黄龙病罹病柑橘叶片内生细菌和内生真菌群落结构多样性分析

刘晓菲; 甘芸; 刘利华; 赵建伟; 黄建成; 郑宇

doi:10.19303/j.issn.1008-0384.2020.01.009

黄龙病罹病柑橘叶片内生细菌和内生真菌群落结构多样性分析

doi: 10.19303/j.issn.1008-0384.2020.01.009

福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2017R1025-4）；福建省农业科学院青年人才创新基金（YC2017-11）；福建省财政专项——福建省农业科学院科技创新团队建设项目（STIT2017-1-8）

详细信息

作者简介:
刘晓菲（1987−），女，硕士，研究实习员，研究方向：植物病理与病害防治（E-mail：lxf_sophie@126.com）

通讯作者:
郑宇（1971−），男，博士，副研究员，研究方向：植物病虫害生物防治（E-mail：ike.zheng@qq.com）

中图分类号: S 435
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出版历程
- 收稿日期: 2019-11-25
- 修回日期: 2019-12-10
- 刊出日期: 2020-01-01

High-throughput Sequencing Analysis on Diversity of Bacterial and Fungal Endophytes on Huanglongbing-infected Citrus Plants

Institute of Plant Protection, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou, Fujian　350013, China

摘要

摘要: 目的比较健康、罹病柑橘叶片在温室条件下内生菌群落结构差异，以及同一植株不同罹病程度叶片的内生菌群落结构差异，分析黄龙病侵染相关的内生细菌和内生真菌种群动态变化，为柑橘黄龙病菌的共培养和病原学研究提供依据。方法通过引物对OI1/OI2c对植株染黄龙病的状态进行检验，以健康和不同罹病程度（无症绿色、斑驳黄化、均匀黄化）的柑橘叶片为样本，通过对细菌16S rRNA和真菌rDNA-ITS序列的高通量测序，将得到的内生细菌和内生真菌的数据进行聚类和多样性分析。结果 4组样品分别通过聚类获得13个门类、22个纲、57个目、101个科、208个属的细菌，以及2个门类、12个纲、24个目、43个科、43个属的真菌。黄龙病菌的相对丰度随着叶片罹病程度的加深而增加。健康组和罹病组共有的细菌优势菌属为甲基细胞菌属、1174-901-12属。Sphingomonas属为健康组的优势菌属。AlphaI cluster属和Hymenobacter属的相对丰度随黄龙病的入侵增加。健康组和罹病组共有的真菌优势菌属为横断孢属，Zasmidium属和Trichomerium属只为罹病组的次优势菌属。Zymoseptoria属的相对丰度与黄龙病菌呈正相关。结论黄龙病菌的侵入改变了植株内生细菌和内生真菌的群落结构和多样性，健康组和罹病组的内生细菌、内生真菌群落结构多样性存在明显差异，其优势及次优势菌属及其相对丰度也有存在差异。高通量测序对植物样本的直接检测能有效避开前人使用传统检测的短板，获得包括难培养、低丰度菌群在内更完整的内生菌群落结构，为更系统分析与黄龙病菌入侵生长相关内生菌和寻找其潜在伴生菌的研究提供依据。
- 黄龙病 /
- 内生细菌 /
- 内生真菌 /
- 群落多样性
Abstract: Objective Diversity of bacterial and fungal endophytes on citrus plants infected with Huanglongbing (HLB) (citrus greening disease by Candidatus Liberibacter) was determined for further study on the pathology. Method The diversities of bacterial and fungal endophytes on healthy and HLB-diseased leaves of citrus plants grown in a greenhouse were compared. In addition, the diversities on the diseased leaves of different severity from a same plant were also compared. The HLB pathogen was confirmed using primer pair of OI1/OI2c. High-throughput sequencing were used to detect the 16S rRNA of bacteria and rDNA-ITS of fungi. Then the abundance and diversity analysis on the endophytes were conducted. Result For the bacterial endophytes, there were 13 phyla, 22 classes, 57 orders, 101 families, and 208 genera found, while for the fungal endophytes, 2 phyla, 12 classes, 24 orders, 43 families, and 43 genera. The relative abundance of Candidatus Liberibacter increased with severity of the disease. Methylocella and 1174-901-12 were the dominant bacteria endophytes on both healthy and diseased plants; whereas, Sphingomonas only on healthy plants. The relative abundances of Alpha cluster and Hymenobacter increased with the disease infection. Strelitziana was the dominant fungal endophyte on both healthy and diseased plants; whereas, Zasmidium and Trichomerium on diseased plants only. The relative abundance of Zymoseptoria increased with the disease infection. Conclusion The infection of HLB by Candidatus Liberibacter bacteria changed the abundance and diversity of bacterial and fungal endophytes on a citrus plant. There were also significant differences on the diversity of bacterial and fungal endophytes between the healthy and the diseased plants. Successful detection of the endophytes using high-throughput sequencing overcame the obstacles on the disease identification due to the unculturable pathogen and endophytes as well as the endophytes with low abundance. It also allowed a systematic study on the microbiology associated with HLB and the cure and prevention of the disease.
- Huanglongbing (citrus greening disease) /
- bacterial endophytes /
- fungal endophytes /
- community diversity

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图 1 染黄龙病叶片采样

注：H：健康组叶片，G：罹病组无症绿色叶片，M：罹病组斑驳黄化叶片，Y：罹病组均匀黄化叶片。

Figure 1. Sampling of HLB-diseased leaves

Note: H: healthy leaves; G: green leaves on diseased plant; M: yellow-mottled leaves on diseased plant; Y: yellow leaves on diseased plant.

下载: 全尺寸图片幻灯片

图 2 各样品黄龙病菌检测电泳结果

注：M：Marker；D1～D9罹病组植株，H1～H9健康组植株，P：阳对照（黄龙病感染植株），N：阴对照（水）。

Figure 2. Electrophoresis of PCR products of HLB pathogens

Note: M, Marker; D1–D9: huanglongbing-affected plants; H1–H9: healthy plants, P: positive control (huanglongbing-confirmed plant); N: negative control (water).

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图 3 各组别的黄龙病菌相对丰度ANOVA分析

注：H：健康组叶片，G：罹病组无症绿色叶片，M：罹病组斑驳黄化叶片，Y：罹病组黄化叶片。

Figure 3. ANOVA for relative abundance of HLB pathogens

Note: H: healthy leaves; G: green leaves on diseased plant; M: yellow-mottled leaves on diseased plant; Y: yellow leaves on diseased plant.

下载: 全尺寸图片幻灯片

图 4 OTU数量与分类分布

注：①a：内生细菌OTU个数分布图，b：内生真菌OTU个数分布图，c：健康组H与罹病组D的内生细菌OTU 韦恩图，d ：健康组H与罹病组D的内生真菌OTU 韦恩图，e：罹病组的细菌OTU韦恩图，f：罹病组的真菌OTU韦恩图。②D：罹病组叶片，包括G、M和Y。

Figure 4. OTU Distribution and Venn graphs

Note: ① a: OTU distribution of bacterial endophytes; b: OTU distribution of fungal endophytes; c: Venn graphs of bacterial endophytes between healthy (H) and diseased (D) plants; d: Venn graphs of fungal endophytes between H and D; e: Venn graphs of bacterium among D; f: Venn graphs of fungi among D. ②D: leaves on diseased plant including G, M and Y.

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图 5 内生菌Beta多样性分析基于bray-curtis算法的PCoA分析

注：a细菌，b真菌。图6同。

Figure 5. Principal coordinates analysis (PCoA) plot based on Bray-Curtis distance

Note: a: bacteria; b: fungi. Same for Fig. 6.

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图 6 属水平下物种丰度排名前十的物种分布

Figure 6. Abundance (top 10) of endophytes in samples at genera level

下载: 全尺寸图片幻灯片

表 1 内生菌alpha多样性指数

Table 1. Alpha diversity index of endophytes

组别 Group	细菌 Bacterium		真菌 Fungi
组别 Group	Shannon	Simpson	Shannon	Simpson
H	2.057	0.253 8	3.222	0.073 6
G	1.733 9	0.447 6	2.649 1	0.182 5
M	2.063 8	0.285 2	2.802 7	0.134 5
Y	2.345 8	0.212 4	2.818 6	0.124 8
注：H：健康组叶片，G：罹病组无症绿色叶片，M：罹病组斑驳黄化叶片，Y：罹病组均匀黄化叶片。 Note: H: healthy leaves; G: green leaves on diseased plant; M: yellow-mottled leaves on diseased plant; Y: yellow leaves on diseased plant.

下载: 导出CSV

表 2 门水平下物种相对丰度大于0.1%门类

Table 2. Abundance of endophytes in samples at phylum level (abundance＞0.1%)

	门 Phylum	H	G	M	Y
细菌 Bacterium	变形菌门 Proteobacteria	94.48%	94.41%	93.97%	93.62%
	放线菌门 Actinobacteria	2.79%	2.50%	1.69%	1.53%
	厚壁菌门 Firmicutes	1.63%	1.58%	2.06%	3.18%
	拟杆菌门 Bacteroidetes	0.55%	0.79%	2.01%	1.31%
真菌 Fungi	子囊菌门 Ascomycota	80.07%	96.34%	83.27%	91.34%
	担子菌门 Basidiomycota	7.23%	2.00%	7.08%	5.66%

下载: 导出CSV

参考文献(31)

[1]	刘利华, 姚锦爱, 种藏文, 等. 柑桔黄龙病亚洲种病原的PCR-SSCP分析 [J]. 福建农业学报, 2005, 20(2):77−79. doi: 10.3969/j.issn.1008-0384.2005.02.004 LIU L H, YAO J A, CHONG Z W, et al. Analysis of Citrus Huanglongbing pathogens from different places by PCR-SSCP assay [J]. Fujian Journal of Agricultural Sciences, 2005, 20(2): 77−79.(in Chinese) doi: 10.3969/j.issn.1008-0384.2005.02.004
[2]	范国成, 刘波, 吴如健, 等. 中国柑橘黄龙病研究30年 [J]. 福建农业学报, 2009, 24(2):183−190. doi: 10.3969/j.issn.1008-0384.2009.02.019 FAN G C, LIU B, WU R J, et al. Thirty years of research on citrus Huanglongbing in China [J]. Fujian Journal of Agricultural Sciences, 2009, 24(2): 183−190.(in Chinese) doi: 10.3969/j.issn.1008-0384.2009.02.019
[3]	宋晓兵, 彭埃天, 陈霞, 等. 柑橘黄龙病病原培养及分子检测技术研究进展 [J]. 广东农业科学, 2013, 40(23):65−69. doi: 10.3969/j.issn.1004-874X.2013.23.017 SONG X B, PENG A T, CHEN X, et al. Research advances of Citrus Huanglongbing pathogen culture and detection technology [J]. Guangdong Agricultural Sciences, 2013, 40(23): 65−69.(in Chinese) doi: 10.3969/j.issn.1004-874X.2013.23.017
[4]	GARCIA-RUIZ F, SANKARAN S, MAJA J M, et al. Comparison of two aerial imaging platforms for identification of Huanglongbing-infected Citrus trees [J]. Computers and Electronics in Agriculture, 2013, 91: 106−115. doi: 10.1016/j.compag.2012.12.002
[5]	郑雪芳, 刘波, 孙大光, 等. 柑橘黄龙病植株内生菌PLFAs多态性研究 [J]. 中国生态农业学报, 2012, 20(7):932−944. doi: 10.3724/SP.J.1011.2012.00932 ZHENG X F, LIU B, SUN D G, et al. Plant endophyte PLFAs polymorphism in Huanglongbing-affected red pomelo plant [J]. Chinese Journal of Eco-Agriculture, 2012, 20(7): 932−944.(in Chinese) doi: 10.3724/SP.J.1011.2012.00932
[6]	BOVÉ J. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus [J]. Journal of Plant Pathology, 2006, 88: 7−37.
[7]	DUAN Y P, ZHOU L J, HALL D G, et al. Complete genome sequence of Citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics [J]. Molecular Plant-Microbe Interactions, 2009, 22(8): 1011−1020. doi: 10.1094/MPMI-22-8-1011
[8]	LIN H, COLETTA-FILHO H D, HAN C S, et al. Draft genome sequence of “Candidatus Liberibacter americanus” bacterium associated with Citrus huanglongbing in Brazil [J]. Genome Announcements, 2013, 1(3): e00275.
[9]	WULFF N A, EVEILLARD S, FOISSAC X, et al. rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with Citrus huanglongbing in Brazil [J]. International Journal of Systematic and Evolutionary Microbiology, 2009, 59(8): 1984−1991. doi: 10.1099/ijs.0.008508-0
[10]	ZHANG S J, FLORES-CRUZ Z, ZHOU L J, et al. 'Candidatus Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections [J]. Molecular Plant-Microbe Interactions, 2011, 24(4): 458−468. doi: 10.1094/MPMI-11-10-0256
[11]	ZHENG Z, DENG X, CHEN J. Draft genome sequence of “Candidatus Liberibacter asiaticus”from California [J]. Genome Announcements, 2014, 2(5): e00999−14.
[12]	ZHENG Z, DENG X, CHEN J. A whole genome sequence of 'Candidatus Liberibacter asiaticus' from Guangdong, China, where HLB was first described [J]. American Phytopathological Society Abstracts, 2014, 104(S3): 137.
[13]	KATOH H, MIYATA S I, INOUE H, et al. Unique features of a Japanese ‘Candidatus Liberibacter asiaticus’ strain revealed by whole genome sequencing [J]. PLoS One, 2014, 9(9): e106109. doi: 10.1371/journal.pone.0106109
[14]	DAVIS M J, MONDAL S N, CHEN H Q, et al. Co-cultivation of ‘Candidatus Liberibacter asiaticus’ with actinobacteria from Citrus with huanglongbing [J]. Plant Disease, 2008, 92(11): 1547−1550. doi: 10.1094/PDIS-92-11-1547
[15]	SECHLER A, SCHUENZEL E L, COOKE P, et al. Cultivation of ‘Candidatus Liberibacter asiaticus’, ‘Ca.L. Africanus’, and ‘Ca.L. Americanus’ associated with huanglongbing [J]. Phytopathology, 2009, 99(5): 480−486. doi: 10.1094/PHYTO-99-5-0480
[16]	赵园园, 明佳佳, 胡承孝, 等. 柑橘黄龙病与树体养分关系的研究进展 [J]. 湖北农业科学, 2015, 54(9):2049−2053. ZHAO Y Y, MING J J, HU C X, et al. Research progress on the relationship between Citrus huanglongbing disease and plant nutrition [J]. Hubei Agricultural Sciences, 2015, 54(9): 2049−2053.(in Chinese)
[17]	TRIVEDI P, HE Z L, VAN NOSTRAND J D, et al. Huanglongbing alters the structure and functional diversity of microbial communities associated with Citrus rhizosphere [J]. The ISME Journal, 2012, 6(2): 363−383. doi: 10.1038/ismej.2011.100
[18]	SAGARAM U S, DEANGELIS K M, TRIVEDI P, et al. Bacterial diversity analysis of huanglongbing pathogen-infected Citrus, using PhyloChip arrays and 16S rRNA gene clone library sequencing [J]. Applied and Environmental Microbiology, 2009, 75(6): 1566−1574. doi: 10.1128/AEM.02404-08
[19]	车玉伶, 王慧, 胡洪营, 等. 微生物群落结构和多样性解析技术研究进展 [J]. 生态环境, 2005, 14(1):127−133. CHE Y L, WANG H, HU H Y, et al. Research progresses on analytical technologies used in microbial community structure and diversity [J]. Ecology and Environment, 2005, 14(1): 127−133.(in Chinese)
[20]	王爱华, 殷幼平, 熊红利, 等. 广西柑橘黄龙病植株韧皮部内生细菌多样性分析 [J]. 中国农业科学, 2010, 43(23):4823−4833. doi: 10.3864/j.issn.0578-1752.2010.23.007 WANG A H, YIN Y P, XIONG H L, et al. Endophytic bacterial diversity analysis of huanglongbing pathogen-infected Citrus phloem tissue in Guangxi [J]. Scientia Agricultura Sinica, 2010, 43(23): 4823−4833.(in Chinese) doi: 10.3864/j.issn.0578-1752.2010.23.007
[21]	李颜方, 殷幼平, 王玉玺, 等. 亚洲韧皮杆菌兼性厌氧型伴生细菌鉴定及优势菌群分析 [J]. 微生物学通报, 2011, 38(9):1362−1370. LI Y F, YIN Y P, WANG Y X, et al. Isolation of facultative anaerobic entophytic bacterial companioned Ca Las infected citrus tissues and evaluation of dominant bacterial populations [J]. Microbiology, 2011, 38(9): 1362−1370.(in Chinese)
[22]	王芳, 殷幼平, 孙丽琴, 等. 柑橘黄龙病罹病植株显症差异组织内生细菌群落结构分析 [J]. 微生物学报, 2014, 54(8):868−875. WANG F, YIN Y P, SUN L Q, et al. Endophytic bacterial community in the symptoms and symptomless tissues of HLB-affected Citrus plant [J]. Acta Microbiologica Sinica, 2014, 54(8): 868−875.(in Chinese)
[23]	李佳, 王中康, 谢攀, 等. 长春花内生细菌多样性与柑橘黄龙病菌的相关性 [J]. 微生物学报, 2012, 52(4):489−497. LI J, WANG Z K, XIE P, et al. Endophytic bacterial community analysis of Catharanthus roseus and its association with huanglongbing pathogen [J]. Acta Microbiologica Sinica, 2012, 52(4): 489−497.(in Chinese)
[24]	熊大维, 金丹凤, 顾斌涛, 等. 柑橘黄龙病赣南脐橙内生菌种群结构分析 [J]. 微生物学通报, 2017, 44(3):631−638. XIONG D W, JIN D F, GU B T, et al. Endophytic bacterial community in Gannan navel orange affected by huanglongbing [J]. Microbiology, 2017, 44(3): 631−638.(in Chinese)
[25]	AMANN R I, LUDWIG W, SCHLEIFER K H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation [J]. Microbiological Reviews, 1995, 59(1): 143−169. doi: 10.1128/MMBR.59.1.143-169.1995
[26]	王绍祥, 杨洲祥, 孙真, 等. 高通量测序技术在水环境微生物群落多样性中的应用 [J]. 化学通报, 2014, 77(3):196−203. WANG S X, YANG Z X, SUN Z, et al. Application of High Throughput Sequencing in the Diversity of Water Microbial Communities [J]. Chemistry, 2014, 77(3): 196−203.(in Chinese)
[27]	马同锁, 田苗珍, 袁红楼, 等. 不同生长环境对2种蔬菜内生菌分布的影响 [J]. 安徽农业科学, 2009, 37(17):7812−7813, 7815. doi: 10.3969/j.issn.0517-6611.2009.17.002 MA T S, TIAN M Z, YUAN H L, et al. Effect of the different growth environmental conditions on the distribution of endophytes in two species of vegetables [J]. Journal of Anhui Agricultural Sciences, 2009, 37(17): 7812−7813, 7815.(in Chinese) doi: 10.3969/j.issn.0517-6611.2009.17.002
[28]	GAGNÉ S, RICHARD C, ROUSSEAU H, et al. Xylem-residing bacteria in alfalfa roots [J]. Canadian Journal of Microbiology, 1987, 33(11): 996−1000. doi: 10.1139/m87-175
[29]	EDGAR R C. UPARSE: highly accurate OTU sequences from microbial amplicon reads [J]. Nature Methods, 2013, 10(10): 996−998. doi: 10.1038/nmeth.2604
[30]	SAKAKI T, TAKESHIMA T, TOMINAGA M, et al. Recurrence of ICA-PCoA aneurysms after neck clipping [J]. Journal of Neurosurgery, 1994, 80(1): 58−63. doi: 10.3171/jns.1994.80.1.0058
[31]	陈燕玲, 唐瑞, 刘洋, 等. 黄龙病菌在柑橘枝条上的分布和多样性分析 [J]. 植物病理学报, 2018, 48(6):728−737. CHEN Y L, TANG R, LIU Y, et al. Distribution and genetic diversity of "Candidatus Liberibacter asiaticus" in citrus canopy shoots [J]. Acta Phytopathologica Sinica, 2018, 48(6): 728−737.(in Chinese)