热带作物学报

基因Gene	引物名称Primer name	序列（5ʹ–3ʹ）Sequence (5ʹ–3ʹ)	片段长度Fragment size/bp	参考文献Reference
16S rDNA	P1	AAGAGTTTGATCCTGGCTCAGGATT	1800	BERND等^[12]
P7	CGTCCTTCATCGGCTCTT
R16F2n	GAAACGACTGCTAAGACT	1200	LEE等^[13]
R16R2	TGACGGGCGGTGTGTACAAACCCCG
rp	rpF1	GGACATAAGTTAGGTGAATTT	1240	LEE等^[14]
rpR1	ACGATATTTAGTTCTTTTTGG
rpF2	TTTTCCCCTACACGTACTTA	1200	MARTINI等^[15]
rpR2	GTTCTTTTTGGCATTAACAT

基因Gene	引物名称Primer name	序列（5ʹ–3ʹ）Sequence (5ʹ–3ʹ)	片段长度Fragment size/bp	参考文献Reference
16S rDNA	P1	AAGAGTTTGATCCTGGCTCAGGATT	1800	BERND等^[12]
P7	CGTCCTTCATCGGCTCTT
R16F2n	GAAACGACTGCTAAGACT	1200	LEE等^[13]
R16R2	TGACGGGCGGTGTGTACAAACCCCG
rp	rpF1	GGACATAAGTTAGGTGAATTT	1240	LEE等^[14]
rpR1	ACGATATTTAGTTCTTTTTGG
rpF2	TTTTCCCCTACACGTACTTA	1200	MARTINI等^[15]
rpR2	GTTCTTTTTGGCATTAACAT

叶下珠黄化植原体和丛枝植原体的分子鉴定

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林兆威 , 牛晓庆 , 唐庆华 , 王晔楠 , 孟秀利 , 宋薇薇 ^*

热带作物学报 | 植物保护与生物安全 2024,45(4): 864-871

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热带作物学报 |植物保护与生物安全 2024 , 45 (4) : 864 -871

叶下珠黄化植原体和丛枝植原体的分子鉴定

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林兆威（1993—），男，硕士，研究实习员，研究方向：热带经济作物主要病害综合防治。

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林兆威（1993—），男，硕士，研究实习员，研究方向：热带经济作物主要病害综合防治。

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林兆威, 牛晓庆, 唐庆华, 王晔楠, 孟秀利, 宋薇薇^*

作者信息

中国热带农业科学院椰子研究所/海南省院士团队创新中心，海南文昌　571339

通讯作者:

^* 宋薇薇（SONG Weiwei），E-mail：songweiwei426@aliyun.com。

Molecular Identification of Phyllanthus urinaria Yellows Phytoplasma and Witches’-broom Phytoplasma

Zhaowei LIN, Xiaoqing NIU, Qinghua TANG, Yenan WANG, Xiuli MENG, Weiwei SONG^*

Affiliations

Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences / Hainan Innovation Center of Academician Team, Wenchang, Hainan 571339, China

出版时间: 2024-04-25 doi: 10.3969/j.issn.1000-2561.2024.04.022

文章导航

摘要

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2022年8月在海南省文昌市一个槟榔黄化病园内，分别发现叶片黄化和丛枝症状的叶下珠，前期检测均为植原体感染。为了明确叶下珠黄化植原体和丛枝植原体的分类鉴定，本研究通过克隆16S rDNA基因和核糖体蛋白（rp）基因，并进行基因序列一致性、系统发育树及虚拟RFLP等分析。结果显示：克隆获得叶下珠黄化植原体16S rDNA片段1246 bp，rp基因1212 bp；叶下珠丛枝植原体16S rDNA片段1827 bp，rp基因1240 bp。基因序列一致性显示，叶下珠黄化植原体与丛枝植原体的16S rDNA基因序列均与16SrⅠ组的植原体一致性高达98%以上，与槟榔黄化植原体海南株系的16S rDNA序列一致性达100%；而二者的rp基因序列均与rpⅠ组的植原体一致性高达99%以上。系统发育树分析显示，叶下珠黄化植原体和丛枝植原体16S rDNA基因均与翠菊黄化组（16SrⅠ）植原体聚于一个大分支，且与16SrⅠ-B亚组的槟榔黄化植原体聚于同一个小分支，亲缘关系接近；而rp基因均与翠菊黄化组（rpⅠ）植原体集聚于一个大分支，且与rpⅠ-B亚组的翠菊黄化植原体聚于同一个小分支，亲缘关系接近。虚拟RFLP分析显示，叶下珠黄化植原体和丛枝植原体的16S rDNA基因序列虚拟RFLP图谱与16SrⅠ-B的洋葱黄化植原体的参考图谱相同，且相似系数为1.00。综上表明，叶下珠黄化植原体和丛枝植原体均属于16SrⅠ-B亚组成员。研究结果可对采用铲除槟榔黄化病中间寄主的防控手段提供理论依据。

关键词

叶下珠 / 黄化 / 丛枝 / 植原体 / 分子鉴定

Abstract

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In August 2022, we found Phyllanthus urinaria with yellows and witches’-broom symptoms, respectively, in areca yellows disease garden in Wenchang city, Hainan province, which were phytoplasma infection. In order to clarify the taxonomic status of the phytoplasma strain in the yellows leaves and witches’-broom of P. urinaria. In this study, we cloned the 16S rDNA gene and ribosomal protein (rp) gene, and analyzed the gene sequence consistency, phylogenetic tree and virtual RFLP. The results showed that the 16S rDNA fragment 1246 bp and rp gene 1212 bp were cloned of P. urinaria yellows phytoplasma, the 16S rDNA fragment 1827 bp and rp gene 1240 bp were cloned of P. urinaria witches’-broom phytoplasma. The gene sequence consistency showed that the 16S rDNA gene sequence of the P. urinaria yellows phytoplasma and the P. urinaria witches’-broom phytoplasma was more than 98% consistent with the phytoplasma of 16SrⅠ group, and 100% consistent with the 16S rDNA sequence of areca yellows phytoplasma Hainan strain; the consistency of them in rp gene sequences was more than 99% to those of the rpⅠ group. The phylogenetic tree analysis showed that the 16S rDNA genes of P. urinaria yellows phytoplasma and P. urinaria witches’-broom phytoplasma were clustered in a large branch with those of aster yellows phytoplasma group (16SrⅠ), and clustered in the same small branch with those of areca yellows phytoplasma of 16SrⅠ-B subgroup, with the close genetic relationship; in the phylogenetic tree of rp gene, they were clustered in a large branch with the aster yellows phytoplasma group (rp Ⅰ), and clustered in the same small branch with those of aster yellows phytoplasma of rpⅠ-B subgroup, with the close genetic relationship. The virtual RFLP analysis showed that the RFLP map obtained by the 16S rDNA gene of the P. urinaria yellows phytoplasma and P. urinaria witches’-broom phytoplasma was the same as the reference map of the onion yellows phytoplasma of 16SrⅠ-B, and the similarity coefficient was 1.00. In summary, the P. urinaria yellows phytoplasma and the P. urinaria witches’-broom phytoplasma belong to 16SrⅠ-B subgroup in classification. The results of this study could provide a theoretical basis for the prevention and control of areca palm yellow disease by eradicating its intermediate host.

Key words

Phyllanthus urinaria / yellows / witches’-broom / phytoplasma / molecular identification

引用本文

林兆威, 牛晓庆, 唐庆华, 王晔楠, 孟秀利, 宋薇薇. 叶下珠黄化植原体和丛枝植原体的分子鉴定. 热带作物学报, 2024 , 45 (4) : 864 -871 . DOI: 10.3969/j.issn.1000-2561.2024.04.022

Zhaowei LIN, Xiaoqing NIU, Qinghua TANG, Yenan WANG, Xiuli MENG, Weiwei SONG. Molecular Identification of Phyllanthus urinaria Yellows Phytoplasma and Witches’-broom Phytoplasma[J]. Chinese Journal of Tropical Crops, 2024 , 45 (4) : 864 -871 . DOI: 10.3969/j.issn.1000-2561.2024.04.022

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叶下珠（Phyllanthus urinaria L.），又名珍珠草、夜合草，是大戟科（Euphorbiaceae Juss）叶下珠属（Phyllanthus）一年生草本植物。在我国主要分布于广西、广东及海南等南方地区。带根全草可入药，具有很高的药用价值。研究发现，叶下珠在抗乙肝病毒（HBV）、抗肿瘤、抗菌、抗内毒素、抗血栓形成及免疫调剂等方面具有较好的药理作用^[1]。近年来，叶下珠常用于临床上抗HBV的研究，对乙型肝炎的治疗有巨大的开发潜力。

植原体（phytoplasma）是存在于植物韧皮部的筛管细胞及介体昆虫的肠道、淋巴及唾液腺等组织内的一类专性菌，主要通过刺吸式介体昆虫进行植物间传播。被植原体侵染的植物常引起丛枝、叶片黄化、花变叶及矮化等症状^[2]。至今已有1000余种植物被植原体侵染，我国已报道100多种植原体病害^[3-4]，其中枣疯病（jujube witches’-broom，JWB）^[5]、小麦蓝矮病（wheat blue dwarf disease，WBD）^[6]、甘蔗白叶病（sugarcane white leaf，SCWL）^[7]及泡桐丛枝病（paulownia witches’-broom，PaWB）^[8]等危害严重，对相关产业造成严重的经济损失。在海南，植原体侵染引起的槟榔黄化病（areca palm yellow leaf disease，YLD）是一种毁灭性病害^[9]，每年因该病造成的经济损失高达20亿元以上^[10]。

2022年8月，本研究团队在海南省文昌市文城镇的1个槟榔园内进行黄化病调查，发现该园有叶片表现黄化的叶下珠和1株表现丛枝的叶下珠，为典型的植原体侵染引起的症状。前期采用荧光定量PCR检测技术（专利申请号：202210933142.8），对叶下珠黄化和丛枝2种症状进行植原体检测，结果发现2种症状均显示植原体阳性，且病原含量分别为6.8×10³、4.6×10⁸ copies/μL。目前国内外尚无叶下珠黄化植原体和丛枝植原体分类鉴定的相关报道，因此，本研究分别对叶下珠黄化、丛枝植原体16S rDNA基因和核糖体蛋白基因（rp）进行克隆，通过分子鉴定的手段明确叶下珠黄化植原体和丛枝植原体的分类地位，并与槟榔黄化植原体16S rDNA进行分析，为槟榔黄化病中间寄主的研究提供理论依据。

1　材料与方法

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1.1　材料

1.1.1　供试材料

待测叶下珠黄化、丛枝样品采自海南省文昌市文城镇（19°33′13″N，110°47′14″E）。叶下珠黄化症状表现为整株黄化，主要为叶肉黄化，叶肉叶脉黄绿相间明显，后期发展为叶肉叶脉黄化（图1A~图1D），该样品编号为HN-Pu2；叶下珠丛枝症状表现为整株丛枝，叶片变小（图1E~图1F），该样品编号为HN-Pu1；健康样品（图1G）采自海南省文昌市文城镇（19°33′14″N，110°47′15″E）。

1.1.2　主要试剂与仪器

pMD18-T载体购自TaKaRa公司；2×Taq PCR Mix、植物基因组DNA提取试剂盒及琼脂糖凝胶回收试剂盒购自TIANGEN公司；DH5α感受态细胞购自上海唯地生物技术有限公司。主要仪器：1~1000 μL移液器、GD120恒温水浴锅、5810R离心机及Biometra TOne 96G梯度PCR等。

1.2　方法

1.2.1　叶下珠叶片总DNA提取

剪取叶片样品0.1 g，参照植物基因组DNA提取试剂盒说明书，进行样品DNA提取，并将提取的DNA置于–20 ℃中保存，备用。

1.2.2　16S rDNA和rp基因的PCR扩增

以待测样品叶片总DNA为模板，健康样品为阴性对照，H₂O为空白对照进行PCR扩增。采用巢氏PCR技术检测叶下珠黄化的植原体，采用一步PCR检测叶下珠丛枝的植原体。16S rDNA和rp基因扩增引物序列见表1，16S rDNA基因的巢氏PCR扩增的第1步反应引物为P1/P7，第2步反应引物为R16F2n/R16R2；rp基因的巢氏PCR扩增的第1步反应引物为rpF1/rpR1，第2步反应引物为rpF2/rpR2；其中，巢氏PCR第1步反应结束后，需将PCR产物稀释50倍作为模板进行第2步反应。所需引物均由生工生物工程（上海）股份有限公司合成。扩增反应体系：2×Taq PCR Mix 12.5 μL，DNA模板为2.0 μL，ddH₂O为8.5 μL，上、下游引物（10 μmol/L）各1.0 μL。引物P1/P7扩增程序为：95 ℃预变性5 min；95 ℃变性1 min，48 ℃退火1 min，72 ℃延伸2 min，共35个循环；72 ℃延伸10 min。引物R16F2n/R16R2扩增程序为：95 ℃预变性5 min；95 ℃变性45 s，55 ℃退火45 s，72 ℃延伸90 s，共35个循环；72 ℃延伸10 min。rp基因的引物rpF1/rpR1和rpF2/rpR2扩增程序参照林兆威等^[11]的方法。

1.2.3　PCR产物纯化、克隆及序列测定

经琼脂糖凝胶回收试剂盒对PCR产物的目标条带进行回收纯化，并将目的片段与pMD18-T载体连接后转至DH5α感受态细胞中，在抗生素平板中进行涂板于37 ℃培养，通过菌落PCR进行阳性验证，将阳性转化子送深圳华大基因股份有限公司测序。

1.2.4　16S rDNA和rp基因序列一致性及系统发育树分析

经测序获得的16S rDNA和rp基因通过NCBI在线blastn程序（https://www.ncbi.nlm.nih.gov/）进行同源性检索分析，在GenBank数据库中下载不同组或亚组的代表性植原体16 rDNA基因序列和rp基因序列，利用MEGA 7.0软件，采用邻接法（neighbor joining），bootstrap复数设置为1000，分别构建16S rDNA和rp基因的系统发育进化树，分析叶下珠黄化植原体和丛枝植原体分类地位，并将16S rDNA和rp基因序列提交至GenBank数据库。

1.2.5　植原体16S rDNA序列虚拟RFLP分析

叶下珠黄化植原体和丛枝植原体的16S rDNA基因通过植原体在线分类鉴定软件iPhyClassifier（https://plantpathology.ba.ars.usda.gov/cgi-bin/re-source/iphyclassifier.cgi）进行相似性分析和虚拟RFLP分析，确定植原体的分类地位^[16]。

2　结果与分析

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2.1　植原体16S rDNA和rp基因片段的获得

从叶下珠黄化样品中扩增植原体16S rDNA基因，获得约1.2 kb的片段；扩增rp基因，获得约1.2 kb的片段。从叶下珠丛枝样品中扩增植原体16S rDNA基因，获得约1.8 kb的片段；扩增rp基因，获得约1.2 kb的片段（图2）。获得的片段大小均与预期目的片段大小基本一致，且健康样品和ddH₂O中未扩增出相应片段。将以上片段进行测序比对，结果为植原体，因此将该叶下珠黄化和丛枝植原体分别暂命名为叶下珠黄化植原体海南株系（PuY-HN-2022）、叶下珠丛枝植原体海南株系（PuWB-HN-2022）。

2.2　16S rDNA、rp基因序列一致性及系统发育树分析

通过克隆基因目的片段，获得叶下珠黄化植原体16S rDNA片段1246 bp（登录号：OP851691），rp基因片段1212 bp（登录号：OP918672）；叶下珠丛枝植原体16S rDNA片段1827 bp（登录号：OP850851），rp基因片段1240 bp（登录号：OP918671）。经基因序列一致性分析，叶下珠黄化植原体和丛枝植原体与16SrⅠ组植原体的16S rDNA序列一致性高达98%以上。其中，叶下珠黄化植原体与欧洲油菜矮缩植原体（登录号：MG599470.1）的16S rDNA序列一致性达100%；叶下珠丛枝植原体与多毡毛苎麻丛枝植原体（登录号：MT708485.1）、柳叶菜花变叶植原体（登录号：AY101386.1）的16S rDNA序列一致性达99.84%；二者均与槟榔黄化植原体海南株系（登录号：MZ971180.1）的16S rDNA序列一致性达100%。叶下珠黄化植原体、丛枝植原体的rp基因序列均与rpⅠ组的植原体一致性高达99%以上，且与洋葱簇生植原体（登录号：GU228514.1）一致性达100%。经系统发育树分析，叶下珠黄化植原体和丛枝原体16S rDNA基因均与翠菊黄化组（16SrⅠ）植原体聚于一个大分支，且与16Sr Ⅰ-B亚组的槟榔黄化植原体（登录号：MZ971180.1）聚于同一个小分支，亲缘关系接近；与其他组植原体亲缘关系较远（图3）。叶下珠黄化植原体和丛枝植原体rp基因均与翠菊黄化组（rpⅠ）植原体聚于一个大分支，且与rpⅠ-B亚组的翠菊黄化植原体（登录号：AY183708.1）聚于同一个小分支，亲缘关系接近；与其他组植原体亲缘关系较远（图4）。根据2004年比较支原体学国际研究规划（International Research Project for Comparative Mycoplasmology，IRPCM）对植原体暂定种的划分规则^[17]，叶下珠黄化植原体和丛枝植原体划为16SrⅠ组。

2.3　虚拟RFLP分析

将叶下珠黄化植原体和丛枝植原体的16S rDNA基因序列进行虚拟限制性片段长度多态性（restriction fragment length polymorphism，RFLP）分析，获得的图谱与16SrⅠ-B的洋葱黄化植原体（登录号：AP006628）的参考图谱相同（图5），且相似系数为1.00。综上，该叶下珠黄化植原体海南株系和叶下珠丛枝植原体海南株系均属于16SrⅠ-B亚组成员。

3　讨论

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由于植原体极难人工分离培养，在植原体的分类鉴定上难以通过形态观察、生理生化指标进行分类鉴定，通常采用16S rDNA基因、rp基因及tuf基因等植原体保守序列进行一致性比较，且植原体的组/亚组的划分标准依据为引物R16F2n/R16R2扩增16S rDNA基因序列的17种限制性内切酶的RFLP分析^[18-19]。本研究通过克隆植原体16S rDNA和rp基因，并对基因序列进行一致性分析、系统发育树分析及16S rDNA虚拟RFLP分析，将叶下珠黄化植原体海南株系和叶下珠丛枝植原体海南株系鉴定为16SrⅠ-B亚组成员。目前关于叶下珠属植物受植原体侵染为害的报道较少，本文为首次报道植原体侵染为害叶下珠。本研究发现，叶下珠均受16SrⅠ-B亚组的植原体侵染，但表现出不同的症状，在前期的植原体检测中发现这2种症状的植原体含量有数量级的差异。因此推测，导致叶下珠表现不同症状的原因与植原体侵染的含量差异有关。

在植原体的各组中，16SrⅠ组成员最多，为29个亚组^[20]，16SrⅠ-B亚组是翠菊黄化组植原体中寄主范围最广的亚组之一。其中，16SrⅠ-B亚组的植原体侵染引起的槟榔黄化病在海南省发生面积占27.89%，对海南槟榔产量造成严重损失^[21]。YU等^[22-23]研究发现，槟榔黄化植原体株系与细圆藤丛枝植原体、辣椒黄化皱缩植原体的亲缘关系很近，同源性可达100%，并推测细圆藤[Pericampylus glaucus（Lam.）Merr.]和辣椒（Capsicum annuum L.）可能是槟榔黄化植原体的中间寄主。本研究发现，叶下珠黄化植原体和丛枝植原体的16S rDNA基因序列与槟榔黄化病植原体海南株系16S rDNA基因序列（登录号：MZ971180.1）的一致性达100%；在系统发育树中，与槟榔黄化植原体的亲缘关系接近；本研究团队前期对该槟榔园进行槟榔黄化病检测时，发现该园的黄化病发生率高，叶下珠黄化和丛枝的发生也在该槟榔园内，因此，是否由感染黄化病槟榔作为传染源引起叶下珠感染将作进一步研究。另外，植原体通常通过刺吸式介体昆虫传播^[24-25]，目前发现槟榔园中椰子坚蚜、长尾粉蚧、黑刺粉虱、蓟马、棘缘蝽、白盾蚧6种昆虫携带植原体^[26]，因此，叶下珠是否会成为槟榔黄化病通过媒介昆虫传播的中间寄主也将作进一步研究。

叶下珠是海南农田、林地常见的草本植物，经上述分析，叶下珠可能是槟榔黄化病田间的中间寄主。植原体寄主的多样性可能会有利于同组或亚组的植原体适应不同的环境，或将不同的寄主植物作为中间介体促进植原体的传播与病害的流行^[27-28]。因此，在槟榔黄化病的防控中，不但要切断媒介昆虫的传播，也应铲除中间寄主作为传播途径的毒源。

基金

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海南省院士创新平台科研专项(YSPTZX202138; YSPTZX202151)
海南省自然科学基金项目(321QN345)

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2024年第45卷第4期

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doi: 10.3969/j.issn.1000-2561.2024.04.022

接收时间：2022-12-12
首发时间：2026-06-23
出版时间：2024-04-25

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收稿日期：2022-12-12
修回日期：2023-01-04

基金

海南省院士创新平台科研专项(YSPTZX202138; YSPTZX202151)

海南省自然科学基金项目(321QN345)

作者信息

中国热带农业科学院椰子研究所/海南省院士团队创新中心，海南文昌　571339

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^* 宋薇薇（SONG Weiwei），E-mail：songweiwei426@aliyun.com。

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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