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.     

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red‐leaf Disease of Salvia miltiorrhiza in China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma‐specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN‐1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII‐A), respectively. The phylogenetic trees showed that LN‐1 phytoplasma clustered with the members of 16SrXII‐A group, including Ca. P. solani. Computer‐simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.     

Molecular study of phytoplasmas associated with pistachio yellows in Iran

A large‐scale survey was conducted on pistachio plants exhibiting foliar symptoms including scorch, little leaf, yellows and reddish in pistachio growing areas in the Qom, Yazd and Qazvin provinces of Iran. Total DNA was extracted from symptomatic and symptomless pistachio and used in nested PCR assays with phytoplasma universal primers. Nested PCR products were obtained for symptomatic plant samples while the symptomless plants yielded no PCR products. Virtual restriction fragment length polymorphism, phylogenetic and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to two ribosomal groups; in particular, “Candidatus Phytoplasma solani” and “Ca. P. phoenicium” were identified. The presence of these phytoplasmas in pistachio is of great phytosanitary significance due to its commercial interest.     

Molecular identification of ‘Candidatus Phytoplasma palmicola’ associated with coconut lethal yellowing in Equatorial Guinea

During the past two decades, a high mortality of coconut palms was observed in the coastal areas of Equatorial Guinea. Reportedly, the palm population has been reduced by 60%–70%, and coconut production has decreased accordingly. To identify the cause of the mortality, a survey was carried out in April 2021 in various localities of the coconut belt. Molecular analyses carried out on 16S rRNA and secA genes detected phytoplasma presence in the majority of the samples. Sequencing and BLAST search of the 16S rRNA gene sequences showed >99% identity of the detected phytoplasmas to ‘Candidatus Phytoplasma palmicola’. The RFLP analyses of 16S ribosomal gene using Tru1I and TaqI enzymes led to assign these phytoplasmas to subgroup 16SrXXII-A. In all samples that tested positive, including one from a hybrid coconut palm and two from oil palm the same phytoplasma was identified. The phylogenetic analyses of 16S rRNA and secA genes confirmed respectively 99.98%–100% and 97.94%–100% identity to ‘Ca. P. palmicola’. RFLP analyses using MboII enzyme on the secA gene amplicon differentiated the phytoplasma found in Equatorial Guinea from those present in Ghana and Ivory Coast. The Equatorial Guinean phytoplasma strain resulted to be identical to the strains from Mozambique, confirming the presence of a geographic differentiation among phytoplasma strains in the coastal areas of Western and Central Africa. The identified phytoplasma is different from the ‘Ca. P. palmicola’ strains found in Ghana and Ivory Coast and represents the first identification a 16SrXXII-A strain in Equatorial Guinea and in Central Africa. Strict monitoring and surveillance procedures for early detection of the pathogen are strongly recommended to reduce its impact and further spread in the country and permit the recovery of coconut plantations.     

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.     

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.     

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.     

泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能和遗传变异比较分析

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

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.     

Phytoplasma Transmission by Heterologous Grafting Influences Viability of the Scion and Results in Early Symptom Development in Periwinkle Rootstock

The stolbur phytoplasma ‘Candidatus Phytoplasma solani’ is responsible for the grapevine disease ‘bois noir’ affecting a number of wine‐growing areas in Europe. Transmission of stolbur phytoplasma to different laboratory hosts can be difficult due to the requirement of transmitting insect vectors or parasite plants. Here, heterologous grafting was used as an alternative technique for transmission of common and strongly symptomatic stolbur genotypes CPsM4_At1 and CPsM4_At6 of ‘Ca. P. solani’ to experimental host plants such as Catharanthus roseus and tomato making phytoplasma strains more accessible for molecular and experimental investigations in different plant species. Transmission was confirmed by quantitative PCR, microscopy and nested PCR followed by marker gene sequencing. In our study, the transmission of different genotypes of ‘Ca. P. solani’ resulted in distinguishable symptom development in the laboratory host C. roseus. Symptom development in grafted rootstock was observed three to 7 weeks after heterologous grafting. Survival of the graft unit was influenced by the presence of ‘Ca. P. solani’ in the scions and was clearly reduced in phytoplasma free scion – rootstock combinations.     

Occurrence of Phytoplasmas Related to Stolbur and to ‘Candidatus Phytoplasma japonicum’ in Woody Host Plants in China

During a survey in a limited area of the Shanxi province in China, phytoplasma symptoms were observed on woody plants such as Chinese scholar tree, apple, grapevine and apricot. The polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses on the phytoplasma 16S ribosomal gene confirmed that symptomatic samples from all these species were infected by phytoplasmas. The molecular characterization of the pathogen, performed also with sequencing of polymerase chain reaction amplified 16S rDNA, showed that the phytoplasmas detected in all plant species tested are closely related with stolbur, but two samples from a Chinese scholar tree were infected with phytoplasmas related to ‘Candidatus Phytoplasma japonicum’. The presence of RFLP polymorphism was found in the 16S rDNA amplicons with three of the six enzymes employed in the majority of phytoplasma strains studied.     

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 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.     

DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

Background

Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.

Methodology/Principal Findings

We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.

Conclusions/Significance

This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.     

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.     

Biological and molecular evidence for the transmission of aster yellows phytoplasma to French marigold (Tagetes patula) by the flatid planthopper Metcalfa pruinosa

Aster yellows (AY) phytoplasmas (Candidatus Phytoplasma asteris) are associated with a number of plant diseases throughout the world. Several insect vectors are responsible for spreading AY diseases resulting in wide distribution and low host specificity. Because the role of sucking insects as vectors of phytoplasmas is widely documented, and the citrus flatid planthopper Metcalfa pruinosa is a phloem feeder, it has been incriminated as a possible vector of phytoplasmas. However, its ability to transfer phytoplasma has not been confirmed. The present work shows that M. pruinosa (Hemiptera: Flatidae), a polyphagous planthopper, is able to vector Ca. P. asteris to French marigold (Tagetes patula). Transmission experiments were conducted in 2017 and 2018 in central Hungary by two approaches: (a) AY-infected M. pruinosa were collected from an area with severe incidence of the disease on T. patula and caged on test plants for an inoculation-access period of 2 weeks, and (b) presumably phytoplasma-free insects were collected from apparently healthy grapevines (Vitis vinifera L.) and fed on AY-infected T. patula plants for 2 weeks prior to being caged on test plants. AY disease symptoms developed on 4 out of 10 and 10 out of 15 test plants, respectively. All phytoplasma-positive marigold and M. pruinosa samples showed identical RFLP patterns and shared 100% 16S rDNA sequence identity with each other and with the aster yellows phytoplasma strain AJ33 (GenBank accession number MK992774). These results indicated that the phytoplasma belonged to the phytoplasma subgroup 16SrI-B Ca. P. asteris. Therefore, the work presented here provides experimental evidence that M. pruinosa is a vector of a 16SrI-B subgroup phytoplasma to T. patula.     

The genome of ‘Candidatus Phytoplasma solani’ strain SA-1 is highly dynamic and prone to adopting foreign sequences

Bacteria of the genus ‘Candidatus Phytoplasma’ are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as ‘Ca. P. solani’, have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of ‘Ca. P. solani’ strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322 bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The ‘Ca. P. solani’ SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.     

Surveys reveal a complex association of phytoplasmas and viruses with the blueberry stunt disease on Canadian blueberry farms

Surveys for phytoplasmas and viruses were conducted during September 2014 and 2015 on highbush blueberry farms in the Région Montérégie, Quebec. Total DNA and RNA were extracted from blueberry bushes showing blueberry stunt (BBS) symptoms and from symptomless blueberry bushes, and utilised as templates for PCR and RT‐PCR assays, respectively. Phytoplasma DNA was amplified with universal phytoplasma primers that target the 16S rRNA, secA and secY genes from 12 out of 40 (30%) plants tested. Based on 16S rRNA, secA and secY gene sequence identity, phylogenetic clustering, actual and in silico RFLP analyses, phytoplasma strains associated with BBS disease in Quebec were identified as ‘Candidatus Phytoplasma asteris’‐related strains, closely related to the BBS Michigan phytoplasma strain (16SrI‐E). The secY gene sequence‐based single nucleotide polymorphism analysis revealed that one of the BBS phytoplasma strains associated with a leaf marginal yellowing is a secY‐I RFLP variant of the subgroup 16SrI‐E. Two viruses were detected in blueberry bushes. The Blueberry Red Ringspot Virus (BRRV) was found in a single infection in the cultivar Bluecrop with no apparent typical BRRV symptoms. The Tobacco Ringspot Virus (TRSV) was found singly infecting blueberry plants and co‐infecting a BBS phytoplasma‐infected blueberry cv. Bluecrop plant. This is the first report of TRSV in the cv. Bluecrop in Quebec. The Quebec BBS phytoplasma strain was identified in the leafhopper Graphocephala fennahi, which suggests that G. fennahi may be a potential vector for the BBS phytoplasma. The BBS disease shows a complex aetiology and epidemiology; therefore, prompt actions must be developed to support focused BBS integrated management strategies.     

‘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 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.