Molecular Diversity and Evolutionary Relatedness of Paulownia Witches'-Broom Phytoplasma in Different Geographical Distributions in China.

To reveal the distribution and transmission pathway of Paulownia witches'-broom (PaWB) disease, which is caused by phytoplasmas related to genetic variation, and the adaptability to the hosts and environments of the pathogenic population in different geographical regions in China, in this study, we used ten housekeeping gene fragments, including rp, fusA, secY, tuf, secA, dnaK, rpoB, pyrG, gyrB, and ipt, for multilocus sequence typing (MLST). A total of 142 PaWB phytoplasma strains were collected from 18 provinces or municipalities. The results showed that the genetic diversity was comparatively higher among the PaWB phytoplasma strains, and substantially different from that of the other 16SrI subgroup strains. The number of gene variation sites for different housekeeping genes in the PaWB phytoplasma strains ranged from 1 to 14 SNPs. Among them, rpoB (1.47%) and dnaK (1.12%) had higher genetic variation, and rp (0.20%) had the least genetic variation. The tuf and rpoB genes showed the fixation of positively selected beneficial mutations in the PaWB phytoplasma populations, and all housekeeping genes except tuf followed the neutral evolutionary model. We found an absence of recombination among PaWB phytoplasma sequence types (STs) for each housekeeping gene except dnaK, and no evidence for such recombination events for concatenated sequences of PaWB phytoplasma strains. The 22 sequence types were identified among the concatenated sequences of seven housekeeping genes (rp, fusA, secY, secA, tuf, dnaK, and rpoB) from 105 representative strains. We analyzed all 22 STs by goeBURST algorithm, forming two clonal complexes (CCs) and three singletons. Among them, ST1, as the primary founder of CC1, had the widest geographical distribution, accounting for 72.38% of all strains, with a high frequency of shared sequence type. The results of phylogenetic analysis of the concatenated sequences further revealed that the 105 strains were clustered into two representative lineages of PaWB phytoplasma, with obvious geographical differentiation. The ST1 strains of highly homogeneous lineage-1 were a widespread and predominant population in diseased areas. Lineage-2 contained strains from Jiangxi, Fujian, and Shaanxi provinces, highlighting the close genetic relatedness of the strains in these regions, which was also consistent with the results of most single-gene phylogenetic analysis of each gene. We also found that the variability in the northwest China population was higher than in other geographical populations; the range of genetic differentiation between the south of the Yangtze River population and the Huang-huai-hai Plain (or southwest China) population was relatively large. The achieved diversity and evolution data, as well as the MLST technique, are helpful for epidemiological studies and guiding PaWB disease control decisions.

Rapid and Efficient Detection of 16SrI Group Areca Palm Yellow Leaf Phytoplasma in China by Loop-Mediated Isothermal Amplification.

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrIB subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64oC for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/µl, namely approximately 53 copies/µl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

The Anatomy and Ultrastructure of the Digestive Tract and Salivary Glands of Hishimonus lamellatus (Hemiptera: Cicadellidae).

In recent years, we found that Hishimonus lamellatus Cai et Kuoh is a potential vector of jujube witches'-broom phytoplasma. However, little is known about the anatomy and histology of this leafhopper. Here, we examined histology and ultrastructure of the digestive system of H. lamellatus, both by dissecting and by semi- and ultrathin sectioning techniques. We found that the H. lamellatus digestive tract consists of an esophagus, a filter chamber, a conical midgut and midgut loop, Malpighian tubules, an ileum, and a rectum. Furthermore, both the basal region of the filter chamber epithelium and the apical surface of the midgut epithelium have developed microvilli. We also identify the perimicrovillar membrane, which ensheaths the microvilli of midgut loop enterocyte, and the flame-like luminal membrane, which covers the microvilli of the conical midgut epithelium. In addition, H. lamellatus has the principal and accessory salivary glands. Our observations also showed that the endoplasmic reticulum, mitochondria, and secretory granules were all highly abundant in the secretory cells of the principal salivary glands, while the accessory glands consist of only one ovate or elbow-like acinus. We also briefly contrast the structure of the gut of H. lamellatus with those of other leafhopper species. These results intend to offer help for the future study on the histological and subcellular levels of phytopathogen-leafhopper relationships, including transmission barriers and the binding sites of pathogens and other microorganisms within their leafhopper vectors.

Brenneria corticis sp. nov., isolated from symptomatic bark of Populus×euramericana canker.

A Gram-stain-negative, facultatively anaerobic, motile bacterial strain, designated gBX10-1-2T, was isolated from symptomatic bark of Populus×euramericana canker in China. Phylogenetic analysis based on its 16S rRNA gene sequence showed that the novel isolate belonged to the genus Brenneria, and shared the highest sequence similarity to Brenneria nigrifluens LMG 2694T (98.3 %). In the phylogenetic trees based on the four housekeeping genes sequences, the novel strain formed a separate branch different from B. nigrifluens LMG 2694T, indicating that the novel strain should be classified as a novel species. The genome sequence-derived average nucleotide identity (ANI) values between the novel isolate and B. nigrifluens LMG 2694T, Brenneria roseaesubsp. roseae FRB 222T and Brenneria roseaesubsp. americana FRB 223T were less than 85 %, lower than the proposed species boundary ANI cut-off value (95-96 %). The DNA G+C content was 56.2 mol%, and the main fatty acids were C16 : 0, C16 : 1ω7c, C18 : 1ω7c and C17 : 0cyclo. Based on the phenotypic and genotypic characteristics, strain gBX10-1-2T represents a novel species of genus Brenneria, for which the name Brenneria corticis sp. nov. is proposed. The type strain is gBX10-1-2T (=CFCC 11842T=KCTC 42840T).

Sphingomonas aeria sp. nov., isolated from air.

A Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented and rod-shaped bacterial strain, designated B093034T, was isolated from air at the foot of Xiangshan mountain, located in Beijing, China. Cells of strain B093034T were oxidase-negative and catalase-positive. Growth was observed at 4-41 °C, at pH 4.5-10.0 and at 0-7 % (w/v) NaCl. The isolate contained Q-10 as the predominant isoprenoid quinone, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0 and C14 : 02-OH as the major fatty acids, sym-homospermidine as the major polyamine, and sphingoglycolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, aminolipid, two unidentified phospholipids and three unidentified polar lipids as the polar lipids. The DNA G+C content was 67.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain B093034T grouped with members of the genus Sphingomonas and was closely related to Sphingomonas sanguinis IFO 13937T (96.49 % similarity), Sphingomonas pseudosanguinis G1-2T (96.37 %), Sphingomonas ginsenosidimutansGsoil 1429T (95.99 %) and Sphingomonas endophytica YIM 65583T (95.78 %). On the basis of the polyphasic evidence presented here, strain B093034T represents a novel species of the genus Sphingomonas, for which the name Sphingomonasaeria sp. nov. is proposed. The type strain is B093034T (=CFCC 13949T=LMG 30133T).

Aureimonas populi sp. nov., isolated from poplar tree bark.

Two novel bacterial strains (4M3-2T and 10-107-7) were isolated from poplar tree bark. The strains were Gram-stain-negative facultative aerobes, and produced short rods that were motile because of polar flagella. A phylogenetic tree was reconstructed based on 16S rRNA gene sequences indicating that the two novel strains are related to species of the genus Aureimonas and Aurantimonas. The two novel strains shared the highest 16S rRNA gene sequence similarities with Aureimonasfrigidaquae CW5 7Y-4T (97.1 %) and Aureimonasaltamirensis DSM 21988T (96.6 %)o. The lipids of the novel strain contain diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine and sulfoquinovosyl diacylglycerol. The presence of a distinct glycolipid (sulfoquinovosyl diacylglycerol) is an important chemotaxonomic feature used to distinguish between species of the genera, Aurantimonas and Aureimonas. Additionally, the DNA-DNA hybridization results indicated that the two novel strains represent a novel taxon distinct from Aureimonas frigidaquae. The results of the 16S rRNA gene sequence analysis, as well as the physiological and biochemical characteristics imply that the two novel strains should be assigned to a novel species, with the proposed name Aureimonas populi sp. nov. The type strain is 4M3-2T (=CFCC 11187T=KCTC 42087T).

Phylogenetic analysis of family Neisseriaceae based on genome sequences and description of Populibacter corticis gen. nov., sp. nov., a member of the family Neisseriaceae, isolated from symptomatic bark of Populus × euramericana canker.

Two Gram-stain negative aerobic bacterial strains were isolated from the bark tissue of Populus × euramericana. The novel isolates were investigated using a polyphasic approach including 16S rRNA gene sequencing, genome sequencing, average nucleotide identity (ANI) and both phenotypic and chemotaxonomic assays. The genome core gene sequence and 16S rRNA gene phylogenies suggest that the novel isolates are different from the genera Snodgrassella and Stenoxybacter. Additionally, the ANI, G+C content, main fatty acids and phospholipid profile data supported the distinctiveness of the novel strain from genus Snodgrassella. Therefore, based on the data presented, the strains constitute a novel species of a novel genus within the family Neisseriaceae, for which the name Populibacter corticis gen. nov., sp. nov. is proposed. The type strain is 15-3-5T (= CFCC 13594T = KCTC 42251T).

Populibacterium corticicola gen. nov., sp. nov., a member of the family Jonesiaceae, isolated from symptomatic bark of Populus × euramericana canker.

Four Gram-stain-positive, aerobic, motile bacterial strains were isolated from the bark tissue of Populus × euramericana canker. Growth occurred between 10 and 37 °C and at pH 6-10, with optimal growth at 28-30 °C and pH 7.0-8.0. Growth occurred at 0-3 % (w/v) salinity. The strains were positive for oxidase and catalase activity. The major fatty acids were anteiso-C15 : 0 and C16 : 0. The phospholipid profiles contained diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, two phospholipids and five glycolipids. The peptidoglycan type was A4α, which is based on l-Lys-d-Ser-d-Asp. The DNA G+C content was 58.5 mol%. Based on 16S rRNA gene sequence analysis, as well as physiological and biochemical characteristics, the strains are considered to represent a novel species of a new genus in the family Jonesiaceae. The name proposed is Populibacterium corticicola gen. nov., sp. nov. The type strain of Populibacterium corticicola is 2D-4T (=CFCC 11886T=KCTC 33576T).

Brenneria populi sp. nov., isolated from symptomatic bark of Populus×euramericana canker.

Five Gran-stain-negative, facultatively anaerobic, motile, bacterial strains were isolated from symptomatic bark tissue of Populus×euramericana canker. Strains grew at 4-41 °C, pH 4-10 and 0-6 % (w/v) salinity. They were positive with respect to catalase activity and negative for oxidase activity, nitrate reduction and the Voges-Proskauer reaction. Analysis of 16S rRNA gene sequences indicated that these five poplar isolates belong to the genus Brenneria, having highest sequence similarity of 95.98 % with Brenneria goodwinii LMG 26270(T). These five isolates formed a single cluster based on multilocus sequence analysis, indicating that they all belong to a single taxon within the genus Brenneria, which was confirmed by DNA-DNA hybridization. The DNA G+C content was 54.9-55.7 mol%, and the main fatty acids were C16 : 0, C18 : 1ω7c, C17 : 0 cyclo and C16 : 1ω7c/iso-C15 : 0 2-OH. Based on these results, we describe a novel species of the genus Brenneria with the proposed name Brenneria populi sp. nov. The type strain is D9-5(T) ( = CFCC 11963(T) = KCTC 42088(T)).

Multilocus sequences confirm the close genetic relationship of four phytoplasmas of peanut witches'-broom group 16SrII-A.

Four witches'-broom diseases associated with Arachis hypogaea (peanut), Crotalaria pallida, Tephrosia purpurea, and Cleome viscosa were observed in Hainan Province, China during field surveys in 2004, 2005, and 2007. In previously reported studies, we identified these four phytoplasmas as members of subgroup 16SrII-A, and discovered that their 16S rRNA gene sequences were 99.9-100% identical to one another. In this study, we performed extensive phylogenetic analyses to elucidate relationships among them. We analyzed sequences of the 16S rRNA gene and rplV-rpsC, rpoB, gyrB, dnaK, dnaJ, recA, and secY combined sequence data from two strains each of the four phytoplasmas from Hainan province, as well as strains of peanut witches'-broom from Taiwan (PnWB-TW), "Candidatus Phytoplasma australiense", "Ca. Phytoplasma mali AT", aster yellows witches'-broom phytoplasma AYWB, and onion yellows phytoplasma OY-M. In the 16S rRNA phylogenetic tree, the eight Hainan strains form a clade with PnWB-TW. Analysis of the seven concatenated gene regions indicated that the four phytoplasmas collected from Hainan province cluster most closely with one another, but are closely related to PnWB-TW. The results of field survey and phylogenetic analysis indicated that Cr. pallida, T. purpurea, and Cl. viscosa may be natural plant hosts of peanut witches'-broom phytoplasma.

[Cloning, expression and characterization of tRNA-isopentenyltransferase genes (tRNA-ipt) from paulownia witches'-broom phytoplasma].

OBJECTIVE: To identify the tRNA-ipt gene of phytoplasmas and analyze the relationship between tRNA-ipt and synthesis of cytokinin as well as pathogenesis in phytoplasmas. METHODS: The paulownia witches'-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E. coli and specific antibody was prepared. Then the growth curve and cytokinin contents of E. coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA respectively. RESULTS: The length of tRNA-ipt genes from PaWB as well as mulberry dwarf, periwinkle virescence and Chinaberry witches'-broom phytoplasmas were 876 bp. All these genes encoded the proteins consisting of 291 amino acids. They contained and indentical motif (GPTASGKT) at N-terminal region related to the ATP or GTP binding sites. Four phytoplasma tRNA-IPTs shared the 99.1-99.5%, amino acid sequence indentity with each other, shared 95.4-99.3% sequence identity with the same group phytoplasmas, whereas the less than 70% identity with 16SrX apple proliferation and 16SrXII Australia grapevine yellows phytoplasmas. The expression of the tRNA-IPT protein and localization in the phloem in phytoplasma-infected paulownia were confirmed by Western blotting and immunofluorescence detection. The determination of growth curve suggested that the growth rate increase of E. coli was affected by the transformation of exogenous tRNA-ipt gene,which might be associated with the cytokinin accumulation. CONCLUSION: This protein was assumed to be involved in the cytokinin synthesis in phytoplasmas as well as other bacteria, which may play an important role in pathogenic processes of phytoplasmas and symptom development.

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.

[Complete sequence of a full-length DNA and molecular characterization of one plasmid from chinaberry (Melia azedarach Z) witches'-broom phytoplasma].

OBJECTIVE: To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization. METHODS: Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain (CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares. Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe. RESULTS: One complete plasmid was sequenced from CWBFq. pCWBFq comprised 4446 bp and had a nucleotide content of 73.5% A + T and encoded six proteins. Protein P2, P3, P4 and P5 of pCWBFq contained 3, 2, 1 and 2 tranmembrane domains respectively, and their predicted signal peptide values were 0.989, 0.505, 0.918 and 0.914 respectively. Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9.6% -85.6% , however, the homology of different SSB proteins was between 74.0% - 89.4%. Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq. In addition, The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy), periwinkle virescence phytoplasma-Hainan stanin (PeVHn), chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma - Puyang strain (MDPy), whereas, no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj), cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc). CONCLUSION: The plasmid encoded a replication associated protein (RepA) and a single-stranded DNA binding protein (SSB), which were for the replication of plasmid. Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localized to the membrane. The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation, among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids. CWBFq, PaWBNy, PeVHn, CWBFz and MDPy possessed distinct plasmids in terms of number and size, whereas there was no plasmid detected in JWBBj, CLYXc and BiWBNc, perhaps as a result of low homology among repA genes in plasmids of JWBBj, CLYXc and BiWBNc.