Molecular detection and characterization of a stolbur phytoplasma associated with Narcissus tazetta phyllody in Iran

During field surveys in 2015, a phytoplasma‐associated disease was identified in Narcissus tazetta plants in Behbahan, Iran. The characteristic symptoms were phyllody and virescence. The presence of phytoplasma in symptomatic plants was confirmed using PCR amplification and sequencing of 16S rRNA, tuf, secY and vmp1 genes. Based on the blastn results, the sequences of 16S rRNA, tuf, secY and vmp1 genes shared, respectively, 99%, 100%, 99% and 99% sequence identity with phytoplasma strains in 16SrXII‐A subgroup. RFLP and phylogenetic analyses using the sequences of 16S rRNA, tuf and secY genes confirmed the assortment of studied strains to 16SrXII‐A phytoplasma subgroup. Sequence comparison of these four genes revealed that all the sequences of 28 strains studied were identical. To the best of our knowledge, the association of “Candidatus Phytoplasma solani” with N. tazetta was demonstrated for the first time in the world.     

Detection and Identification of Two Phytoplasmas (16SrIII‐B and 16SrXII‐A) From Alfalfa (Medicago sativa) in Serbia

Plants of alfalfa (Medicago sativa) exhibiting general stunting, proliferation and phyllody associated with leaf yellowing and reddening were observed in three localities of Central Serbia. Phytoplasma strains belonging to 16SrIII‐B and 16SrXII‐A groups were detected and identified by RFLP and sequence analysis of 16S rDNA. Stolbur phytoplasma tuf gene RFLP analysis showed the presence of the TufAY‐b‐type phytoplasma subgroup in 80% of symptomatic samples. This is the first report of 16SrIII‐B and 16SrXII‐A phytoplasma groups affecting alfalfa in Serbia.     

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

A search for phytoplasma-associated diseases was conducted for the first time in the main grapevine-growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf-type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'-related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.     

Detection and Identification of Phytoplasmas in Pomegranate Trees with Yellows Symptoms

Symptoms resembling those associated with phytoplasma presence were observed in pomegranate (Punica granatum L.) trees in June 2012 in the Aegean Region of Turkey (Ayd?n province). The trees exhibiting yellowing, reduced vigour, deformations and reddening of the leaves and die‐back symptoms were analysed to verify phytoplasma presence. Total nucleic acids were extracted from fresh leaf midribs and phloem tissue from young branches of ten symptomatic and five asymptomatic plants. Nested polymerase chain reaction assays using universal phytoplasma‐specific 16S rRNA and tuf gene primers were performed. Amplicons were digested with Tru1I, Tsp509I and HhaI restriction enzymes, according to the primer pair employed. The phytoplasma profiles were identical to each other and to aster yellows (16SrI‐B) strain when digestion was carried out on 16Sr(I)F1/R1 amplicons. However, one of the samples showed mixed profiles indicating that 16SrI‐B and 16SrXII‐A phytoplasmas were present when M1/M2 amplicons were digested, the reamplification of this sample with tuf cocktail primers allowed to verify the presence of a 16SrXII‐A profile. One pomegranate aster yellows strain AY‐PG from 16S rRNA gene and the 16SrXII‐A amplicon from tuf gene designed strain STOL‐PG were directly sequenced and deposited in GenBank under the Accession Numbers KJ818293 and KP161063, respectively. To our knowledge, this is the first report of 16SrI‐B and 16SrXII‐A phytoplasmas in pomegranate trees.     

Identification of ‘Candidatus Phytoplasma asteris' (16SrI‐B) Causing Witches' Broom Disease of Mallotus japonicus in Korea

Mallotus japonicus with witches' broom disease were observed in Jeollabuk‐do, Korea. A phytoplasma from the infected leaves was identified, based on the 16S rDNA, 16S‐23S intergenic spacer region, and fragment of rp operon and tuf gene sequences. The 16S rDNA sequences exhibited maximum (99.7%) similarity with Iranian lettuce phytoplasma, the rp operon sequences exhibited 100% similarity with Goldenrain stunt phytoplasma, and the tuf gene sequences exhibited 99.8% similarity with Japanese spurge yellows phytoplasma. Results of the sequence analysis and phylogenetic studies confirmed that the phytoplasma associated with M. japonicus in Korea was an isolate of Aster Yellows group (subgroup16SrI‐B).     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines,bindweeds and vegetables

Aim: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Materials and Results: Phytoplasma isolates belonging to stolbur subgroup 16SrXII‐A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf‐a type and 68% by tuf‐b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf‐b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions. Conclusions: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Significance and Impact of the Study: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.     

Identification of ‘Candidatus Phytoplasma solani’ Associated with Tree Peony Yellows Disease in China

Tree peony (Paeonia suffruticosais) plants with yellowing symptoms suggestive of a phytoplasma disease were observed in Shandong Peninsula, China. Typical phytoplasma bodies were detected in the phloem tissue using transmission electron microscopy. The association of a phytoplasma with the disease was confirmed by polymerase chain reaction (PCR) using phytoplasma universal primer pair R16mF2/R16mR1 followed by R16F2n/R16R2 as nested PCR primer pair. The sequence analysis indicated that the phytoplasma associated with tree peony yellows (TPY) was an isolate of ‘Ca. Phytoplasma solani’ belonging to the stolbur (16SrXII) group. This is the first report of a phytoplasma associated with tree peony.     

Molecular Characterization of Aster Yellows Subgroup 16SrI‐B Phytoplasma in Verbena × hybrida

Symptoms resembling phytoplasma disease were observed on Verbena × hybrida in Alanya, Turkey, during October 2013. Infected plants were growing as perennials in a flower border and showed symptoms of discoloured flowers, poor flower clusters, inflorescences with a small number of developed flowers and thickened fruit stalks. Electron microscopy examination of the ultra‐thin sections revealed polymorphic bodies in the phloem tissue of leaf midribs. The phytoplasma aetiology of this disease was confirmed by polymerase chain reaction of the 16S rRNA gene, the 16–23S rRNA intergenic spacer region and the start of the 23S rRNA gene using universal phytoplasma‐specific primer pair P1A/P7A, two ribosomal protein (rp) genes (rpl22 and rps3) (the group‐specific primer pair rp(I)F1A/rp(I)R1A) and the Tuf gene (group‐specific fTufAy/rTufAy primers) generating amplicons of 1.8 kbp, 1.2 kbp and 940 bp, respectively. Comparison of the amplified sequences with those available in GenBank allowed classification of the phytoplasma into aster yellows subgroups 16SrI‐B, rpI‐B and tufI‐B. This is the first report about molecular detection and identification of natural infection of the genus Verbena by phytoplasma and occurrence of the aster yellows group phytoplasma on an ornamental plant in Turkey.     

First Report of Aster Yellows Phytoplasma (16SrI‐B) Associated with Witches' Broom Disease of Melia azedarach var. japonica in Korea

Melia azedarach var. japonica trees with leaf yellowing, small leaves and witches' broom were observed for the first time in Korea. A phytoplasma from the symptomatic leaves was identified based on the 16Sr DNA sequence as a member of aster yellows group, ribosomal subgroup 16SrI‐B. Sequence analyses of more variable regions such as 16S–23S intergenic spacer region, secY gene, ribosomal protein (rp) operon and tuf gene showed 99.5?100% nucleotide identity to several GenBank sequences of group 16SrI phytoplasmas. Phylogenetic analysis confirmed that the Melia azedarach witches' broom phytoplasma belongs to aster yellows group.     

Solanum malacoxylon, a New Natural Host of Stolbur Phytoplasma

Stolbur phytoplasma infection has been reported, for the first time, in a new host, Solanum malacoxylon, growing in the Botanical Garden of Milan University. This shrub, native of South America, synthesizes vitamin D compounds, important for biomedical and biotechnological purposes. Pathogen detection was performed by light and transmission electron microscopy, and confirmed by molecular diagnosis, based on PCR and restriction fragment length polymorphic (RFLP) analysis of the phytoplasmal 16S rRNA and tuf genes. By means of enzymatic restriction and phylogenetic analysis on these genes, it was found that the phytoplasma belongs to the Stolbur group, taxonomic subgroup 16SrXII‐A, thus indicating S. malacoxylon as an additional host for this pathogen. Solanum malacoxylon could be then involved in the natural Stolbur phytoplasma spreading throughout South American areas, where this wild plant grows endemically.     

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 identification of diverse ‘Candidatus Phytoplasma’ species associated with grapevine decline in Iran

Grapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

Comparative Molecular Analyses of Phytoplasmas infecting Sophora japonica cv. golden and Robinia pseudoacacia

Phytoplasmas were detected in Sophora japonica cv. golden and Robinia pseudoacacia with diseased branches of witches'‐broom collected in Haidian district, Beijing, China. Phytoplasma cells were observed in phloem sieve elements of symptomatic S. japonica cv. golden by transmission electron microscopy. The presence of phytoplasmas was further confirmed by sequence determination of partial gene sequences of 16S rDNA, rp (ribosomal protein) and secY. Phylogenetic trees and virtual restriction fragment length polymorphism (RFLP) analyses indicated that the phytoplasmas causing S. japonica cv. golden witches'‐broom (SJGWB) and R. pseudoacacia witches'‐broom (RPWB) belong to the 16SrV (elm yellows) group, and they are most closely related to subgroup 16SrV‐B, rpV‐C and secYV‐C jujube witches'‐broom (JWB) phytoplasma. Comparative analyses indicated that the phytoplasma of RPWB was closer to the JWB and that R. pseudoacacia might serve as an alternative host plant of JWB phytoplasma.     

Survival,fecundity, and body mass of Amplicephalus curtulus influenced by ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) infection

The leafhopper Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) can transmit ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) from a native Chilean shrub, Ugni molinae Turcz. (Myrtaceae), to ryegrasses. A recent study showed that this phytoplasma reduced the total protein content and the activity of detoxifying enzymes in A. curtulus, which could also affect its vector fitness. This study evaluated the effect of ‘Ca. Phytoplasma ulmi’ on the longevity, fecundity, and body mass of A. curtulus. Both females and males were exposed to ‘Ca. Phytoplasma ulmi’‐infected plants for 96 h, whereas a control group remained unexposed. Quartiles from adult emergence to 75% (t₇₅), 50% (t₅₀), and 25% (t₂₅) survival rates were determined for each leafhopper survival distribution. The dry weight was also established at the end of the experiment. The adult lifespan of phytoplasma‐infected males and females was significantly lower than that of the uninfected leafhoppers in quartile survival distributions t₅₀ and t₂₅. The phytoplasma‐infected males and females lived 3 and 4 weeks less than uninfected ones in the last quartile, respectively. Fecundity was established by number of nymphs per female (in four periods) in phytoplasma‐infected and uninfected assays. In general, the weekly pattern of the number of nymphs per phytoplasma‐infected female was lower than that of uninfected leafhoppers; it was 37% lower at the end of the experiment. Phytoplasma‐infected females weighed significantly less (11%) than uninfected individuals. Phytoplasma‐infected males weighed 8% less than uninfected ones, but this difference was not significant. Our data indicated that ‘Ca. Phytoplasma ulmi’ negatively affected the fitness of A. curtulus, and nymphs produced by phytoplasma‐infected females varied over time, which may influence the disease dynamics in nature or in field crops.     

Detection and Identification of ‘Candidatus Phytoplasma prunorum’, ‘Candidatus Phytoplasma mali’ and ‘Candidatus Phytoplasma pyri’ in Stone Fruit Trees in Poland

A survey was made to determine the incidence of phytoplasmas in 39 sweet and sour cherry, peach, nectarine, apricot and plum commercial and experimental orchards in seven growing regions of Poland. Nested polymerase chain reaction (PCR) using the phytoplasma‐universal primer pairs P1/P7 followed by R16F2n/R16R2 showed the presence of phytoplasmas in 29 of 435 tested stone fruit trees. The random fragment length polymorphism (RFLP) patterns obtained after digestion of the nested PCR products separately with RsaI, AluI and SspI endonucleases indicated that selected Prunus spp. trees were infected by phytoplasmas belonging to three different subgroups of the apple proliferation group (16SrX‐A, ‐B, ‐C). Nucleotide sequence analysis of 16S rDNA fragment amplified with primers R16F2n/R16R2 confirmed the PCR/Restriction Fragment Length Polymorphism (RFLP) results and revealed that phytoplasma infecting sweet cherry cv. Regina (Reg), sour cherry cv. Sokowka (Sok), apricots cv. Early Orange (EO) and AI/5, Japanese plum cv. Ozark Premier (OzPr) and peach cv. Redhaven (RedH) was closely related to isolate European stone fruit yellows‐G1 of the ‘Candidatus Phytoplasma prunorum’ (16SrX‐B). Sequence and phylogenetic analyses resulted in the highest similarity of the 16S rDNA fragment of phytoplasma from nectarine cv. Super Queen (SQ) with the parallel sequence of the strain AP15 of the ‘Candidatus Phytoplasma mali’ (16SrX‐A). The phytoplasma infecting sweet cherry cv. Kordia (Kord) was most similar to the PD1 strain of the ‘Candidatus Phytoplasma pyri’ (16SrX‐C). This is the first report of the occurrence of ‘Ca. P. prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ in naturally infected stone fruit trees in Poland.     

Infection of ‘Candidatus Phytoplasma ulmi’ reduces the protein content and alters the activity of detoxifying enzymes in Amplicephalus curtulus

It has been reported that insecticide‐detoxifying enzymes such as glutathione S‐transferases (GST) and esterases are affected by microbial infections in hemipteran insect vectors. The total protein content, and GST and α‐ and β‐esterase activities were quantified in ‘Candidatus Phytoplasma ulmi’‐infected and uninfected adults of Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) at 25, 35, and 45 days after the acquisition access period (AAP) in the head‐thorax and abdomen sections. The total protein content was lower in phytoplasma‐infected leafhoppers 25, 35, and 45 days after the AAP. Thirty‐five days after the AAP, the GST and β‐esterase activities had increased (26 and 69%, respectively) compared to the control. However, 45 days after the AAP, the phytoplasma‐infected leafhoppers displayed lower GST (87%) and β‐esterase (253%) activities than the uninfected individuals. On the other hand, the α‐esterase activity proved to be unaffected by the phytoplasma infection. Forty‐five days after the AAP, females had a higher phytoplasma titer (46%) in their head‐thorax than in their abdomen sections, whereas males showed a higher titer in their abdomens (75%). In addition, the GST and β‐esterase activities in the abdomen were affected negatively by 96–98% as a result of the increasing ‘Ca. Phytoplasma ulmi’ titer. These results indicate that an infection of ‘Ca. Phytoplasma ulmi’ alters the metabolic activities of A. curtulus.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Identification of phytoplasmas from Neoaliturus haematoceps associated with sesame phyllody disease in southwestern Turkey

Phytoplasmas were detected based on nested PCR of the F2nR2 region of the 16S rDNA from Neoaliturus haematoceps (Mulsant and Rey) (Family: Cicadellidae). A total of 65 insect samples collected from sesame fields in Antalya, Turkey, during 2012–2014 were tested for phytoplasma detection. Phytoplasmas detected in fifteen samples showed an amplicon approximately 1250 bp in size using the universal primers of P1/P7 and R16F2n/R16R2. Identification of the phytoplasmas by sequence analysis revealed three different 16S rDNA phytoplasma groups: the peanut witches’‐broom, group II; clover proliferation, group VI; and pigeon pea witches’‐broom, group IX. The molecular characterization of subgroups was determined by sequence analysis and PCR‐RFLP using the restriction enzymes RsaI and TaqI. Restriction profiles of the subgroups were also confirmed using the iPhyclassifier program. BLAST and PCR‐RFLP analyses classified the subgroups as II‐D, VI‐A and IX‐C. This is the first report of molecular detection of three 16S rDNA subgroups of phytoplasmas, II‐D, VI‐A and IX‐C, from N. haematoceps in Turkey. This study also supports earlier studies of sesame phyllody phytoplasmas by N. haematoceps.