First Report of a 16SrI‐C Phytoplasma Infecting Celery (Apium graveolens) with Stunting,Bushy Top and Phyllody in the Czech Republic

Apium graveolens L. plants showing stunting, purplish/whitening of new leaves, flower abnormalities and bushy tops were observed in South Bohemia (Czech Republic) during 2011 and 2012. Transmission electron microscopy observations showed phytoplasmas in phloem sieve tube elements of symptomatic but not healthy plants. Polymerase chain reactions with universal and group‐specific phytoplasma primers followed by restriction fragment length polymorphism analyses and sequencing of 16S rDNA enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐C. Identical analyses of the ribosomal protein genes rpl22 and rps3 were used for further classification and revealed affiliation of the phytoplasmas with the rpIC subgroups. This is the first report of naturally occurring clover phyllody phytoplasma in A. graveolens in both the Czech Republic and worldwide.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

Detection and Identification of Aster Yellows Group Phytoplasma (16SrI‐C) Associated with Peach Red Leaf Disease

In 2011, typical symptoms suggestive of phytoplasma infection such as reddening of leaves were observed in peach trees in Fuping, Shaanxi Province, China. Phytoplasma‐like bodies were observed by transmission electron microscope in the petiole tissues of symptomatic peach trees. Products of c. 1.2 kb were generated from all symptomatic peach leaf samples by a nested polymerase chain reaction using phytoplasma universal primer pairs P1?P7 and R16F2n?R16R2, whereas no such amplicon was obtained from healthy samples. Results of phylogenetic analysis and restriction fragment length polymorphism suggested that the phytoplasma associated with such peach red leaf disease was a member of subgroup 16SrI‐C. To our knowledge, this is the first record of 16SrI‐C subgroup phytoplasma occurred in peach tree in China.     

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.     

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.     

Multigene Sequence Analysis of Aster Yellows Phytoplasma Associated with Primrose Yellows

Primula acaulis (L.) Hill. plants showing stunting, leaf‐yellowing and virescence were first discovered in the Czech Republic. Polymerase chain reactions with subsequent restriction fragment length polymorphism analyses and sequencing enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐B, tufI‐B, rpI‐B, groELIB‐III and SecY‐IB subgroups. Phylogeny of the 16S rRNA gene sequences as well as sequence analysis of several chromosomal regions, such as the 16S‐23S ribosomal operon, ribosomal proteins, spc ribosomal protein operon, genes for elongation factor EF‐Tu, molecular chaperonin large subunit GroEL, immunodominant membrane protein, ribosome recycling factor, urydilate kinase, ATP‐ and Zn²⁺‐dependent proteases not only confirmed its affiliation with the ‘Candidatus Phytoplasma asteris’ species but also enabled its detailed molecular characterization. The less researched regions of phytoplasma genome (amp, adk, hflB, pyrH‐frr genes) could be valuable as additional markers for phytoplasma through differentiation especially within the 16SrI‐B ribosomal subgroup.     

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).     

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.     

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).     

Association of Elm Yellows Subgroup 16SrV‐B Phytoplasma with a Disease of Hovenia dulcis

Japanese raisin (Hovenia dulcis) trees with typical phytoplasma‐like symptoms were observed for the first time in South Korea. The disease, named Japanese raisin witches’ broom, is progressively destructive. The cause of the graft‐transmissible disease was confirmed by electron microscopy and molecular studies. The 16S rDNA sequence analysis showed that the phytoplasma was closely related to the elm yellows (EY) group, ribosomal subgroup 16SrV‐B. The 16S‐23S rDNA intergenic spacer region, fragment of rp operon and secY gene sequences had 96–99% similarity with members of EY phytoplasma. Based on the sequence analyses and phylogenetic studies, it was confirmed that the phytoplasma infecting Japanese raisin trees in Korea belongs to the EY group.     

Detection and Molecular Identification of Aster Yellows Phytoplasma in Date Palm in Egypt

Phytoplasma‐like symptoms were detected in date palm trees (Phoenix dactylifera L.) in Al‐Giza Governorate in Egypt. Symptoms varied from leaf chlorotic streaks, stunting and marked reduction in fruit and stalk sizes. Direct and nested PCR of symptomatic samples using P1/P7 and R16F2n/R16R2n primers, respectively, of the 16S rRNA gene, resulted in a DNA amplification product of c. 1.3 kbp. Symptomless samples collected from the same location and the healthy control produced no product upon amplification. Products were cloned into TOPO TA vector for sequencing. Data generated were deposited in the GenBank (Accession KF826615 ). A BLAST search showed that the sequence of the 16SrRNA gene shared ‘Candidatus Phytoplasma asteris’ (16SrI group) with other isolates. Phylogenetic analysis revealed that the isolate clustered with the date palm phytoplasma causing Al‐Wijam disease in Saudi Arabia.     

Asparagus officinalis: A New Host of ‘Candidatus Phytoplasma asteris’

Asparagus officinalis plants with severe fasciation of some spears were observed in southern Bohemia between 1998 and 2007. Nucleic acids extracted from these and asymptomatic plants were assayed with nested polymerase chain reaction (PCR) using the phytoplasma‐specific universal ribosomal primers P1/P7 and R16F2n/R2. The restriction profiles obtained from digestion of the PCR products with five endonucleases (AluI, HhaI, KpnI, MseI and RsaI) were identical in all phytoplasmas infecting asparagus in the Czech Republic and indistinguishable from those of phytoplasmas in the aster yellows group (subgroup 16SrI‐B). Sequence analysis of 1754 bp of the ribosomal operon indicated that the closest related phytoplasmas were those associated with epilobium phyllody and onion yellows. This is the first report of the natural occurrence of ‘Candidatus Phytoplasma asteris’ in A. officinalis.     

Detection and Identification of an Elm Yellows Group Phytoplasma Associated with Camellia in China

In 2012, yellowing of camellias was observed in Tai'an in Shandong province, China. Transmission electron microscopy (TEM) revealed phytoplasma in the phloem sieve tube elements of symptomatic plants. A specific fragment of phytoplasma 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal phytoplasma primers P1/P7 followed by R16F2n/R16R2. Sequence and restriction fragment length polymorphism (RFLP) analyses allowed us to classify the detected phytoplasma into the elm yellows (EY) group (16SrV), subgroup 16SrV‐B. Sequence analyses of the ribosomal protein (rp) gene confirmed a close relationship with phytoplasmas belonging to the rpV‐C subgroup. Thus, the phytoplasma associated with yellows disease in camellia, designated as ‘CY’, is a member of the 16SrV‐B subgroup. This is the first report of phytoplasma associated with camellia.     

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 ‘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 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 of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

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 and Biological Analysis of Potato virus M (PVM) Isolates from the Czech Republic

The sequences of the 3′‐terminal region of four Czech Potato virus M isolates VIRUBRA 4/007, VIRUBRA 4/009, VIRUBRA 4/016 and VIRUBRA 4/035 were determined and compared with sequences of PVM isolates available in GenBank. Among the Czech isolates, VIRUBRA 4/007 and 4/016 as well as VIRUBRA 4/016 and 4/035 showed the highest nucleotide identity (93%). Isolates VIRUBRA 4/007, 4/016 and 4/035 were most similar to the PV0273 isolate from Germany and to the wild isolate from Russia. Interestingly, isolate VIRUBRA 4/009 significantly differed from the other three Czech isolates and was the only European isolate that showed the highest nucleotide identity with American isolates. Moreover, the PVM isolates from the Czech Republic and Germany differed in their host range. Phylogenetic analysis based on ORF5 coding for coat protein showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on molecular and biological analysis of the genome sequences of PVM isolates from the Czech Republic.