New 16Sr subgroups and distinct single nucleotide polymorphism lineages among grapevine Bois noir phytoplasma populations

Bois noir (BN) is an insect-transmitted grapevine yellows disease caused by phytoplasmas belonging to the stolbur subgroup 16SrXII-A. In Italy, increasing prevalence of stolbur phytoplasma strains in vineyards suggests progressive spread of the disease and potential for heavy impacts on the wine industry. In this study, we investigated the genetic diversity of stolbur phytoplasma strains in BN phytoplasma populations. Nucleotide sequences of 16S rRNA genes from stolbur phytoplasma strains affecting vineyards in the Lombardy region of Italy and stolbur phytoplasma 16S rDNA sequences retrieved from GenBank were subjected to virtual restriction fragment length polymorphism analysis. Calculation of virtual restriction similarity coefficients revealed the presence of new subgroups in group 16SrXII (stolbur phytoplasma group). Representative strains of confirmed new subgroups 16SrXII-F (XII-F) and XII-G and tentative new subgroups XII-A1 through XII-A19, XII-H, XII-I, and XII-J as well as known subgroup XII-A were from grapevines; strains representing three additional tentative new subgroups (XII-K, XII-L and XII-M) were from other plant hosts. Nucleotide sequence alignments identified no less than nine genetically distinct 16S rDNA single nucleotide polymorphism lineages from grapevine, indicating a high degree of genetic heterogeneity within BN phytoplasma populations. The findings open new opportunities for in-depth studies of the distribution of grapevine-associated 16SrXII phytoplasma strains in weeds, insect vector populations and grapevines from vineyards located in different geographic areas.     

First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome

Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.     

A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group

An azalea little leaf (AzLL) disease characterised by abnormally small leaves, yellowing and witches'‐broom growth symptoms was observed in suburban Kunming, southwest China. Transmission electron microscopic observations of single‐membrane‐bound, ovoid to spherical bodies in phloem sieve elements of diseased plants and detection of phytoplasma‐characteristic 16S rRNA gene sequence in DNA samples from diseased plants provided evidence linking the disease to infection by a phytoplasma. Results from restriction fragment length polymorphism, phylogenetic and comparative structural analyses of multiple genetic loci containing 16S rRNA, rpsS, rplV, rpsC and secY genes indicated that the AzLL phytoplasma represented a distinct, new 16Sr subgroup lineage, designated as 16SrI‐T, in the aster yellows phytoplasma group. The genotyping also revealed that the AzLL phytoplasma represented new rp and secY gene lineages [rp(I)‐P and secY(I)‐O, respectively]. Phylogenetic analyses of secY and rp gene sequences allowed clearer distinctions between AzLL and closely related strains than did analysis of 16S rDNA.     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).     

Molecular Identification and Phylogenetic Analysis of Sugarcane Yellow Leaf Phytoplasma (SCYLP) in Egypt

Samples of sugarcane leaves were collected from different commercial fields and breeding stations in Egypt. Aetiology of sugarcane phytoplasma disease was investigated using nested PCR. Phytoplasma‐specific primers (P1/P7 and R16F2n/R16R2) were used to amplify a fragment of the 16S rRNA gene. Sequencing and restriction fragment length polymorphism analyses revealed that the tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group. Phylogenetic analyses of 60 screened accessions of 16S ribosomal RNA gene sequences of Candidatus phytoplasmas comprising those collected from Egypt (this study) and those extracted from GenBank showed that they split into two distinct clusters. All the phytoplasmas form a stable phylogenetic subcluster, as judged by branch length and bootstrap values of 100% in the 16S group cluster. Results of phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. Conversely, based on the analysis of the 16S‐23S region, examined isolates segregated into four different clusters suggesting a notable heterogeneity between them. These results are the first record of the presence of phytoplasma in association with sugarcane yellow leaf in Egypt.     

Detection and characterisation of a phytoplasma associated with cucumber (Cucumis sativus) regional yellows disease in Iran

Abstract

A phytoplasma was detected in cucumber (Cucumis sativus), exhibiting regional yellows symptoms in leaves, stem and fruits, that was grown in the greenhouse near Tehran (Iran). Since this is a previously undescribed disease, the name cucumber regional yellows have been tentatively assigned to it. Based on in silico RFLP and phylogenetic analysis of PCR-amplified 16S rDNA sequences, the phytoplasma associated with regional yellows disease was identified as a new member of phytoplasma 16S rRNA group VI (16SrVI-A) with closest relationships to zucchini phyllody phytoplasma (KP119494). According to our results, cucumber regional yellows phytoplasma could be designated as a subgroup VI-A.     

First Report of Aster Yellows Phytoplasma (16SrI‐B) Associated with Witches' Broom Disease of Melia azedarach var. japonica in Korea

Melia azedarach var. japonica trees with leaf yellowing, small leaves and witches' broom were observed for the first time in Korea. A phytoplasma from the symptomatic leaves was identified based on the 16Sr DNA sequence as a member of aster yellows group, ribosomal subgroup 16SrI‐B. Sequence analyses of more variable regions such as 16S–23S intergenic spacer region, secY gene, ribosomal protein (rp) operon and tuf gene showed 99.5?100% nucleotide identity to several GenBank sequences of group 16SrI phytoplasmas. Phylogenetic analysis confirmed that the Melia azedarach witches' broom phytoplasma belongs to aster yellows group.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Identification of ‘Candidatus Phytoplasma asteris' (16SrI‐B) Causing Witches' Broom Disease of Mallotus japonicus in Korea

Mallotus japonicus with witches' broom disease were observed in Jeollabuk‐do, Korea. A phytoplasma from the infected leaves was identified, based on the 16S rDNA, 16S‐23S intergenic spacer region, and fragment of rp operon and tuf gene sequences. The 16S rDNA sequences exhibited maximum (99.7%) similarity with Iranian lettuce phytoplasma, the rp operon sequences exhibited 100% similarity with Goldenrain stunt phytoplasma, and the tuf gene sequences exhibited 99.8% similarity with Japanese spurge yellows phytoplasma. Results of the sequence analysis and phylogenetic studies confirmed that the phytoplasma associated with M. japonicus in Korea was an isolate of Aster Yellows group (subgroup16SrI‐B).     

First Report of Aster Yellows Phytoplasma in Goldenrain Tree (Koelreuteria paniculata) in Korea

Aster yellows phytoplasma was detected for the first time in goldenrain tree (Koelreuteria paniculata) growing in Sinpyeong‐myeon, Jeollabuk‐do, South Korea. DNA was extracted from the infected leaf samples and part of the 16S rDNA, rp operon and tuf gene were amplified using R16F2n/R2 and gene‐specific primers. The sequence analysis showed that the phytoplasma was closely related (99%) to members of the Aster Yellows (AY) group, and belonging to 16Sr I, subgroup B. Moreover, the 16S rDNA sequences of the isolate showed 88–96% identity with members of other 16Sr and undesignated groups. Based on the sequence identity and phylogenetic studies, it was confirmed that phytoplasma infecting goldenrain tree in South Korea belongs to the AY group.     

Association of Elm Yellows Subgroup 16SrV‐B Phytoplasma with a Disease of Hovenia dulcis

Japanese raisin (Hovenia dulcis) trees with typical phytoplasma‐like symptoms were observed for the first time in South Korea. The disease, named Japanese raisin witches’ broom, is progressively destructive. The cause of the graft‐transmissible disease was confirmed by electron microscopy and molecular studies. The 16S rDNA sequence analysis showed that the phytoplasma was closely related to the elm yellows (EY) group, ribosomal subgroup 16SrV‐B. The 16S‐23S rDNA intergenic spacer region, fragment of rp operon and secY gene sequences had 96–99% similarity with members of EY phytoplasma. Based on the sequence analyses and phylogenetic studies, it was confirmed that the phytoplasma infecting Japanese raisin trees in Korea belongs to the EY group.     

Molecular Identification of a ‘Candidatus Phytoplasma ziziphi’‐related Strain Infecting Amaranth (Amaranthus retroflexus L.) in China

Amaranth (Amaranthus retroflexus L.) is a common weed that grows vigorously in orchards, roadside verges, fields, woods and scrubland in China. In 2009, phytoplasma disease surveys were made in orchards in Beijing, China, and stem/leaf tissues were collected from asymptomatic amaranths. Direct PCR using universal phytoplasma primers P1/P7 detected 16S rRNA gene sequences in every DNA sample extracted from the symptomless amaranths. Sequence alignment and phylogenetic analyses of the 16S rRNA gene determined that the amaranth phytoplasma strain was related to ‘Candidatus Phytoplasma ziziphi’. Furthermore, virtual RFLP pattern analysis showed that the amaranth phytoplasma belonged to the 16SrV‐B subgroup. This is the first report of symptomless plants containing a ‘Candidatus Phytoplasma ziziphi’‐related strain.     

Detection of Aster Yellows Phytoplasma (16SrI) Associated with Prickly Ash (Zanthoxylum schinifolium S. et Z.) witches' Broom Disease in Korea

Prickly ash trees with shortened internodes, proliferation of shoots, phyllody and witches' brooms were observed for the first time in Korea. A phytoplasma was detected in infected trees by polymerase chain reaction amplification of 16S rDNA, 16S–23S intergenic spacer region and the fragment of rp operon sequences. The 16S rDNA sequences exhibited maximum (99.6%) similarity with Iranian lettuce phytoplasma, and the sequences of rp operon exhibited maximum (100%) similarity with golden rain phytoplasma. Based on the sequence analysis and phylogenetic studies, it was confirmed that phytoplasma infecting prickly ash trees in Korea belongs to the aster yellows group (subgroup 16SrI‐B).     

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.     

Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.     

Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.