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

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

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.     

Molecular Identification and Phylogenetic Analysis of Sugarcane Yellow Leaf Phytoplasma (SCYLP) in Egypt

Samples of sugarcane leaves were collected from different commercial fields and breeding stations in Egypt. Aetiology of sugarcane phytoplasma disease was investigated using nested PCR. Phytoplasma‐specific primers (P1/P7 and R16F2n/R16R2) were used to amplify a fragment of the 16S rRNA gene. Sequencing and restriction fragment length polymorphism analyses revealed that the tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group. Phylogenetic analyses of 60 screened accessions of 16S ribosomal RNA gene sequences of Candidatus phytoplasmas comprising those collected from Egypt (this study) and those extracted from GenBank showed that they split into two distinct clusters. All the phytoplasmas form a stable phylogenetic subcluster, as judged by branch length and bootstrap values of 100% in the 16S group cluster. Results of phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. Conversely, based on the analysis of the 16S‐23S region, examined isolates segregated into four different clusters suggesting a notable heterogeneity between them. These results are the first record of the presence of phytoplasma in association with sugarcane yellow leaf in Egypt.     

Detection and Characterization of Phytoplasmas Infecting Ornamental and Weed Plants in Iran

During field surveys in 2004, ornamental and weed plants showing symptoms resembling those caused by phytoplasmas were observed in Mahallat (central Iran). These plants were examined for phytoplasma infections by polymerase chain reaction (PCR) assays using universal phytoplasma primers directed to ribosomal DNA (rDNA). All affected plants gave positive results. The detected phytoplasmas were characterized and differentiated through restriction fragment length polymorphism (RFLP) and sequence analysis of PCR‐amplified rDNA. The phytoplasmas detected in diseased Asclepias curassavica and Celosia argentea were identified as members of clover proliferation phytoplasma group (16SrVI group) whereas those from the remaining plants examined proved to be members of aster yellow phytoplasma group (16SrI group) (‘Candidatus Phytoplasma asteris’). In particular, following digestion with AluI, HaeIII and HhaI endonucleases, the phytoplasma detected in Limonium sinuatum showed restriction profiles identical to subgroup 16SrI‐C; phytoplasmas from Gomphocarpus physocarpus, Tanatacetum partenium, Lactuca serriola, Tagetes patula and Coreopsis lanceolata had the same restriction profiles as subgroup 16SrI‐B whereas Catharanthus roseus‐ and Rudbeckia hirta‐infecting phytoplasmas showed restriction patterns of subgroup 16SrI‐A. This is the first report on the occurrence of phytoplasma diseases of ornamental plants in Iran.     

“Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma mali” strains infecting sweet and sour cherry in the Czech Republic

A survey for phytoplasma diseases was conducted in a sweet and sour cherry germplasm collection and in cherry orchards within the Czech Republic during 2014–2015. Phytoplasmas were detected in 21 symptomatic trees. Multiple infections of cherry trees by diverse phytoplasmas of 16SrI and 16SrX groups and 16SrI‐A, 16SrI‐B, 16SrI‐L, 16SrX‐A subgroups were detected by restriction fragment length polymorphism (RFLP). Nevertheless, phylogenetic analysis placed subgroups 16SrI‐B and 16SrI‐L inseparable together onto one branch of phylogenetic tree. This is the first report of subgroups 16SrI‐A and 16SrI‐L in Prunus spp., and subgroup 16SrX‐A in sour cherry trees. Additionally, novel RFLP profiles for 16SrI‐A and 16SrI‐B‐related phytoplasmas were found in cherry samples. Phytoplasmas with these novel profiles belong, however, to their respective 16SrI‐A or 16SrI‐B phylogenetic clades.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Occurrence of 16SrIV Subgroup A Phytoplasmas in Roystonea regia and Acrocomia mexicana Palms with Lethal Yellowing‐like Syndromes in Yucatán,Mexico

The lethal yellowing (LY) disease and LY‐type syndromes affecting several palm species are associated with 16SrIV phytoplasmas in the Americas. In Mexico, palms of the species Roystonea regia and the native Acrocomia mexicana were found to exhibit LY‐type symptoms, including leaf decay, starting with mature leaves, necrosis and atrophy of inflorescences. DNA extracts obtained from these palms could be amplified by nested‐PCR using phytoplasma‐universal primer pair P1/P7 followed by LY‐group‐specific primer pair LY16Sr/LY16Sf. Blast analysis of the sequences obtained revealed an identity of 100% for R. regia and 99.27% for A. mexicana with 16SrIV‐A strain associated with LY in Florida, USA (Acc. AF498309 ). Computer‐simulated RFLP analysis showed that the patterns for the phytoplasma DNA of the two palm species were highly similar to that for 16SrIV subgroup A strain. A neighbour‐joining tree was constructed, and the sequences of the two palm species clustered in the same clade of group 16SrIV subgroup A. The results therefore support that LY‐type syndromes observed in palms of R. regia and A. mexicana in the Yucatan region of Mexico are associated with 16SrIV subgroup A phytoplasmas.     

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 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.     

The phytoplasma associated with purple woodnettle witches'‐broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group

In October 2013, a new disease affecting purple woodnettle, Oreocnide pedunculata, plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches'‐broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of polymerase chain reaction with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettles. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, rplV‐rpsC region revealed that purple woodnettle witches'‐broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rpI group and was designated as 16SrI‐AH and rpI‐Q, respectively, herein. RFLP analysis based on tuf gene region revealed that the PWWB belongs to tufI‐B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the tufI group. Taken together, our analyses based on 16S rRNA and rplV‐rpsC region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a Candidatus Phytoplasma asteris‐related strain in 16SrI‐AH, rpI‐Q and tufI‐B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas.     

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.     

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.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

‘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.