Host Of Phytoplasma Research Articles

Molecular characterization of a phytoplasma affiliated with the 16SrVII group representative of the novel 16SrVII-F subgroup.

Vernonia brasiliana is a wild perennial shrub frequently found in pasture areas. Plants of this species have been observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, deformed leaves and leaf chlorosis. The present study confirmed the presence of phytoplasmas in association with affected plants. Sequencing of the 16S rRNA gene, computer-simulated RFLP analysis and phylogenetic analysis revealed that one of the phytoplasmas identified was representative of novel subgroup. The sequence identity scores between the novel strain and those of previously described 'CandidatusPhytoplasma fraxini'-related strains was 99 %, while similarity coefficient values were lower than 0.97. These findings provide support to delineate the phytoplasma found in vernonia plants as a reference phytoplasma for a novel subgroup designated 16SrVII-F. This representative of the novel subgroup was denominated VbSP phytoplasma (Vernonia brasiliana Shoot Proliferation; GenBank KX342018). The results of the present study revealed V. brasiliana to be a host of phytoplasmas, evidenced a novel phytoplasma associated with phytoplasmal disease in Brazil and extended the knowledge of the genetic diversity existing within the 16SrVII group.

Occurrence of a subgroup 16SrIII-J phytoplasma in non-symptomatic Brachiaria decumbens cultivated in a grazing area

Brachiaria spp. are the most important and widely used warm season grasses for pasture, but are also important weeds for most economic crops in the tropics. During phytoplasma surveys in pasture fields, asymptomatic plants of Brachiaria decumbens were collected for inspection. Nested PCR detected phytoplasmas in more than 90% of sampled plants. Based on computer-simulated RFLP and a similarity coefficient calculation, a subgroup 16SrIII-J phytoplasma was molecularly characterized. Phylogenetic analysis was consistent with virtual RFLP identification. Representatives of this subgroup have been identified in diverse Brazilian regions encompassing various commercial crops. Our study revealed B. decumbens as a new host and reservoir of 16SrIII-J phytoplasma.

Two distinct 16SrIII phytoplasma subgroups are associated with shoot proliferation in Vernonia brasiliana, a wild species inhabiting the Brazilian savanna

Vernonia brasiliana is a wild species frequently found in Brazilian savanna areas, also known as “cerrado” areas. In pasture fields, shrubs were observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, mild leaf chlorosis and deformed leaves. Molecular detection revealed the presence of phytoplasmas in approximately 80% of the symptomatic plants. Identification by computer-simulated RFLP allowed the classification of phytoplasma strains as representatives of ‘Candidatus Phytoplasma pruni’, taxonomic subgroups 16SrIII-B and 16SrIII-J. Moreover, phylogenetic analysis evidenced perfect agreement with virtual RFLP. Both phytoplasmas found in diseased vernonia plants have been reported in diverse commercial species cultivated in Brazil, including many vegetables and an extensive number of crops. These findings identify V. brasiliana as a new host of phytoplasmas affiliated with the subgroups 16SrIII-B and 16SrIII-J and suggest this species as a probable reservoir and possible inoculum source of phytoplasmas.

Differential distribution of phytoplasma during phyllody progression in sesame (Sesamum indicum L.) under field conditions - An important consideration for effective sampling of diseased tissue

Phytoplasmas are wall-less plant pathogenic bacteria that are transmitted through sap-feeding insects. Phytoplasma colonization in the host plant has been shown to depend on the affecting phytoplasma group and the properties of the host plant. Such studies on phytoplasma colonization are scanty in the agriculturally important annuals that are suffering severe yield losses worldwide. In this regard, the oilseed crop ‘sesame’, which is a common annual host of phytoplasma and generally shows symptoms of phyllody on infection, was selected for the study. Vegetative tissues were collected from progressive stages of sesame phyllody from pre/post-blooming infection. Phytoplasma was initially detected using nested-PCR and transmission electron microscopy. A TaqMan based qPCR assay was then performed to detect and understand the in planta distribution of phytoplasma in sesame under field conditions. Results revealed a wide range of phytoplasma titer across the organs of phyllody affected sesame plants i.e. 22 cells to 4.2 × 107 cells/μg plant DNA. The lowest titer was observed in the roots, whereas the maximum number of phytoplasma cells was observed in the leaves. Phytoplasma concentration positively correlated with disease severity during the initial three stages of disease progression; however, a decline was observed at a later stage. In addition, significant variation was observed in the upper and lower plant parts,and the pattern showed a reversal with respect to early and late stages of disease. The study indicates that the lower plant parts of sesame are advantageous for phytoplasma detection at early stages. However, for experiments with pre-requisite of high phytoplasma abundance, leaves from later stages of phyllody will be beneficial. The outcomes will prove useful in effective sampling of sesame tissues for research and quarantine purposes. To our knowledge, this is the first comprehensive report of the in planta distribution of phytoplasma in an annual indeterminate crop plant.

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.

Wild plants could play a role in the spread of diseases associated with phytoplasmas of pigeon pea witches'-broom group (16SrIX)

Phytoplasmas are cell wall-less prokaryotic parasites associated with diseases affecting hundreds of plant species. In the Middle East, phytoplasmas of taxonomic subgroups 16SrIX-B and -C are the etiological agents of important diseases of almond and sesame, and are transmitted from plant-to-plant by leafhoppers and planthoppers. In the present study, surveys on wild plants as reservoirs of 16SrIX phytoplasmas have been carried out in North and South Lebanon. During field surveys, leaf samples were collected from 261 wild plants belonging to 41 species within 25 families. PCR-based analyses allowed the detection of 16SrIX phytoplasmas in the leaf samples of 24 plants belonging to 12 species. Eight of such species have been reported as phytoplasma hosts for the first time. Molecular characterization by nucleotide sequence analysis of 16S rRNA and rplV-rpsC genes revealed that the infected wild plant species harbored 16SrIX-C phytoplasma strains genetically undistinguishable. In silico enzymatic digestion of 16S rDNA sequences of phytoplasma strains, described in previous works, allowed the recognition of two new tentative subgroups (16SrIX-G and -H) within group 16SrIX. Results of the present study highlighted the wide host range of 16SrIX-C phytoplasma, associated with almond broomings, an almond witches’- broom-like disease in Iran, and with sesame phyllody in Iran and Turkey. The potential adaptation of 16SrIX-C phytoplasma to numerous wild plants highlights the elevated risk of its spread throughout the Middle East and neighbouring geographic regions.

Cacti and succulent plant species as phytoplasma hosts: A review

The trade in ornamental cacti and succulent plants has been developed into an important industry, and it is accountable for the intercontinental commercialization of many new and old species. Several cacti are hosts for phytoplasmas and this could cause a severe threat to agriculture. Despite its deleterious effects, phytoplasma infection is considered useful, and as an added ornamental value in some cacti and succulent species. Diseases associated with phytoplasmas presence and their interaction with cacti and succulent species are reviewed.

A subgroup 16SrIII-J phytoplasma associated with Aegiphila verticillata, a typical wild plant of Brazilian savanna region, exhibiting witches’ broom symptoms

Aegiphila verticillata (parrot fruit) is a wild plant that commonly grows in Brazilian savanna fields, which are also denominated Cerrado. Trees and shrubs of this species were observed exhibiting profuse shoot proliferation that produced small leaves with mild chlorosis and flowers and fruit of reduced size which fell off prematurely. In the present study, PCR assays demonstrated the association of a phytoplasma with naturally affected plants. Identification performed with group-specific primers indicated this phytoplasma as belonging to the 16SrIII group. Molecular analysis based on virtual RFLP patterns and similarity coefficient calculations allowed to classify the phytoplasma as a representative of the 16SrIII-J subgroup. Phylogenetic analysis confirmed this result and evidenced that this phytoplasma was closely related to the reference strain for the 16SrIII-J subgroup. The disease was denominated Aegiphila witches’ broom (AegWB). In South America, representatives of the 16SrIII-J subgroup are frequently found in association with diseases of phytoplasmal etiology that occur in several wild and cultivated species. This study reveals for the first time that A. verticillata, a typical plant from the Brazilian Cerrado region, is a host of phytoplasmas, specifically of a member of the 16SrIII-J subgroup.

Molecular and Scanning Electron Microscopic Proof of Phytoplasma Associated With Areca Palm Yellow Leaf Disease in Sri Lanka

HomePlant DiseaseVol. 99, No. 11Molecular and Scanning Electron Microscopic Proof of Phytoplasma Associated With Areca Palm Yellow Leaf Disease in Sri Lanka PreviousNext DISEASE NOTES OPENOpen Access licenseMolecular and Scanning Electron Microscopic Proof of Phytoplasma Associated With Areca Palm Yellow Leaf Disease in Sri LankaC. Kanatiwela-de Silva, M. Damayanthi, R. de Silva, M. Dickinson, N. de Silva, and P. UdagamaC. Kanatiwela-de SilvaSearch for more papers by this author, M. DamayanthiSearch for more papers by this author, R. de SilvaSearch for more papers by this author, M. DickinsonSearch for more papers by this author, N. de SilvaSearch for more papers by this author, and P. UdagamaSearch for more papers by this authorAffiliationsAuthors and Affiliations C. Kanatiwela-de Silva , Department of Zoology, Faculty of Science, University of Colombo, Sri Lanka; M. Damayanthi , Sri Lanka Institute of Nanotechnology, Nanotechnology & Science Park, Mahenwatte, Pitipana, Homagama, Sri Lanka; R. de Silva , Crop Protection Division, Coconut Research Institute, Lunuwila, Sri Lanka; M. Dickinson , School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK; N. de Silva , Sri Lanka Institute of Nanotechnology, Nanotechnology & Science Park, Mahenwatte, Pitipana, Homagama, Sri Lanka; and P. Udagama , Department of Zoology, Faculty of Science, University of Colombo, Sri Lanka. Published Online:21 Sep 2015https://doi.org/10.1094/PDIS-01-15-0072-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat The areca palm, Areca catechu L., of the Arecaceae family, is an important commercial crop in the world, where the drupe, commonly called the areca nut, is mainly used in betel quid chewing in many Asian and Oceanic countries. Areca palms have a low commercial value in Sri Lanka; they are largely grown in wet and intermediate zones on a total area of 11,968 ha in home gardens, as mixed cultivations, and seldom as small scale commercial cultivations. Although areca yellow leaf disease (AYLD) was not previously reported in Sri Lanka, it is a serious disease in neighboring India, reducing yield by much as 50% (Manimekalai et al. 2010). After an occurrence of Weligama coconut leaf wilt disease (WCLWD) in the southern province of Sri Lanka (Perera et al. 2012), areca palms with similar symptoms were noted in the vicinity of the WCLWD-affected plantations. Foliar yellowing of areca palms, the most conspicuous symptom, began from the inner whorl and spread to the outer parts of the crown. Chlorosis was observed on almost all leaves in the whorl. The association of a phytoplasma strain with this yellowing disease was investigated by molecular characterization based on the secA gene in conjunction with scanning electron microscopy (SEM). Symptomatic palms were selected from the vicinity of WCLWD-affected plantations in the Matara District. Symptomless, apparently healthy areca palms were selected from the Kandy District of the central province of the island. Total DNA was extracted from midrib of areca pinnae using the small scale DNA extraction method (Zhang et al. 1998). Total DNA derived from symptomatic and apparently healthy leaf samples were analyzed by primary PCR using a phytoplasma-specific universal primer pair, SecAfor1/SecArev3 (Hodgetts et al. 2008) in the primary PCR, and a 1/10 dilution of this amplicon used as the template to perform the nested PCR using RicesecAfor2/RiceseAcrev3 primers specific for phytoplasma of the 16SrXI and 16SrXIV groups (Bekele et al. 2011). Twelve of 15 symptomatic samples yielded an amplicon of 420 bp; no amplicon was generated by DNA templates from symptomless plants (n = 15). Analysis of these partial secA sequences (NCBI Accession No. KM978910) of this characteristic band of AYLD confirmed the association of a phytoplasma strain that shared 99% nucleotide homology with that of ‘Candidatus Phytoplasma cynodontis’ reference strain and 100% identity with WCLWD phytoplasma (NCBI Accession No. KM978909). Observations of transverse sections of midribs of pinnae of infected spear leaves by SEM revealed the presence of spherical, pleomorphic phytoplasma bodies ranging in diameter from 100 to 1000 nm. These appeared as separate or clustered particles, adhering to the inner surface of sieve tubes of diseased plants. The uneven distribution of phytoplasma bodies in sieve tubes as well as a developmental stage of the phytoplasma (i.e., those undergoing budding) was observed. Phytoplasma bodies were categorically absent in transverse sections of midrib of pinnae of healthy areca leaf samples. To our knowledge, this is the first report of AYLD in Sri Lanka as well as the detection of a 16SrXIV group phytoplasma strain associated with this disease. It is plausible that the same phytoplasma strain is associated with both AYLD and WCLWD. Thus, identification of areca palm as an alternative host of WCLWD phytoplasma will not only increase the complexity of the epidemiology of this disease, but will be more important in implementing control strategies for WCLWD.

Molecular characterization of ‘Candidatus Phytoplasma asteris’ subgroup I-B associated with sesame phyllody disease and identification of its natural vector and weed reservoir in India

An incidence of 12 to 35 % sesame phyllody (SP) disease was observed in sesame fields at Kushinagar and Gorakhpur districts in Uttar Pradesh and Indian Agricultural Research Institute, New Delhi, India during July-October 2013. The association of phytoplasma with sesame phyllody plants at all the three locations and a weed, Sclerocarpus africanus Jacq (SA) showing little leaf and witches’ broom symptoms at Gorakhpur was confirmed by direct and nested PCR amplification of phytoplasma 16S rDNA. Out of four predominant feeding leafhopper species in the symptomatic sesame fields, viz. Empoasca prima (Distant), Exitianus indicus (Distant), Hishimonus phycitis (Distant) (HP) and Cofana unimaculata (Signoret), only H. phycitis was found to be associated with phytoplasma in nested PCR assays with phytoplasma universal primer pair R16F2n/R16R2. Transmission test and population dynamics study further confirmed that H. phycitis was the proven natural and potential vector to transmit the SP phytoplasma from diseased to healthy sesame plants in transmission assays. BLAST analysis of 1.25 kb 16SrDNA partial sequences of nested PCR products obtained from symptomatic SP plants, weed (SA) and the leafhopper (HP) revealed 99 % sequence identities among themselves and 99 % identity with other reported strains of ‘Ca. P. asteris’ (16Sr I group). Phylogenetic analysis also suggested the closest phylogenetic relationship of SP, SA and HP phytoplasmas with those of ‘Ca. P. asteris’ group. RFLP analysis of R16F2n/R16R2 primed 16S rDNA sequences of SP, SA and HP phytoplasma isolates using iPhyClassifier online tool with 17 selected restriction enzymes confirmed the association of ‘Candidatus Phytoplasma asteris’ subgroup B with SP, SA and HP in India. The identity of natural vector and new alternative host of SP phytoplasma in the present study suggested that these may act as potential/natural source for secondary spread of sesame phyllody phytoplasma.

Molecular Determination and Characterization of Phytoplasma 16S rRNA Gene in Selected Wild Grasses from Western Kenya

Napier grass (Pennisetum purpuruem) production for zero grazing systems has been reduced to rates of up to 90% in many smallholder fields by the Napier stunt (Ns) disease caused by phytoplasma sub-group 16SrXI in western Kenya. It is hypothesized that several other wild grasses in Kenya could be infected by phytoplasmas that would otherwise pose a significant threat to Napier, other important feeds and food crops. This study therefore sought to detect and identify phytoplasma strains infecting wild grasses in western Kenya using 16S ribosomal RNA (ribonucleic acid) gene as well as identify wild grass species hosting phytoplasmas in 646 wild grass samples that were collected in October 2011 and January 2012 during a random crossectional survey conducted in Bungoma and Busia counties of western Kenya. DNA was extracted and nested polymerase reaction (nPCR) used to detect phytoplasmas. Two sub-groups of phytoplasmas were detected in eight grass species observed to grow near infected Napier fields. Only one of the two phytoplasmas reported was related to the Ns phytoplasma. There was a strong association between proportions of phytoplasma infection and the grass species collected (p = 0.001). C. dactylon, D. scalarum, B. brizantha, poverty grass and P. maximum had high proportions of infection and were abundantly distributed in western Kenya hence considered wild phytoplasma hosts. E. indica and C. ciliaris were scarcely distributed and had low infection rates. There was statistically significant difference in proportions of infection per location of survey (p = 0.001). Phytoplasma subgroups 16SrXI and 16SrXIV were the only phytoplasma genotypes distributed among wild grasses in western Kenya. Phytoplasma subgroup 16SrXIV predominantly infects only C. dactylon and B. brizantha grasses while phytoplasma subgroup 16SrXI is broad spectrum and infects a large number of wild grasses. In general, there is a diversity of wild grasses hosting phytoplasmas in western Kenya. These host grasses may be the reason for the high rates observed in the spread of Ns disease in western Kenya by acting as reservoirs for Ns phytoplasma.

Molecular biology and pathogenicity of phytoplasmas

AbstractPhytoplasmas are a large group of plant‐pathogenic wall‐less, non‐helical, bacteria associated with diseases, collectively referred to as yellows diseases, in more than a thousand plant species worldwide. Many of these diseases are of great economic importance. Phytoplasmas are difficult to study, in particular because all attempts at culturing these plant pathogens under axenic conditions have failed. With the introduction of molecular methods into phytoplasmology about two decades ago, the genetic diversity of phytoplasmas could be elucidated and a system for their taxonomic classification based on phylogenetic traits established. In addition, a wealth of information was generated on phytoplasma ecology and genomics, phytoplasma–plant host interactions and phytoplasma–insect vector relationships. Taxonomically, phytoplasmas are placed in the class Mollicutes, closely related to acholeplasmas, and are currently classified within the provisional genus ‘Candidatus Phytoplasma’ based primarily on 16S rDNA sequence analysis. Phytoplasmas are characterised by a small genome. The sizes vary considerably, ranging from 530 to 1350 kilobases (kb), with overlapping values between the various taxonomic groups and subgroups, resembling in this respect the culturable mollicutes. The smallest chromosome, about 530 kb, is known to occur in the Bermuda grass white leaf agent ‘Ca. Phytoplasma cynodontis’. This value represents the smallest mollicute chromosome reported to date. In diseased plants, phytoplasmas reside almost exclusively in the phloem sieve tube elements and are transmitted from plant to plant by phloem‐feeding homopteran insects, mainly leafhoppers and planthoppers, and less frequently psyllids. Most of the phytoplasma host plants are angiosperms in which a wide range of specific and non‐specific symptoms are induced. Phytoplasmas have a unique and complex life cycle that involves colonisation of different environments, the plant phloem and various organs of the insect vectors. Furthermore, many phytoplasmas have an extremely wide plant host range. The dynamic architecture of phytoplasma genomes, due to the occurrence of repetitive elements of various types, may account for variation in their genome size and adaptation of phytoplasmas to the diverse environments of their plant and insect hosts. The availability of five complete phytoplasma genome sequences has made it possible to identify a considerable number of genes that are likely to play major roles in phytoplasma–host interactions. Among these, there are genes encoding surface membrane proteins and effector proteins. Also, it has been shown that phytoplasmas dramatically alter their gene expression upon switching between plant and insect hosts.

Phytoplasma associated with Chinese tallow tree yellowing disease in China represents a new 16SrIII subgroup

SummaryRecently, samples of the Chinese tallow tree (Sapium sebiferum) displaying yellowing symptoms were collected from a grove in Tai'an, Shandong Province, China. The association of phytoplasma with yellowing disease was ascertained using nested polymerase chain reaction (PCR) of the 16S rRNA gene by using the phytoplasma‐specific universal primer pair P1/P7, followed by R16F2n/R16R2 as nested primers, and rp genes primed using rpL2F3/rp(I)R1A followed by rp(III)‐FN/rp(I)R1A. The sequence and phylogenetic analyses of the 16S rRNA gene and rp genes revealed that the phytoplasma associated with the Chinese tallow tree belonged to the 16SrIII group (the X‐disease group). Computer‐simulated and gel‐based restriction fragment length polymorphism (RFLP) analyses revealed that the RFLP patterns were different from the reference patterns of all previously established 16SrIII subgroups, with the maximum similarity coefficient exceeding the threshold for delineation of a new subgroup RFLP pattern type within the 16SrIII group. Thus, the phytoplasma associated with the Chinese tallow tree yellowing disease, designated as ‘CTTY’, represents a new subgroup (16SrIII‐Y). This study shows the Chinese tallow tree as a new host of phytoplasma belonging to the 16SrIII group in China and worldwide.

Optimization of virus‐induced gene silencing in Catharanthus roseus

Catharanthus roseus (periwinkle) contains abundant alkaloids and is the only commercial source of two cancer drugs, vinblastine and vincristine. The plant is also used as an experimental host for phytoplasmas because it can be infected by most phytoplasmas and shows distinctive symptoms. Abundant gene sequences for periwinkle have become available through high‐throughput transcriptome sequencing, so periwinkle has become an ideal model for studies of alkaloid metabolism and plant–phytoplasma interactions. Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) for functional genomics studies has been established, but the duration of the system was too short for plant–phytoplasma interaction studies. To improve the system, several factors were analysed, including culture temperature, plant age, leaf age, organ preferences and virus distribution, for their effects on VIGS efficacy. A new agroinfiltration method was also used for inoculating entire seedlings. High temperature (30°C) strongly inhibited VIGS, but low temperature (16°C) inhibited plant growth. VIGS efficacy was reduced in old leaves but was not affected by plant age. VIGS occurred in all examined organs, including roots, stems, leaves and flowers, but was most effective in leaves and flowers. The TRV‐based system required the existence of TRV2 to maintain gene silencing, so TRV2 concentration may be used as an indicator of successful gene silencing. The optimized VIGS system was useful for studies of plant–phytoplasma interactions and conferred gene silencing for more than 6 months.

First Report of a Group 16SrVII-C Phytoplasma Associated with Shoot Proliferation of Sunn Hemp (Crotalaria juncea) in Brazil.

Sunn hemp (Crotalaria juncea) is widely grown in tropical and subtropical regions. In Brazil, this species is commonly used for green manure, since this legume is an efficient nitrogen fixer that produces organic residues for soil improvement. In July of 2012, C. juncea exhibiting intense shoot proliferation, leaf malformation, shortened internodes, and generalized yellowing were found in an experimental field located in Piracicaba, State of São Paulo, Brazil. The incidence was about 1 to 2% and the diseased plants were distributed at random. Since these symptoms are indicative of infection by phytoplasmas, the present study aimed to detect and identify the phytoplasma. Four symptomatic and two asymptomatic plants were sampled. Small segments of leaf veins were prepared for microscopy, as previously reported (1), and observations were made using a Jeol (Akishima/Japan) model Jem-1011 transmission electron microscope. Total DNA was extracted from leaves using a commercial kit (DNeasy Plant Mini, Qiagen Inc.), and nested PCR assays were performed with primers, P1/Tint followed by R16F2n/R16R2 (2). The initial assumption that disease symptoms were associated with phytoplasma was confirmed by PCR amplification of 1.2 kb DNA fragments from the 16S rDNA gene. In contrast, no amplicon was generated with PCR using template DNA from asymptomatic plants. The phytoplasma detected from each symptomatic sample was considered to be an isolate. PCR products were purified and cloned in Escherichia coli DH5α, using the pGEM-T Easy Vector System I (Promega). Three isolates were selected and the cloned 16S rDNA sequences from three colonies of each isolate were sequenced. Since no sequence polymorphisms were found, a majority consensus sequence was selected for each isolate. These sequences were identical and one of them, designated CrSP-Br01 (crotalaria shoot proliferation) with 1,249 bp (GenBank Accession KC756947), was used as representative of the sunn hemp phytoplasma. The 16S rDNA nucleotide sequence of this phytoplasma shared 100% sequence identity with the reference phytoplasma for subgroup VII-C (Argentinian Alfalfa witches'-broom phytoplasma, AY147038). According to the in silico RFLP analysis for delineation of subgroups (3), which is based on virtual RFLP patterns and similarity coefficient calculation, the C. juncea phytoplasma was classified as a member of group 16SrVII, subgroup C. Phylogenetic analysis supported that this phytoplasma is closely related to the representative of subgroup 16SrVII-C, since both phytoplasmas emerged from the same branch. Transmission electron microscopic examination revealed the presence of phytoplasmas by visualization of pleomorphic and round bodies 100 to 400 nm in diameter, in the phloem vessels of symptomatic plants. The present study reports the first occurrence of a 16SrVII-C phytoplasma in Brazil. In addition, C. juncea was identified as a new host for phytoplasmas belonging to this subgroup.

First report of a ‘Candidatus Phytoplasma asteris’ isolate affecting macadamia nut trees in Cuba

DNA was extracted from 0.5 g of leaf midribs of twelve macadamia plants collected (Murray & Thomsom, 1980), and used as a template for a nested PCR assay. Universal primer pairs that target the phytoplasma 16S rRNA gene, P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995) were used for the first reaction, and R16F2n/R16R2 (Gundersen & Lee, 1996) for the nested reaction. Nested PCR products of expected size (approximately 1250 bp) were obtained from ten symptom-bearing plants. PCR products were purified (Wizard SV Gel and PCR Clean-Up System, Promega, Madison, WI, USA), cloned (pGEMT-Easy Vector, Promega), and two individual clones per infected plant were sequenced using Macrogene Inc. Sequencing Service. The R16F2n/R16R2 sequences were subjected to in silico restriction fragment length polymorphism (RFLP) analysis with endonucleases AluI, BfaI, BstUI, HaeIII, HhaI, HinfI, HpaII, MseI and Tsp509 (pDRAW32 AcaClone Software, http://www.acaclone.com). Phylogenetic relationships were established between the macadamia phytoplasma and those of 16SrI and other phytoplasma groups (Mega 5.0, USA). The R16F2n/R16R2 sequences of phytoplasmas detected in the symptom-bearing samples were 100% identical to each other. The consensus sequence (1262 nt) of the macadamia phytoplasma was deposited in GenBank (Accession No. KC513772) and showed 99% of sequence identity with those of the 16SrI group ‘Candidatus Phytoplasma asteris’, including phytoplasma strains AY-CVB (AY265212), ACLR-AY (AY265211) and Baigah periwinkle little leaf phytoplasma (DQ266089), all members of subgroup 16SrI-F. The virtual RFLP profile of the macadamia phytoplasma was identical to those of the 16SrI-F subgroup, which suggests that the phytoplasma strain detected in Cuban macadamia trees may be a member of this subgroup. The results of sequence and RFLP analyses were confirmed by phylogeny (Fig. 2) since the macadamia phytoplasma grouped within the cluster corresponding to the 16SrI group, closely related to those of subgroup 16SrI-F. The group 16SrI was first recorded in Cuba in 1999 (Arocha et al., 1999) and since then in several other plant species. However, this is the first report of a phytoplasma of group 16SrI associated with symptoms in macadamia nut trees, a possible new host for 16SrI phytoplasmas. These results have a significant impact for Cuban agriculture since phytoplasma group 16SrI is well known by its widest plant host range and the most complex epidemiology worldwide, and macadamia may become an important future export crop for the Cuban fruit industry.

Gene expression and biochemical changes of carbohydrate metabolism in in vitro micro-propagated apple plantlets infected by ‘Candidatus Phytoplasma mali’

'Candidatus Phytoplasma mali' (Ca. P. mali) is the disease agent causing apple proliferation (AP), which has detrimental effects on production in many apple growing areas of Central and Southern Europe. The present study investigated transcriptional and biochemical changes related to the sugar metabolism as well as expression of pathogenesis-related (PR) protein genes in invitro micro-propagated AP-infected and healthy control plantlets with the aim of shedding light on host plant response to 'Ca. P. mali' infection. Expression changes between infected and control plantlets were detected by quantitative real-time PCR analysis. The most significant transcriptional changes were observed for genes coding for pathogenesis-related proteins and for heat shock protein 70, as well as for some genes related to the sugar metabolism, such as a sorbitol transporter (SOT5), hexokinase, sucrose-phosphate synthase or granule bound starch synthase. Furthermore, biochemical analyses revealed that infected plantlets were characterized by a significant accumulation of starch and sucrose, while hexoses, such as glucose and fructose, and sorbitol were present at lower concentrations. In summary, the present analysis provides an overview of a gene set that is involved in response to phytoplasma infection and, therefore, it may help for a better understanding of the molecular mechanisms involved in phytoplasma-host plant interaction in apple.

A subgroup 16SrIII-B phytoplasma identified in honeyweed plants with leaf deformation in Brazil

Leonurus sibiricus (honeyweed) plants displaying small, shriveled and mildly chlorotic leaves were observed in commercial cassava fields. Based on nested PCR assays using a group-specific primer pair, computer-simulated RFLP analysis, similarity coefficient calculation and phylogenetic analysis, the phytoplasma identified in these plants was classified as a representative of subgroup 16SrIII-B. This is the first report of honeyweed as a host of phytoplasmas.

Natural occurrence, identification and transmission of the phytoplasma associated with flax phyllody and stem fasciation in Pakistan

Flax plants (Linum usitatissimum) of the white (album) flower variety exhibiting typical phytoplasma-like symptoms were found for the first time in Pakistan during 2011. The symptoms included floral virescence, phyllody, little leaf, stunting and stem fasciation. Light microscopy of hand-cut stem sections treated with Dienes’ stain showed blue areas in the phloem region of symptomatic plants. To confirm phytoplasma infection, total DNA was extracted separately from five plants showing virescence/phyllody and from five others showing fasciation, and was amplified by nested PCR using universal 16S rDNA phytoplasma primers P1/P7 followed by R16F2n/R16R2. All samples from plants with virescence/phyllody and fasciation yielded a 1,250 bp PCR product, and identical RFLP profiles using the enzymes AluI and HpaII. Direct sequencing of the 16S rDNA of one representative PCR amplicon (GenBank Accession No. JX567504 for phyllody and Accession No. JX567505 for fasciation) showed highest sequence identity (99%) with 16SrII ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas, and phylogenetic analysis placed the phytoplasma in subgroup 16SrII-D. Disease was successfully transmitted by grafting and by the leafhopper Orosius albicinctus. To our knowledge, flax is a new natural host for 16SrII-D phytoplasmas in Pakistan.

'Candidatus Phytoplasma brasiliense' (16SrXV-A Subgroup) Associated with Cauliflower Displaying Stunt Symptoms in Brazil.

Cauliflower stunt, caused by a phytoplasma of the group 16SrIII-J, was reported in the beginning of 2012 and has occurred with high incidences of infected plants (up to 90%) in crops located in the state of São Paulo in the southeast region of Brazil (3). Diseased plants exhibit general stunting, malformation of inflorescence, reddening leaves, and vessel necrosis (3). Further investigations with plants displaying identical symptoms collected in Nova Bassano, state of Rio Grande do Sul, Brazilian south region, have revealed the presence of a phytoplasma distinct from 16SrIII-J subgroup. Four symptomatic plus four asymptomatic samples were assayed from a field, and the presence of phytoplasma was evidenced by nested PCR assays performed with primers P1/Tint followed by R16F2n/16R2 in three affected plants, which amplified genomic fragments of 1.2 kb from the 16S rRNA gene. No amplification occurred in non-affected samples. Nested PCR products analyzed by conventional RFLP (2) using the enzymes AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI pointed to the presence of a phytoplasma belonging to group 16SrXV-A in all three phytoplasma-positive samples. Virtual RFLP analysis based on restriction patterns, derived from in silico digestion with 17 endonucleases (4), confirmed the previous results obtained from those samples by conventional RFLP. The 16S rDNA sequences of this phytoplasma identified in cauliflower (GenBank Accession No. JN818845) shared 99% sequence similarity with the reference phytoplasma for subgroup 16SrXV-A (Hibiscus witches'-broom phytoplasma, AF147708), designated 'Candidatus Phytoplasma brasiliense.' Analysis of putative restriction sites showed excellent identity between the phytoplasma studied here and the reference phytoplasma. In addition, the arrangement of branches of a phylogenetic tree constructed with phytoplasmas representing diverse 16Sr groups and subgroups supported that the phytoplasma found in cauliflower is closed related to the representative of the subgroup 16SrXV-A. Association of distinct phytoplasmas with the same kind of disease is not rare and the present pathosystem constitutes a new example. Members of this subgroup have been described almost exclusively in Brazil and previously reported in Sida sp., periwinkle, and hibiscus (1). In some European countries, as well as in the United States and Canada, phytoplasmas belonging to group 16SrI has been associated with this type of disease, which has been reported for various species of the genus Brassica, as published in previous works (3). However, a representative of the group 16SrVI was described in infected plants in Iran (3). Although the 16SrIII-J phytoplasma is currently the most important agent of cauliflower stunt in Brazil, and members of 16SrI are prevalent in other countries, this study revealed that a 16Sr XV-A phytoplasma may be also associated with this important disease of brassicas. Besides, the findings here reported expand the natural host range, including cauliflower as new host for phytoplasmas affiliated with 16SrXV-A.