Jujube Witches'-broom Phytoplasma Research Articles

The 'Candidatus Phytoplasma ziziphi' effectors SJP1/2 negatively control leaf size by stabilizing the transcription factor ZjTCP2 in jujube.

Phytoplasmas manipulate host plant development to benefit insect vector colonization and their own invasion. However, the virulence factors and mechanisms underlying small-leaf formation caused by jujube witches' broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small-leaf formation in jujube (Ziziphus jujuba). In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized the transcription factor ZjTCP2. Overexpression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f_1 was significantly reduced in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern to the expression of its target, ZjTCP2, which was consistent with the pattern in diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced leaf size. Constitutive expression of the miRNA319f_1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small-leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and the protein level, providing new insights into small-leaf formation caused by phytoplasmas in woody plants.

'Candidatus Phytoplasma ziziphi' Changes the Metabolite Composition of Jujube Tree Leaves and Affects the Feeding Behavior of Its Insect Vector Hishimonus hamatus Kuoh.

Hishimonus hamatus Kuoh is a leafhopper species native to China that feeds on Chinese jujube leaves. This leafhopper species has been verified to transmit jujube witches' broom (JWB) disease, caused by phytoplasma, a fatal plant pathogen, which belongs to the phytoplasma subgroup 16SrV-B. The transmission of JWB phytoplasma largely relies on the feeding behavior of piercing-sucking leafhoppers. However, the specific mechanisms behind how and why the infection of JWB influences the feeding behavior of these leafhoppers are not fully understood. To address this, a study was conducted to compare the feeding patterns of H. hamatus when feeding JWB-infested jujube leaves to healthy leaves using the electrical penetration graph (EPG) technique. Then, a widely targeted metabolome analysis was performed to identify differences in the metabolite composition of JWB-infected jujube leaves and that of healthy jujube leaves. The results of EPG analyses revealed that when feeding on JWB-infected jujube leaves, H. hamatus exhibited an increased frequency of phloem ingestion and spent longer in the phloem feeding phase compared to when feeding on healthy leaves. In addition, the results of metabolomic analyses showed that JWB-infected leaves accumulated higher levels of small-molecular carbohydrates, free amino acids, and free fatty acids, as well as lower levels of lignans, coumarins and triterpenoids compared to healthy leaves. The above results indicated that the H. hamatus preferentially fed on the phloem of infected leaves, which seems to be linked to the transmission of the JWB phytoplasma. The results of metabolomic analyses partially imply that the chemical compounds might play a role in making the infected leaves more attractive to H. hamatus for feeding.

Jujube witches' broom phytoplasmas inhibit ZjBRC1-mediated abscisic acid metabolism to induce shoot proliferation.

Jujube witches' broom (JWB) phytoplasmas parasitize the sieve tubes of diseased phloem and cause an excessive proliferation of axillary shoots from dormant lateral buds to favour their transmission. In previous research, two JWB effectors, SJP1 and SJP2, were identified to induce lateral bud outgrowth by disrupting ZjBRC1-mediated auxin flux. However, the pathogenesis of JWB disease remains largely unknown. Here, tissue-specific transcriptional reprogramming was examined to gain insight into the genetic mechanisms acting inside jujube lateral buds under JWB phytoplasma infection. JWB phytoplasmas modulated a series of plant signalling networks involved in lateral bud development and defence, including auxin, abscisic acid (ABA), ethylene, jasmonic acid, and salicylic acid. JWB-induced bud outgrowth was accompanied by downregulation of ABA synthesis within lateral buds. ABA application rescued the bushy appearances of transgenic Arabidopsis overexpressing SJP1 and SJP2 in Col-0 and ZjBRC1 in the brc1-2 mutant. Furthermore, the expression of ZjBRC1 and ABA-related genes ZjHB40 and ZjNCED3 was negatively correlated with lateral main bud outgrowth in decapitated healthy jujube. Molecular evidence showed that ZjBRC1 interacted with ZjBRC2 via its N-terminus to activate ZjHB40 and ZjNCED3 expression and ABA accumulation in transgenic jujube calli. In addition, ZjBRC1 widely regulated differentially expressed genes related to ABA homeostasis and ABA signalling, especially by binding to and suppressing ABA receptors. Therefore, these results suggest that JWB phytoplasmas hijack the ZjBRC1-mediated ABA pathways to stimulate lateral bud outgrowth and expansion, providing a strategy to engineer plants resistant to JWB phytoplasma disease and regulate woody plant architecture to promote crop yield and quality.

Influences of Jujube Witches' Broom (JWB) Phytoplasma Infection and Oxytetracycline Hydrochloride Treatment on the Gene Expression Profiling in Jujube.

Jujube witches' broom disease (JWB), caused by Candidatus Phytoplasma ziziphi, is the most destructive phytoplasma disease threatening the jujube industry. Tetracycline derivatives treatments have been validated to be capable of recovering jujube trees from phytoplasma infection. In this study, we reported that oxytetracycline hydrochloride (OTC-HCl) trunk injection treatment could recover more than 86% of mild JWB-diseased trees. In order to explore the underlying molecular mechanism, comparative transcriptomic analysis of healthy control (C group), JWB-diseased (D group) and OTC-HCl treated JWB-diseased (T group) jujube leaves was performed. In total, 755 differentially expressed genes (DEGs), including 488 in 'C vs. D', 345 in 'D vs. T' and 94 in 'C vs. T', were identified. Gene enrichment analysis revealed that these DEGs were mainly involved in DNA and RNA metabolisms, signaling, photosynthesis, plant hormone metabolism and transduction, primary and secondary metabolisms, their transportations, etc. Notably, most of the DEGs identified in 'C vs. D' displayed adverse change patterns in 'D vs. T', suggesting that the expression of these genes was restored after OTC-HCl treatment. Our study revealed the influences of JWB phytoplasma infection and OTC-HCl treatment on gene expression profiling in jujube and would be helpful for understanding the chemotherapy effects of OTC-HCl on JWB-diseased jujube.

The Jujube TCP Transcription Factor ZjTCP16 Regulates Plant Growth and Cell Size by Affecting the Expression of Genes Involved in Plant Morphogenesis

Jujube production is threatened by jujube witches’ broom (JWB) disease, which is caused by JWB phytoplasma. The jujube TCP transcription factor (TF) ZjTCP16 may be involved in the interaction of jujube plants with JWB phytoplasma. In this study, qRT-PCR proved that the expression pattern of ZjTCP16 was altered by JWB phytoplasma. The gene functions of ZjTCP16 were analyzed by its overexpression in Arabidopsis and jujube, as well as knock-down in. The overexpression of ZjTCP16 in Arabidopsis and jujube resulted in dwarfism and small leaves, while the zjtcp16 CRISPR mutants were higher than the WT. Microscopic observation of paraffin sections of jujube stems showed that ZjTCP16 affected the size of cells. The interactions of ZjTCP16 with ZjAS2 and ZjLOB in both the cytoplasm and nucleus were demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Yeast one-hybrid (Y1H) assays and qRT-PCR further confirmed that ZjTCP16 affected the expression of genes involved in leaf morphogenesis and cell proliferation (ZjAS1, ZjKNAT1, ZjKNAT2 and ZjKNAT6) at the mRNA level through the ZjAS2 and ZjLOB pathways. In conclusion, ZjTCP16 regulates plant growth and cell size by altering the expression pattern of morphogenesis-related genes in jujube.

Genome-wide identification of jujube witches’ broom phytoplasma effectors revealed the role of SJP3 in inducing phyllody

Jujube witches’ broom (JWB), associated with ‘Candidatus Phytoplasma ziziphi’, is a devastating disease in Chinese jujube (Ziziphus jujuba Mill.). One of the symptoms in plants infected with phytoplasmas is the change of floral organs into leaf-like structures, known as phyllody. Effector proteins secreted by phytoplasmas are key to inducing this and other witches’ broom symptoms. However, the phytoplasma effector proteins that induce phyllody in jujube remain unknown. We identified 43 candidate secreted JWB phytoplasma proteins (SJPs) in the JWB phytoplasma genome. Eight of the predicted candidate effector genes exhibited high transcript levels in infected flower buds. Among the eight candidate effectors, SJP3 experimentally showed the potential to induce phyllody. SJP3 expression in infected jujube was at a high level in young leaves and flowers. Ectopic expression of SJP3 in Arabidopsis induced leaf-like flowers and extra secondary flowers and significantly changed the transcript levels of PISTILLATA (PI), SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). The expression levels of these MADS-box homologous genes were also changed in infected jujube flower buds and all four whorls of the floral organs. The data showed that the effector SJP3 disrupts expression of several MADS-box genes associated with floral organ identity and flowering time and is likely key to inducing phyllody in phytoplasma-infected jujube. This study provides new insight into understanding the pathogenesis of JWB disease induced by phytoplasma.

Jujube witches' broom phytoplasma effectors SJP1 and SJP2 induce lateral bud outgrowth by repressing the ZjBRC1-controlled auxin efflux channel.

Comprehensively controlling phytoplasma-associated jujube witches' broom (JWB) disease is extremely challenging for the jujube industry. Although the pathogenesis of phytoplasma disease has been highlighted in many plant species, the release of lateral buds from dormancy under JWB phytoplasma infection has not been characterized in woody perennial jujube. Here, two 16SrV-B group phytoplasma effectors, SJP1 and SJP2, were experimentally determined to induce witches' broom with increased lateral branches. In vivo interaction and subcellular localization analyses showed that both SJP1 and SJP2 were translocated from the cytoplasm to the nucleus to target the CYC/TB1-TCP transcription factor ZjBRC1. The N- and C-terminal coiled-coil domains of SJP1 and SJP2 were required for the TCP-binding ability. ZjBRC1 bound directly to the auxin efflux carrier ZjPIN1c/3 promoters and down-regulated their expression to promote the accumulation of endogenous auxin indole-3-acetic acid in jujube calli. Furthermore, JWB phytoplasma infection suppressed ZjBRC1 accumulation and induced ZjPIN1c/3 expression to stimulate lateral bud outgrowth. Therefore, SJP1 and SJP2 stimulate lateral bud outgrowth, at least partly, by repressing the ZjBRC1-controlled auxin efflux channel in jujube, representing a potential strategy for comprehensive phytoplasma-associated disease control and a resource for gene editing breeding to create new cultivars with varying degrees of shoot branching.

Genome-wide identification and expression of the lipoxygenase gene family in jujube (Ziziphus jujuba) in response to phytoplasma infection

Chinese jujube (Ziziphus jujuba) is an important fruit tree in China. Jujube witches' broom (JWB) disease, caused by JWB phytoplasma (‘Candidatus phytoplasma ziziphi’), is the most devastating infectious disease of the jujube tree. Lipoxygenase (LOX) plays important roles in the jasmonic acid (JA) biosynthesis pathway and plant defense to biotic stresses. In this study, we identified 13 LOX genes in the jujube genome distributed on 5 chromosomes. The ZjLOX proteins were classified into two categories based on phylogenetic analysis. We tracked gene duplication events and found two sets of tandem repeats on chromosome 2. We predicted the promoters of the ZjLOXs using PlantCARE and found a large number of cis elements that may respond to phytohormones, light, and biological stresses. Transcriptomic, proteomic, and qRT-PCR expression analyses showed that ZjLOX02, ZjLOX05, ZjLOX06 and ZjLOX08 may play important roles in the interaction between jujube and phytoplasma. This study provides a foundation for better understanding both the molecular evolution of the jujube LOX gene family and the response of the ZjLOX genes to JWB phytoplasma infection.

Molecular cloning and expression of a novel eukaryotes elongation factor1A gene (ZjeEF-1α) from Chinese jujube in response to phytoplasma infection

Eukaryotic translation elongation factor 1A (eEF1A) in plant is a multifunctional protein. In our previous study, we identified a 651-bp EST sequence of eEF-1α from a suppression subtractive hybridization (SSH) library of Chinese jujube (Ziziphus jujuba Mill.), which was established under jujube witches' broom (JWB) phytoplasma stress. Here we isolated the eEF1A gene (ZjeEF-1α, JF488063) from Chinese jujube by single oligonucleotide nested (SON)-PCR and rapid amplification of cDNA ends (RACE). ZjeEF-1α contains an open reading frame (ORF) of 1344 bp, encoding a polypeptide of 447 amino acids with an estimated molecular mass of 49.29 kDa and a theoretical pI of 9.10. ZjeEF-1α shared over 92% similarity with eEF-1α proteins from other 24 eukaryotes including plants and animals. Search Pfam predicted that ZjeEF-1A contains three typical eEF1A functional domains. Moreover, the corresponding genomic sequence of ZjeEF-1α was also sequenced, in which a 115-bp intervening sequence with splicing site GT-AG was found. Real-time quantitative PCR (RT-qPCR) and western blotting assays revealed that the expression of ZjeEF-1α was enhanced in resistant cultivar ‘Xingguang’ and decreased in sensitive cultivar ‘Zanhuangdazao’ at early stages of phytoplasma infection. It is proved that ZjeEF-1A expression is early response to phytoplasma infection and its possible functions in resistant process to JWB phytoplasma are discussed.

16S rDNA sequence analysis of witches’ broom phytoplasma isolates from Chinese jujube in North China

Phytoplasmas are agriculturally important pathogens for more than one thousand plant species. Jujube witches’ broom (JWB), a plant disease caused by phytoplasma, is highly fatal in Chinese jujube, an important fruit tree and medicinal crop in Asia. The 16S rDNA of 15 JWB isolates from 4 provinces in North China were amplified, cloned and sequenced. The isolates were over 99.8 % homologous, indicating that JWB phytoplasma is highly conserved. Phylogenetic analysis of the 16S rDNA sequences from those isolates, together with the representative phytoplasma sequences from other host species, showed that all JWB isolates belong to a subgroup of 16Sr V group and are closely related to Elm yellows (EY) phytoplasma. Virtual RFLP and single-nucleotide polymorphism (SNP) analysis of 16S rDNA also revealed that JWB isolates have a relatively low level of genetic diversity and that their SNP sites exhibit a strong bias toward A/G variation when compared with other phytoplasmas. The high similarity among JWB isolates from different regions and cultivars is consistent with the conserved symptoms of the disease and the wide infectivity of JWB phytoplasmas. This result provides valuable information to better understand the genetic diversity of JWB phytoplasmas.

Comparative Molecular Analyses of Phytoplasmas infecting Sophora japonica cv. golden and Robinia pseudoacacia

AbstractPhytoplasmas were detected in Sophora japonica cv. golden and Robinia pseudoacacia with diseased branches of witches'‐broom collected in Haidian district, Beijing, China. Phytoplasma cells were observed in phloem sieve elements of symptomatic S. japonica cv. golden by transmission electron microscopy. The presence of phytoplasmas was further confirmed by sequence determination of partial gene sequences of 16S rDNA, rp (ribosomal protein) and secY. Phylogenetic trees and virtual restriction fragment length polymorphism (RFLP) analyses indicated that the phytoplasmas causing S. japonica cv. golden witches'‐broom (SJGWB) and R. pseudoacacia witches'‐broom (RPWB) belong to the 16SrV (elm yellows) group, and they are most closely related to subgroup 16SrV‐B, rpV‐C and secYV‐C jujube witches'‐broom (JWB) phytoplasma. Comparative analyses indicated that the phytoplasma of RPWB was closer to the JWB and that R. pseudoacacia might serve as an alternative host plant of JWB phytoplasma.

Migration and Distribution of Graft-inoculated Jujube Witches'-broom Phytoplasma within a Cantharanthus roseus Plant

Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea(Received on February 14, 2012; Revised on April 29, 2012; Accepted on May 2, 2012)Periwinkle seedlings (Cantharanthus roseus) were ino-culated with jujube witches’- broom (JWB) phytoplasmavia grafting to analyze the migration of JWB phyto-plasmas within the host plant. The phytoplasmas weredetected using nested polymerase chain reaction (PCR)and fluorescence microscopy. Fluorescence microscopywas a simple and easy method of detecting phytoplasmas;however, it was not sufficiently sensitive to detect verylow phytoplasma concentrations. Therefore, the migra-tion of JWB phytoplasma was investigated throughPCR. The first migration of JWB phytoplasma from aninfected tissue to healthy tissues occurred late. Aftergrafting, the phytoplasmas moved from the inoculatedtwig (or scion) to the main stem, which took 28 days.Afterward, the phytoplasma migrated faster and tookless than 4 days to spread into the roots from the mainstem. All twigs were then successively colonized by theJWB phytoplasmas from the bottom to the top. JWBphytoplasma was detected via nested PCR in all parts ofthe periwinkle seedling 82 days after inoculation. Basedon these results, the inoculated JWB phytoplasmaappeared to migrate downward to the roots along themain stem during the early stages, and then continuedto move upward, colonizing twigs along the way untilthey reached the apex.Keywords : grafting, migration, nested PCR, periwinkle,phytoplasmaAmong the diseases of jujube (Zizyphus jujube Mill.) trees,the jujube witches’-broom (JWB) disease is the mostserious and destructive. JWB is a systemic disease, with theinfected trees showing typical symptoms of phytoplasmassuch as witches’-broom, phyllody, and yellows. The patho-gen, Candidatus Phytoplasma ziziphi, is usually present inhigh density in plant tissues with or without visible externalsymptoms, even though it is limited to the sieve elements ofthe plant (Jung et al., 2003; Kuske and Kirkpatrick, 1992;Nakashima and Hayashi, 1995). However, phytoplasma issometimes difficult to detect even in tissues that displaydistinct external symptoms. For example, phytoplasmacells were observed via transmission electron microscopy(TEM) in young inflorescences of coconut palms, but not inmature inflorescences, leaves, or stems with symptoms ofthe lethal yellows disease (Parthasarathy, 1974). This obser-vation may indicate the involvement of phytoplasma-produced or phytoplasma-induced substances in symptomexpression. The metabolism of infected plants is known tobe disturbed by phytoplasmas, resulting in the degenerationof infected sieve elements (Credi, 1994; Douglas, 1993).Phytoplasmas are obligate endoparasites of plant cellsand cannot be mechanically transmitted, thereby the studyof their biological and phytopathological characteristicshave been hindered (Kirkpatrick, 1992; McCoy et al., 1989)for a long time. After the discovery of phytoplasma as aplant pathogen, most studies focused on new diseases,including the means of transmission and disease control.Due to the fastidious nature of phytoplasmas, their di-stribution in the organs of an individual plant has not yetbeen well elucidated, even though they can be detectedfrom the phloem sieve tubes of an infected plant. Fortu-nately, the progress of molecular biological techniques suchas polymerase chain reaction (PCR) has made the detectionof relatively low concentrations of phytoplasmas possibleeven in plants with extremely mild symptoms or asympto-matic (Ahrens and Seemuller, 1992; Christensen et al.,2004; Garcia-Chapa, 2003; Lee et al., 1995). Several studies on the localization and migration ofphytoplasmas within infected plant tissues have been con-ducted (Cha and Tattar, 1991; Seemuller et al., 1984; Yi etal., 2001). About a decade ago, Wei et al. (2000) verifiedthat mulberry dwarf phytoplasma is distributed to an entirevein and petiole of a mulberry leaf by using nested PCR. Adynamic analysis of onion yellows phytoplasmas in Chry-santhemum coronarium was also conducted (Wei et al.,2004). However, little is still known on the distribution andmigration of phytoplasmas in plant organs, including roots,buds, flowers, and fruits. Hence, findings on the within-plant distribution of phytoplasmas and their migration from

Reverse-cleft in vitro micrografting of Ziziphus jujuba Mill. Infected with jujube witches’ broom (JWB)

Jujube witches’ broom (JWB) is a destructive disease, caused by a phytoplasma, of Chinese jujube (Ziziphus jujuba Mill.), an important traditional fruit in China and South Korea. Micrografting of plantlets to detect those affected with JWB is difficult due to differences in stem diameter between the scion and rootstock. In this study, an in vitro micrografting technique, reverse-cleft graft, suitable for rootstock thinner than scion was developed and optimized. The JWB phytoplasma was successfully transmitted into the scion from diseased rootstock using this technique as determined by polymerase chain reaction. Micrografted plantlets of Chinese jujube were incubated on Murashige and Skoog medium without plant growth regulators. The survival frequencies of 78–91%, depending on grafting combinations, were obtained.

Molecular Identification of a New Phytoplasma Strain Associated with the First Observation of Jujube Witches'-Broom Disease in Northeastern China.

Jujube (Zizyphus jujuba Mill.) is a deciduous tree that is native to northern Africa and Syria. Because of its tolerance to a broad range of climatic conditions, jujube has attained a wide natural distribution from southeastern Europe to eastern Asia (3). Jujube has a long history of cultivation, especially in Asia, for its valuable medicinal properties, strong wood, and nutritious fruits. Jujube trees are susceptible to phytoplasma infections and develop jujube witches'-broom (JWB) disease. To date, JWB diseases have been reported in Korea, Japan, and central China (1,4). In this communication, we describe a new phytoplasma strain associated with the first observation of JWB disease in northeastern China. In the summer of 2006, six jujube trees exhibiting pronounced witches'-broom symptoms were observed in suburban Dalian, Liaoning Province. The trees developed dense clusters of highly proliferating branches with shortened internodes. Leaves on the affected branches were chlorotic and significantly reduced in size. A DNA segment characteristic of phytoplasma rRNA partial operons was amplified from DNA samples extracted from leaves of all diseased trees in polymerase chain reactions (PCR) using phytoplasma-universal primer pair P1/P7 (2). No PCR product was obtained from DNA samples extracted from two symptomless jujube trees in the same region. The PCR-amplified DNA segment, spanning a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene was cloned and sequenced to achieve 4× coverage per base position in sequencing both strands (GenBank Accession No. EF661852). Results from analysis of the sequence data indicated that the six jujube trees were infected by a phytoplasma of elm yellows group (16SrV), to which other reported JWB phytoplasma strains belong. However, the JWB phytoplasma strain identified in the current study, hereby designated as JWB-DL, displayed sequence variations within the partial rRNA operon compared with those of other JWB strains (GenBank Accession Nos. AY072722, AF305240, and AY197661), indicating that JWB-DL is a distinct strain. To further characterize the JWB-DL phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F2A/rpR1 (2), cloned, and sequenced (GenBank Accession No. EF661581). The nucleotide sequence of the JWB-DL phytoplasma rplV-rpsC locus is identical to that of hemp fiber witches'-broom phytoplasma (GenBank Accession No. EF029093) rather than to those of JWB phytoplasma strains described previously. To our knowledge, this is the first report of a JWB disease in northeastern China, and JWB-DL represents a new, distinct 'Candidatus Phytoplasma ziziphi'-related strain.

First Report of Witches'-Broom Disease in a Cannabis spp. in China and Its Association with a Phytoplasma of Elm Yellows Group (16SrV).

Hemp fiber plants (Cannabis spp.) spread naturally in almost every climate zone in China and have a long history of cultivation in the country (1). While hemp stalks provide high-quality fibers for making ropes, clothes, and paper products, hemp seeds are a rich source of edible oil. During the summer of 2004, a disease characterized by witches'-broom symptoms was observed in wild hemp fiber plants growing in suburban Taian, Shandong, China. The diseased plants developed clusters of highly proliferating branches with much shortened internodes and leaves on the affected branches were significantly reduced in size. Phytoplasma infection was suspected in this hemp fiber witches'-broom (HFWB) disease because of the typical symptoms and because of its geographic location where other phytoplasmal diseases such as jujube witches'-broom (JWB), paulownia witches'-broom (PaWB), paper mulberry witches'-broom (PMWB), and Chinese wingnut witches'-broom (CWWB) diseases were previously reported (3,4). Total DNA was extracted from leaves of four diseased and four nearby healthy looking hemp fiber plants. Nested PCR were carried out on the DNA samples using phytoplasma universal 16S rDNA primers (P1A/16S-SR and R16F2n/R16R2) (2). Results revealed that all examined diseased plants were infected by phytoplasma, whereas nearby healthy looking plants were phytoplasma free. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA R16F2n/R16R2 fragment indicated that the phytoplasma associated with HFWB disease belongs to subgroup 16SrV-B of the elm yellows (EY) phytoplasma group. Nucleotide sequence analysis of the cloned HFWB phytoplasma partial rRNA operon (GenBank Accession No. EF029092), spanning a near full-length 16S rRNA gene and a partial 16S-23S rRNA intergenic spacer, suggested that HFWB phytoplasma is most closely related to JWB and PMWB phytoplasmas, both members of subgroup16SrV-B. To further characterize the HFWB phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F1A/rp(V)R1A (2), cloned, and sequenced (GenBank Accession No. EF029093). The nucleotide sequence of the HFWB phytoplasma rplV and rpsC locus is nearly identical (99.9%) to that of JWB phytoplasma. To our knowledge, this is the first report of a phytoplasmal disease in Cannabis spp. Since HFWB and JWB phytoplasmas share extremely high sequence identity and share the same eco-geographic location, further investigation is warranted to determine whether these two phytoplasmas are actually one species that can infect both plants, an issue having important implications in managing both diseases.

Specific Primer for Detection of Jujube Witches' Broom Phytoplasma Group (16SrV) in Korea

In order to diagnose and differentiate jujube witches` broom (JWB) phytoplasma rapidly, oligonucleotide primer pair, 16Sr(V) F/R, for polymerase chain reactions (PCRs) was designed on the basis of 16S rRNA sequences of JWB phytoplasma. The PCR employing phytoplasma universal primer pair P1/P7 consistently amplified DNA in all tested phytoplasma isolates. But no phytoplasma DNA was detected from healthy jujube seedlings. The nested PCR, the primer pair 16S(V) F/R, about 460 bp fragment, amplified DNA in all tested JWB and related phytoplasmas including ligustrum witches` broom phytoplasma of the 16S rRNA group V, but no DNA amplification was detected from other phytoplasma strains such as groups 16SrI (Aster yellows) and 16SrXII (Stolbur group) in which mulberry dwarf phytoplasma and chrysanthemum witches` broom phytoplasma belong to, respectively. The same results were obtained from both Korean and Chinese isolates of JWB phytoplasma. Nested-PCR using phytoplasma universal primer pair P1/P7 and 16SrV group-specific primer pair 16S(V) F/R could detect group V phytoplasmas rapidly and easily, in particular JWB phytoplasma.

First Report of Witches'-Broom Disease of Broussonetia papyrifera and Its Association with a Phytoplasma of Elm Yellows Group (16SrV)

Broussonetia papyrifera, commonly known as paper mulberry, is an ornamental tree that is native to northeastern Asia. Because of its fast-growing nature and tolerance of dust, smoke, and high temperatures, paper mulberry is an important component of the biological diversity in natural ecosystems as well as a favorable shade tree in the region. In September of 2003, a disease characterized by pronounced witches'-broom symptoms was observed in paper mulberry trees growing near a jujube (Ziziphus jujuba) orchard and in home gardens located in Taian, Shandong, China. The diseased trees developed dense clusters of highly proliferating branches with shortened internodes. Leaves on the affected branches were chlorotic and greatly reduced in size. Phytoplasma infection was first suspected in this paper mulberry witches'-broom (PMWB) disease because the disease occurred in an area where other phytoplasmal diseases, including jujube witches'-broom (JWB) disease and paulownia witches'-broom (PaWB) disease, are common (4). Results from nested polymerase chain reactions (PCR), performed using phytoplasma-universal 16S rDNA primers (P1/P7 and R16F2n/R16R2) (1,2,3), revealed that all seven diseased trees tested contained phytoplasma, whereas PCR assay of comparable leaf samples from three nearby symptomless paper mulberry trees were negative. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA indicated that all diseased trees contained the same phytoplasma and that the PMWB phytoplasma belongs to the subgroup B of the elm yellows (EY) phytoplasma group (16SrV-B). Nucleotide sequence analysis of the cloned PMWB phytoplasma partial rRNA operon (GenBank Accession No. AY576685), spanning a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that PMWB phytoplasma is most closely related to JWB phytoplasma, a member of the subgroup16SrV-B. To our knowledge, this is the first report of a paper mulberry witches'-broom disease and the first report of its association with a phytoplasma. Further work is underway to determine whether the PMWB phytoplasma is distinct from previously characterized phytoplasmas included in group 16SrV and to assess impacts of the phytoplasma on the ecosystems in the region.

'Candidatus phytoplasma ziziphi', a novel phytoplasma taxon associated with jujube witches'-broom disease.

Phylogenetic relationships of five jujube witches'-broom (JWB) phytoplasma isolates from four different districts, and other phytoplasmas, were investigated by 16S rDNA PCR amplification and sequence analysis. The 16S rDNA sequences of any pair of the five isolates of JWB phytoplasmas were > 99.5% similar. The JWB phytoplasma 16S rDNA sequences were most closely related to that of the elm yellows (EY) phytoplasma in 16S-group VIII. Phylogenetic analysis of the 16S rDNA sequences from the JWB phytoplasma isolates, together with sequences from most of the phytoplasmas archived in GenBank, produced a tree in which the JWB isolates clustered as a discrete subgroup. The uniqueness of the JWB phytoplasma appears to be correlated with a specific insect vector (Hishimonus sellatus) and the host plant (Zizyphus jujuba), or with a specific geographical distribution. The unique properties of the JWB phytoplasma sequences clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma ziziphi'.

A Leafhopper (Hishimonus sellatus) Transmits Phylogenetically Distant Phytoplasmas: Rhus Yellows and Hovenia Witches' Broom Phytoplasma

Phytoplasmas causing a severe decline of three tree species, i.e., Rhus javanica, Hovenia tomentella and Zizyphus jujuba, in Japan were examined for their transmissibility by a leafhopper species Hishimonus sellatus, and for their phylogenetic relatedness. By H. sellatus, Rhus yellows (RhY) phytoplasma was transmissible to white clover and periwinkle seedlings, causing typical symptoms in these plants. Jujube witches' broom (JWB) phytoplasma was also transferred to the host plant, Z. jujuba, by the leafhopper. Because JWB phytoplasma was transmitted to Hovenia tomentella and caused the same symptoms as Hovenia witches' broom (HWB), JWB phytoplasma may be very closely related to HWB phytoplasma. RFLP analysis of the PCR products of 16S rDNA revealed that RhY phytoplasma belongs to the Aster yellows (AY) group, and JWB and HWB phytoplasmas belong to a different group (possibly Elm yellows group). Thus, we found that one species of leafhopper can carry phylogenetically distant phytoplasmas.

Genetic Similarity Between Jujube Witches¡？Broom and Mulberry Dwarf Phytoplasmas Transmitted by Hishimonus sellatus Uhler

Using phytoplasma universal primer pair Pl and P7, a fragment of about 1.8 kb nucleotide sequences of 16S rRNA gene and 16S-23S rRNA intergenic spacer region, and a portion of 23S rRNA gene of jujube witches`broom (JWB) and mulberry dwarf(MD) phytoplasmas were determined. The nucleotide sequences of JWB and MD were 1,850 bp and 1,831 bp long, respectively. The JWB phytoplasma sequence was aligned with the homologous sequence of MD phytoplasma. Twenty-eight base insertions and nine base deletions were found in the JWB phytoplasma sequence compared with that of MD phytoplasma. The similarity of the aligned sequences of JWB and MD was 84.8%. The near-complete 16S rRNA gene DNA sequences of JWB and MD were 1,529 bp and 1,530 bp in length, respectively, and revealed 89.0% homology. The 16S-23S rRNA intergenic spacer region DNA sequences were 263 bp and 243 bp in lengths respectively, while homology was only 70% and the conserved tRNA-lle gene of JWB and MD was located into the intergenic space region between 16S-23S rRNA gene. The nucleotide sequences were 77 bp long in both JWB and MD, and showed 97.4% sequence homology. Based on the phylogenetic analysis of the two phytoplasmas, the JWB phytoplasma belongs to the Elm yellow phytoplasma group (16S rV), whereas, the MD phytoplasma belongs to the Aster yellow group (16S rI).