Molecular detection of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in Palestine.

Citrus hosts various phytopathogens that have impacted productivity, including viroids. Missing data on the status of viroids in citrus in Palestine were not reported. This study was aimed to detect any of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in the Palestinian National Agricultural Research Center (NARC) germplasm collection Field inspections found symptoms such as leaf epinasty; vein discoloration, and bark cracking on various citrus varieties. RT-PCR revealed a significant prevalence of CVd-IV; CEVd and CVd-III (47%, 31%, and 22%; respectively). CVd-III variants with 91.3% nucleic acid sequence homology have been reported. The sequence of each viroid were deposited in GenBank as (OP925746 for CEVd, OP902248 and OP902249 for CVd-III-PS-1 and -PS-2 isolates, and OP902247 for CVd-IV). This was the first to report three of citrus viroids in Palestine, appealing to apply of phytosanitary measures to disseminate healthy propagating materials free from viroids.

Identification of lactobacilli by pheS and rpoA gene sequence analyses.

The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Reclassification of Lactobacillus amylophilus LMG 11400 and NRRL B-4435 as Lactobacillus amylotrophicus sp. nov.

The taxonomic position of six Lactobacillus amylophilus strains isolated from swine waste-corn fermentations was reinvestigated. All strains were included in a multilocus sequence analysis (MLSA) study for species identification of Lactobacillus using the genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA). Partial pheS and rpoA gene sequences showed that strains LMG 11400 and NRRL B-4435 represent a separate lineage that is distantly related to the type strain of L. amylophilus, LMG 6900T, and to three other strains of the species. The MLSA data showed that the two strains LMG 11400 and NRRL B-4435 constituted a distinct cluster, sharing 100% pheS and rpoA gene sequence similarity. The other reference strains clustered together with the type strain of L. amylophilus, LMG 6900T, and were clearly differentiated from strains LMG 11400 and NRRL B-4435 (80 and 89% pheS and rpoA gene sequence similarity, respectively). The 16S rRNA gene sequences of the latter two strains are 100% identical, with the nearest phylogenetic neighbour L. amylophilus LMG 6900T showing only 97.2% 16S rRNA gene sequence similarity. Further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the two strains represent a single, novel Lactobacillus species, for which the name Lactobacillus amylotrophicus sp. nov. is proposed. The type strain is LMG 11400T (=NRRL B-4436T=DSM 20534T).

Reclassification of Lactobacillus brevis strains LMG 11494 and LMG 11984 as Lactobacillus parabrevis sp. nov.

A polyphasic study revealed taxonomic heterogeneity among reference strains of the species Lactobacillus brevis. Representative strains of L. brevis and related taxa were investigated by partial sequence analysis of the housekeeping gene encoding the alpha-subunit of phenylalanyl-tRNA synthase (pheS). Species-specific clusters were delineated for all taxa studied except for two L. brevis strains, LMG 11494 and LMG 11984, respectively isolated from cheese and wheat, which occupied a distinct position. Their phylogenetic affiliation was determined using 16S rRNA gene sequence analysis and it was found that both strains (with 99.9 % gene sequence similarity between them) belonged to the Lactobacillus buchneri group, with nearest neighbours Lactobacillus hammesii and L. brevis (gene sequence similarities of 99.2 and 98.1 %, respectively). Further genotypic and phenotypic studies, including fluorescent amplified fragment length polymorphism, DNA-DNA hybridization and DNA G+C content, clearly demonstrated that the two strains represent a single novel taxon for which the name Lactobacillus parabrevis sp. nov. is proposed (type strain LMG 11984(T)=ATCC 53295(T)).

Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.

The applicability of a multilocus sequence analysis (MLSA)-based identification system for lactobacilli was evaluated. Two housekeeping genes that code for the phenylalanyl-tRNA synthase alpha-subunit (pheS) and RNA polymerase alpha-subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the alpha-subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8-100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA-DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.

Enterococcus silesiacus sp. nov. and Enterococcus termitis sp. nov.

Three enterococci constituted two aberrant branches after numerical analysis of (GTG)5-PCR fingerprints: analogous patterns were found for two water isolates, strains W213 and W442T, and a separate position was found for an isolate from the gut of a termite, strain LMG 8895T. 16S rRNA gene sequence analysis classified all three strains in the Enterococcus faecalis species group. Further sequencing analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase alpha-subunit) and whole-cell-protein analysis confirmed a distinct position for the two water isolates and the termite strain, respectively. DNA-DNA hybridization experiments and distinct phenotypic features between the strains studied and representatives of the E. faecalis species group confirmed novel species status, respectively, for the two water isolates, strains W213 and W442T, and for strain LMG 8895T. The names Enterococcus silesiacus sp. nov. and Enterococcus termitis sp. nov. are proposed for the novel taxa, with W442T (= CCM 7319T = LMG 23085T) and LMG 8895T (= CCM 7300T) as the respective type strains.

Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980).

Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99.3 to 99.8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S-23S rRNA internally transcribed spacer (ITS) sequences revealed 98.3-100% similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA-DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70% and confirmed that these names are synonyms.

Reclassification of Enterococcus flavescens Pompei et al. 1992 as a later synonym of Enterococcus casseliflavus (ex Vaughan et al. 1979) Collins et al. 1984 and Enterococcus saccharominimus Vancanneyt et al. 2004 as a later synonym of Enterococcus italicus Fortina et al. 2004.

The taxonomic relatedness between the species Enterococcus casseliflavus and Enterococcus flavescens and between Enterococcus italicus and Enterococcus saccharominimus was investigated. Literature data had already indicated the synonymy between E. casseliflavus and E. flavescens, but this observation had not been formally published. Additional evidence that the two taxa represent a single species was provided by comparison of the partial sequences for three housekeeping genes, phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the alpha subunit of ATP synthase (atpA). Additional genomic data derived from DNA-DNA hybridization demonstrated that the two species are synonymous. For E. italicus and E. saccharominimus, two recently described taxa, a high 16S rRNA gene sequence similarity of >99% and analogous phenotypic features indicated a close taxonomic relatedness. The same multilocus sequence analysis scheme for the three housekeeping genes was also applied for E. italicus and E. saccharominimus and indicated possible conspecificity, an observation that was also confirmed by a high DNA-DNA hybridization value (>or=78%). Data from the present study led to the proposal that E. flavescens should be reclassified as a later synonym of E. casseliflavus and that E. saccharominimus should be reclassified as a later synonym of E. italicus.

Enterococcus devriesei sp. nov., associated with animal sources.

The taxonomic position of two bovine strains, LMG 13603 and LMG 14595, assigned to the species Enterococcus raffinosus on the basis of biochemical features, was reinvestigated. Both reference strains and two other isolates, 6/1 (=LMG 22829) originating from a charcoal-broiled river lamprey and IE38.4 (=LMG 22830) from the air of a poultry slaughter by-product processing plant, occupied a clearly separate position, on the basis of sequence analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase alpha-subunit), relative to the type strain of E. raffinosus and all other enterococcal species with validly published names. 16S rRNA gene sequencing of strains LMG 13603, LMG 14595, 6/1 and IE38.4 confirmed their phylogenetic position in the Enterococcus avium species group, there being more than 99 % 16S rRNA gene sequence similarity to most members of the group, including E. raffinosus, and revealed Enterococcus pseudoavium as the closest phylogenetic relative (99.8-99.9 %). Further phenotypic and genotypic analyses using whole-cell-protein electrophoresis, (GTG)(5)-PCR fingerprinting, ribotyping and DNA-DNA hybridization experiments demonstrated that all four strains represent a novel enterococcal species, for which the name Enterococcus devriesei sp. nov. is proposed. The type strain is LMG 14595T (=CCM 7299T).

Enterococcus canintestini sp. nov., from faecal samples of healthy dogs.

The taxonomic position of strain LMG 13590(T), originally isolated from dog faeces and classified as Enterococcus dispar in the BCCM/LMG Bacteria Catalogue, was reinvestigated. This strain and 12 recent isolates from faecal samples of healthy dogs occupied a clearly separate position when investigated with multilocus sequence analysis (MLSA) of the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS). The 16S rRNA gene sequence of one representative strain showed highest similarities of 98-99% with E. dispar LMG 13521(T), Enterococcus canis LMG 12316(T) and Enterococcus asini LMG 18727(T). A further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the 13 enterococcal dog faecal strains represent a single, novel Enterococcus species for which the name Enterococcus canintestini sp. nov. is proposed. The type strain is LMG 13590(T) (=CCM 7285(T)).

Enterococcus aquimarinus sp. nov., isolated from sea water.

Two enterococcal strains LMG 16607(T) and LMG 16612 originating from sea water were analysed in a polyphasic taxonomic study. Both strains, assigned as Enterococcus sp. in the BCCM/LMG culture collection, possessed analogous protein profiles, but these were different from all other enterococcal species. 16S rRNA gene sequence analysis of one strain showed the highest similarity, 96.9-96.1%, with its closest phylogenetic neighbours Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus saccharominimus and Enterococcus italicus. Further genomic analysis by (GTG)(5)-PCR fingerprinting and sequence analysis of the housekeeping gene phenylalanyl-tRNA synthase (pheS) and distinct biochemical features confirmed that the two strains represent a novel enterococcal species for which the name Enterococcus aquimarinus sp. nov. is proposed. The type strain is LMG 16607(T) (=CCM 7283(T)).

Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes.

The aim of this study was to evaluate the use of RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase (pheS) gene sequences as species identification tools for enterococci. Ninety-six representative strains comprising all currently recognized Enterococcus species were examined. rpoA gene sequences generated a robust classification into species groups similar to the one based on 16S rRNA gene sequence analysis. On the other hand, the pheS gene is a fast-evolving clock even better suited for species delineation than the rpoA gene, but not for recognition of species groups within Enterococcus as determined by both rpoA and 16S rRNA genes. All enterococcal species were clearly differentiated on the basis of their rpoA and pheS sequences. Evaluation of intraspecies variation showed that both rpoA and pheS genes have a high degree of homogeneity among strains of the same species. Strains of the same enterococcal species have at least 99% rpoA and 97% pheS gene sequence similarity, whereas, different enterococcal species have at maximum 97% rpoA and 86% pheS gene sequence similarity. It was concluded that both genes can be used as reliable tools for identification of clinical and environmental species of Enterococcus and are efficient screening methods for the detection of novel species. The sequence data obtained in this study were compared to the available atpA and 16S rRNA gene sequences. The MLSA approach to Enterococcus taxonomy provides portable, highly reproducible data with lower costs for rapid identification of all enterococcal species.