、J19I(12) nitrogenase (nifH) gene、TNO_elv_81h1 　dinitrogenase reductase (nifH) gene、TNO_elv_81h1 dinitrogenase reductase (nifH) gene、HP-46 nitrogenase (nifH) gene、HP-75 　nitrogenase (nifH) gene

孝顺竹叶	HP-215 nitrogenase (nifH) gene、HP-5 nitrogenase (nifH) gene、HP-218 nitrogenase (nifH) gene、CY1-01 nitrogenase iron 　protein (nifH) gene、HP-56 nitrogenase (nifH) gene、HP-226 nitrogenase (nifH) gene、HP-157 nitrogenase (nifH) gene

孝顺竹根	isolate DGGE gel band 18 nitrogenase iron protein (nifH) gene、isolate DGGE gel band 12 nitrogenase iron protein (nifH) gene、　HP-226 nitrogenase (nifH) gene、partial nifH gene for dinitrogenase reductase、HP-90 nitrogenase (nifH) gene、partial nifH 　gene for dinitrogenase reductase、HP-237 nitrogenase (nifH) gene、HP-246 nitrogenase (nifH) gene、HP-246 nitrogenase 　(nifH) gene、HP-51 nitrogenase (nifH) gene、C13L7NH26 dinitrogenase reductase (nifH) gene、isolate DGGE gel band 20 　nitrogenase iron protein (nifH) gene、b1-HA3-1 nitrogenase iron protein (nifH) gene、431 nitrogenase iron protein (nifH) gene

狭叶青苦竹叶	TC30 NifH (nifH) gene、C12L6CK9 dinitrogenase reductase (nifH) gene、C13L6NH2 dinitrogenase reductase (nifH) gene、　C12L7NH35 dinitrogenase reductase (nifH) gene、nifH gene for nitrogenase iron protein(partial cds)、C13L10NH15 　 dinitrogenase reductase (nifH) gene、C13L8CK17 dinitrogenase reductase (nifH) gene、HP-75 nitrogenase (nifH) gene

狭叶青苦竹根	isolate DGGE gel band 14 nitrogenase iron protein (nifH) gene、Rb3_NifH_48 nitrogenase gene、C12L8CK4 dinitrogenase 　reductase (nifH) gene、WK-A5 nitrogenase iron protein (nifH) gene、486 nitrogenase iron protein (nifH) gene、C13L8NL22 　dinitrogenase reductase (nifH) gene、C13L6CK40 dinitrogenase reductase (nifH) gene、99 nitrogenase iron protein (nifH) 　gene、HP-201 nitrogenase (nifH) gene、HP-2 nitrogenase (nifH) gene、243 nitrogenase iron protein (nifH) gene、　C12L10NL20 dinitrogenase reductase (nifH) gene、C13L10NL22 dinitrogenase reductase (nifH) gene、isolate DGGE gel band 　11 nitrogenase iron protein (nifH) gene、C12L7CK25 dinitrogenase reductase (nifH) gene、HP-159 nitrogenase (nifH) gene、　HP-215 nitrogenase (nifH) gene、HP-90 nitrogenase (nifH) gene、C12L7NH35 dinitrogenase reductase (nifH) gene、　b1-HA3-1 nitrogenase iron protein (nifH) gene

类群	样品来源	NCBI中最相近菌种(登录号)	序列相似性/%
慢生根瘤菌属 Bradyrhizobium	MG38	Bradyrhizobium sp.(KC356360.1)	98
	QG11	Bradyrhizobium sp.(AB079616.1)	99
	QG255	Bradyrhizobium sp.(LT859959.1)	99
	XG32	Bradyrhizobium sp.(CU234118.1)	99

Dechlorosoma	MG31	Dechlorosoma suillum(CP003153.1)	98

固氮螺菌属 Azospirillum	XG434	Azospirillum canadense strain(GU256446.1)	99

肠杆菌属 Enterobacter	XY47	Enterobacter sp.(KC989924.1)	99

Kosakonia	MG6	Kosakonia sacchari strain(KR075981.1)	99
Kosakonia	XG52	Kosakonia sacchari strain(KR075959.1)	99

变形杆菌属 Gammaproteobacteria	XG255	Immundisolibacter cernigliae strain (CP014671.1)	99

红长菌属 Rubrivivax	MG16	Rubrivivax gelatinosus strain(KF800058.1)	98

Azohydromonas	XG66	Azohydromonas australica(AB188121.1)	100