Paulownia Witches' Broom Research Articles

Long Non-Coding RNAs Responsive to Witches’ Broom Disease in Paulownia tomentosa

Paulownia witches’ broom (PaWB) disease caused by phytoplasmas is a fatal disease that leads to considerable economic losses. Long non-coding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in posttranscriptional and transcriptional regulation. However, lncRNAs and their functional roles remain poorly characterized in Paulownia. To identify lncRNAs and investigate their roles in the response to PaWB phytoplasmas, RNA sequencing was performed for healthy Paulownia tomentosa, PaWB-infected P. tomentosa, and for healthy and PaWB-infected P. tomentosa treated with 100 mg L−1 rifampicin. A total of 28,614 unique mRNAs and 3693 potential lncRNAs were identified. Comparisons between lncRNAs and coding genes indicated that lncRNAs tended to have shorter transcripts and fewer exon numbers, and displayed significant expression specificity. Based on our comparison scheme, 1063 PaWB-related mRNAs and 110 PaWB-related lncRNAs were identified; among them, 12 PaWB-related candidate target genes that were regulated by nine PaWB-related lncRNAs were characterized. This study provides the first catalog of lncRNAs expressed in Paulownia and gives a revealing insight into the molecular mechanism responsible for PaWB.

Quantitative proteome-level analysis of paulownia witches' broom disease with methyl methane sulfonate assistance reveals diverse metabolic changes during the infection and recovery processes.

Paulownia witches’ broom (PaWB) disease caused by phytoplasma is a fatal disease that leads to considerable economic losses. Although there are a few reports describing studies of PaWB pathogenesis, the molecular mechanisms underlying phytoplasma pathogenicity in Paulownia trees remain uncharacterized. In this study, after building a transcriptome database containing 67,177 sequences, we used isobaric tags for relative and absolute quantification (iTRAQ) to quantify and analyze the proteome-level changes among healthy P. fortunei (PF), PaWB-infected P. fortunei (PFI), and PaWB-infected P. fortunei treated with 20 mg L−1 or 60 mg L−1 methyl methane sulfonate (MMS) (PFI-20 and PFI-60, respectively). A total of 2,358 proteins were identified. We investigated the proteins profiles in PF vs. PFI (infected process) and PFI-20 vs. PFI-60 (recovered process), and further found that many of the MMS-response proteins mapped to “photosynthesis” and “ribosome” pathways. Based on our comparison scheme, 36 PaWB-related proteins were revealed. Among them, 32 proteins were classified into three functional groups: (1) carbohydrate and energy metabolism, (2) protein synthesis and degradation, and (3) stress resistance. We then investigated the PaWB-related proteins involved in the infected and recovered processes, and discovered that carbohydrate and energy metabolism was inhibited, and protein synthesis and degradation decreased, as the plant responded to PaWB. Our observations may be useful for characterizing the proteome-level changes that occur at different stages of PaWB disease. The data generated in this study may serve as a valuable resource for elucidating the pathogenesis of PaWB disease during phytoplasma infection and recovery stages.

Comparative Proteomic Analysis of Paulownia fortunei Response to Phytoplasma Infection with Dimethyl Sulfate Treatment

Paulownia fortunei is a widely cultivated economic forest tree species that is susceptible to infection with phytoplasma, resulting in Paulownia witches' broom (PaWB) disease. Diseased P. fortunei is characterized by stunted growth, witches' broom, shortened internodes, and etiolated and smaller leaves. To understand the molecular mechanism of its pathogenesis, we applied isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography coupled with tandem mass spectrometry approaches to study changes in the proteomes of healthy P. fortunei, PaWB-infected P. fortunei, and PaWB-infected P. fortunei treated with 15 mg·L−1 or 75 mg·L−1 dimethyl sulfate. We identified 2969 proteins and 104 and 32 differentially abundant proteins that were phytoplasma infection responsive and dimethyl sulfate responsive, respectively. Based on our analysis of the different proteomes, 27 PaWB-related proteins were identified. The protein-protein interactions of these 27 proteins were analyzed and classified into four groups (photosynthesis-related, energy-related, ribosome-related, and individual proteins). These PaWB-related proteins may help in developing a deeper understanding of how PaWB affects the morphological characteristics of P. fortunei and further establish the mechanisms involved in the response of P. fortunei to phytoplasma.

Identification and dynamic expression profiling of microRNAs and target genes of Paulownia tomentosa in response to Paulownia witches’ broom disease

Paulownia witches’ broom (PaWB) is a disastrous epidemic disease in Paulownia tomentosa trees in many parts of the world. Infected Paulownia tomentosa trees are generally characterized by witches’ brooms, proliferating branches, yellowing and withering leaves, and branch dieback. In previous studies, we found that by treating seedlings with a certain concentration of methyl methanesulphonate (MMS), PaWB could be cured. In this study, three small RNA libraries from healthy, PaWB-infected, and PaWB-infected Paulownia tomentosa treated with MMS were constructed and sequenced by Illumina sequencing and identified approximately 50 conserved and novel microRNAs (miRNAs) in each library. We investigated the expression patterns of the miRNAs in three libraries and found that 20 conserved miRNAs were significantly differently expressed in the comparison (healthy vs. PaWB-infected); nine of these conserved miRNAs were also significantly differently expressed in another comparison (PaWB-infected vs. PaWB-infected MMS-treated). The target genes of these identified miRNAs were found through degradome sequencing platform and functionally annotated. Some of the target genes encoded transcription factors, including auxin response factors, which are known regulators of plant defense responses. Further, we found that the expression levels of some of the miRNA sequences changed in response to MMS treatment and were close to their expression levels in the healthy plantlets. Our results indicate possible roles for Paulownia miRNAs associated with plants infected with PaWB and may provide available information for improving the prevention and treatment of PaWB disease in the future.

Identification of microRNAs and their targets in Paulownia fortunei plants free from phytoplasma pathogen after methyl methane sulfonate treatment.

MicroRNAs (miRNAs) play major roles in plant responses to various biotic and abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. Paulownia witches' broom (PaWB) disease caused by phytoplasmas reduces Paulownia production worldwide. In this study, we investigated the miRNA-mediated plant response to PaWB phytoplasma by Illumina sequencing and degradome analysis of Paulownia fortunei small RNAs (sRNAs). The sRNA and degradome libraries were constructed from healthy and diseased P.fortunei plants and the plants free from phytoplasma pathogen after 60mgL(-1) methyl methane sulfonate treatment. A total of 96 P.fortunei-conserved miRNAs and 83 putative novel miRNAs were identified. Among them, 37 miRNAs (17 conserved, 20 novel) were found to be differentially expressed in response to PaWB phytoplasma infection. In addition, 114 target genes for 18 of the conserved miRNA families and 33 target genes for 15 of the novel miRNAs in P.fortunei were detected. The expression patterns of 14 of the PaWB phytoplasma-responsive miRNAs and 12 target genes were determined by quantitative real-time polymerase chain reaction (qPCR) experiments. A functional analysis of the miRNA targets indicated that these targeted genes may regulate transcription, stress response, nitrogen metabolism, and various other activities. Our results will help identify the potential roles of miRNAs involved in protecting P.fortunei from diseases.

Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa.

BackgroundPaulownia witches’ broom (PaWB) is a fatal disease of Paulownia caused by a phytoplasma. In previous studies, we found that plants with PaWB symptoms would revert to a healthy morphology after methyl methane sulfonate (MMS) treatment. To completely understand the gene expression profiles of the Paulownia-phytoplasma interaction, three high-throughput sequencing technologies were used to investigate changes of gene expression and microRNAs (miRNAs) in healthy Paulownia tomentosa plantlets, PaWB-infected plantlets, and PaWB-infected plantlets treated with 60 mg · L−1 MMS.MethodsTranscriptome, miRNAs and degradome sequencing were performed to explore the global gene expression profiles in the process of Paulownia tomentosa with phytoplasma infection.ResultsA total of 98,714 all-unigenes, 62 conserved miRNAs, and 35 novel miRNAs were obtained, among which 902 differentially expressed genes (DEGs) and 24 miRNAs were found to be associated with PaWB disease. Subsequently, the target genes of these miRNAs were predicted by degradome sequencing. Interestingly, we found that 19 target genes of these differentially expressed miRNAs were among the 902 DEGs. The targets of pau-miR156g, pau-miR403, and pau-miR166c were significantly up-regulated in the P. tomentosa plantlets infected with phytoplasma. Interaction of miRNA -target genes mediated gene expression related to PaWB were identified.ConclusionsThe results elucidated the possible roles of the regulation of genes and miRNAs in the Paulownia-phytoplasma interaction, which will enrich our understanding of the mechanisms of PaWB disease in this plant.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2074-3) contains supplementary material, which is available to authorized users.

Plant-Pathogen Interaction-Related MicroRNAs and Their Targets Provide Indicators of Phytoplasma Infection in Paulownia tomentosa × Paulownia fortunei.

Paulownia witches’ broom (PaWB) caused by a phytoplasma, has caused extensive losses in the yields of paulownia timber and resulted in significant economic losses. However, the molecular mechanisms in Paulownia that underlie the phytoplasma stress are poorly characterized. In this study, we use an Illumina platform to sequence four small RNA libraries and four degradome sequencing libraries derived from healthy, PaWB-infected, and PaWB-infected 15 mg·L−1 and 30 mg·L−1 methyl methane sulfonate (MMS)-treated plants. In total, 125 conserved and 118 novel microRNAs (miRNAs) were identified and 33 miRNAs responsive to PaWB disease were discovered. Furthermore, 166 target genes for 18 PaWB disease-related miRNAs were obtained, and found to be involved in plant-pathogen interaction and plant hormone signal transduction metabolic pathways. Eleven miRNAs and target genes responsive to PaWB disease were examined by a quantitative real-time PCR approach. Our findings will contribute to studies on miRNAs and their targets in Paulownia, and provide new insights to further understand plant-phytoplasma interactions.

Transcriptome analysis of the genes related to the morphological changes of Paulownia tomentosa plantlets infected with phytoplasma

Paulownia witches’ broom (PaWB) caused by phytoplasma is a destructive disease of Paulownia in China and has been studied over the past 40 years. Recently, our research team found that methyl methanesulfonate (MMS) could help Paulownia plants recover from the symptoms of Paulownia witches’ broom in which the expression levels of genes related to photosynthesis, carbohydrate and nitrogen metabolism, plant-pathogen interaction, circadian rhythm changed. However, the molecular mechanisms underlying the interaction between PaWB and phytoplasma are still not fully understood. Here, high-throughput mRNA sequencing and de novo assembly were conducted in order to investigate gene expression profiles in three Paulownia tomentosa samples (healthy plantlets, diseased plantlets, and diseased plantlets treated with MMS. A total of 85,545 all-unigene were obtained, 2540 of which were significantly differentially expressed among the three samples. Some genes associated with folate and fatty acid synthesis, signal transduction pathways of plant hormones were identified, and their expression patterns were further validated using quantitative real-time PCR. To our knowledge, this is the first transcriptome-based study to demonstrate variations of gene expression related to morphological changes in metabolic pathways of phytoplasma-infected P. tomentosa plants. The information provided in this study will help understand the molecular pathogenesis of the Paulownia plant in response to phytoplasma infection.

Discovery of microRNAs and transcript targets related to witches' broom disease in Paulownia fortunei by high-throughput sequencing and degradome approach.

Paulownia witches' broom (PaWB) caused by the phytoplasma is a devastating disease of Paulownia trees. It has caused heavy yield losses to Paulownia production worldwide. However, knowledge of the transcriptional and post-transcriptional regulation of gene expression by microRNAs (miRNAs), especially miRNAs responsive to PaWB disease stress, is still rudimentary. In this study, to identify miRNAs and their transcript targets that are responsive to PaWB disease stress, six sequencing libraries were constructed from healthy (PF), PaWB-infected (PFI), and PaWB-infected, 20mgL(-1) methyl methane sulfonate-treated (PFI20) P. fortunei seedlings. As a result, 95 conserved miRNAs belonging to 18 miRNA families, as well as 122 potential novel miRNAs, were identified. Most of them were found to be a response to PaWB disease-induced stress, and the expression levels of these miRNAs were validated by quantitative real-time PCR analysis. The study simultaneously identified 109 target genes from the P. fortunei for 14 conserved miRNA families and 24 novel miRNAs by degradome sequencing. Furthermore, the functions of the miRNA targets were annotated based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The results presented here provide the groundwork for further analysis of miRNAs and target genes responsive to the PaWB disease stress, and could be also useful for addressing new questions to better understand the mechanisms of plant infection by phytoplasma in the future.

Phenylpropanoid metabolism, hormone biosynthesis and signal transduction-related genes play crucial roles in the resistance of Paulownia fortunei to paulownia witches’ broom phytoplasma infection

Paulownia witches’ broom (PaWB) caused by an obligate biotrophic plant pathogen called phytoplasma, is a devastating disease of paulownia trees over a large part of the world. However, little is known about the molecular mechanisms that underlie phytoplasma pathogenicity in paulownia or about the mode of interactions with host plants. In this study, genome-wide gene expression profiling was used to compare healthy, phytoplasma-infected, and both phytoplasma-infected and 20 mg L−1 methyl methane sulfonate (MMS) treated Paulownia fortunei plants using high-throughput mRNA sequencing analysis. A total of 6571 and 1377 differentially expressed unigenes were identified in the phytoplasma-infected plants versus healthy plants and in 20 mg L−1 MMS-treated plants versus phytoplasma-infected plants, respectively. Expression changes of 16 candidate differentially expressed unigenes were validated by qRT-PCR, indicating significant differences among the three P. fortunei samples. Our analysis showed that dramatic changes occurred in the gene expression profile of P. fortunei after PaWB phytoplasma infection and MMS treatment. The transcription of a large number of genes related to the plant–pathogen interaction, including phenylpropanoid metabolism, hormone biosynthesis and signaling, defense and/or pathogenesis, and signal transduction, were significantly up-regulated in the phytoplasma-infected paulownia and then returned to the levels in the healthy controls after MMS treatment. Our systematic analysis provides comprehensive transcriptomic data about P. fortunei trees infected by PaWB phytoplasma. The findings will help unraveling the molecular mechanisms of plant-phytoplasma interactions and may pave the way for engineering P. fortunei trees with improved properties.

Plant-pathogen interaction, circadian rhythm, and hormone-related gene expression provide indicators of phytoplasma infection in Paulownia fortunei.

Phytoplasmas are mycoplasma-like pathogens of witches’ broom disease, and are responsible for serious yield losses of Paulownia trees worldwide. The molecular mechanisms of disease development in Paulownia are of considerable interest, but still poorly understood. Here, we have applied transcriptome sequencing technology and a de novo assembly approach to analyze gene expression profiles in Paulownia fortunei infected by phytoplasmas. Our previous researches suggested that methyl methane sulfonated (MMS) could reverse the effects of the infection. In this study, leaf samples from healthy, infected, and both infected and methyl methane sulfonate treated plants were analyzed. The results showed that the gene expression profile of P. fortunei underwent dramatic changes after Paulownia witches’ broom (PaWB) phytoplasma infection. Genes that encoded key enzymes in plant-pathogen interaction processes were significantly up-regulated in the PaWB-infected Paulownia. Genes involved in circadian rhythm and hormone-related genes were also altered in Paulownia after PaWB infection. However, after the PaWB-infected plants were treated with MMS, the expression profiles of these genes returned to the levels in the healthy controls. The data will help identify potential PaWB disease-resistance genes that could be targeted to inhibit the growth and reproduction of the pathogen and to increase plant resistance.

Morphological changes of Paulownia seedlings infected phytoplasmas reveal the genes associated with witches' broom through AFLP and MSAP.

Paulownia witches' broom (PaWB) caused by phytoplasma might result in devastating damage to the growth and wood production of Paulownia. To study the effect of phytoplasma on DNA sequence and to discover the genes related to PaWB occurrence, DNA polymorphisms and DNA methylation levels and patterns in PaWB seedlings, the ones treated with various concentration of methyl methane sulfonate (MMS) and healthy seedlings were investigated with amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). Our results indicated that PaWB seedlings recovered a normal morphology, similar to healthy seedlings, after treatment with more than 20 mg·L−1 MMS; Phytoplasma infection did not change the Paulownia genomic DNA sequence at AFLP level, but changed the global DNA methylation levels and patterns; Genes related to PaWB were discovered through MSAP and validated using quantitative real-time PCR (qRT-PCR). These results implied that changes of DNA methylation levels and patterns were closely related to the morphological changes of seedlings infected with phytoplasmas.

Identification of genes related to Paulownia witches' broom by AFLP and MSAP.

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L−1 MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

Discovery of Genes Related to Witches Broom Disease in Paulownia tomentosa × Paulownia fortunei by a De Novo Assembled Transcriptome

In spite of its economic importance, very little molecular genetics and genomic research has been targeted at the family Paulownia spp. The little genetic information on this plant is a big obstacle to studying the mechanisms of its ability to resist Paulownia Witches’ Broom (PaWB) disease. Analysis of the Paulownia transcriptome and its expression profile data are essential to extending the genetic resources on this species, thus will greatly improves our studies on Paulownia. In the current study, we performed the de novo assembly of a transcriptome on P. tomentosa × P. fortunei using the short-read sequencing technology (Illumina). 203,664 unigenes with a mean length of 1,328 bp was obtained. Of these unigenes, 32,976 (30% of all unigenes) containing complete structures were chosen. Eukaryotic clusters of orthologous groups, gene orthology, and the Kyoto Encyclopedia of Genes and Genomes annotations were performed of these unigenes. Genes related to PaWB disease resistance were analyzed in detail. To our knowledge, this is the first study to elucidate the genetic makeup of Paulownia. This transcriptome provides a quick way to understanding Paulownia, increases the number of gene sequences available for further functional genomics studies and provides clues to the identification of potential PaWB disease resistance genes. This study has provided a comprehensive insight into gene expression profiles at different states, which facilitates the study of each gene’s roles in the developmental process and in PaWB disease resistance.

Transcriptomic Analysis of Paulownia Infected by Paulownia Witches'-Broom Phytoplasma

Phytoplasmas are plant pathogenic bacteria that have no cell wall and are responsible for major crop losses throughout the world. Phytoplasma-infected plants show a variety of symptoms and the mechanisms they use to physiologically alter the host plants are of considerable interest, but poorly understood. In this study we undertook a detailed analysis of Paulownia infected by Paulownia witches’-broom (PaWB) Phytoplasma using high-throughput mRNA sequencing (RNA-Seq) and digital gene expression (DGE). RNA-Seq analysis identified 74,831 unigenes, which were subsequently used as reference sequences for DGE analysis of diseased and healthy Paulownia in field grown and tissue cultured plants. Our study revealed that dramatic changes occurred in the gene expression profile of Paulownia after PaWB Phytoplasma infection. Genes encoding key enzymes in cytokinin biosynthesis, such as isopentenyl diphosphate isomerase and isopentenyltransferase, were significantly induced in the infected Paulownia. Genes involved in cell wall biosynthesis and degradation were largely up-regulated and genes related to photosynthesis were down-regulated after PaWB Phytoplasma infection. Our systematic analysis provides comprehensive transcriptomic data about plants infected by Phytoplasma. This information will help further our understanding of the detailed interaction mechanisms between plants and Phytoplasma.

First Report of Elm Yellows Phytoplasma Infecting Clover in China.

In the summer of 2008, phyllody and enlarged petioles resembling symptoms of phytoplasma infection were observed on clover (Trifolium repens) plants in lawns on the campus of Northwest A&F University. Typical phytoplasma-like bacteria were observed in the phloem cells when ultra-thin sections from leaf midrib tissues were examined with transmission electron microscopy. Nested PCR assays were used to verify the association of phytoplasma with the disease. Total DNA was extracted from the phloem of leaf midribs from 20 symptomatic plants and six symptomless plants using the modified CTAB method (1). Using the phytoplasma universal primer pair R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (4), PCR products of 1.4 and 1.2 kb were amplified, respectively, from symptomatic plants only. Jujube witches'-broom (JWB) and paulownia witches'-broom (PaWB) phytoplasma DNA samples served as controls and were used to study group relationships. After sequencing of the 16S rDNA fragment (GenBank Accession No. FJ436792), a BLAST search determined that the clover phytoplasma shared closest homology (99.6%) with JWB (GenBank Accession No. FJ154846) phytoplasmas compared with lesser identity (90.4%) with PaWB (GenBank Accession No. EF199937). Subsequent restriction fragment length polymorphism analysis of the PCR-amplified 1.2-kb 16S rDNA R16(1)F1/R1 fragment indicated that the phytoplasma associated with the disease belongs to subgroup 16SrV-B of the elm yellows phytoplasma group. Clover phyllody phytoplasma were previously reported to infect clover in Canada (GenBank Accession No. L33762) (3) and Italy (GenBank Accession No. X77482) (2). The phytoplasma reported here shared 86.7 and 90.0% identity with the clover phyllody phytoplasma above, respectively, much lower than that with Elm yellows phytoplasma group. To our knowledge, this is the first report of Elm yellows phytoplasma infecting clover in China.

First Report of Syringa oblata and S. reticulata Leafroll Disease in China.

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China.

First Report of Paulownia Witches'-Broom Phytoplasma in China.

Paulownia witches'-broom (PaWB) is one of the most important diseases affecting Paulownia tomentosa trees in China. According to 2006 statistics, the disease has affected 880,000 ha of trees for timber production causing billions of dollars in economic losses. During the spring and summer of 2006, a survey was done in Shaanxi Province to confirm phytoplasma infection of paulownia trees exhibiting symptoms of witches'-broom, stunting, yellowing, and proliferating secondary shoots. Foliage samples were collected from 24 symptomatic and 8 symptomless paulownia plants in eight different production fields. Total DNA was extracted from 0.5 g of leaf midrib and stem phloem tissue with a modified cetyltrimethylammoniumbromide (CTAB) method (3). Resulting DNA extracts were analyzed by a nested PCR assay using phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/ R16R2 (1), which amplified a 1.4-kb and a 1.2-kb product, respectively, from symptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, BfaI, HinfI, and TaqI endonuclease (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup D (16SrI-D) phytoplasma strains. A 1.2-kb 16S rDNA sequence (GenBank Accession No. DQ851169) derived from representative strain PaWB-Shaanxi was identical (100%) to that of PaWB phytoplasma (L27033), a known subgroup 16SrI-D strain from Taiwan (2). The agreement between the RFLP analysis and sequence data confirms that PaWB from Shaanxi is a member of subgroup 16SrI-D. To our knowledge, this is the first report of PaWB disease being present in China and of its association with the 16SrI-D subgroup.

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.

First Report of Witches'-Broom Disease of Chinese Wingnut in China and its Association with a Phytoplasma of Aster Yellows Group (16SrI).

Pterocarya stenoptera C. DC., commonly known as Chinese wingnut, is a fast-growing deciduous tree with tough bark and attractive foliage. Because of its tolerance of compact and nutritionally poor soil, drought, and heat, Chinese wingnut is an important component of the biological diversity in natural ecosystems and is a favorable shade tree in China. Chinese wingnut has also been used as a rootstock for walnuts because of its high resistance to soilborne Phytophthora spp. In the spring of 2004, a disease characterized by witches'-broom symptoms was observed affecting Chinese wingnut trees growing in suburban Taian, Shandong, China. The diseased trees developed dense clusters of highly proliferating branches with shortened internodes, leaves on the affected branches were significantly smaller, and some branches and twigs suffered dieback. Phytoplasma infection was suspected as the cause of this Chinese wingnut witches'-broom (CWWB) disease because the disease occurred in an area where phytoplasmal diseases, such as paulownia witches'-broom (PaWB) and jujube witches'-broom (JWB), are common (3). Nested polymerase chain reactions (PCR) were performed on DNA samples extracted from leaves of six diseased trees using phytoplasma-universal 16S rDNA primers (R16mF2/R16mR1 and R16F2n/ R16R2) (1,2). Results revealed that all diseased trees examined were infected by phytoplasma, whereas PCR assays of leaf samples from two nearby symptomless Chinese wingnut trees were negative. Subsequent restriction fragment length polymorphism analysis of the PCR-amplified 16S rDNA indicated that all diseased trees contained the same phytoplasma and that the CWWB phytoplasma belongs to subgroup B of the "Candidatus Phytoplasma asteris" (AY) group (16SrI). Nucleotide sequence analysis of a 16S rRNA gene cloned from CWWB phytoplasma (GenBank Accession No. AY831966) suggested that this phytoplasma is closely related to, but distinct from, PaWB phytoplasma, another member of group16SrI. To our knowledge, this is the first report of Chinese wingnut witches'-broom disease and of its association with a phytoplasma. Further work is being undertaken to examine the ecological and evolutionary relationship between CWWB phytoplasma and other phytoplasmas in the region and to assess the impact of CWWB on walnut rootstock selection.