Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues

The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain).     

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.     

Occurrence and Characterization of a 16SrII‐D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran

During 2010–2013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches’ broom, stunting, flower virescence and phyllody. The disease causative agent was dodder transmitted from symptomatic parsley to periwinkle and from periwinkle to periwinkle by grafting inducing phytoplasma‐type symptoms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally inoculated periwinkle plants in direct polymerase chain reaction (PCR) using phytoplasma primer pair P1/P7 or nested PCR using the same primer pair followed by R16F2n/R16R2 primers. Restriction fragment length polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd province belong to 16SrII‐D phytoplasma subgroup. This is the first report of association of a 16SrII‐related phytoplasma with PrWB disease in Iran.     

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.     

First report of Echinococcus granulosus G8 in Eurasia and a reappraisal of the phylogenetic relationships of 'genotypes' G5-G10

In this study, we investigated the presence of the larval stage of the tapeworm Echinococcus granulosus in wild ungulates in Estonia, genetically characterized E. granulosus isolates using mitochondrial gene sequences and used the sequence data, together with those available in a public database, to infer the phylogenic relationships of E. granulosus 'genotypes' G5-G10. While 0.8% of the 2038 moose (Alces alces) examined were found to be infected with E. granulosus, the parasite was not detected in other wild ungulates, such as roe deer (Capreolus capreolus: 1044 specimens examined) and wild boar (Sus scrofa: 442 specimens). Genetic analyses of concatenated atp6, nad1 and cox1 gene (1028 bp) sequences revealed that 2 novel E. granulosus haplotypes, namely E8 (11 samples: 69%) and E10 (5 samples: 31%), grouped with E. granulosus G8 and G10, respectively, are present in Estonia. This is the first record of an E. granulosus G8 in Eurasia. Phylogenetic analyses, using 4 different methods, demonstrated with considerable statistical support that E. granulosus G6/7 forms a subgroup together with G10, whereas G8 is a sister taxon to G6/7-G10.     

Further molecular discrimination of Spanish strains of Echinococcus granulosus

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.     

Detection of Phytoplasma from deferent infected hosts in Jordan based on PCR amplification of the 16S rDNA sequences

Sixteen leaf samples, both healthy and Phytoplasma diseased, were collected from different plants such as grape, peach, almond, tomato, paper, squash, apple and pear in northern Jordan. Extracted DNA from diseased grape, peach, almond, tomato, paper and squash plus from infected periwinkle (Catharanthus roseus) samples were amplified with the Phytoplasma universal 16S rDNA sequences primer pairs. Extracted DNA samples from healthy and diseased apple and pear plants were not amplified with the same primer pairs. All the PCR-amplified DNA samples show a common band with size of 558 bp, indicating Phytoplasma pathogens as a disease-causative agent for grape, peach, almond, tomato, paper and squash plants. The restriction fragment length polymorphisms of Alu1 enzyme for the amplified 16S rDNA sequences shows the same DNA fragment patterns indicating no or a limited diversity among the DNA of the detected Phytoplasma pathogens.     

Niger Seed (Guizotia abyssinica), a New Host of ‘Candidatus Phytoplasma asteris’ in Iran

Shrubs of niger seed with phyllody and internode elongation symptoms suggestive of phytoplasma infections occurred in the central regions of Iran. Phytoplasma was detected by polymerase chain reaction (PCR) and nested PCR amplifications using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2. Using aster yellows group–specific primer pair rp(I)F1A/rp(I)R1A, a fragment of 1212 bp of the rp genes was amplified from DNA samples of infected plants. Random fragment length polymorphism (RFLP) analyses of R16F2n/R16R2‐amplified products using the CfoI restriction enzyme confirmed that Iranian niger seed phyllody phytoplasma is associated with aster yellows group phytoplasmas. Sequence analyses of the partial rp genes fragment indicated that the Iranian niger seed phyllody phytoplasma, which was collected from central regions of Iran, is related to ‘Candidatus Phytoplasma asteris’. This is the first report of a phytoplasma infecting the niger seed plant.     

Description of Echinococcus granulosus genotypes in human hydatidosis in a region of southern Chile

INTRODUCTION: Echinococcus granulosus species has a wide variety in both geography and hosts; indeed, 10 genotypes have been reported in studies on material of animal origin. The aim of this study was to genotype E. granulosus obtained from human hydatid cysts. MATERIALS AND METHODS: The hydatid fluid and sand was collected from patients who underwent surgery for hepatic and pulmonary hydatidosis at Hospital Regional in Temuco, Chile, between 2004 and 2005. Two PCR systems were used: PCR Eg 9 and PCR Eg 16. The RsaI enzyme was used for RFLP. The genotype was confirmed using the sequence of one fragment of 366 bp from a mitochondrial gene (cox1). RESULTS: The DNA of protoscolices from 24 samples was analyzed, 4 of them from pulmonary cysts and 20 from hepatic cysts. The 366 bp fragment was amplified in 20 out of 24 samples (83.3%). Enzymatic digestion revealed the presence of 3 possible genotypes: in 20 out of 21 samples (95,2%), a restriction was observed corresponding to the G1 or G7 genotypes; in the remaining sample genotype G4 or G7 was observed. Sequencing confirmed the presence of G1 genotype for 19 samples and G6 genotype for the remaining sample (G4 or G7 according to PCR-RFLP). CONCLUSION: The PCR-RFLP technique enabled three possible genotypes present (G1 or G7, G4 or G7) to be established. Sequencing allowed us to decisively identify the G1 and G6 genotypes in our study group. Previous studies agree with the identification of the G1 genotype in our country. We consider it significant that the G6 genotype is present in Chile for its epidemiological implications.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Genotypic characterization of Iranian camel (Camelus dromedarius) isolates of Echinoccocus granulosus

In the present study, Echinoccocus granulosus isolates collected from camels (Camelus dromedarius) in eastern Iran were characterized based on the nucleotide sequences of mitochondrial CO1 and NDI genes. The molecular results for camel isolates demonstrated that at least 2 different genotypes are present, i.e., a buffalo genotype (G3) and the camel genotype (G6). Although the sequences of the Iranian camel genotype (G6) are completely homologous to the reference sequence of G6 (M84666) of E. granulosus , a nucleotide mutation (C to T at position 168) was detected in the CO1 sequences of the Iranian G3 isolates (HM626405) when compared with the reference G3 genotype (M84663). The findings of the present study represent the demonstration of the buffalo strain in camels. As previously reported, humans can be infected by this genotype; accordingly, the epidemiological importance of this genotype in the camel population should be considered in further studies.     

Genetic characterization of Echinococcus granulosus in camels, cattle and sheep from the south-east of Iran indicates the presence of the G3 genotype

Echinococcus granulosus, the aetiologic agent of cystic echinococcosis (CE), is one of the most important zoonotic helminthes worldwide. Isolates of the parasite show considerable genetic variation in different intermediate hosts. Several genotypes and species are described in different eco-epidemiological settings. This study investigated E. granulosus genotypes existing in livestock and humans from the province of Kerman, located in south-eastern Iran, using sequencing data of cox1 and nad1 mitochondrial genes. Fifty-eight E. granulosus isolates, including 35 from sheep, 11 from cattle, 9 from camels and 3 from goats, were collected from slaughterhouses throughout Kerman. One human isolate was obtained from a surgical case of CE. Mitochondrial cox1 and nad1 regions were amplified using polymerase chain reaction (PCR) and 38 isolates were sequenced. Genotypes G1 (73.7%), G3 (13.2%) and G6 (13.1%) were identified from the isolates. G1 was the most common genotype from sheep (86.7%), cattle (80%), camels (44.4%) and goats (100%). Sheep, cattle and camels were also found to be infected with the G3 genotype (buffalo strain). The human isolate was identified as the G6 genotype. Results showed that the G3 genotype occurred in different animal hosts in addition to G1 and G6 genotypes.     

Development of a SCAR marker and a strain‐specific genomic marker for the detection of the biocontrol agent strain CPA‐8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA‐8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA‐8 that was sequenced and used to design SCAR (sequence‐characterised amplified regions) primer pairs. The SCAR‐4 marker amplified a semi‐specific fragment of 665 bp not only for the strain CPA‐8 but also for other 12 strains whose morphology was completely different from CPA‐8. Another approach was developed to obtain a strain‐specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA‐8. Our results revealed that these two molecular markers, SCAR‐4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA‐8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA‐8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.     

恶性疟原虫红内期Pf332抗原基因未知序列的测定及分析

测定恶性疟原虫红内期Pf332抗原 (Ag332 )基因的未知序列 ,并进行序列分析 .根据非洲恶性疟原虫Palo alto株Pf332基因的G1片段序列 ,设计 1对引物 ,从中国恶性疟原虫海南株 (FCC1 HN)基因组DNA中扩增出P332 1片段 .Pf332基因中经常出现SVTEEI短肽的编码序列 ,据此分别设计非特异的正、反义寡核苷酸引物 (NSP1、NSP2 ) ,应用低严谨PCR(LSPCR)分别扩增出P332 1邻近的未知序列片段P332 up1和P332 dow1.根据恶性疟原虫Palo alto株Pf332基因G1片段上、下游的G9和C1片段序列以及测定的P332 up1和P332 dow1序列 ,分别设计 2对特异引物继续扩增邻近的未知序列片段P332 up2和P332 dow2 .根据P332 dow2片段的 3'端序列 ,设计 2条特异引物分别与非特异引物NSP2行LSPCR和巢式PCR ,扩增出P332 dow2邻近的未知序列片段P332 dow3.对获得的Pf332基因片段进行序列测定 ,并用分子生物学软件辅助进行序列分析 .序列测定和拼接结果显示 ,共获得了连续 6 14 4bp的恶性疟原虫FCC1 HN株Pf332基因序列 .序列分析表明 ,所获得的 614 4bp序列位于Pf332基因的编码区内 ,不含内含子 ,编码 2 0 4 8个氨基酸残基 ,包含 5个氨基酸残基重复区 .对恶性疟原虫FCC1 HN株Pf332基因 6 14 4bp序列的测定和分析 ,为获得Pf332全基因     

Mitochondrial DNA-like sequence in the nuclear genome of an akodontine rodent.

Initial amplification and sequencing of a 366-bp fragment of the cytochrome b gene by a conserved primer pair (MVZ 03 and MVZ 04) revealed a nonfunctional copy of the gene with two deletions (one of which is 17 bp in length and the other of which is 3 bp in length) in Chroeomys jelskii, a South American akodontine rodent. By means of an alternative primer to MVZ 03--namely, MVZ 05--from the region of the tRNA for glutamic acid, a functional copy of cytochrome b was subsequently amplified. Both primer pairs amplify functional sequence when applied to purified mitochondrial DNA (mtDNA). Restriction-endonuclease digestion of purified mtDNA from C. jelskii did not reveal any additional sets of bands that would suggest heteroplasmy in the mitochondrial genome. When probed with both functional and nonfunctional gene fragments, MboI restriction digests revealed the same pattern, providing further evidence that the nonfunctional copy must be located in the nucleus. Observed differences in the mitochondrial and nuclear sequences from two populations are consistent with a faster rate of change in mtDNA than in nuclear DNA.     

Detection and Molecular Characterization of a 16SrII‐A* Phytoplasma in Grapefruit (Citrus paradisi) with Huanglongbing‐like Symptoms in China

In the year 2010, in a survey in Guangxi Province, China, to detect and characterize phytoplasmas in a huanglongbing (HLB)‐infected grapefruit (Citrus paradisi) orchard, 87 leaf samples with symptoms of blotchy mottle were collected from symptomatic grapefruit trees, and 320 leaf samples from symptomless trees adjacent to the symptomatic trees. Nested polymerase chain reaction (PCR) using universal phytoplasma primer set P1/P7 followed by primer set fU5/rU3 identified 7 (8.0%) positive samples from symptomatic samples but none from symptomless samples. Of the 87 symptomatic samples, 77 (88.5%) were positive for ‘Candidatus Liberibacter asiaticus’ and 5 for both phytoplasma and ‘Ca. L. asiaticus’. Sequence analysis indicated that seven 881‐bp amplicons, amplified by nested phytoplasma primer sets P1/P7 and fU5/rU3, shared 100.0% sequence identity with each other. Genome walking was then performed based on the 881 bp known sequences, and 5111 bp of upstream and downstream sequences were obtained. The total 5992 bp sequences contained a complete rRNA operon, composed of a 16S rRNA gene, a tRNA^Ile gene, a 23S rRNA gene and a 5S rRNA gene followed by eight tRNA genes. Phylogenetic analysis and virtual restriction fragment length polymorphism analysis confirmed the phytoplasma was a variant (16SrII‐A*) of phytoplasma subgroup 16SrII‐A. As phytoplasmas were only detected in blotchy‐mottle leaves, the 16SrII‐A* phytoplasma identified was related to HLB‐like symptoms.     

Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.     

Association of a Phytoplasma with Pistachio Witches’ Broom Disease in Iran

Pistachio is an important crop in Iran, which is a major producer and exporter of pistachio nuts. The occurrence of a new disease of pistachio trees, characterized by the development of severe witches’ broom, stunted growth and leaf rosetting, was observed in Ghazvin Province. A phytoplasma was detected in infected trees by polymerase chain reaction (PCR) amplification of rRNA operon sequences. Nested PCR with primer pairs P1/P7 and R16F2n/R16R2 was used for specific detection of the phytoplasma in infected trees. To determine its taxonomy, the random fragment length polymorphism (RFLP) pattern and sequence analysis of the amplified rRNA gene were studied. Sequencing of the amplified products of the phytoplasma 16S rRNA gene indicated that pistachio witches’ broom (PWB) phytoplasma is in a separate 16S rRNA group of phytoplasmas (with sequence homology 97% in Blast search). The unique properties of the DNA of the PWB phytoplasma indicate that it is a representative of a new taxon.     

仙人掌丛枝病植原体CWB1株系16SrRNA基因克隆及序列分析

对表现丛枝症状的仙人掌植株总DNA进行植原体 1 6SrRNA基因PCR扩增 ,得到一条约 1 5kb的特异片段 ,表明植株中有植原体存在 ,将此植原体株系命名为CWB1。把此特异片段与pGEM Teasy载体连接并转化到大肠杆菌JM1 0 9感受态细胞中 ,通过PCR鉴定、限制性内切酶 (EcoRI)酶切分析及核苷酸序列分析 ,均表明克隆成功。序列分析结果显示 ,此株系的 1 6SrRNA基因全长 1 489个碱基 ,与属于植原体 1 6SrⅡ C亚组的Fababeanphyllody植原体同源率最高 ,为 99 7%。通过 1 6SrRNA基因核酸序列同源性比较 ,认为该株系属于 1 6SrⅡ C亚组 ,基本确定了其分类地位。     

Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran

Evaluation of phytoplasmas infection was conducted in the pistachio-growing areas of Iran (Rafsanjan in Kerman province) in early autumn of 2011. A total of 30 pistachio trees collected from a pistachio orchard in Rafsanjan showing Psylla damage symptoms and 10 samples with different abnormal symptoms from miscellaneous orchards were tested for the presence of phytoplasma. By using nested PCR with primer pairs P1/P7 and internal primer sets R16F2N/R16R2 and fU3-rU5, amplified fragment of expected size was observed in some trees with deformation and yellowing symptoms. On the basis of nucleotide sequence analysis of 16S?rDNA amplified by PCR, this phytoplasma was classified in group 16S?rII. In addition, we observed false positive reaction in three trees of Ahmadaghaei cultivar by using primer sets R16F2N/R16R2 and sequence analysis of ~1250bp PCR product indicated that amplified fragment was related to Schinus terebinthifolius; a species of flowering plant in the Anacardiaceae family.