Association of two groups of phytoplasma with various symptoms in some wooden and herbaceous plants

During 2015–2016, wooden and herbaceous plants growing in parks, boulevards, fields, gardens and forests in Khuzestan province, southwestern Iran, were visually inspected for symptoms resembling phytoplasma. Fifty‐one symptomatic samples from nine different species and one symptomless sample from each plant were collected. Leaf midribs, petioles and the parts of stem cambium were separated and freeze‐dried. Total DNA was extracted using CTAB‐based method and tested for phytoplasma using a nested PCR assay. The expected size amplicons of 16S rDNA were sequenced and compared to those of reference phytoplasmas by BLASTn search and phylogenetic analysis. The consensus 16S rDNA sequence of the detected phytoplasma in narrow cattail related to reference phytoplasma group 16SrVI, “Candidatus Phytoplasma trifolii” while in the other plants were related to reference phytoplasma subgroup 16SrII–D, “Candidatus Phytoplasma aurantifolia.” All isolates showed 98%–99% sequence identity to members of their reference groups. To our knowledge, this is the first report of “Candidatus Phytoplasma aurantifolia”‐related strains infecting the plants of Acacia salicina, Alternanthera ficoidea, Melaleuca citrine, Citrus aurantium throughout the world and Celosia christata in Iran. Furthermore, this study is the first to report the association of a “Candidatus Phytoplasma trifolii”‐related strain with Typha angustifolia worldwide.     

Multilocus genotyping of a ‘Candidatus Phytoplasma aurantifolia’‐related strain associated with cauliflower phyllody disease in China

A new cauliflower disease characterised by the formation of leaf‐like inflorescences and malformed flowers occurred in a seed production field located in Yunnan, a southwest province of China. Detection of phytoplasma‐characteristic 16S rRNA gene sequences in DNA samples from diseased plants linked the cauliflower disease to phytoplasmal infection. Results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence indicated that the cauliflower‐infecting agent is a ‘Candidatus Phytoplasma aurantifolia’‐related strain and is a new member of the peanut witches'‐broom phytoplasma group, subgroup A (16SrII‐A). Multilocus genotyping showed close genetic relationship between this cauliflower phytoplasma and a broad host range phytoplasma lineage found only in East Asia thus far. Molecular markers present in the secY and rp loci distinguished this phytoplasma from other members of the subgroup 16SrII‐A.     

Detection and Characterization of Phytoplasma Associated with Big Bud Disease of Tomato in China

Symptomatic tomato plants exhibiting big bud, proliferation and small leaves of lateral shoots, purplish top leaves, phyllody, enlarged pistils, hypertrophic calyxes and small and polygonal fruit were collected in Yunnan Province of China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants by transmission electron microscopy. The presence of phytoplasma in collected samples was further analysed and identified by PCR and virtual computer‐simulated restriction fragment length polymorphism (virtual RFLP). A 1.2 kb product was amplified by PCR with universal primers R16F2n/R16R2. Sequence comparisons revealed that the tested strains shared 99% 16S rRNA gene sequence similarity with members of ‘Candidatus Phytoplasma aurantifolia’ (16SrII group). Phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences confirmed that the phytoplasma is a member of the 16SrII group. This is the first report of 16SrII group phytoplasma infecting tomato in China.     

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.     

Transmission of the Phytoplasma Associated with Bunchy Top Symptom of Papaya by Empoasca papayae Oman

Transmission tests were conducted with field‐collected Bunchy Top Symptoms (BTS) phytoplasma‐infected specimens of Empoasca papayae. BTS developed in all eight inoculated papayas 3 months later. The BTS phytoplasma was identified in six of eight inoculated papayas, whose partial 16S rRNA sequence (GenBank Accession no. FJ6492000 ) was 99.9% identical with those from the collected papayas (GenBank Accession no FJ649198 ) and E. papayae (GenBank Accession no. FJ649199 ), all of which are members of group 16SrII, ‘Candidatus Phytoplasma aurantifolia’. Results confirmed the ability of E. papayae to transmit the BTS phytoplasma.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Molecular Identification of a 16SrII‐A Group‐Related Phytoplasma Associated with Cinnamon Yellow Leaf Disease in China

Chinese cinnamon (Cinnamomum cassia Presl), an evergreen tree native to China, is a multifaceted medicinal plant. The stem bark of cinnamon is used worldwide in traditional and modern medicines and is one of the most popular cooking spices. In recent years, cinnamon with pronounced yellow leaf symptoms has been observed in their natural habitat in Hainan, China. Phytoplasmas were detected from symptomatic cinnamon trees via polymerase chain reaction using phytoplasma universal primers P1/P7 followed by R16F2n/R16R2. No amplification products were obtained from templates of asymptomatic cinnamon trees. These results indicated a direct association between phytoplasma infection and the cinnamon yellow leaf (CYL) disease. Sequence analysis of the CYL phytoplasma 16S rRNA gene determined that CYL phytoplasma is a ‘Candidatus Phytoplasma australasiae’‐related strain. Furthermore, virtual restriction fragment length polymorphism pattern analysis and phylogenetic studies showed that CYL phytoplasma belongs to the peanut witches’‐broom (16SrII) group, subgroup A. This is the first report of a 16SrII group phytoplasma infecting cinnamon under natural conditions.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Detection and Identification of a 16SrII Group Phytoplasma Causing Clover Little Leaf Disease in Iran

White clover plants showing little leaf and leaf reddening symptoms were observed in Isfahan Province in central Iran. Restriction fragment length polymorphism analyses of nested PCR‐amplified fragments from Iranian clover little leaf phytoplasma isolates and representative phytoplasmas from other phytoplasma groups using AluI, CfoI, KpnI and RsaI restriction enzymes indicated that the clover phytoplasma isolates are related to the peanut WB group. Sequence analyses of partial 16S rRNA fragments showed that Iranian clover little leaf phytoplasma has 99% similarity with soybean witches'‐broom phytoplasma, a member of the peanut WB (16SrII) phytoplasma group. This is the first report of clover infection with a phytoplasma related to the 16SrII group.     

Molecular Detection and Identification of a 16SrVI Group Phytoplasma Associated with Tomato Big Bud Disease in Xinjiang,China

In 2010, tomato plants with big bud symptoms were observed in Xinjiang, China. PCR products of approximately 1.2 and 2.8 kb were amplified from infected tomato tissues but not from asymptomatic plants. A comparison of 16S rDNA sequences showed that the casual tomato big bud (TBB) phytoplasma was closely (99%) related to the ‘Candidatus Phytoplasma trifolii’ (16SrVI group). The TBB phytoplasma clustered into one branch with the Loofah witches'‐broom phytoplasma according to the 23S rDNA analysis but with no other member of the 16SrVI group. The cause of TBB symptoms was identified as ‘Ca. Phytoplasma trifolii' (16SrVI group) by PCR, virtual RFLP and sequencing analyses. This is the first report of a phytoplasma related to ‘Ca. Phytoplasma trifolii' causing TBB disease in China.     

Molecular characterization of phytoplasma diseases of pepper in Turkey

During autumn, an extensive survey was conducted in pepper (Capsicum annum L.) in intensive cultivation areas of four provinces in southeastern Turkey (Adana, Kahramanmara?, Mersin and ?anl?urfa) in order to identify the causal agent (s) of phytoplasma‐like symptoms (chlorosis, little‐leaf, short internodes and stunting). DNA amplification by PCR and RFLP analysis using EcoRI restriction enzyme confirmed the presence of phytoplasmas in ?anl?urfa and Mersin, and consequently their possible association with the symptoms. Sequencing and phylogenetic analysis revealed that the isolate from ?anl?urfa had 99% sequence identity with “Candidatus Phytoplasma trifolii” (16SrVI) and is a member of the clover proliferation group (16SrVI‐A). Additionally, the isolate from Mersin had 96% sequence identity with “Candidatus Phytoplasma asteris” (16SrI). Importantly, gene sequence of the Mersin isolate shared <97.5% similarity to previously discovered “Ca. Phytoplasma” species. Consequently, the phytoplasma detected from Mersin could represent a new “Ca. Phytoplasma” species and to our knowledge, this is the first report of asteris‐like phytoplasmas infecting pepper in Turkey.     

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.     

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).     

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

Royal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.     

Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, Iran

In 2002, garden beet witches’ broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR‐amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII‐E, in the peanut witches’ broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722 ) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII‐E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma.     

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.     

Molecular detection and identification of phytoplasmas associated with Catharanthus roseus,Pinus eldarica,and Petunia hybrida in southeastern Iran's urban green spaces

Given the potential for urban green spaces to provide fresh and healthy environments for humans, exploring the issues that threaten plants in these places is crucial. Phytoplasma-related symptoms were encountered on some plants in urban green spaces in the province of Kerman, southeastern Iran, between 2017 and 2019. Affected periwinkles and petunias exhibited phytoplasma disease symptoms, including virescence, phyllody, and witches'-broom. However, ball or disc-like shoot proliferation symptoms were noticed on the trunks and branches of pine trees. PCR was performed with phytoplasma-detecting universal primers, targetting and amplifying the 16S rRNA gene, and determining whether phytoplasmas are implicated in the symptomatic plants. The infection of the symptomatic plants was confirmed using nested-PCR amplification of expected DNA sizes for phytoplasmas. No product, however, was amplified from sampled symptomless plants. The sequencing of nested-PCR products was performed to obtain sequences encasing the standard F2nR2 fragments. The resulted sequences were submitted to iPhyClassifier, the universal phytoplasma classification platform, for the taxonomic assignment of the found phytoplasmas compared with previously identified ‘Candidatus Phytoplasma’ species, groups, and subgroups. The results revealed that phytoplasma strains related to the species ‘Ca. P. trifolii’ (16SrVI-A subgroup) infect periwinkles and pines. However, strains from the species ‘Ca. P. aurantifolia’ (16SrII-D subgroup) and ‘Ca. P. phoenicium’ (16SrIX-C subgroup) were found in petunias and periwinkles, respectively. To the best of our knowledge, phytoplasmas from the 16SrVI-A and 16SrII-D subgroups are the first reported to infect these plants in Kerman province, while a related strain from the subgroup 16SrIX-C is the first recorded to infect periwinkles in Iran and the second in the world.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

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.     

Molecular Characterization of Phytoplasmas Associated with Potato Purple Top Disease in Iran

Potato plants with symptoms suggestive of potato purple top disease (PPTD) occurred in the central, western and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7 followed by primer pairs R16F2n/R16R2 and fU5/rU3 for phytoplasma detection. Using primer pairs R16F2n/R16R2 and fU5/rU3 in nested PCR, the expected fragments were amplified from 53% of symptomatic potatoes. Restriction fragment length polymorphism (RFLP) analysis using AluI, CfoI, EcoRI, KpnI, HindIII, MseI, RsaI and TaqI restriction enzymes confirmed that different phytoplasma isolates caused PPTD in several Iranian potato‐growing areas. Sequences analysis of partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma solani’, ‘Ca. Phytoplasma astris’ and ‘Ca. Phytoplasma trifolii’ are prevalent in potato plants showing PPTD symptoms in the production areas of central, western and north‐western regions of Iran, although ‘Ca. Phytoplasma solani’ is more prevalent than other phytoplasmas. This is the first report of phytoplasmas related to ‘Ca. Phytoplasma astris’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ causing PPTD in Iran.