Comparison of the ribosomal RNA operon from Texas Phoenix decline and lethal yellowing phytoplasmas

A comparison was made of the two palm yellows phytoplasmas affecting palms to determine if the entire ribosomal RNA operon portion of the phytoplasma genome, or portions thereof, could account for the observed palm host differences. Polymerase chain reaction (PCR) was used to amplify a 5.0?kb DNA fragment consisting of the entire ribosomal RNA operon from a subgroup 16SrIV-D phytoplasma that causes Texas Phoenix palm decline (TPD) in cabbage (Sabal palmetto) palm in west central Florida and from a subgroup 16SrIV-A phytoplasma that causes lethal yellowing (LY) in coconut (Cocos nucifera) palm in Jamaica. Before the PCR reaction, we sequenced by 454 sequencing a draft genome of the coconut LY phytoplasma, strain LYFL, that infects C. nucifera in Florida, and obtained from this draft sequence both copies of the entire ribosomal operon. Sequence analysis of the ribosomal RNA operons from both the LY and TPD phytoplasmas revealed the gene composition and orientation for the operons to be 5′16S rRNA-tRNA^Ile-23S rRNA-5S rRNA3′ and a tRNA^Val3′ downstream of the 5S rRNA gene. Based on molecular comparisons using the sequences of the ribosomal RNA operon, the TPD (16SrIV-D) strain was 98?% similar to the LY (16SrIV-A) strains.     

Phytoplasmas associated with disease of coconut in Malaysia: phylogenetic groups and host plant species

Coconut palm ( Cocos nucifera ), oil palm ( Elaeis guineensis ), Bermudagrass ( Cynodon dactylon ) and Madagascar periwinkle ( Catharanthus roseus ) with symptoms indicative of phytoplasma disease were collected from different locations in Malaysia. PCR assays employing phytoplasma universal rRNA gene primers P1/P7 alone or P1/P7 followed by R16F2n/R16R2 detected phytoplasmas in eight out of 20 Malayan Red Dwarf (MRD), nine out of 12 Malayan Yellow Dwarf (MYD) and 12 out of 12 Malayan Tall (MT) coconut palms displaying coconut yellow decline symptoms. Positive detections were also obtained from six out of six oil palm seedlings showing symptoms of yellowing and necrosis, from 10 out of 10 Bermudagrass samples with white leaf symptoms, and from eight out of eight periwinkle plants showing phyllody, virescence, little leaf, proliferation and foliar yellowing. Phytoplasmas were not detected in any of the symptomless plants tested. Sequencing and phylogenetic analysis of PCR products determined that phytoplasmas infecting both MRD and MT coconuts and Bermudagrass in Serdang, Selangor State, were all members of the 16SrXIV ' Candidatus Phytoplasma cynodontis' group, whereas isolates in periwinkle in Serdang were all members of the 16SrI ' Ca. Phytoplasma asteris' group. However, the phytoplasmas detected in MYD coconuts and oil palms from Banting, Selangor State, and in periwinkle from Putrajaya were collectively very similar (99%), but shared <97·5% similarity with 16S rDNA sequences of all other known phytoplasmas, indicating that they represent a novel taxonomic group. Thus, at least two phylogenetically distinct phytoplasmas are associated with the coconut yellow decline syndrome in Malaysia, both of which were also detected in other plant species.     

Detection of lethal yellowing phytoplasma in embryos from coconut palms infected with Cape St Paul wilt disease in Ghana

This study investigated the potential of seed transmission of Cape St. Paul wilt disease (CSPWD) in coconuts. PCR amplification was used to assess the distribution of phytoplasmas in parts of West African Tall (WAT) palms infected with CSPWD. Employing phytoplasma universal primer pair P1/P7 in standard PCR, or followed with a nested PCR using CSPWD–specific primer pair G813f/AwkaSR, phytoplasma infection was detected in the trunks, peduncles, spikelets, male and female flowers of four infected WAT coconut palms. Through nested PCR, phytoplasma was also detected in four of 19 embryo DNA samples extracted individually from fruits harvested from three of the four infected palms and was confirmed as CSPWD by cloning and sequencing. Subsequently, CSPWD phytoplasma was again detected in five of 33 embryos from nine infected palms, and in one of eight fruits from two symptomless palms. Fruits from infected palms recorded higher percentage germinations in two field nurseries (average of 71·0%) compared to fruits from healthy palms (average of 57·6%), and matured fruits that had dropped from infected palms showed the same levels of germination as those harvested directly from the palms. This indicates that infected fruits retain the ability to germinate whether harvested or dropped. No phytoplasmas were detected in any of the resulting seedlings and plantlets obtained through embryo in-vitro culture. Therefore, although phytoplasma DNA can be detected in embryos, there is as yet no evidence that the pathogen is seed transmitted through to the seedling to cause disease in progeny palms.     

Detection and molecular characterization of phytoplasma associated with chickpea phyllody disease in south India

Chickpea (Cicer arietinum L.) plants showing typical symptoms of infection by a phytoplasma that causes phyllody disease have been commonly observed in recent years in parts of south India. The symptoms included pale green leaves, bushy appearance due to excessive stunting of shoots, reduced internodal length and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by polymerase chain reaction (PCR) from infected samples, as well as by sequencing and phylogenetic analysis. First round PCR and nested-PCR protocols were standardized for improved efficiency and reliability of the diagnostic protocols. Using the primers P1/P7 and R16F2n/R16R2, 1,800?bp and 1,200?bp size products were amplified in first round PCR and nested-PCR protocols, respectively. The PCR product was cloned and sequenced and compared with the reference phytoplasma sequences from the database (NCBI). The Indian chickpea phyllody phytoplasma 16S rDNA sequences shared the highest nucleotide identity (>98%) with the 16S rII group phytoplasma candidates, also infecting chickpea from Australia and Pakistan. This is the first report of a phytoplasma of the 16SrII-group infecting chickpea from India. The genetic similarities and the potential threat of this new disease to chickpea cultivation in India are discussed.     

Genetic diversity of Ganoderma in oil palm plantings

Isolates of Ganoderma spp. associated with basal stem rot of oil palm were obtained by sampling from two palm plantings in Malaysia. Members of a subset of these were compared using a combination of somatic incompatibility and molecular genetic analyses. Incompatibility interactions between heterokaryons in culture indicated that Ganoderma generally occurred as numerous distinct strains, with compatible strains usually confined to individual palms. Analysis of restriction fragment length polymorphisms (RFLPs) in presumptive mitochondrial DNA (mtDNA) revealed heterogeneity among isolates from neighbouring palms, and from within individual palms. The combination of mtDNA variability and somatic incompatibility suggests that Ganoderma BSR does not spread by direct root-to-root contact between palms. The results may indicate infection spread through basidiospore dispersal or through contact with a heterogeneous long-term residual inoculum in debris.     

Kerala wilt disease phytoplasma: Phylogenetic analysis and identification of a vector, Proutista moesta

Kerala wilt disease of coconut palm is a major threat of coconut production in Kerala caused by phytoplasma. The genomic DNA purified from the insect tissues of Proutista moesta (PM) and Stephanitis typica (ST) was subjected to PCR assay using the primer combination P1/P6, P1/P7 and P4/P7. The amplified products resolved a prominent band of 650 bp for the universal primer P4/P7 and no bands were noticed for the primer pairs P1/P6 and P1/P7 combination. Since P4/P7 amplifies the 16S–23S intergenic spacer region of 16SrRNA gene, the PCR product 650 bp of the insect PM indicate the phytoplasma DNA. The presence of 650 bp for the primer P4/P7 in the genomic DNA isolated from P. moesta indicates the vectoral ability of the insect. No sign of amplification was noticed in the case of ST for the three sets of primers suggesting the inability of this insect as vector. The amplified product 650 bp from the genomic DNA of KWD palms as well as the insect tissues of P. moesta was gel purified and sequenced. The sequential similarity of 650 bp of both KWD phytoplasma and the insect phytoplasma supports the transmission of phytoplasma through the vector PM. Moreover, the sequence of 650 bp was compared with other sequences of 26 coconut phytoplasmas so far reported internationally and a cladogram was prepared for determining the phylogenetic status. It is obvious from the cladogram that the KWD disease phytoplasma is evolutionarily closest to coconut phytoplasma of coconut lethal yellowing of Mexican palms within the group 16SrIV. Phylogenetically, KWD phytoplasma is grouped in the new subgroup 16SrIV-C subsequent to the groups 16SrIV-A and 16SrIV-B for Mexican coconut lethal yellowing and Tanzanian coconut lethal decline, respectively. The restriction enzyme analysis of the PCR product 650 bp using the enzymes AluI, BclI, HindIII and RsaI further supports the phytoplasmic nature of DNA. This data records the first finding of the vector of Kerala wilt disease by detecting KWD phytoplasma in insect tissue of PM by PCR based methods. Moreover, the study reveals the phylogenetic status of KWD phytoplasma compared to other coconut phytoplasmas internationally.     

Evaluation of Malaysian oil palm progenies for susceptibility,resistance or tolerance to Fusarium oxysporum f. sp. elaeidis and defence‐related gene expression in roots

Vascular wilt of oil palm caused by Fusarium oxysporum f. sp. elaeidis (Foe) is a devastating disease in West and Central Africa. As the oil palm industry in southeast Asia is still expanding, so is the oil palm germplasm collection through the importation of seed and pollen from Africa, the centre of diversity for Elaeis guineensis. There is a risk of inadvertent spread of the disease on contaminated seed or pollen. Regular re‐evaluation of the reaction of currently grown palm genotypes towards Foe is clearly required for biosecurity. This study has demonstrated that four Malaysian oil palm progenies, three in current or recent commercial use, are highly susceptible to infection by at least one of two African isolates of Foe, representing different countries, aggressiveness and vegetative compatibility groups. Symptoms and reduction of palm growth generally reflected the extent and intensity of systemic colonization by Foe. Progeny PK 5463 expressed partial resistance to Foe isolate F3, but not to isolate 16F, displaying significantly milder symptoms and supporting less widespread vascular colonization. This relatively incompatible interaction was used to study expression of potential defence‐related genes during root infection when compared to a susceptible palm–isolate combination. The only significant response was an early up‐regulation of chitinase in resistant palms. The research revealed at least one progeny–isolate differential interaction, and the associated resistance expression suggests a component of tolerance, because colonization by Foe was systemic in both compatible and incompatible combinations.     

Promising primers for detection of phytoplasma causing coconut lethal yellowing disease in Mozambique

Coconut is one of the main cash crop in Mozambique, which occupied the second position after Tanzania in coconut production in Africa. Coconut production was drastically affected by the occurrence of a devastating Coconut Lethal Yellowing Disease (CLYD) epidemics, which reduced significantly the coconut yields. CLYD symptoms triggered upon phytoplasma infection, i.e. premature fruit dropping, necrosis of the inflorescence and progressive yellowing of the leaves, are used to identify infected trees. However, the diagnostic based uniquely on symptoms is not conclusive to confirm infection, and needs to be confirmed by molecular methods. In this study, three previously described reference primers for phytoplasma detection were tested on infected samples collected in Mozambique. Since those primers gave incongruent results, 20 new primer pairs targeting the 16S rDNA region, were newly designed. To evaluate their performance in detecting coconut infecting phytoplasma, 108 samples were tested and selected positive samples confirmed by sequencing. Our results showed a new primer pair more accurate and reliable compared to the reference pairs for CLYD detection in Mozambique. Moreover, the new primer pair was able to detect a new putative phytoplasma variant in Mozambique. Therefore, this study makes an important contribution to CLYD phytoplasma molecular diagnostics and its causative agent, giving insights that may be applied to the study of CLYD phytoplasma infection systems.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Strawflower Yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Changes in carbohydrate metabolism in coconut palms infected with the lethal yellowing phytoplasma

ABSTRACT Lethal yellowing (LY), a disease caused by a phytoplasma, is the most devastating disease affecting coconut (Cocos nucifera) in Mexico. Thousands of coconut palm trees have died on the Yucatan peninsula while plantations in Central America and on the Pacific coast of Mexico are severely threatened. Polymerase chain reaction assays enable identification of incubating palm trees (stage 0+, phytoplasma detected but palm asymptomatic). With the development of LY, palm trees exhibit various visual symptoms such as premature nut fall (stage 1), inflorescence necrosis (stages 2 to 3), leaf chlorosis and senescence (stages 4 to 6), and finally palm death. However, physiological changes occur in the leaves and roots prior to onset of visual symptoms. Stomatal conductance, photosynthesis, and root respiration decreased in stages 0+ to 6. The number of active photosystem II (PSII) reaction centers decreased during stage 2, but maximum quantum use efficiency of PSII remained similar until stage 3 before declining. Sugar and starch concentrations in intermediate leaves (leaf 14) and upper leaves (leaf 4) increased from stage 0- (healthy) to stages 2 to 4, while root carbohydrate concentrations decreased rapidly from stage 0- to stage 0+ (incubating phytoplasma). Although photosynthetic rates and root carbohydrate concentrations decreased, leaf carbohydrate concentrations increased, suggesting inhibition of sugar transport in the phloem leading to stress in sink tissues and development of visual symptoms of LY.     

Detection and identification of a ‘<Emphasis Type="Italic">Candidatus</Emphasis> phytoplasma aurantifolia’-related strain (16SrII-A subgroup) associated with <Emphasis Type="Italic">corchorus aestuans</Emphasis> phyllody in China

In September 2015, a phyllody that is typical of phytoplasma infection was observed on Corchorus aestuans plants in Haikou, Hainan Province, China. Total DNA from symptomatic and asymptomatic plants was extracted for molecular diagnosis. On the basis of sequence analysis and phylogenetic trees based on 16S rDNA and rp genes, the phyllody phytoplasma was ascertained to be related to ‘Candidatus phytoplasma aurantifolia’. To the best of our knowledge, this is the first report of a phytoplasma infecting C. aestuans in the world.     

‘Candidatus Phytoplasma aurantifolia’-related strain associated with tomato yellows disease in China

A new disease of tomato plants with typical phytoplasma disease symptoms such as stunting, yellows, auxiliary shoot proliferation and phyllody was observed in Yunnan Province, southwest China in 2011. By a nested-PCR, phytoplasma were detected using the phytoplasma universal primers specific for 16S rDNA. The results of the 16S rDNA sequencing, computer-simulated RFLP patterns and phylogenetic analysis indicated that the phytoplasma associated with the diseased tomato plants belongs to subgroup A of the peanut witches’-broom group. This is the first report of a 16SrII-A phytoplasma associated with a new tomato disease in China. This new disease was named tomato yellows.     

Detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma brasiliense’ in a new geographic region and existence of two genetically distinct populations’

‘Candidatus Phytoplasma brasiliense’, a phytoplasma taxon associated with hibiscus witches’ broom disease was first described in 2001 in Brazil. In September 2007, a peach tree (Prunus persica) displaying yellowing symptoms reminiscent of phytoplasma infection was sampled in Guba region of Azerbaijan. A phytoplasma was detected in the diseased peach tree by nested PCR amplification of its 16S rDNA with universal primers for phytoplasmas. Phylogenetical analyses of the amplified 16S rDNA showed that the phytoplasma infecting the peach tree corresponded to ‘Ca. P. brasiliense’, a species never reported in Euro-Mediterranean area. To set up a detection assay, cloning of a ‘Ca. P. brasiliense’ DNA fragment was undertaken by comparative RAPD. The amplified dnaK-dnaJ genetic locus was used to design a nested PCR assay able to amplify all ‘Ca. P. brasiliense’ isolates of the subgroup 16SrXV-A without amplifying the related members of the group 16SrII. This assay also allowed confirming the first detection of ‘Ca. P. brasiliense’ in diseased basil collected in south Lebanon.     

Mapping the spread of apricot chlorotic leaf roll (ACLR) in southern France and implication of Cacopsylla pruni as a vector of European stone fruit yellows (ESFY) phytoplasmas

An epidemiological study on European stone fruit yellows (ESFY) phytoplasmas infecting Prunus fruit trees was carried out from 1994 to 2000 in Languedoc-Roussillon (southern France). The spread of the disease was monitored for 7 years by visual observation of symptoms and by PCR detection of the phytoplasma in an experimental orchard planted with apricot hybrid seedlings. This indicated that aerial vectors were responsible for disease spread, and that transmission rates were low at the beginning of the spread. Seventy thousand homopteran insects were captured within and in the surroundings of highly ESFY-infected apricot orchards, of which about 10 000 were used in PCR and nested-PCR assays with universal ribosomal and ESFY-specific nonribosomal primers to detect ESFY phytoplasmas. The other insects were confined in cages for trials of transmission to test plants. ESFY phytoplasmas could not be detected by PCR in any of the leafhopper species captured but could be detected in the psyllid Cacopsylla pruni caught on Prunus domestica and Prunus cerasifera rootstock suckers of apricot trees and on Prunus spinosa . Nested PCR revealed ESFY phytoplasmas in one individual of the deltocephalid Synophropsis lauri captured on an apricot tree. Transmission trials confirmed the role of Cacopsylla pruni as the ESFY phytoplasma vector in France. When apricot seedlings were used as bait plants from April to November during two consecutive years, no natural transmission could be demonstrated. However, one out of 50 apricot seedlings left for the whole year in the orchard became infected. An early spring ESFY infection is in agreement with both the natural transmission results and the life cycle of Cacopsylla pruni .     

Transmission of the coconut cadang-cadang viroid to six species of palm by inoculation with nucleic acid extracts

Seedlings of Areca catechu (betel nut palm), Corypha elata (buri palm), Adonidia merrillii (manila palm), Elaeis guineensis (oil palm), Chrysalidocarpus lutescens (palmera) and Oreodoxa regia (royal palm) were inoculated with nucleic acid extracts from coconut palms with cadang-cadang disease. Within 2 years of inoculation, analysis using a ³²P-labelled DNA probe complementary to the coconut cadang-cadang viroid (CCCV) showed that RNA sequences identical to CCCV were present in the inoculated seedlings. Electrophoresis in polyacrylamide gels showed that these palms also contained an RNA with mobility identical to CCCV. Four to five years after inoculation, the infected palms of four species were usually stunted compared with uninoculated palms, while betel nut and palmera were not stunted. Yellowing of leaflets was observed with defined spots or mottling of the older fronds in all except betel nut palms. All infected palms showed mild or severe yellow-leaf spotting. These results widen the known host range and. hence, the potential number of viroid reservoir species in the field.     

广东桉树黄化(丛枝)病植原体分子鉴定与检测

从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总DNA,采用植原体通用引物与巢式引物进行PCR和巢式PCR扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA基因及部分16～23S rRNA基因间隔区序列.序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank登录号:AY101386和AY566302)同源率为99.9%,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6%和99.3%.该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-Gd).建立了桉树植原体巢式PCR检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品.     

Detection and molecular characterization of a phytoplasma affecting Prunus persica L. in Jujuy, Argentina

Peach (Prunus persica L.) plants with symptoms of yellowing, reddening, curling and leaf necrosis, premature defoliation and internode shortening were observed in production fields in Jujuy province (Argentina). A phytoplasma was detected by PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 in all the symptomatic samples analysed. The RFLP profile of PCR products, amplified with R16F2n/R16R2 primers, shows that this phytoplasma, named Argentinean Peach Yellows (ArPY), belongs to subgroup 16Sr III-B. The phylogenetic analysis of the 1244 bp 16S rDNA cloned sequence, grouped the ArPY phytoplasma into the X-disease group with a closer relationship with CFSD, PssWB and ChTDIII phytoplasmas. This is the first report of a phytoplasma infecting peach trees in Argentina.     

A set of novel RNAs transcribed from the chloroplast genome accumulates in date palm leaflets affected by brittle leaf disease

Brittle leaf disease or maladie des feuilles cassantes (MFC) is a lethal disorder of date palms that has assumed epidemic proportions in the oases of southern Tunisia. After a prolonged period during which palms are declining, the disease ends with the death of the palms. Whereas no pathogen could ever be associated with the disease, leaflets of affected palms have been previously shown to be deficient in manganese. Analysis of RNA preparations from leaflets of MFC-affected palms revealed the presence of a set of novel RNAs (MFC-RNAs) of sense and antisense polarities, which are homologous to various regions of the date palm chloroplast genome, such as the regions containing genes rrn5S-trnR(ACG) and trnM(CAU)-atpE. In the RNA preparations obtained from leaflets of affected palms, some of these RNAs are present as double-stranded species (MFC-dsRNAs), as witnessed by results from cellulose chromatography, end labeling, RNase digestion, and northern hybridization with strand specific probes. These MFC-RNAs represent a novel type of host-derived RNAs, and their presence in MFC-affected date palms is of diagnostic value.     

First report of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma malaysianum’ associated with <Emphasis Type="Italic">Elaeocarpus</Emphasis> yellows of <Emphasis Type="Italic">Elaeocarpus zollingeri</Emphasis>

“Elaeocarpus yellows” (ELY) is a widely reported phytoplasma disease of Elaeocarpus zollingeri trees in Japan. The phytoplasma associated with ELY (ELY phytoplasma) had not been identified at the species level because its 16S rRNA sequence had yet to be reported. Here, we report the results of a sequence analysis based on 16S rRNA and secA gene sequences, which showed that the ELY phytoplasma is related to ‘Candidatus Phytoplasma malaysianum’. To our knowledge, this is the first report showing the occurrence of ‘Ca. P. malaysianum’ outside Malaysia and the infection of E. zollingeri by the phytoplasma.