Beet leafhopper (Hemiptera: Cicadellidae) transmits the Columbia Basin potato purple top phytoplasma to potatoes, beets, and weeds

Experiments were conducted to determine whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits the purple top phytoplasma to potato, Solanum tuberosum L.; beets, Beta vulgaris L.; and selected weed hosts. The beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was identified as the causal agent of the potato purple top disease outbreaks that recently occurred in the Columbia Basin of Washington and Oregon. The phytoplasma previously was found to be associated almost exclusively with the beet leafhopper, suggesting that this insect is the probable vector of BLTVA in this important potato-growing region. Eight potato cultivars, including 'Russet Burbank', 'Ranger Russet', 'Shepody', 'Umatilla Russet', 'Atlantic', 'FL-1879', 'FL-1867', and 'FL-1833', were exposed for a week to BLTVA-infected beet leafhoppers. After exposure, the plants were maintained outdoors in large cages and then tested for BLTVA by using polymerase chain reaction after 6 to 7 wk. The leafhoppers transmitted BLTVA to seven of the eight exposed potato cultivars. Sixty-four percent of the exposed plants tested positive for the phytoplasma. In addition, 81% of the BLTVA-infected potato plants developed distinct potato purple top disease symptoms. Beet leafhoppers also transmitted BLTVA to beets and several weeds, including groundsel, Senecio vulgaris L.; shepherd's purse, Capsella bursa-pastoris (L.) Medik); kochia, Kochia scoparia (L.) Schrad; and Russian thistle, Salsola kali L. This is the first report of transmission of BLTVA to potatoes, beets, and the above-mentioned four weed species. Results of the current study prove that the beet leafhopper is a vector of the potato purple top disease.     

Beet leafhopper (Hemiptera: Cicadellidae) settling behavior, survival, and reproduction on selected host plants

Experiments were conducted to determine the settling behavior, survival, and reproduction of the beet leafhopper, Circulifer tenellus (Baker), when maintained on selected host plants. This leafhopper was recently identified in the Columbia Basin of Washington and Oregon as the probable vector of the beet leafhopper-transmitted virescence agent phytoplasma, causal agent of several vegetable crop diseases, including potato purple top. Plants selected for study were sugar beet, Beta vulgaris L.; radish, Raphanus sativus L.; dry bean, Phaseolus vulgaris L.; potato, Solanum tuberosum L.; carrot, Daucus carota L.; and tomato, Lycopersicon esculentum Mill. Leafhopper adults were confined on caged plants, and settling behavior was observed during a 72-h period and survival was monitored for 40 d. Also, oviposition and nymphal production were investigated by maintaining leafhoppers for approximately 90 d on each of the selected plants. Sixty to 100% of leafhoppers settled on all studied plants during the first 5 h, but settling on bean and tomato declined sharply thereafter. Leafhopper mortality was very high on bean and tomato, with 95 and 65% of the leafhoppers, respectively, dying in about a week. In contrast, 77, 90, and 95% of leafhoppers maintained on potato, sugar beet, and radish, respectively, survived until the end of the 40-d experimental period. Beet leafhopper oviposition and nymphal production and development only occurred on sugar beet, radish, and potato; reproduction was lower on potato.     

Molecular characterization of ‘Clover proliferation’ phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches’ broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of ‘clover proliferation’ (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of ‘clover proliferation’ subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.     

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.     

Molecular Characterization of a Phytoplasma Associated with Potato Witches’‐broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’‐broom disease including witches’‐broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’‐broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’‐broom disease in Iran. This is the first report of potato witches’‐broom disease in Iran.     

Detection and Identification of Group 16SrVI Phytoplasma in Willows in China

In 2010 and 2011, willow proliferation disease was observed in Erdos, Inner Mongolia, China. The phytoplasma‐specific 16S rRNA gene fragment of 1.2 kb was amplified by a nested PCR with universal primer pair P1/P7 followed by R16F2n/R2. Phylogenetic and virtual RFLP analyses revealed that the phytoplasma associated with willow proliferation was a member of subgroup 16SrVI‐A. The field survey indicated that the incidence of willow proliferation in Erdos was approximately 36.84%. To our knowledge, this is the first record of group 16SrVI phytoplasma infecting willow in China.     

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.     

Development of molecular markers and a diagnostic tool for investigation of coinfections by and interactions between potato purple top and potato witches'‐broom phytoplasmas in tomato

Columbia Basin potato purple top (PPT) phytoplasma and Alaska potato witches'‐broom (PWB) phytoplasma are two closely related but mutually distinct pathogenic bacteria that infect potato and other vegetable crops. Inhabiting phloem sieve elements and being transmitted by phloem‐feeding insect vectors, both pathogens are affiliated with ‘Candidatus Phytoplasma trifolii’ and are members of the clover proliferation phytoplasma group (16SrVI). The polyphagous nature and wide geographic distribution of their insect vectors make mixed infection inevitable. In this study, we experimentally constituted a simultaneous PPT and PWB phytoplasma infection in tomato (Solanum lycopersicum) and developed a sensitive diagnostic tool to investigate mixed infections by and in planta interactions of the two phytoplasmas. The distribution and relative abundance of the two co‐infecting phytoplasmas were monitored over a 45‐day post‐infection time course and for three serial passages in planta. Our results revealed that dual infections of the two phytoplasmas induce a new symptom unseen in infection by either phytoplasma alone. Our results also raised an interesting question as to whether the two phytoplasmas differ in ability of competitive dominance under co‐infection conditions. The molecular markers and the diagnostic tool devised in this study should be useful for further investigations of the interactions between the two closely related phytoplasmas in their hosts.     

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.     

Beet curly top virus transmission by artificially fed and injected beet leafhoppers (Circulfer tenellus)

The transmission of beet curly top virus (BCTV) by leafhoppers, Circulifer tenellus, fed virus through Parafilm® membranes was compared with their transmission when injected with virus from phloem exudates of Amsinckia douglasiana. Virus uptake from ³²P-labelled test solutions and the resulting virus transmission, as measured by an infectivity index, varied widely. By contrast, insects injected with virus transmitted with similar efficiencies. If insects were fasted for 3, 5, or 7 h before a 6 h acquisition access period on test solutions, their ³²P, and presumably virus uptake, was greater than that of nonfasted insects and their variability in virus transmission decreased. The proportion of insects transmitting curly top virus, after fasting and given a 6 h acquisition access period, was similar to that of insects injected with virus. Maximum liquid uptake by the beet leafhopper occurred with a 12% sucrose solution.     

Occurrence,Molecular Characterization and Vector Transmission of a Phytoplasma Associated with Rapeseed Phyllody in Iran

Symptoms of rapeseed phyllody were observed in rapeseed fields of Fars, Ghazvin, Isfahan, Kerman and Yazd provinces in Iran. Circulifer haematoceps leafhoppers testing positive for phytoplasma in polymerase chain reaction (PCR) successfully transmitted a rapeseed phyllody phytoplasma isolate from Zarghan (Fars province) to healthy rapeseed plants directly after collection in the field or after acquisition feeding on infected rapeseed in the greenhouse. The disease agent was transmitted by the same leafhopper from rape to periwinkle, sesame, stock, mustard, radish and rocket plants causing phytoplasma‐type symptoms in these plants. PCR assays using phytoplasma‐specific primer pair P1/P7 or nested PCR using primers P1/P7 followed by R16F2n/R2, amplified products of expected size (1.8 and 1.2 kbp, respectively) from symptomatic rapeseed plants and C. haematoceps specimens. Restriction fragment length polymorphism analysis of amplification products of nested PCR and putative restriction site analysis of 16S rRNA gene indicated the presence of aster yellows‐related phytoplasmas (16SrI‐B) in naturally and experimentally infected rapeseed plants and in samples of C. haematoceps collected in affected rapeseed fields. Sequence homology and phylogenetic analysis of 16S rRNA gene confirmed that the associated phytoplasma detected in Zarghan rapeseed plant is closer to the members of the subgroup 16SrI‐B than to other members of the AY group. This is the first report of natural occurrence and characterization of rapeseed phyllody phytoplasma, including its vector identification, in Iran.     

Transmission of sugarcane white leaf phytoplasma by Yamatotettix flavovittatus, a new leafhopper vector

Sugarcane white leaf disease is caused by plant pathogenic phytoplasmas that are transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). To determine whether there are other insect vectors that transmit this disease pathogen, leafhopper species in sugarcane, Saccharum officinarum L., fields in northeastern Thailand were monitored by using light traps. Sixty-nine leafhopper species from family Cicadellidae were found. Using nested polymerase chain reaction (PCR) with specific primers, a 210-bp amplified DNA fragment corresponding to phytoplasma associated with sugarcane white leaf disease was detected from 12 species of leafhoppers [Balclutha rubrostriata (Melichar), Balclutha sp., Bhatia olivacea (Melichar), Exitianus indicus Distant, Macrosteles striifrons Anufriew, Matsumuratettix hiroglyphicus (Matsumura), Recilia distincta (Motschulsky), Recilia dorsalis (Motschulsky), Recilia sp., Thaia oryzivora Ghauri, Yamatotettix flavovittatus Matsumura, and Xestocephalus sp.]. The percentage of individual infection with phytoplasma varied from 5% in B. olivacea to 35% in Xestocephalus sp. The most abundant leafhopper species, i.e., E. indicus, Y. flavovittatus, and M. hiroglyphicus were used in transmission tests to determine their vector status for the sugarcane white leaf phytoplasma transmission. Infected insects were reared on healthy plants and specific PCR followed by sequencing of the amplicons was used to determine whether the phytoplasma was transmitted to the plants. The results showed that both Y. flavovittatus and M. hiroglyphicus, but not E. indicus, can transmit sugarcane white leaf phytoplasma to healthy sugarcane plants. The transmission efficiency of M. hiroglyphicus (55%) was higher than that of Y. flavovittatus (45%). We conclude that Y. flavovittatus is a newly discovered vector for sugarcane white leaf disease, in addition to M. hiroglyphicus. These two species peak at different times of the year and therefore complement each other in the transmission of the phytoplasma. Because there are no known alternative host plants for the sugarcane white leaf, management of the disease will necessarily require the control of both Y. flavovittatus and M. hiroglyphicus.     

Characterisation of phytoplasma (16SrVI) associated with little leaf disease of Portulaca grandiflora – An in silico analysis

A new severe little leaf disease was observed on P. grandiflora, popular as Moss-rose Purslane, widely grown in temperate zones. Characteristic symptoms, ultrastructural studies, antibiotic response and amplification of 16S ribosomal DNA fragments (about 1.5 kb) by polymerase chain reaction (PCR) from infected samples, suspect the involvement of phytoplasma as a pathogen. Nested PCR product, 1.2 kb, with primer pairs R16F2n/R16R2 used for cloning and sequencing. Comparision of the 16S rRNA gene sequences showed that the causal, PLL phytoplasma, is very close (98%) to Indian brinjal little leaf (EF186820) and “Candidatus Phytoplasma trifolii” (AY390261), 16SrVI group phytoplasmas, previously reported from India and Canada respectively. Here, the status of PLL (EF651786) is verified by computer-simulated restriction fragment length polymorphism analysis of 16S rRNA genes of the F2n/R2 sequences of closely related strains of the 16SrVI group using 17 restriction enzymes.     

Multiple phytoplasmas associated with potato diseases in Mexico

In recent years, the potato crop in Mexico has been notably affected by diseases recognized as potato purple top (PPT) in foliage and potato hair sprouts (PHS) in germinating tubers. In both cases, these syndromes reduce production by affecting viability of the tubers used as seeds. There is evidence indicating that phytoplasmas are associated with these syndromes. This study presents data on the molecular detection, characterization, and ecology of the pathogens related to PPT and PHS. Restriction fragment length polymorphism (RFLP) and sequence analysis indicated that PPT phytoplasma belongs to the 16SrI group and PHS phytoplasma fits in the 16SrII group. In this paper, we report that the two different phytoplasmas have been found coexisting in the same potato plant, which demonstrates the presence of mixed infection in the field. These phytoplasmas were also detected in weeds surrounding potato fields; therefore they should be considered as alternative hosts or natural reservoirs of PPT and PHS phytoplasmas.     

Differential acquisition of chrysanthemum yellows phytoplasma by three leafhopper species

Chrysanthemum yellows (CY) phytoplasma has been transmitted with three leafhopper species: Euscelidius variegatus (Kirschbaum), Macrosteles quadripunctulatus (Kirschbaum) and Euscelis incisus (Kirschbaum): the first two species are reported as CY phytoplasma vectors for the first time. Leafhoppers were allowed to acquire the pathogen from the following source plants: Apium graveolens L., Catharanthus roseus L., Chrysanthemum carinatum Schousboe L. and C. frutescens L. DNA extracted from healthy or inoculative leafhoppers-exposed plants were analyzed by dot-blot and Southern hybridizations with a molecular probe constructed onto a fragment of European aster yellows phytoplasma DNA. The three leafhopper species were able to transmit CY phytoplasma after acquisition on chrysanthemum, but only M. quadripunctulatus and E. variegatus transmitted after feeding on periwinkle, and none acquired it from celery. All plant species tested were susceptible to CY, but while chrysanthemum and periwinkle were suitable for both inoculation and acquisition, celery did not seem to be a good source of phytoplasma for further inoculations. It is concluded that host plants influence leafhoppers' vectoring ability, possibly due to the different feeding behaviour of the insects on diverse plant species. Since CY, like several other phytoplasmas, can be transmitted by different insect species, it is likely that a close transmission specificity probably does not exist between phytoplasmas and their leafhopper vectors.     

Vector status of three leafhopper species for Australian lucerne yellows phytoplasma

Abstract   The leafhoppers Orosius argentatus (Evans), Austroagallia torrida (Evans) and Batracomorphus angustatus (Osborn) were used in transmission tests to determine their vector status for the phytoplasma associated with Australian lucerne yellows (ALuY). Caged, seed-grown lucerne plants were monitored for foliar symptom expression after feeding by leafhoppers transferred from ALuY symptomatic lucerne plants. Twelve of 25 plants developed phytoplasma disease-like symptoms including stunting and yellowing. The most pronounced foliar symptoms were displayed by five plants that had been fed on by O. argentatus and four plants that had been fed on by A. torrida. One plant, fed on by O. argentatus , showed the distinctive root symptoms of ALuY . A phytoplasma was identified by electron microscopy in two plants fed on by O. argentatus and one by A. torrida. For each group of plants that had been fed on by a single leafhopper species, one plant was phytoplasma positive as determined by the polymerase chain reaction (PCR) using universal primers. The phytoplasma detected by PCR in the plant fed on by A. torrida was identified by restriction fragment length polymorphism (RFLP) analysis as the tomato big bud (TBB) phytoplasma. The PCR product from two plants fed on by B. angustatus and O. argentatus were too faint for RFLP analysis. PCR assays were conducted on DNA extracted from the head and thorax of each leafhopper species from transmission tests and from field-collected insects, but no phytoplasma DNA was detected. These findings suggest O. argentatus is a vector of the ALuY pathogen and A. torrida is a vector of the TBB phytoplasma.     

Parasitization of beet leafhopper eggs, Circulifer tenellus, in California

Parasitoids attacking eggs of beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), were surveyed at eight sites in southern and central California for 2 years. One site was an insecticide‐free sugar beet field, and the remaining sites were all uncultivated and supported weedy vegetation. At each site, host plants of beet leafhopper were collected and stored until parasitoids emerged from the leafhopper eggs in the vegetation. Vegetation samples included both naturally occurring host plants and sugar beet outplants that were first infested with beet leafhopper eggs in the laboratory and then placed in the field for 2 days. Parasitism generally was highest in summer and lowest in winter. Beet leafhopper eggs were parasitized by the mymarids (Hymenoptera: Mymaridae) Anagrus nigriventris Girault, Polynema eutettexi Girault, P. longipes (Ashmead), Polynema sp., Gonatocerus capitatus Gahan and two Gonatocerus spp. from the litoralis species group, and the trichogrammatids (Hymenoptera: Trichogrammatidae) Aphelinoidea zarehi Triapitsyn, Walker and Bayoun, A. turanica Trjapitzin, A. roja Triapitsyn, Walker and Bayoun, A. anatolica Nowicki and Paracentrobia sp. near P. subflava (Girault). The most dominant were A. nigriventris, A. zarehi and Paracentrobia sp. The intensity of parasitism varied greatly among the sites with peak levels ranging from 13% to 82%. Species composition also varied among sites, especially between the sugar beet field where A. nigriventris was by far the most dominant parasitoid and the uncultivated sites where A. zarehi and/or Paracentrobia sp. usually dominated. Within the uncultivated sites, parasitoid species composition also varied between the sugar beet outplants and the naturally occurring vegetation; A. nigriventris was much more common in the former than in the latter.     

The Association of Clover Proliferation Phytoplasma with Stolbur Disease of Pepper in Spain

A phytoplasma disease, `stolbur', affects pepper ( Capsicum annuum ) in Spain. Affected plants have short internodes, green flowers buds and other symptoms that are characteristic of phytoplasma-induced diseases. Herein the detection and classification of the phytoplasma that may cause the disease is reported. DNA amplification by polymerase chain reaction, sequencing and phylogenetic analysis indicate that this phytoplasma should be classified in the clover proliferation group 16SrVI, a group that is clearly distinct from the stolbur group 16SrXII.     

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.