广东番茄巨芽病植原体的分子鉴定

2022年, 对在广东省湛江市廉江市田间发现的疑似番茄巨芽病病株, 利用分子生物学方法对其相关植原体进行了鉴定。以番茄病株叶片总DNA为模板, 利用植原体16S rRNA基因通用引物R16mF2/R16mR1进行PCR扩增, 获得了广东番茄巨芽病植原体(TBB-GD-2022)16S rRNA基因片段(1 430 bp, GenBank登录号为ON102780)。16S rRNA基因序列相似性分析显示, TBB-GD-2022与16SrⅡ组植原体菌株的相似性较高, 为96.82%~100%, 其中与隶属于16SrⅡ-V亚组的6个植原体株系相似性为100%。系统进化分析显示, TBB-GD-2022与16SrⅡ组各植原体株系聚类在一个大分支, 并与16SrⅡ-V亚组成员聚类在一个小分支, 亲缘关系较近。16S rRNA 基因相似系数分析表明, TBB-GD-2022与16SrⅡ-V亚组的参照株系‘Praxelis clematidea’ phyllody phytoplasma (GenBank登录号:KY568717) 的相似系数为1.00。上述研究结果表明, 广东番茄巨芽病植原体隶属16SrⅡ-V亚组成员。本文首次报道在广东发现番茄巨芽病, 通过其16S rRNA序列分析进一步确定了其相关植原体的分类地位, 为该病害的防控提供了科学依据。     

杏褪绿卷叶植原体新疆分离物16S rDNA基因克隆与序列分析

利用植原体16S rDNA基因通用引物对新疆轮台县疑似杏褪绿卷叶病植株总DNA进行巢氏PCR检测,扩增出大小约1.2 kb的特异性条带。对扩增产物克隆和测序,确定特异片段大小为1248 bp。序列同源性比较和系统进化分析表明,新疆杏褪绿卷叶植原体不同分离株16S rDNA基因序列同源性极高,达到99.8%~100%。与16SrⅤ组成员的同源性达到98.2%以上,其中与16SrⅤ-B亚组的枣疯病植原体山东宝山分离株,甜樱桃绿化植原体山东分离株同源性最高,达到99.4%~99.6%。进一步虚拟RFLP分析,结果表明该植原体属于榆树黄化组(16SrⅤ)的一个新的亚组,与其相似性最高的是16SrⅤ-B亚组,相似系数为0.94。本研究首次报道了新疆杏褪绿卷叶植原体16S rDNA的序列,确定了其分类地位,为杏褪绿卷叶病的早期诊断和检测提供了基础。     

金莲花绿变植原体的分子鉴定

本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。     

植原体引起雪花木小叶病在中国的首次报道

2022年首次在广州市发现园林植物雪花木小叶病病株, 采用分子生物学技术对其进行植原体的种类鉴定。以雪花木叶片总DNA为模板, 利用植原体16S rRNA通用引物P1/P7进行PCR扩增, 获得广东雪花木小叶病植原体(BLL-GD2022)16S rRNA基因片段(1 811 bp, GenBank登录号为OQ625536)。16S rRNA序列相似性显示, BLL-GD2022与16SrVI组植原体株系的相似性最高, 为97.05%~99.83%, 其中与隶属于16SrVI-D亚组的10个植原体株系相似性为99.21%~99.83%。系统进化分析显示, BLL-GD2022与16SrVI组各植原体株系聚类在一个大分支, 其中与16SrVI-D亚组成员聚类在一个小分支, 亲缘关系最近。基于16S rRNA序列的iPhyClassifier限制性内切酶虚拟RFLP分析表明, BLL-GD2022与16SrVI-D亚组的参考株系Brinjal little leaf phytoplasma (GenBank登录号为X83431)的酶切图谱一致, 相似系数为1.00。基于上述研究结果, 明确广州市雪花木小叶病植原体隶属16SrVI-D亚组成员。本研究首次在园林植物雪花木上检测到植原体, 通过16S rRNA序列分析明确为16SrVI-D亚组成员, 为开展16SrVI-D亚组植原体在蔬菜、花卉和园林植物的发生监测及病害防控提供科学依据。     

小麦蓝矮病植原体16S rDNA序列分析研究

 小麦蓝矮病是我国西北地区冬小麦上一种重要病害。本研究利用植原体16S rDNA通用引物对小麦蓝矮病患病植株全DNA进行nest-PCR扩增,获得1.2 kb的特异片段,并对扩增产物进行核苷酸序列测定,从分子水平证明了小麦蓝矮病的病原是植原体。利用最大简约法构建了16S rDNA系统演化树,系统演化关系分析表明:小麦蓝矮病植原体应该归属于翠菊植原体(Candidatus Phytoplasma asteris);小麦蓝矮病植原体与三叶草变叶病植原体(CPh)关系密切,被聚类为同一亚组(16Sr I-C),但是它们在寄主范围和传播介体等生物学性状方面差异很大。     

海南省木豆丛枝病植原体的分子检测及鉴定

 利用植原体通用引物R16mF2/R16mR1和rp (Ⅱ) F1/rp (Ⅱ) R1对海南木豆丛枝病植原体16S rDNA和部分核糖体蛋白(ribosomal protein,rp)基因序列进行PCR扩增、克隆和测序。获得海南木豆丛枝病植原体16S rDNA基因片段为1430bp,rp基因片段为1170bp。核苷酸同源性比较和系统进化树构建表明,引起海南木豆丛枝病的植原体应属于16SrⅡ组中的亚组ⅲ。本研究首次从分子水平确定了引起我国海南木豆丛枝病的病原物为植原体,明确了其分类地位,为该病害流行学研究和防治提供了理论依据。     

菊花绿变病植原体在中国的发生及分子鉴定

对内蒙古农业大学校园内表现花器绿变症状的菊花样品进行采集和DNA提取,应用植原体16S rRNA基因和rp基因的引物进行巢式PCR扩增,从感病样品中分别扩增得到了长度均约为1.2 kb的片段。序列一致性分析表明,菊花绿变植原体16S rRNA基因与翠菊黄化植原体匈牙利风信子株系(GenBank登录号MN080271)、印度玉米株系(KY565571)、印度繁缕株系(KC623537)和印度马铃薯株系(KC312703)的核酸一致性最高,为99.9%,rp基因序列与翠菊黄化植原体立陶宛洋葱株系(GU228514)的核酸一致性最高,为99.8%。基于16S rRNA基因和rp基因构建系统进化树时发现,菊花绿变植原体均与16SrI-B亚组成员聚为一起。16S rRNA基因相似性系数分析表明,菊花绿变植原体与洋葱黄化植原体(AP006628)的相似性系数最高为1.00,洋葱黄化植原体(AP006628)在分类上属于16SrI-B亚组。因此,我们可以确定该菊花绿变植原体属于16SrI-B亚组。这是我国首次报道菊花绿变病的发生。     

广东花生丛枝病植原体的分子鉴定

2020年在广东省湛江市遂溪县田间发现表现明显丛枝?小叶, 类似植原体感染症状的花生病株?本研究利用分子生物学技术对其病原进行鉴定?以花生病叶的总DNA为模板, 利用植原体16S rRNA和SecY基因通用引物进行PCR扩增, 获得广东花生丛枝病植原体(PnWB-GDSX-2020)16S rRNA基因片段(1 430 bp, GenBank登录号为MZ427281)和SecY基因片段(1 709 bp, GenBank登录号为MZ437794)?序列一致性和系统进化分析显示, PnWB-GDSX-2020的16S rRNA序列与16SrⅡ-A?16SrⅡ-D和16SrⅡ-V亚组植原体一致性最高, 亲缘关系最近; 进一步利用iPhyClassifier对16S rRNA序列进行在线虚拟RFLP分析, 结果显示, PnWB-GDSX-2020的虚拟RFLP 图谱与16SrⅡ-V亚组的参照株系‘Praxelis clematidea’ phyllody phytoplasma (GenBank登录号:KY568717) 酶切图谱一致, 相似系数为1.00?因此, PnWB-GDSX-2020属于16SrⅡ-V亚组成员?所获得的PnWB-GDSX-2020 Sec Y基因序列与花生丛枝植原体的一致性最高, 亲缘关系最近?本文确定了广东花生丛枝病相关植原体的分类地位, 为当地病害诊断?检测以及防控提供科学依据?     

宁夏马铃薯僵顶植原体的分子鉴定

通过透射电子显微镜,在从宁夏回族自治区固原市彭阳县红河镇采集的表现叶片上卷、红叶、气生薯症状的马铃薯样品叶脉韧皮部筛管细胞内观察到大量直径为500~700 nm的球形植原体粒子。以提取的感病和健康马铃薯叶片总DNA为模板,应用植原体16S rRNA基因和rp基因通用引物进行PCR扩增,从感病样品中扩增得到了长度均约为1.2 kb的片段。对获得基因核酸一致性比较分析表明,马铃薯僵顶植原体宁夏株系16S rRNA基因与‘Candidatus Phytoplasma fragariae’槭树株系(MK501642)16S rRNA基因核酸一致性最高,为99.7%,rp基因与‘Ca.P.fragariae’云南马铃薯YN-2G株系(KJ144889)rp基因核酸一致性最高,为100%;基于16S rRNA基因和rp基因构建系统进化树发现,马铃薯僵顶植原体宁夏株系与16SrⅫ-E亚组成员聚在一起。基于透射电镜观察和基因序列比较分析,证明宁夏发生的马铃薯僵顶病与植原体侵染相关,该植原体在分类地位上属于植原体16SrⅫ-E亚组。     

丁香卷叶病植原体的分子鉴定

 通过透射电子显微镜,在表现卷叶、褪绿症状的丁香(Syringa oblata)样品的叶脉韧皮部筛管细胞内观察到大量植原体粒子。应用植原体16S rRNA基因通用引物对P1/P7和R16F2n/R16R2对表症丁香植株总DNA进行巢式PCR扩增,得到了约1.2 kb的目标片段,通过对扩增片段进行测序、系统发育分析和同源性分析,结果表明,该片段长度为1 246 bp,在系统发育进化树上与翠菊黄化组（Candidatus Phytoplasma asteris）成员是聚集在一起的,与该组成员同源性均在98%以上。用16Sr RNAⅠ组和Ⅴ组特异引物确定了该病害非混合侵染所致,相似性系数和RFLP分析表明该植原体属于16SrⅠ B亚组。这是国内关于翠菊黄化组植原体在丁香上感染的首次报道。     

安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测

 Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.     

云南芒果变叶病植原体的分子鉴定

 植原体（phytoplasma）是一类没有细胞壁,不能离体培养的原核生物,对四环素敏感,主要存在于植物筛管细胞中。植原体主要通过叶蝉、飞虱等取食植物韧皮部的昆虫传播,也可通过菟丝子寄生和嫁接等方式传播。目前,全世界已发现1 000多种由植原体引起的植物病害,我国大陆已报道100余种与之相关的病害\[1\]。由植原体引起的病害症状主要表现为植株花器病态、小叶、丛枝、黄化等,从而导致植物产量和品质明显下降。     

Differential detection and genetic relatedness of phytoplasmas in papaya

The genetic relatedness of phytoplasmas associated with dieback (PDB), yellow crinkle (PYC) and mosaic (PM) diseases in papaya was studied by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and 16S rRNA/23S rRNA spacer region (SR). RFLP and SR sequence comparisons indicated that PYC and PM phytoplasmas were identical and most closely related to members of the faba bean phyllody strain cluster. By comparison the PDB phytoplasma was most closely related to Phormium yellow leaf (PYL) phytoplasma from New Zealand and the Australian grapevine yellows (AGY) phytoplasma from Australia. These three phytoplasmas cluster with the stolbur and German grapevine yellows (VK) phytoplasmas within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify gene products from members of the AY strain cluster, also amplified a DNA product from the PDB phytoplasma but not from either the PYC or PM phytoplasmas. Primers deduced from the 16S rRNA/SR selectively amplified rDNA sequences from the PDB and AGY phytoplasmas but not from other members of the stolbur strain cluster. Similarly, primers designed from 16S rRNA/SR amplified rDNA from the PYC and PM phytoplasmas but not from the PDB phytoplasma. These primers may provide for more specific detection of these pathogens in epidemiological studies.     

臭矢菜丛枝病植原体的分子鉴定研究

 本实验采用DAPI荧光显微镜、PCR、克隆和测序等技术,对海南臭矢菜丛枝病样进行了检测和鉴定。以染病臭矢菜总DNA为模板应用3对植原体特异性引物进行PCR扩增,获得PCR产物为16S rDNA(1 430 bp)、16S-23S rDNA(358bp)、rp DNA(1 294 bp)。应用DNA回收试剂盒获得了3个PCR扩增片断的纯化产物,并克隆到DH5α大肠杆菌中测序。应用DNAMAN和MEGA软件对获得的序列与NCBI数据库中植原体序列进行同源性分析和构建系统发育树。结果显示臭矢菜丛枝病植原体与花生丛枝病植原体序列同源性最高,16S rDNA的序列同源性为99.9%,16S-23S rDNA高达100%,rp为99.7%,因而将臭矢菜丛枝病植原体归为花生丛枝组(16SrⅡ),根据16S rDNA的RFLP分析,将其归为16SrⅡ-A亚组。     

Molecular detection and identification of phytoplasma associated with pepper witches’ broom in China

Pepper witches’ broom (PWB) disease was observed in a field in Yangling, Shaanxi Province, China. The result of mechanical inoculation test for this disease was negative. Phytoplasma-like bodies were observed in ultrathin sections of petiole tissues of symptomatic samples. 16S rRNA gene and tuf gene of phytoplasma were amplified from the total DNA of symptomatic samples. Phylogeny analysis of the 16S rRNA gene and tuf gene suggested that the pepper witches’ broom associated phytoplasma belongs to the subgroup 16SrI-B, which was confirmed by the RFLP analysis of the 16S rRNA gene. The phytoplasma subgroup 16SrI-B was also detected in the vector Cicadella viridis trapped from the infected field. To our knowledge, this is the first report of 16SrI-B phytoplasma causing pepper witches’ broom in China.     

Association of 'Candidatus Phytoplasma australiense' with green petal and lethal yellows diseases in strawberry

The identity of phytoplasmas detected in strawberry plants with green petal (SGP) and lethal yellows (SLY) diseases was determined by RFLP analysis of the 16S rRNA gene and adjacent spacer region (SR). RFLP and sequence comparisons indicated that the phytoplasmas associated with SGP and SLY were indistinguishable and were most closely related to ' Candidatus Phytoplasma australiense', the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases. This taxon lies within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify only members of the AY strain cluster, amplified a DNA product from the SGP and SLY phytoplasmas. Primers deduced from the 16S rRNA/SR of P. australiense that amplify only members of this taxon amplified rDNA sequences from the SGP and SLY phytoplasmas. Primers that selectively amplify members of the faba bean phyllody (FBP) phytoplasma group, the most commonly occurring phytoplasma group in Australia, did not amplify rDNA from the SGP and SLY phytoplasmas.     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.