Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.     

Analysis of the genus Petagnaea Caruel (Apiaceae), using new molecular and literature data

In Southern Italy, an endemic monotypic genus belonging to family Apiaceae occurs: Petagnaea (P. gussonei), relict of Tertiary flora, belonging to subfamily Saniculoideae. At present, P. gussonei is an endangered species and is included in various lists of species deserving special protection. The genus belongs to scapose hemicryptophytes and shares a sciaphilous habitat (hygrophilous woodland). This study is aimed at doing a complete contribution about the evolutionary history of Petagnaea, using molecular markers as plastidial DNA (cpDNA), nuclear ribosomal DNA (rDNA) and data present in literature. We used nucleotide sequences from four regions of the chloroplast genome (rps16 intron, trnL^(UAA) intron, atpB-rbcL intergenic spacer, and partial matK gene) to investigate possible haplotypes in Petagnaea populations. To have an idea of the molecular relationships of all populations of P. gussonei, the internal transcribed spacer (ITS) sequences, already employed in recent studies, were obtained for 18 populations. These sequences in combination with other Saniculoideae ITS sequences available from GenBank have been used for a further phylogenetic analysis. The results agree with the current classification of Saniculoideae in placing P. gussonei in tribe Saniculeae, since P. gussonei is in basal position to Sanicula. According to intraspecific chloroplast DNA diversity, no different haplotypes were detected. In addition to molecular data, morphology, cytology, phytochemistry and conservation status have been considered in the discussion.     

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).     

A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex

We constructed a two-locus database, comprising partial translation elongation factor (EF-1α) gene sequences and nearly full-length sequences of the nuclear ribosomal intergenic spacer region (IGS rDNA) for 850 isolates spanning the phylogenetic breadth of the Fusarium oxysporum species complex (FOSC). Of the 850 isolates typed, 101 EF-1α, 203 IGS rDNA, and 256 two-locus sequence types (STs) were differentiated. Analysis of the combined dataset suggests that two-thirds of the STs might be associated with a single host plant. This analysis also revealed that the 26 STs associated with human mycoses were genetically diverse, including several which appear to be nosocomial in origin. A congruence analysis, comparing partial EF-1α and IGS rDNA bootstrap consensus, identified a significant number of conflicting relationships dispersed throughout the bipartitions, suggesting that some of the IGS rDNA sequences may be non-orthologous. We also evaluated enniatin, fumonisin and moniliformin mycotoxin production in vitro within a phylogenetic framework.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.     

Suitability of chloroplast LSU rDNA and its diverse group I introns for species recognition and phylogenetic analyses of lichen-forming Trebouxia algae

To date, species identification of lichen photobionts has been performed principally on the basis of microscopic examinations and molecular data from nuclear-encoded genes. In plants, the chloroplast genome has been more readily exploited than the nuclear genome for systematic investigations. At the present time, very little information is available about the chloroplast genome of lichen-forming algae. For this reason, we have sequenced a portion of the gene encoding for the chloroplast large sub-unit rRNA (LSU rDNA) as a new molecular marker. Sequencing of the chloroplast LSU rDNAs revealed the existence of an unusual diversity of group I introns (a total of 31) within 15 analyzed Trebouxia species. The number, sequence and insertion site of these introns were very different among species, contributing to their recognition. A relatively large intron-free portion of the chloroplast LSU rDNA and part of the nuclear ribosomal cistron (18S–5.8S–26S) between the nuclear internal transcribed spacers (nrITS) were subjected to phylogenetic analyses. The obtained results indicate that data combination from both nuclear and chloroplast sequences can improve phylogenetic accuracy. Herein, we propose the suitability of both intronic and exonic sequences of the chloroplast LSU rDNA for species recognition, and an exonic sequence spanning from position 879 to 1837 in the Escherichia coli 23S rDNA for phylogenetic analyses of Trebouxia phycobionts.     

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).     

Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions

A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome. The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method. The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5′ and 3′ ends of the maize 26 S rDNA have been located by comparison with the E. coli sequence. The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA. In all cases, there are numerous regions of 70% or higher homology.     

Phylogenetic analysis of the insect order Odonata using 28S and 16S rDNA sequences: a comparison between data sets with different evolutionary rates

Molecular phylogenetic analyses were conducted for the insect order Odonata with a focus on testing the effectiveness of a slowly evolving gene to resolve deep branching and also to examine: (i) the monophyly of damselflies (the suborder Zygoptera); and (ii) the phylogenetic position of the relict dragonfly Epiophlebia superstes. Two independent molecular sources were used to reconstruct phylogeny: the 16S rRNA gene on the mitochondrial genome and the 28S rRNA gene on the nuclear genome. A comparison of the sequences showed that the obtained 28S rDNA sequences have evolved at a much slower rate than the 16S rDNA, and that the former is better than the latter for resolving deep branching in the Odonata. Both molecular sources indicated that the Zygoptera are paraphyletic, and when a reasonable weighting for among‐site rate variation was enforced for the 16S rDNA data set, E. superstes was placed between the two remaining major suborders, namely, Zygoptera and Anisoptera (dragonflies). Character reconstruction analysis suggests that multiple hits at the rapidly evolving sites in the 16S rDNA degenerated the phylogenetic signals of the data set.     

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.     

Specific Detection of Bradyrhizobium and Rhizobium Strains Colonizing Rice (Oryza sativa) Roots by 16S-23S Ribosomal DNA Intergenic Spacer-Targeted PCR

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

Differentiation of petroleum hydrocarbon-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. based on PCR-RFLP of the 16S-23S rDNA intergenic spacer region

Genetic diversity among 43 petroleum hydrocarbon-degrading Pseudomonas belonging to four different species and the type strain Pseudomonas aeruginosa MTCC1034 was assessed by using restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified 16S–23S rDNA intergenic spacer regions (ISRs) polymorphism. PCR amplification from all Pseudomonas species yielded almost identical ISR amplicons of “?” 800 bp and in nested PCR of “?” 550 bp. The RFLP analysis with MboI and AluI revealed considerable intraspecific variations within the Pseudomonas species. The dendrogram constructed on the basis of the PCR-RFLP patterns of 16S–23S rDNA intergenic spacer regions differentiated all the species into seven different clusters.     

Transfer of Two Burkholderia and An Alcaligenes Species to Ralstonia Gen. Nov.

Based on the results of phenotypic characterization, cellular lipid and fatty acid analysis, phylogenetic analysis of 16S rDNA nucleotide sequences and rRNA-DNA hybridization, Burkholderia pickettii, Burkholderia solanacearum and Alcaligenes eutrophus are transferred to the new genus Ralstonia, and Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb, nov., and R. eutropha (Davis 1969) comb. nov. are proposed. The type species of the new genus is R. pickettii. Type strain of R. pickettii is ATCC 27511^T, of R. solanacearum is ATCC 10696^T, and of R. eutropha is ATCC 17697^T.     

Presence of two mitochondrial genomes in the mytilid Perumytilus purpuratus: Phylogenetic evidence for doubly uniparental inheritance

This study presents evidence, using sequences of ribosomal 16S and COI mtDNA, for the presence of two mitochondrial genomes in Perumytilus purpuratus. This may be considered evidence of doubly uniparental mtDNA inheritance. The presence of the two types of mitochondrial genomes differentiates females from males. The F genome was found in the somatic and gonadal tissues of females and in the somatic tissues of males; the M genome was found in the gonads and mantle of males only. For the mitochondrial 16S region, ten haplotypes were found for the F genome (nucleotide diversity 0.004), and 7 haplotypes for the M genome (nucleotide diversity 0.001), with a distance Dxy of 0.125 and divergence Kxy of 60.33%. For the COI gene 17 haplotypes were found for the F genome (nucleotide diversity 0.009), and 10 haplotypes for the M genome (nucleotide diversity 0.010), with a genetic distance Dxy of 0.184 and divergence Kxy of 99.97%. Our results report the presence of two well-differentiated, sex-specific types of mitochondrial genome (one present in the male gonad, the other in the female gonad), implying the presence of DUI in P. purpuratus. These results indicate that care must be taken in phylogenetic comparisons using mtDNA sequences of P. purpuratus without considering the sex of the individuals.     

Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus

According to the conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic 16S-23S rDNA intergenic spacers, namely, IGS^GLAV, IGS^GLV, IGS^lA, IGS^G and IGS^A; while the strain CG021 has the same types of IGSs except lacking IGS^A. Among these five IGS types, IGS^GLAV is the biggest type, including the gene cluster of tRNA^Glu - tRNA^Lys - tRNA^Ala - tRNA^Val; IGS^GLV includes that of tRNA^Glu-tRNA^Lys-tRNA^Val; IGS^AG, tRNA^Ala-tRNA^Glu; IGS^IA, tRNA^Ile-tRNA^Ala; IGS^G, tRNA^Glu and IGS^A, tRNA^Ala. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non-coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。