泰安枣疯病植原体的分子鉴定

【目的】对3个枣疯病病原物泰安株系进行分子鉴定。【方法】采用植原体通用引物对R16F2n/R16R2,通过直接PCR技术,扩增枣疯病植原体16S rDNA基因,通过16S rDNA基因序列分析和在线模拟16S rDNA-RFLP分析,并将其16S rDNA基因序列提交到GenBank数据库。【结果】3个枣疯病病原物16S rDNA基因片段与16SrⅤ-B亚组中枣疯病植原体(AB052876和AF279272)、樱桃致死黄化植原体(AY197659)及杏卷叶植原体(FJ572660)的同源性高达99.5%99.7%,分别命名为枣疯病植原体泰安圆铃1号株系(Jujube witches’-broom phytoplasma strain Yuanling1,JWB-Yuanling1,TA)、枣疯病植原体泰安鲁北冬枣株系(Jujube witches’-broom phytoplasma strain Lubeidongzao,JWB-Lubeidongzao,TA)和枣疯病植原体泰安大白铃株系(Jujube witches’-broom phyto-plasma strain Dabailing,JWB-Dabailing,TA),基因登录号分别为:HM989946、HM989947和HM989948。【结论】3个枣疯病植原体泰安株系均归属于16SrⅤ-B亚组。     

新疆小叶白蜡丛枝病植原体的鉴定及16S rRNA基因序列分析

【目的】对新疆小叶白蜡丛枝病植原体进行检测,通过其16S rRNA基因分析确定其分类地位。【方法】利用苯胺蓝和4′,6-二脒基-2-苯基吲哚(DAPI)染色,在荧光显微镜下观察新疆小叶白蜡嫩茎横切片;采用植原体16S rRNA基因的通用引物对P1/P7和R16F2n/R16R2进行直接和巢式PCR扩增,对得到的16S rRNA基因的序列进行RFLP和构建系统进化树分析。【结果】表现丛枝病症状的新疆小叶白蜡中存在植原体,暂命名为Fraxinus sogdianaBunge witches’broom phytoplasma(Fraxinus sogdianaBunge WB);其16S rRNA基因的序列GenBank登录号为KF061042,RFLP图谱与16Sr V-B亚组的枣疯病植原体相同,系统进化地位与枣疯病菌株AB052876相同。【结论】新疆小叶白蜡丛枝病植原体为16Sr V-B亚组成员。     

枣疯病植原体tuf和rp基因的克隆与序列分析

【目的】枣疯病是一种重要的植原体病害,本研究旨在明确北京及河北地区枣疯病植原体的分类地位,为枣疯病在亚组水平上分类提供一定的参考依据。【方法】利用植原体通用引物fTufu/rTufu和rp(v)F1A/rp(v)R1A对北京和河北地区枣疯病植原体延伸因子tuf基因和核糖体蛋白基因(rp)进行PCR扩增并进行核苷酸序列测定及相似性分析。【结果】获得北京地区JWB-XFSZ株系、JWB-XFDO株系以及河北地区JWB-TXSZ株系的tuf基因片段均为824 bp;北京地区JWB-XFSZ株系的rp基因片段为1196 bp。经序列相似性比较表明:tuf基因与16SrV组的葡萄黄叶病(Flavescence dorée)相似性最高,为92.84%,而与已经公布的其它地区(陕西杨凌)的枣疯病植原体tuf基因相似性较低,为57.29%;关于rp基因,北京地区枣疯病JWB-XFSZ株系与16SrV组的枣疯病泰山株系(JWB-Taishan)以及大麻丛枝病植原体(HFWB)相似性最高,均为99.83%,与16SrV组的成员相似性均在96%以上。【结论】北京与河北地区枣疯病植原体具有较高的相似性,而在tuf基因水平上,与陕西地区枣疯病植原体具有较大的差异;本研究中北京与河北两地区枣疯病植原体归属于16SrV组。     

泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能和遗传变异比较分析

【目的】探究泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能差异及其遗传多样性。【方法】利用热不对称交错式PCR(TAIL-PCR)扩增枣疯病植原体tuf基因上游未知序列,利用启动子探针载体pSUPV4构建了泡桐丛枝和枣疯病植原体tuf基因上游序列的大肠杆菌异源表达体系,分析泡桐丛枝、苦楝丛枝、莴苣黄化、桑萎缩、长春花绿变等16SrI组和枣疯病、樱桃致死黄化、重阳木丛枝等16SrV组株系tuf基因上游调控序列的遗传变异特征和启动子活性。【结果】泡桐丛枝等16SrI组植原体株系tuf基因和其上游fus A基因之间的间区序列长129-130 bp,预测有完整的启动子保守结构。泡桐丛枝植原体tuf基因上游130 bp片段具有启动子活性,此间区序列在5种35株16SrI组株系中存在4种变异类型;枣疯病植原体等16SrV组株系fusA和tuf基因间区长53-54 bp,未预测到完整启动子结构。枣疯病植原体tuf基因上游144 bp和346 bp片段均未检测到启动子活性,fus A和tuf基因间区序列在3种20株16SrV组株系中存在2种变异类型。fus A-tuf基因间区序列相对保守,基于此序列构建的进化树可清晰区分不同组别的植原体株系。【结论】研究方法和结果为深入研究植原体基因表达与调控、揭示植原体生长繁殖规律及其致病机理等奠定了良好的基础。     

坡柳(Dodonaea viscosa L.)丛枝植原体新病原的分子鉴定

从四川攀枝花芒果园中表现为丛枝、小叶和黄花等症状的坡柳植株发病样品中,利用植原体16S rDNA基因的通用引物R16m F2/R16m R1和R16F2n/R16R2,对发病植株总DNA进行巢式PCR检测,同时设计植原体抗原膜蛋白基因(AntMP)的保守引物AntMP-F/AntMP-R进行PCR验证。结果显示,坡柳样品巢式PCR的第一轮、第二轮DNA条带大小分别为1 400 bp和1 200 bp左右,经NCBI序列相似性比较均为植原体16S rDNA序列,Gen Bank登录号为KT957205和KT957206;PCR结果显示抗原膜蛋白基因大小约600 bp,与目标基因大小一致。将测得的16S rDNA基因序列与Gen Bank数据库中登录的16SrⅠ~ⅩⅤ组植原体16S rDNA序列进行同源性比对,构建系统进化树,结果显示四川坡柳丛枝植原体(DOVI-SC)属于16SrⅠ组(即翠菊黄花组),与已报道的5个16SrⅠ组(AY101386,AY566302,AY389822,L33760和KP662119)同属一个组。利用植原体亚组分类鉴定软件iPhy Classfier对获得的2条植原体16S rDNA序列进行虚拟RFLP分析,结果显示KT957205,KT957206与16SrⅠ-B亚组洋葱黄化植原体(NC-005303)相似度分为1.0、0.97,归属于16SrⅠ-B亚组。本研究对引起四川坡柳丛枝的植原体病原进行检测,为坡柳植原体病害的早期诊断、快速检测以及预防措施制定提供科学线索。     

多重PCR法区分枣园两种菱纹叶蝉及检测其体内枣疯病植原体

【目的】目前发现,北京枣园中的凹缘菱纹叶蝉Hishimonus sellatus(Uhler)和片突菱纹叶蝉Hishimonus lamellatus Cai混同发生。已知凹缘菱纹叶蝉可以传播枣疯病,而片突菱纹叶蝉是否携带枣疯病植原体尚待证明。正确鉴别区分枣园中菱纹叶蝉的种类并测定其体内感染枣疯病植原体情况有助于阐明田间枣疯病的流行规律,从而提出有效的预防枣疯病及其媒介昆虫措施显得十分重要。传统形态学鉴定两种菱纹叶蝉种类的方法局限于雄性成虫外生殖器,本研究目的在于建立一种快速的分子生物学方法,在区分枣园中两种枣菱纹叶蝉的同时,可检测虫体内的枣疯病植原体。【方法】以凹缘菱纹叶蝉和片突菱纹叶蝉的COI基因以及枣疯病植原体的16S r DNA为扩增目标,分别设计引物,建立一种包含3对引物的多重PCR体系。测试该多重PCR体系对叶蝉总DNA的灵敏度、准确性,以及当两种叶蝉DNA同时存在时的辨别能力和对枣疯病植原体16S r DNA的灵敏度。【结果】该多重PCR可以准确区分凹缘菱纹叶蝉和片突菱纹叶蝉,并对虫体内枣疯病植原体实现检测,其对昆虫总DNA的灵敏度达到0.012 ng,对枣疯病植原体16S r DNA模板的灵敏度达到900拷贝。【结论】该方法极大方便了对枣菱纹叶蝉的田间种群发生动态及虫体中枣疯病植原体感染的监测。     

假臭草从枝病植原体鉴定与多样性分析

研究假臭草丛枝病植原体的多样性,并确定其分类地位,对于利用假臭草丛枝病植原体对假臭草进行生物防治具有重要的意义.本研究采集了海南省8个地区的假臭草丛枝病样品,采用PCR以及巢式PCR方法扩增了假臭草丛枝病植原体的16S rDNA序列片段,进一步选用AfaⅠ、Alu Ⅰ、EcoR Ⅰ、HaeⅢ、HpaⅡ、HhaⅠ、HinfⅠ、Kpn Ⅰ、Sau3A Ⅰ、Taq Ⅰ和Xsp Ⅰ等11种限制性内切酶对巢式PCR产物进行酶切分析(RFLP),并对16S rDNA序列进行测序,确定假臭草丛枝病植原体多样性和生物学地位.结果发现:假臭草丛枝病的病原确为植原体;8个地区的假臭草丛枝病植原体的酶切图谱基本一致,与已知植原体的相似度为0.26～0.97;8个地区假臭草丛枝病植原体的序列同源性均在99.4％以上,应为同种植原体;假臭草丛枝病植原体与16S rRNAⅡ-A组的花生丛枝病(PnWB)的同源性最高达到99.1％,说明假臭草丛枝病植原体在分类学地位上应归属于植原体16S rRNAⅡ-A组.     

基于16S rDNA基因的谷斑皮蠹PCR检测技术

【目的】谷斑皮蠹是一种重要的检疫性害虫,在新疆周边多个国家分布,口岸检疫人员多次从进境货物中截获谷斑皮蠹,该虫对新疆的农业生产极具威胁。【方法】以谷斑皮蠹的16S rDNA基因为靶序列,用昆虫通用引物对4种供试皮蠹进行PCR扩增,将扩增产物进行克隆和测序,用生物软件设计检测谷斑皮蠹的特异性引物与探针。【结果】设计的特异性引物(TG-SNP-F/TG-SNP-R)及所建立的常规PCR方法能有效检测出谷斑皮蠹,其扩增产物的片段大小为250 bp,灵敏度为3 ng·μL~(-1)。设计的特异性引物(TG-F/TG-R)和探针(TG-probe),以及所建立的实时荧光PCR方法,对谷斑皮蠹的检测特异性强,灵敏度达0.8 fg·μL~(-1)。【结论】建立的常规PCR方法和实时荧光PCR检测方法能够对谷斑皮蠹进行准确鉴定,为口岸检疫人员检测进境货物中携带的谷斑皮蠹提供技术支持。     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

PCR反应条件对16S rRNA基因扩增子测序准确性的影响

【背景】16S rRNA基因扩增子测序技术是一种不依赖培养而获得样本中细菌种群结构、相对丰度等信息的方法。高通量测序技术实验步骤较多,每一步骤细微的差别都可能在最终的测序结果中放大,并造成测序结果与实际情况的偏差。【目的】基于MiSeq测序平台,探讨PCR反应体系中扩增引物序列、退火温度、模板起始量、扩增循环数和变性时间等5个因素对16S rRNA基因测序结果的影响。【方法】对mock DNA的16S rRNA基因扩增子进行测序,分别分析不同的扩增引物、退火温度、模板起始量、循环数和变性时间对数据准确性的影响。【结果】不同的扩增引物对检测结果有较大的影响,采用的4组引物中,引物B (V3–V4,341F/806R)的准确性最好,引物A(V3–V4,341F/805R)次之。比较不同退火温度(52、55和60℃)对检测准确性的影响,退火温度60℃的结果最接近理论值。模板起始量(2、10和50 ng)的检测结果显示,mock DNA起始量为2ng的结果准确性最高。相较于其他3组(15+18、25+8和30+8),循环数为(20+8)的检测结果最接近mock DNA的理论值。不同变性时间(3...     

Detection and Identification of an Elm Yellows Group Phytoplasma Associated with Camellia in China

In 2012, yellowing of camellias was observed in Tai'an in Shandong province, China. Transmission electron microscopy (TEM) revealed phytoplasma in the phloem sieve tube elements of symptomatic plants. A specific fragment of phytoplasma 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal phytoplasma primers P1/P7 followed by R16F2n/R16R2. Sequence and restriction fragment length polymorphism (RFLP) analyses allowed us to classify the detected phytoplasma into the elm yellows (EY) group (16SrV), subgroup 16SrV‐B. Sequence analyses of the ribosomal protein (rp) gene confirmed a close relationship with phytoplasmas belonging to the rpV‐C subgroup. Thus, the phytoplasma associated with yellows disease in camellia, designated as ‘CY’, is a member of the 16SrV‐B subgroup. This is the first report of phytoplasma associated with camellia.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

Characterization of putative membrane protein genes of the 'Candidatus Phytoplasma asteris', chrysanthemum yellows isolate

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches' broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the 'Candidatus Phytoplasma asteris'. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of 'Ca. Phytoplasma asteris' from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.     

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.     

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.     

Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province,Saudi Arabia

Typical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400?bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.     

Artemisia vulgaris,a new host of 16SrV-C phytoplasma related strains infecting black alder in Poland

Yellowing of leaf tissue and strongly deformed shoots were observed in common mugwort (Artemisia vulgaris L.) growing in a nature reserve in Southern Poland. Similar foliage chlorosis together with abnormal shoot proliferation was noticed on alder tree (Alnus glutinosa Gaertn.) growing next to the common mugwort. DNA specific fragments coding 16S rRNA and ribosomal proteins (rp) were amplified from mugwort and alder samples using direct and nested PCR (Polymerase Chain Reaction) assays. Phylogenetic relationships inferred from 16S and rps3 genes indicated that strains infecting mugwort and alder were most closely related to phytoplasmas of subgroups 16SrV-C and 16SrV-D. Based on the restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA, the investigated phytoplasma strains were classified to subgroup 16SrV-C. Two sequence variants of the rps3 gene which differed by a single nucleotide were detected in all analysed samples by pairwise analysis of the aligned reads. Taking into account that this single-nucleotide polymorphism (SNP) occurs among 16SrV-C and 16SrV-D related phytoplasmas and that the phytoplasmas have a single copy of rp operon, we concluded that each plant species was infected by two distinct, closely related phytoplasma strains. To the best of our knowledge, this is the first report of group 16SrV-C related phytoplasmas infecting common mugwort worldwide, adding a new host species that is possibly linked to the spread of the alder pathogen in Eastern Europe. Although alder yellows phytoplasma has been frequently found in Europe, this is the first detection of phytoplasmas associated with alder in Poland.     

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.