Candidatus Phytoplasma Phoenicium Research Articles

CONTRIBUTION TO THE PRODUCTION SCHEME OF LOCAL CERTIFIED PROPAGATING MATERIAL OF ALMOND: IN VITRO SANITATION AND MICROPROPAGATION

Tissue culture techniques, alone or coupled with heat treatment, have largely contributed to the eradication of viruses from a wide range of infected plants and less for elimination of phytoplasma. In this study we present the elimination of both Prunus necrotic ringspot virus and Candidatus Phytoplasma phoenicium from two infected local almond accessions by using different tissue culture techniques combined with heat therapy. Shoot tip culture coupled or not with thermotherapy was the most effective to eliminate PNRSV in 100% of regenerated shootlets, while both shoot tip and stem cutting cultures associated to heat treatment were all suitable for phytoplasma elimination from regenerated shootlets. This free-pathogens material has been multiplied for successive subcultures and then rooted in vitro before being hardened in the glasshouse conditions. At this stage elimination of infectious agents has been confirmed prior to use the freed material as certified mother plants for further propagation. These results showed the important potential of traditional tissue culture techniques to be used in the certification program for local germplasm.

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.

Specific PCR and real-time PCR assays for detection and quantitation of ‘Candidatus Phytoplasma phoenicium’

Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with ‘Candidatus Phytoplasma phoenicium’. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of ‘Ca. P. phoenicium’ and for epidemiological studies. The developed qPCR assay herein uses a TaqMan® probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against ‘Ca. P. phoenicium’ and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of ‘Ca. P. phoenicium’ were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 105 to 106 genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

Asymmetrasca decedens (Cicadellidae, Typhlocybinae), a natural vector of ‘Candidatus Phytoplasma phoenicium’

Abstract‘Candidatus Phytoplasma phoenicium’ is associated with a lethal disease of almond, peach and nectarine named almond witches'‐broom disease (AlmWB). The disease spread rapidly in Lebanon from coastal areas to elevations exceeding 1200 m, killing over 150,000 trees in a span of two decades. The mode of spread suggested the involvement of efficient vector(s) and Asymmetrasca decedens (Hemiptera, Cicadellidae) was suspected as it is the most abundant leafhopper species present in Lebanese stone fruit orchards. Living A. decedens specimens were collected from fields heavily infested by AlmWB and used in transmission trials on healthy peach almond hybrid GF‐677 and peach GF‐305 seedlings with an inoculation‐access period of 30 days. PCR analysis supported by sequencing showed that A. decedens is a carrier of the phytoplasma, and that the phytoplasma was detected in insect salivary glands and in some inoculated GF‐677 and GF‐305 seedlings. One year post‐inoculation, ‘Ca. P. phoenicium’ was detected in newly emergent leaves of inoculated seedlings. However, the characteristic symptoms of witches'‐broom were not observed. PCR amplified fragments from phytoplasma‐positive seedlings and from A. decedens samples showed 99.9% nucleotide identity in their 16S RNA region and phylogenetic analysis using a neighbour jointing tree confirmed that the phytoplasmas detected in both insects and inoculated seedlings belonged to 16SrIX‐B (D). The present manuscript is the first known report for a leafhopper vector of ‘Ca. P. phoenicium’ and shows that the incubation period of the disease in plants may be longer than 1 year. The importance of phytosanitary control measures, the adoption of a national strategy and regional cooperation in order to contain the further spread of the disease are discussed.

Molecular Study of a New Disease of Peach in Iran Associated with a Phytoplasma

In recent years, a disease has been reported to affect peach trees in Kurdistan province of Iran causing serious losses to the production. Main symptoms of disease include leaf stunting and yellowing, which lead to failure in fruit production at harvest. For diagnosis of disease and identification of the causal agent, symptomatic leaf samples were collected in Kurdistan orchards during summer 2010 and were carried to the laboratory. Total DNA was extracted from plant samples according to the standard procedures and indexed by grafting and nested PCR using phytoplasma generic primers, P1/P7 and R16F2n/R2. PCR products were characterized by RFLP technique and direct sequencing. The 16S rDNA sequences were compared with those of other phytoplasmas in GenBank. Phytoplasma rDNA was amplified from 20 out 35 samples. The 16S rDNA sequences of the phytoplasma were identified in Peach samples which showed 98% similarity to that of “Candidatus Phytoplasma phoenicium” which is considered to be the causal agent of Almond witches’ broom. Phylogenetic analysis of 16S rDNA sequences placed peach strains in Almond witches broom isolate as a member of pigeon pea witches broom (PPWB) group. Further studies on the epidemiology of “Ca. Phytoplasma phoenicium” and its vector(s) in Iran are recommended in order to identify new natural hosts and develop successful disease management programs.

First Report of a 16SrIX Group ('Candidatus Phytoplasma phoenicium'-Related) Phytoplasma Associated with a Chrysanthemum Disease.

In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region.

Association of Pigeon pea witches' broom phytoplasma (16Sr IX) infecting Phlox drummondii in India

Phlox is a genus of 67 species of perennial and annual plants in the family Polemoniaceae. Phlox are popular ornamental plants, and traditionally used to treat aches, colds, anaemia, diarrhoea and eczema. During a survey at the Indian Agricultural Research Institute Campus, New Delhi, from 2010 to 2011, symptoms including extensive yellowing and stunting, proliferation of shoots, little leaves and reduced size of flowers were observed in approximately 17% of plants of Phlox drummondii (Fig. 1). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from leaf midribs and stems of three different infected plants. Healthy-looking plants without symptoms were also collected to use as a negative control using the phytoplasma enrichment procedure (Ahrens & Seemüller, 1). The DNA isolated from periwinkle infected with toria phyllody phytoplasma (group 16SrIX, pigeon pea witches' broom phytoplasma; Azadvar et al., 2) and maintained in greenhouse was used as positive control. Total DNA was used as a template for nested PCR using universal primers that target the phytoplasma 16S rRNA gene: P1/P7 (Deng & Hiruki, 3; Schneider et al., 5) and R16F2n/R16R2 (Gundersen & Lee, 4). Expected size amplicons of ~1.8 kb and ~1.2 kb, respectively, were amplified from all the three symptomatic P. drummondii plants (Fig. 2). However, no PCR products were obtained from the healthy-looking plant samples. The amplicon of ~1.2 kb was sequenced from a representative sample, and the consensus sequence obtained was submitted to GenBank (Accession No. KC178678). BLAST analysis of the partial 16S rDNA sequence of the P. drummondii phytoplasma revealed the highest sequence identity (99%) with that of phytoplasma members of group 16SrIX, ‘Candidatus Phytoplasma phoenicium’, the former Pigeon pea witches' broom group. Phylogenetic analysis (MEGA version 4.0, USA) confirmed the closest phylogenetic relationship of the P. drummondii phytoplasma 16S rDNA sequence with those of the 16SrIX phytoplasma group (Fig. 3). Hence, the P. drummondii phytoplasma was identified as a 16SrIX isolate. Phytoplasmas of group 16SrI ‘Candidatus Phytoplasma asteris’ subgroup M (16SrI-M) have been previously associated with P. drummondii in Lithuania (Samuitienė et al., 6). However, this is the first world report of the association of the 16SrIX group with a phytoplasma disease on P. drummondii. The first author is thankful to Department of Science and Technology, New Delhi, India for providing financial assistance under WOS ‘A’ scheme. The authors wish to express sincere thanks to Head, Division of Plant Pathology and Director, Indian Agricultural Research Institute for providing laboratory facilities.

Phytoplasmas associated with Almond witches’ broom disease: An overview

Almond is affected by several phytoplasma diseases but almond witches' broom (AlmWB) is the most destructive one. The disease spread rapidly, killed about a hundred thousands trees in recent years. Association of phytoplasma with AlmWB disease thatbelonged to the pigeon pea witches’-broom (PPWB) group has been established and the name ‘Candidatus Phytoplasma phoenicium’ (16sIX-B) is proposed for it. The phytoplasma was graft-transmissible to almond, plum and peach seedlings. A specific PCR test was developed to distinguish the almond phytoplasma from others in the PPWB group. The specific PCR will be particularly useful for identification of both the insect vector(s) and reservoir plant(s) of the almond phytoplasma. For the moment only very few data is available on the extent of the disease, on its biology and epidemiology. However, this disease could represent a serious threat for almond-producing countries. Key words: Almond witches broom, phytoplasma, peach, almond.

Identification of new 16SrIX subgroups, -F and -G, among ‘ Candidatus Phytoplasma phoenicium ’ strains infecting almond, peach and nectarine in Lebanon

Almond witches’-broom is a lethal almond disease associated with ‘ Candidatus Phytoplasma phoenicium ’, subgroups 16SrIX-B and -D. In Lebanon, where the disease was first reported, analogous diseases affecting peach and nectarine were recently associated with presence of ‘ Ca. P. phoenicium ’. In the present study, genetic diversity among 24 ‘ Ca. P. phoenicium ’ strains infecting almond, peach and nectarine plants in diverse geographic regions of Lebanon was investigated by virtual restriction fragment length polymorphism (RFLP) analysis of 16S rDNA nucleotide sequences. Calculation of virtual restriction similarity coefficients revealed the presence of two new subgroups, -F and -G, in group 16SrIX that were confirmed by real RFLP analyses. Obtained results open new opportunities for in-depth studies of the distribution of ‘ Ca. P. phoenicium ’ strains in plant hosts and insect vector populations from different geographic areas of Lebanon.

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 omanense', associated with witches'-broom of Cassia italica (Mill.) Spreng. in Oman

Samples from plants of Cassia italica exhibiting typical witches'-broom symptoms (Cassia witches'-broom; CWB) were examined for the presence of plant pathogenic phytoplasmas by PCR amplification using universal phytoplasma primers. All affected plants yielded positive results. RFLP analyses of rRNA gene products indicated that the phytoplasmas detected were different from those described previously. Phylogenetic analysis of 16S rRNA gene sequences confirmed that CWB represents a distinct lineage and shares a common ancestor with 'Candidatus Phytoplasma phoenicium'. Molecular comparison revealed that the 16S rRNA gene sequences of the four CWB strains (IM-1, IM-2, IM-3 and IM-4) identified in symptomatic C. italica samples were nearly identical (99.6-100 % similarity). The closest relatives were members of the pigeon pea witches'-broom phytoplasma ribosomal group (16SrIX; 95-97 % sequence similarity). On the basis of unique 16S rRNA gene sequences and biological properties, the phytoplasma associated with witches'-broom of C. italica in Oman represents a coherent but discrete novel phytoplasma, 'Candidatus Phytoplasma omanense', with GenBank/DDBJ/EMBL accession number EF666051 representing the reference strain.

Attempts to eliminate Candidatus phytoplasma phoenicium from infected Lebanese almond varieties by tissue culture techniques combined or not with thermotherapy

Elimination of Candidatus phytoplasma phoenicium from two infected Lebanese varieties of almond by using different tissue culture techniques is reported. Except for the oxytetracycline therapy which totally inhibited the development of explants, stem cutting cultures associated with thermotherapy, shoot tip cultures associated or not with thermotherapy, and shoot tip micrografting were all suitable, either for shoot regeneration or for elimination of phytoplasma from the two varieties. However, stem cutting culture coupled with thermotherapy seemed to be the most effective for regeneration of phytoplasma-free plantlets.

'Candidatus phytoplasma phoenicium' sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran.

Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches'-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches'-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called 'almond brooming' in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name 'Candidatus Phytoplasma phoenicium' is proposed for the phytoplasma associated with almond witches'-broom in Lebanon and Iran.