Phytoplasma Phoenicium Research Articles

First report of ‘Candidatus Phytoplasma phoenicium’ (16SrIX-B) and ‘Candidatus Phytoplasma aurantifolia’ (16SrII-C) related strains on greengage trees

During 2021 surveys, greengage witches’-broom (GWB) and decline (GD) diseases were respectively observed in Fars and Yazd provinces of Iran. Presence of phytoplasma in the symptomatic plants was confirmed by PCR amplifications of 16S rRNA gene. Sequence analyses showed affiliation of the GWB and GD phytoplasmas to‘Candidatus Phytoplasma phoenicium’ (16SrIX-B) and‘Ca. P. aurantifolia’ (16SrII-C) related strains, respectively. This is the first report of phytoplasma diseases of greengage in Iran and probably in the world.

First Report of ‘Candidatus Phytoplasma phoenicium’ on Almond in Southern Italy

First Report of ‘<i>Candidatus</i> Phytoplasma phoenicium’ on Almond in Southern Italy

‘Candidatus Phytoplasma phoenicium’ and its phytosanitary status definition for the Russian Federation

‘<i>Candidatus</i> Phytoplasma phoenicium’ and its phytosanitary status definition for the Russian Federation

‘Candidatus Phytoplasma phoenicium’ associated with apricot yellows and peach witches’ broom in Iran

‘<i>Candidatus</i> Phytoplasma phoenicium’ associated with apricot yellows and peach witches’ broom in Iran

Genetic diversity of ‘Candidatus Phytoplasma phoenicium’ strain populations associated with almond witches’ broom in Lebanon and Iran

Genetic diversity of ‘<i>Candidatus</i> Phytoplasma phoenicium’ strain populations associated with almond witches’ broom in Lebanon and Iran

First report of a ‘Candidatus Phytoplasma phoenicium‘‐related strain associated with Bidens alba phyllody in Iran

Bidens alba (family Asteraceae), commonly known as shepherd’s needle, is grown in tropical and subtropical regions of North and South America, Asia and Africa (Hall et al ., 2009). Some studies indicate that it can be used as a remedy for…

First report of a ‘Candidatus Phytoplasma phoenicium‘‐related strain (16Sr IX) associated with Salix witches' broom in Iran

In Iran Salix acmophylla, S. aegyptiaca, S. alba and S. babylonica are trees traditionally grown in urban areas. Over the last few years witches' broom symptoms (Fig. 1) were observed in 12 out of 20 trees of S. alba growing along the Chalus Road in Alborz province, Iran. The symptomatology observed suggested the presence of a phytoplasma; there have been previous reports of an aster yellows group phytoplasma (16SrI-B) in Salix tetradenia (black mountain willow) in China (Muo et al., 6), a clover proliferation phytoplasma (16SrVI) in Salix bebbiana, S. discolor, S. exigua and S. petiolaris in Canada (Khadhair & Hiruki, 5), and a stolbur group phytoplasma (16SrXII) in Salix babylonica in Spain (Alfaro-Fernandez et al., 1). Leaf samples from the twelve trees (S. alba) showing witches' broom symptoms and five asymptomatic trees were collected in different areas in Alborz province. Leaf tissue was subjected to DNA extraction immediately after collection according to the procedure described by Doyle & Doyle (2). The partial 16Sr DNA was amplified with phytoplasma universal primers using primers P1/tint in the first round. The resultant PCR products were diluted with sterile distilled water (1:29) prior to nested PCR using primers R16F2/R2 (Gundersen & Lee, 4). Nested PCR gave positive results from all twelve Salix trees with witches' broom symptoms. No PCR products were obtained from the five asymptomatic Salix trees or negative controls. The PCR product from one randomly selected positive Salix sample (reference BT18) was cloned, sequenced and submitted to GenBank (Accession No. KX500119). It showed a 99% sequence identity to the reference isolate of ‘Candidatus P. phoenicum’ (AF515636), and is therefore a 'Ca. P. phoenicum'-related strain. Phylogenetic analysis with selected reference strains indicated that the phytoplasma clustered together with member strains of 'Ca. P. phoenicium' (16SrIX) (Fig. 2). Other hosts of ‘Ca. P. phoenicium’ include almond in Lebanon, and numerous hosts including almond, grapevine, peach, pistachio, Bidens alba and Chrysanthemum morifolium in Iran (Ghayeb Zamharir et al., 3). To our knowledge this is the first report of a ‘Ca. P. phoenicum‘-related strain associated with Salix worldwide.

Erratum to: Transmission of ‘Candidatus Phytoplasma asteris’ (16SrI) by Osbornellus horvathi (Matsumura 1908) co-infected with “Ca. Phytoplasma phoenicium” (16SrIX)

Erratum to: Transmission of ‘Candidatus Phytoplasma asteris’ (16SrI) by Osbornellus horvathi (Matsumura 1908) co-infected with “Ca. Phytoplasma phoenicium” (16SrIX)

A cixiid survey for natural potential vectors of ‘Candidatus Phytoplasma phoenicium’ in Lebanon and preliminary transmission trials

AbstractAlmond witches'‐broom (AlmWB) disease, associated with ‘Candidatus Phytoplasma phoenicium’, is an emerging threat with real risk of introduction in Euro‐Mediterranean Countries. Its rapid spread over large geographical areas suggests the presence of efficient insect vector(s). In the present work, a survey on cixiids was carried out in Lebanon in the years 2010–2013 in AlmWB‐infested almond and nectarine orchards. Insects were collected by means of different methods, identified with a stereo microscope, and analysed for phytoplasma identification through 16S rDNA PCR‐based amplification and nucleotide sequence analyses. Preliminary transmission trials were performed with the most abundant species. A list of the cixiid genera and species present in the studied area is given as well as some information about their biology. ‘Ca. Phytoplasma phoenicium’ strains were detected in the genera Cixius, Tachycixius, Eumecurus and Hyalesthes. Preliminary trials revealed that Tachycixius specimens were able to transmit the detected strains to healthy peach potted seedlings. Further studies are required to better clarify the taxonomic status and the bio‐ethology of collected planthoppers and deeply study their role as phytoplasma vectors.

EvaGreen Real-time PCR protocol for specific ‘Candidatus Phytoplasma mali’ detection and quantification in insects

In this paper the validation and implementation of a Real-time PCR protocol based on ribosomal protein genes has been carried out for sensitive and specific quantification of ‘Candidatus (Ca.) Phytoplasma mali’ (apple proliferation phytoplasma, APP) in insects. The method combines the use of EvaGreen® dye as chemistry detection system and the specific primer pair rpAP15f-mod/rpAP15r3, which amplifies a fragment of 238 bp of the ribosomal protein rplV (rpl22) gene of APP. Primers specificity was demonstrated by running in the same Real-time PCR ‘Ca. Phytoplasma mali’ samples with phytoplasmas belonging to the same group (16SrX) as ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma prunorum’, and also phytoplasmas from different groups, as ‘Ca. Phytoplasma phoenicium’ (16SrIX) and Flavescence dorée phytoplasma (16SrV). ‘Ca. Phytoplasma mali’ titre in insects was quantified using a specific approach, which relates the concentration of the phytoplasma to insect 18S rDNA. Absolute quantification of APP and insect 18S rDNA were calculated using standard curves prepared from serial dilutions of plasmids containing rplV-rpsC and a portion of 18S rDNA genes, respectively. APP titre in insects was expressed as genome units (GU) of phytoplasma per picogram (pg) of individual insect 18S rDNA. ‘Ca. Phytoplasma mali’ concentration in examined samples (Cacopsylla melanoneura overwintered adults) ranged from 5.94 × 102 to 2.51 × 104 GU/pg of insect 18S rDNA. Repeatability and reproducibility of the method were also evaluated by calculation of the coefficient of variation (CV%) of GU of phytoplasma and pg of 18S rDNA fragment for both assays. CV less than 14% and 9% (for reproducibility test) and less than 10 and 11% (for repeatability test) were obtained for phytoplasma and insect qPCR assays, respectively. Sensitivity of the method was also evaluated, in comparison with conventional 16S rDNA-based nested-PCR procedure. The method described has been demonstrated reliable, sensitive and specific for the quantification of ‘Ca. Phytoplasma mali’ in insects. The possibility to study the trend of phytoplasma titre in the vectors will allow a deepen investigation on the epidemiology of the disease.

EMERGING BACTERIAL AND PHYTOPLASMA DISEASES OF FRUIT TREES THAT ARE OR MAY BECOME A THREAT FOR THE MEDITERRANEAN BASIN: NOTES ON EPIDEMIOLOGY, RISKS, PREVENTION AND MANAGEMENT ON FIRST OCCURRENCE

In this contribution the epidemiology, management and main risks of three emerging bacterial diseases of Citrus approaching the Mediterranean basin, their causal organisms and vectors will be highlighted: 1) Citrus huanglongbing or Citrus greening, caused by the heat tolerant “Candidatus” Liberibacter asiaticus and heat sensitive “Candidatus” L. africanus; both forms and their respective psyllid vectors Diaphorina citri and Trioza erytreae are present in the Arabian peninsula, while huanglongbing has been recently reported in Iran, Mali, Ethiopia and Somalia and T. erytreae in some Atlantic Ocean Islands; 2) leaf scorch and leaf scald diseases of grape and diverse fruit and ornamental trees, caused by Xylella fastidiosa, of which local possible vectors such as Cicadella viridis and Philaenus spumarius occur in Europe or the Mediterranean basin; 3) citrus canker, caused by Xanthomonas citri pv. citri, the most severe form, the so-called Asiatic, is present in Irak, Iran, Oman, Somalia, UAE, Saudi-Arabia, Yemen and Reunion. Information on outbreaks and possible emerging character of some other bacterial pathogens (Xanthomonas citri pv. mangiferaeindicae on mango, approaching like X. c. pv. citri the Mediterranean basin, and Phytoplasma phoenicium on almond destroying thousands of trees in Lebanon) will be also reported. It will be argued that the three first diseases mentioned are emerging threats, with real risks of introduction and in some cases closely approaching the Mediterranean basin.

Molecular detection and phylogeny of phytoplasmas affecting cycads in India

Cycads are an ancient group of gymnosperms, often considered to be living fossils. Recently, abnormal yellowing symptoms were observed on two cycad species of the order Cycadales: Cycas revoluta (family Cycadaceae), and Zamia furfuracea (family Zamiaceae), grown as ornamental plants in gardens of the North Delhi region, India. C. revoluta exhibited yellowing symptoms on fully developed leaves in all the whorls on the rachis and the leaf lamina (Fig. 1a). Z. furfuracea showed a pale yellow colour in the leaf lamina and little leaf symptoms (Fig. 1b). Genomic DNA was isolated from the leaf midribs of five symptom-bearing and two symptomless C. revoluta plants, and three symptom-bearing and one symptomless Z. furfuracea plants, using the CTAB method (Saghai-Maroof et al., 1987). DNA was used as a template in nested-PCR with phytoplasma universal 16S ribosomal DNA primers P1/P7 (Deng & Hiruki, 1991) and fU5/rU3 primers (Lorenz et al., 1995). The amplicons were resolved on a 1.2% agarose gel, extracted, purified (QIAquick Gel Extraction Kit, QIAGEN, USA) and sequenced. Phylogenetic relationships were evaluated using the 16S rDNA sequences of phytoplasmas identified from C. revoluta (Cycas yellowing phytoplasma) and Z. furfuracea (Zamia yellowing phytoplasma), and from 18 other 16S rDNA sequences from GenBank reference phytoplasmas using the maximum parsimony method of MEGA version 4.01. The bootstrap consensus tree was inferred from 1000 replicates using Acholeplasma laidlawaii as the outgroup. Amplicons of desired size (~880bp) were obtained in all symptom-bearing plants while no amplification was observed for the symptomless ones. BLAST analysis of the phytoplasma 16S rDNA sequences from both Cycas and Zamia yellowing phytoplasmas showed 99% sequence identity with those of members of phytoplasma group 16SrII (‘Candidatus Phytoplasma aurantifolia’). The phylogenetic tree supported the sequence comparison analysis since the 16S rDNA sequences of Cycas and Zamia yellowing phytoplasmas (GenBank Accession Nos. HQ625434 and HQ625433, respectively) clustered in the same phylogenetic branch of 16SrII phytoplasmas (Fig. 2). Previous reports indicate that gymnosperms in India are highly sensitive to phytoplasma infection. ‘Ca. Phytoplasma pini’ (AJ632155, AJ310849), group 16SrV (Schneider et al., 2005) and a ‘Ca. Phytoplasma phoenicium’-related phytoplasma (AF515637), group16SrIX-E (Davis et al., 2010) have been associated with leaf abnormalities. Moreover, a ‘Ca. Phytoplasma trifolii’-related strain (FJ002570), group 16SrVI (Gupta et al., 2010) has been associated with Araucaria heterophylla little leaf and stunting. This is the first record of cycads (Cycadales) being infected by phytoplasmas of group 16SrII, the most widespread phytoplasma group in the region. The fact that gymnosperms are increasingly grown as ornamentals in India and have been identified as natural hosts for phytoplasmas, including those of 16SrII, has epidemiological impact possibly important for future disease control in the country.

Molecular characterization of a phytoplasma of group 16SrIX related to 'Ca. Phytoplasma phoenicium' in periwinkle in Brazil

Periwinkle (Catharanthus roseus), a tropical perennial plant, was found to be infected by a phytoplasma. Plants exhibiting virescence, phyllody and variegation symptoms were collected in the states of Minas Gerais and São Paulo, Brazil. The phytoplasma was transmitted by grafting from an infected periwinkle plant to healthy plants and by dodder to a citrus plant. Phytoplasma isolates from periwinkle plants from Brazil had the 16S rDNA gene sequenced and were classified in the 16SrIX group, subgroup A, belonging to the 'Candidatus P. phoenicium' species.

First report of a new subgroup 16SrIX‐E (‘Candidatus Phytoplasma phoenicium’‐related) phytoplasma associated with juniper witches’ broom disease in Oregon, USA

Western juniper (Juniperus occidentalis) is a native tree indigenous to parts of Oregon, Washington, Idaho, Nevada and California (USA). The tree has increased in density since settlement of these areas, raising concern over loss of understory plants, decreased wildlife habitat and increased soil erosion. A newly recognized disease, juniper witches’ broom (JunWB), affecting at least 1% of trees in central Oregon, is characterized by abnormal proliferation of shoots, reduced size of leaves, shortened internodes, and growths having a ball-like appearance. DNA was extracted from leaf samples from ball-like growths and used as template in polymerase chain reactions primed by primer pair P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995). DNA fragments of 1·8 kb amplified from two samples were sequenced and the sequences deposited in GenBank (Accession Nos. GQ925918 and GQ925919). RFLP patterns of 16S rDNA, observed as virtual patterns using iPhyClassifier (Zhao et al., 2009), indicated that JunWB phytoplasma represents a new subgroup lineage, designated 16SrIX-E. 16S rDNA sequence similarity confirmed that JunWB is a ‘Ca. Phytoplasma phoenicium’-related phytoplasma. JunWB is one of three phytoplasmas found thus far to infect gymnosperms and is the only phytoplasma known to infect Juniperus sp. Occurrence of three distinct phytoplasmas, JunWB phytoplasma (this study), a group 16SrIII strain, and ‘Ca. Phytoplasma pini’ (Schneider et al., 2005), in gymnosperms of two different families (Pinaceae and Cupressaceae, Division Coniferae) in Europe and North America, suggests that phytoplasmal infection of conifers may be more common than previously envisioned.

Characterization of Phytoplasmas Associated With Almond Diseases in Iran

AbstractIn recent years, almond witches’‐broom disease has been prevalent in almond growing areas in the centre and south of Iran. Furthermore, almond trees showing different symptoms of phytoplasma diseases such as little leaf, leaf rolling, dieback of branches, rosette and yellowing were observed in the central regions of Iran. DNA isolated from symptomatic almond trees was used to amplify 16S rDNA and 16S‐23S rDNA intergenic spacer (IS) fragments by nested polymerase chain reaction (PCR) using phytoplasma universal primer pairs (P1/P7, R16F2/R2, PA2F/R and NPA2F/R). Phytoplasmas were detected in symptomatic almonds in two major almond‐growing regions, Isfahan and ChaharMahal‐O‐Bakhtiari. Restriction fragment length polymorphism analyses of nested PCR products using endonuclease enzymes HpaII and TaqI revealed that phytoplasmas associated with infected almonds are genetically different. Sequence analyses of amplified fragments of 16S rDNA and IS region indicated that the almond phytoplasmas in Iran are closely related to ‘Candidatus (Ca.) Phytoplasma aurantifolia’, ‘Ca. Phytoplasma phoenicium’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’. The phytoplasmas related to ‘Ca. Phytoplasma aurantifolia’ were more prevalent than other phytoplasmas in the central regions of Iran.

First report of Almond witches’ broom phytoplasma1 (‘Candidatus Phytoplasma phoenicium’) causing a severe disease on nectarine and peach trees in Lebanon

Symptoms of shoot proliferation characteristic of phytoplasma diseases were observed on nectarine (Prunus persica var. nucipersica) and peach (P. persica) trees in the Sarada plain, south of Lebanon. The presence of phytoplasmas in the two orchards visited was confirmed by nested polymerase chain reaction using universal primers. The amplified DNA fragments were cloned and sequenced. Blast analysis of over 1000 nucleotides demonstrated the presence of ‘Candidatus Phytoplasma phoenicium’ which is considered to be the causal agent of Almond witches’ broom. This phytoplasma which belongs to the pigeon pea witches’ broom group (16SrIX) can be devastating since Almond witches’ broom has killed thousands of almond trees in Lebanon and Iran. Previous reports indicated that Almond witches’ broom may be transmitted by grafting to peach and nectarine under experimental conditions. This is the first report of a natural and epidemic spread of ‘Ca. Phytoplasma phoenicium’ in peach and nectarine. Farmers in the region were advised to eradicate the infected trees immediately. Further studies on the epidemiology of ‘Ca. Phytoplasma phoenicium’ and its vector(s) are recommended in order to develop successful eradication or disease management programmes.

'Candidatus Phytoplasma', a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects.

The trivial name 'phytoplasma' has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus 'Candidatus (Ca.) Phytoplasma'. Given the diversity within 'Ca. Phytoplasma', several subtaxa are needed to accommodate organisms that share <97.5% similarity among their 16S rRNA gene sequences. This report describes the properties of 'Ca. Phytoplasma', a taxon that includes the species 'Ca. Phytoplasma aurantifolia' (the prokaryote associated with witches'-broom disease of small-fruited acid lime), 'Ca. Phytoplasma australiense' (associated with Australian grapevine yellows), 'Ca. Phytoplasma fraxini' (associated with ash yellows), 'Ca. Phytoplasma japonicum' (associated with Japanese hydrangea phyllody), 'Ca. Phytoplasma brasiliense' (associated with hibiscus witches'-broom in Brazil), 'Ca. Phytoplasma castaneae' (associated with chestnut witches'-broom in Korea), 'Ca. Phytoplasma asteris' (associated with aster yellows), 'Ca. Phytoplasma mali' (associated with apple proliferation), 'Ca. Phytoplasma phoenicium' (associated with almond lethal disease), 'Ca. Phytoplasma trifolii' (associated with clover proliferation), 'Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), 'Ca. Phytoplasma ziziphi' (associated with jujube witches'-broom), 'Ca. Phytoplasma oryzae' (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97.5% similar to those of other 'Ca. Phytoplasma' species, are characterized by distinctive biological, phytopathological and genetic properties. These include 'Ca. Phytoplasma pyri' (associated with pear decline), 'Ca. Phytoplasma prunorum' (associated with European stone fruit yellows), 'Ca. Phytoplasma spartii' (associated with spartium witches'-broom), 'Ca. Phytoplasma rhamni' (associated with buckthorn witches'-broom), 'Ca. Phytoplasma allocasuarinae' (associated with allocasuarina yellows), 'Ca. Phytoplasma ulmi' (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being <97.5% similar to that of any other 'Ca. Phytoplasma' species, are not presently described as Candidatus species, due to their poor overall characterization.