Identical 16S rDNA Sequences Research Articles

First Report of Soft Rot Caused by Pectobacterium polaris on Lettuce in China.

Lettuce (Lactuca sativa) is a leafy vegetable that belongs to the family Asteraceae. It is widely cultivated and consumed around the world. In May 2022, lettuce plants (cv. 204) showing soft rot symptoms were observed in greenhouses in Fuhai District (25°18'N, 103°6'E), Kunming City, Yunnan Province, China. The disease incidence in three greenhouses (0.3 ha in size) was between 10% to 15%. The lower parts of the outer leaves showed brown and water-soaked symptoms, but at the same time the roots were asymptomatic. Sclerotinia species can cause soft decay on lettuce leaves, known as lettuce drop, which can produce symptoms partially resembling those of bacterial soft rot (Subbarao 1998). The absence of white mycelium or black sclerotia on the leaf surfaces of diseased plants indicated that Sclerotinia species were not responsible for the disease. Instead, it is more likely that bacterial pathogens were the cause. Fourteen diseased plants were sampled from three greenhouses, and potential pathogens were isolated from the leaf tissues of six plant individuals. Leaf samples were cut into pieces ca. 0.5 cm in length. The pieces were then surface-sterilized by dipping in 75% ethanol for 60 sec, followed by three successive rinses using sterile distilled water. The tissues were immersed in 250 µl of 0.9% saline in 2 mL microcentrifuge tubes and gently pressed down with grinding pestles for 10 sec. The tubes were let stand still for 20 min. Aliquots (20 μl) 100-fold dilutions of the tissue suspensions were plated onto Luria-Bertani (LB) plates and incubated at 28°C for 24 h. Three single colonies were picked from each LB plate and restreaked five times for purity. After purification, eighteen strains were obtained, and nine of these were identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991). Six out of nine strains (6/9) belonged to the genus Pectobacterium (OP968950-OP968952, OQ568892- OQ568894), two strains (2/9) belonged to the genus Pantoea (OQ568895 and OQ568896), and one strain (1/9) belonged to Pseudomonas sp. (OQ568897). Since the Pectobacterium strains shared identical 16S rDNA sequence, strains CM22112 (OP968950), CM22113 (OP968951) and CM22132 (OP968952) were selected as representative strains for further testing. The 16S rDNA sequences of Pectobacterium strains were 100% identical to that of the P. polaris strain NIBIO 1392 (NR_159086.1). To identify the strains to the species level, multilocus sequence analysis (MLSA) was performed using sequences of six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (OP972517-OP972534) (Ma et al. 2007; Waleron et al. 2008). Phylogenetic analysis showed that the strains clustered with P. polaris type strain NIBIO1006T (Dees et al. 2017). They were all capable of utilizing citrate, which is an important biochemical feature in distinguishing P. polaris from its most closely related sister species P. parvum (Pasanen et al. 2020). Lettuce plants (cv. 204), at the rosette stage, were inoculated with the strains CM22112 and CM22132 by injecting 100 µl of bacterial suspensions (107 CFU·mL-1) into the lower parts of the leaf; for the controls, 100 µl of saline was used instead. Inoculated plants were incubated at room temperature (23°C) and 90% relative humidity. Five days after inoculation, only the bacteria-inoculated lettuce showed severe soft rot symptoms. Similar results were observed in two independent experiments. Bacterial colonies were obtained from the infected lettuce leaves, which showed identical sequences to P. polaris strains CM22112 and CM22132. Therefore, these strains fulfilled Koch's postulates for lettuce soft rot. P. polaris is prevalent on potato in many countries (Dees et al. 2017). To our knowledge, this is the first report of P. polaris causing soft rot on lettuce in China. This disease could seriously affect the appearance and saleability of lettuce. Further research on the epidemiology and management strategies of the disease is needed.

First Report of Rhizome Rot Caused by Pectobacterium brasiliense on Ginger in China.

In August 2020, ginger (Zingiber officinale) rhizomes (cv. Mianjiang) showing soft rot symptoms were observed in a field in Tayang Village, Fengrun District, Tangshan, Hebei Province (North China). The disease incidence in that field (15 ha in size) was more than 20%. Symptomatic rhizomes (brown and water-soaked) were surface-sterilized in 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Rhizomes were cut into pieces ca. 0.5 cm in length, and then were soaked in 500 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the lysogeny broth (LB) medium. And LB plates were incubated at 28°C for 24 h. Five single colonies were picked from each LB plate and restreaked three times for purity. Endophytic bacteria were also isolated from asymptomatic rhizomes as control. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R (Weisburg et al. 1991) and sequenced. The results of BLASTN against NCBI nr of the 16S rDNA amplicons suggested that the most isolates (8/10) obtained from the rotten rhizomes belonged to the genus Pectobacterium, and few isolates (2/10) were Enterobacter spp.. Only Enterobacter spp. were isolated from asymptomatic rhizomes. Since all Pectobacterium isolates showed identical 16S rDNA sequence, thus, only two isolates were selected for further analysis. Pectobacterium isolates TS20HJ1 and TS20HJ2 (MZ853520, MZ853521) represent isolates from two plant individuals. To determine the species of the rhizome rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes acnA, icdA, mdh, proA and rpoS (MZ994717-MZ994726) (Ma et al. 2007; Waleron et al. 2008), and a phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between the two isolates. The result of phylogenetic analysis showed that the ginger rhizome isolates clustered with P. brasiliense type strain IBSBF1692T (Duarte et al. 2004; Nabhan et al. 2012). Ginger seedlings (cv. Mianjiang) were inoculated with the isolate TS20HJ1 by injecting 10 µl of bacterial suspensions (108 CFU·mL-1) into the rhizomes, or injected with 10 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Ten days after inoculation, only the bacteria-inoculated rhizomes showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected rhizomes and were identified with MLSA gene sequencing, fulfilling Koch's postulates. P. brasiliense causes soft rot of a wide range of economically important crops (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing rhizome rot of ginger in China. The rhizome rot caused 20-25% yield loss on average in Tangshan region in 2020, which poses a significant threat to the local ginger farming. Further research on epidemiology and disease management options is needed.

Differences at Species Level and in Repertoires of Secondary Metabolite Biosynthetic Gene Clusters among Streptomyces coelicolor A3(2) and Type Strains of S. coelicolor and Its Taxonomic Neighbors

Streptomyces coelicolor A3(2) is used worldwide for genetic studies, and its complete genome sequence was published in 2002. However, as the whole genome of the type strain of S. coelicolor has not been analyzed, the relationship between S. coelicolor A3(2) and the type strain is not yet well known. To clarify differences in their biosynthetic potential, as well as their taxonomic positions, we sequenced whole genomes of S. coelicolor NBRC 12854T and type strains of its closely related species—such as Streptomyces daghestanicus, Streptomyces hydrogenans, and Streptomyces violascens—via PacBio. Biosynthetic gene clusters for polyketides and non-ribosomal peptides were surveyed by antiSMASH, followed by bioinformatic analyses. Type strains of Streptomyces albidoflavus, S. coelicolor, S. daghestanicus, S. hydrogenans, and S. violascens shared the same 16S rDNA sequence, but S. coelicolor A3(2) did not. S. coelicolor A3(2) and S. coelicolor NBRC 12854T can be classified as Streptomycesanthocyanicus and S. albidoflavus, respectively. In contrast, S. daghestanicus, S. hydrogenans, and S. violascens are independent species, despite their identical 16S rDNA sequences. S. coelicolor A3(2), S. coelicolor NBRC 12854T, S. daghestanicus NBRC 12762T, S. hydrogenans NBRC 13475T, and S. violascens NBRC 12920T each harbor specific polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) gene clusters in their genomes, whereas PKS and NRPS gene clusters are well conserved between S. coelicolor A3(2) and S. anthocyanicus JCM 5058T, and between S. coelicolor NBRC 12854T and S. albidoflavus DSM 40455T, belonging to the same species. These results support our hypothesis that the repertoires of PKS and NRPS gene clusters are different between different species.

Interventions designed to control postpasteurization contamination in high-temperature, short-time-pasteurized fluid milk processing facilities: A case study on the effect of employee training, clean-in-place chemical modification, and preventive maintenance programs

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions.

Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.

Mesorhizobium delmotii and Mesorhizobium prunaredense are two new species containing rhizobial strains within the symbiovar anthyllidis

Eight mesorhizobial symbiotic strains isolated from Anthyllis vulneraria root-nodules were studied and compared taxonomically with defined Mesorhizobium species. All strains presented identical 16S rDNA sequences but can be differentiated by multilocus sequence analysis of housekeeping genes (recA, atpD, glnII and dnaK). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses separate these strains in two groups and a separate strain. Levels of DNA–DNA relatedness were less than 55% between representative strains and their closest Mesorhizobium reference relatives. The two groups containing four and three strains, respectively, originating from border mine and non-mining areas in Cévennes, were further phenotypically characterized. Groupings were further supported by average nucleotide identity values based on genome sequencing, which ranged from 80 to 92% with their close relatives and with each other, confirming these groups represent new Mesorhizobium species. Therefore, two novel species Mesorhizobium delmotii sp. nov. (type strain STM4623T=LMG 29640T=CFBP 8436T) and Mesorhizobium prunaredense sp. nov. (type strain STM4891T=LMG 29641T=CFBP 8437T) are proposed. Type strains of the two proposed species share accessory common nodulation genes within the new symbiovar anthyllidis as found in the Mesorhizobium metallidurans type strain.

A broad-range survey of ticks from livestock in Northern Xinjiang: changes in tick distribution and the isolation of Borrelia burgdorferi sensu stricto.

BackgroundBorreliosis is highly prevalent in Xinjiang Uygur Autonomous Region, China. However, little is known about the presence of Borrelia pathogens in tick species in this region, in addition Borrelia pathogens have not been isolated from domestic animals.MethodsWe collected adult ticks from domestic animals at 19 sampling sites in 14 counties in northern Xinjiang from 2012 to 2014. Ticks were identified to species by morphology and were molecularly analysed by sequences of mitochondrial 16S rDNA gene; 4–8 ticks of each species at every sampling site were sequenced. 112 live adult ticks were selected for each species in every county, and were used to culture Borrelia pathogens; the genotypes were then determined by sequences of the 5S-23S rRNA intergenic spacer and the outer surface protein A (ospA) gene.ResultsA total of 5257 adult ticks, belonging to four genera and seven species, were collected. Compared with three decades ago, the abundance of the five common tick species during the peak ixodid tick season has changed. Certain tick species, such as Rhipicephalus turanicus (Rh. turanicus), was found at Jimusaer, Yining, Fukang, and Chabuchaer Counties for the first time. Additionally, the sequence analyses showed that the Hyalomma asiaticum (Hy. asiaticum), Haemaphysalis punctata (Ha. punctata), and Dermacentor marginatus (D. marginatus) that were collected from different sampling sites (≥3 sites) shared identical 16S rDNA sequences respectively. For the tick species that were collected from the same county, such as Hy. asiaticum from Shihezi County and Rh. turanicus from Yining County, their 16S rDNA sequences showed genetic diversity. In addition, sixteen Borrelia isolates were found in Hy. asiaticum, Ha. punctata, D. marginatus and Rh. turanicus, which infested cattle, sheep, horse and camel in Yining, Chabuchaer, Shihezi and Shawan Counties. All of the isolates were genetically identified as B. Burgdorferi sensu stricto.ConclusionsWarmer and wetter climate may have contributed to the altered distribution and abundance of the five most common ticks in northern Xinjiang. The genetic analyses showed that certain tick species, such as Hy. asiaticum or Rh. turanicus, exhibit genetic commonness or diversity. Additionally, this study is the first to isolate B. burgdorferi sensu stricto in Hy. asiaticum asiaticum, H. punctata, D. nuttalli and D. marginatus ticks from domestic animals. These ticks may transmit borreliosis among livestock.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1021-0) contains supplementary material, which is available to authorized users.

The physicochemical conditions of isolation source determine the occurrence of Pseudomonas fluorescens group species

Pseudomonas fluorescens group species are indigenous bacteria in many different environments. The physicochemical conditions related to their selection to different surface and subsurface environments are poorly understood. We isolated 35 P. fluorescens group strains under the pesticide stress from mesocosms of surface soils and groundwater-monitoring pipe deposit slurries, and from a few of their initially sterilized counterparts, which were colonized during 599–625 days of laboratory incubation. In addition, strains were isolated from mesocosms of drilling sediment slurries. Based on the 16S rDNA analysis, strains belonged to five species. The statistical analysis of relationships between the isolates and physicochemical conditions in their isolation mesocosms revealed the typical habitats of five species. Pseudomonas IC038 and Pseudomonas LAB-23-like strains were common in mesocosms of deposit/sediment slurries low in growth substrates. Pseudomonas marginalis-like isolates preferred surface soil mesocosms rich in nutrients. Pseudomonas veronii and Pseudomonas mandelii-like strains grew in mesocosms of surface soils and subsurface deposit/sediment slurries, which indicated that they either had a versatile metabolism to adapt to different environments, or differed in substrate specificities. The initially sterilized mesocosms were mainly colonized with the same species as their non-sterilized counterparts, while all P. fluorescens group species tolerated atrazine and/or terbutryn. Our results on habitats were in agreement with the isolation sources of strains with identical 16S rDNA sequences in databases. The observed differences in the distribution patterns of the P. fluorescens group species indicate that species selection should be carefully done for man-made ecosystem applications, such as stable bioremediation.

Genome and metabolic network of "Candidatus Phaeomarinobacter ectocarpi" Ec32, a new candidate genus of Alphaproteobacteria frequently associated with brown algae.

Rhizobiales and related orders of Alphaproteobacteria comprise several genera of nodule-inducing symbiotic bacteria associated with plant roots. Here we describe the genome and the metabolic network of “Candidatus Phaeomarinobacter ectocarpi” Ec32, a member of a new candidate genus closely related to Rhizobiales and found in association with cultures of the filamentous brown algal model Ectocarpus. The “Ca. P. ectocarpi” genome encodes numerous metabolic pathways that may be relevant for this bacterium to interact with algae. Notably, it possesses a large set of glycoside hydrolases and transporters, which may serve to process and assimilate algal metabolites. It also harbors several proteins likely to be involved in the synthesis of algal hormones such as auxins and cytokinins, as well as the vitamins pyridoxine, biotin, and thiamine. As of today, “Ca. P. ectocarpi” has not been successfully cultured, and identical 16S rDNA sequences have been found exclusively associated with Ectocarpus. However, related sequences (≥97% identity) have also been detected free-living and in a Fucus vesiculosus microbiome barcoding project, indicating that the candidate genus “Phaeomarinobacter” may comprise several species, which may colonize different niches.

Ultrafine Cellulose Fibers Produced by Asaia bogorensis, an Acetic Acid Bacterium

The ability to synthesize cellulose by Asaia bogorensis, a member of the acetic acid bacteria, was studied in two substrains, AJ and JCM. Although both strains have identical 16S rDNA sequence, only the AJ strain formed a solid pellicle at the air-liquid interface in static culture medium, and we analyzed this pellicle using a variety of techniques. In the presence of cellulase, glucose and cellobiose were released from the pellicle suggesting that it is made of cellulose. Field emission electron microscopy allowed the visualization of a 3D knitted structure with ultrafine microfibrils (approximately 5-20 nm in width) in cellulose from A. bogorensis compared with the 40-100 nm wide microfibrils observed in cellulose isolated from Gluconacetobacter xylinus, suggesting differences in the mechanism of cellulose biosynthesis or organization of cellulose synthesizing sites in these two related bacterial species. Identifying these differences will lead to a better understanding of cellulose biosynthesis in bacteria.

Bacterial communities in Asian dust-containing snow layers on Mt. Tateyama, Japan

Asian dust particles transported from Asian desert areas are trapped in spring snow on the western plateau of Mt. Tateyama, Japan. We investigated bacterial communities in snow layers containing Asian dust, and other underlying snow layers, on Mt. Tateyama during 2008 and 2009. Several genera of bacteria were present among isolates obtained from Asian dust-containing snow layers, including the presumed dust-associated bacterial genus Bacillus. In particular, we detected isolates with identical 16S rDNA sequences (AB500941) over 2 consecutive years, and these isolates were most closely related to Bacillus subtilis. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that bacterial community profiles in the Asian dust-containing snow layers were similar over the 2 years. The bacterial communities differed between the Asian dust-containing layers and other layers. Total bacterial cell counts ranged from 6.85×10² to 2.39×105 cells ml-1, with higher values in the Asian dust-containing layers than in the other layers. A backward trajectory analysis showed that the possible source of Asian dust was similar each year. The bacterial community composition of the Asian dust-containing layers presumably reflected the long-distance transportation of microorganisms from dust source regions.

우리나라에서 분리한 Xanthomonas arboricola pv. pruni의 유전적 다양성

Xanthomonas arboricola pv. pruni, the causal agent of bacterial shot holes in stone fruits, was known to have a low population diversity. To investigate the genetic characteristics of X. arboricola pv. pruni isolated in Korea, three strains which have identical 16S rDNA sequences - including type strain (LMG852), Japanese isolate (MAFF301420) and Korean isolate (XWD1) - were analysed based on the nucleotide sequences of three DNA regions and RAPD pattern. No sequence diversity among the three strains was found within the ITS, glnA and atpD gene sequences. However, five of 756 nucleotides of the atpD gene determined (accession number FJ429319) were different from those of the French strain available from the Genbank database. RAPD analyses performed with 40 different arbitrary primers revealed that two strains isolated from Korea and Japan showed similarity in their band patterns distinguished by type strain. These results suggest that Korean and Japanese strains are very close and belong to a population with a low genetic diversity, and might have a different origin from strains found in West Europe.

Characterization of Streptomyces spp. Isolated from the Sea Surface Microlayer in the Trondheim Fjord, Norway

The water surface microlayer is still poorly explored, although it has been shown to contain a high density of metabolically active bacteria, often called bacterioneuston. Actinomycetes from the surface microlayer in the Trondheim fjord, Norway, have been isolated and characterized. A total of 217 isolates from two separate samples morphologically resembling the genus Streptomyces have been further investigated in this study. Antimicrobial assays showed that about 80% of the isolates exhibited antagonistic activity against non-filamentous fungus, Gram-negative, and Gram-positive bacteria. Based on the macroscopic analyses and inhibition patterns from the antimicrobial assays, the sub-grouping of isolates was performed. Partial 16S rDNAs from the candidates from each subgroup were sequenced and phylogenetic analysis performed. 7 isolates with identical 16S rDNA sequences were further studied for the presence of PKS type I genes. Sequencing and phylogenetic analysis of the PKS gene fragments revealed that horizontal gene transfer between closely related species might have taken place. Identification of unique PKS genes in these isolates implies that de-replication can not be performed based solely on the 16S rDNA sequences. The results obtained in this study suggest that streptomycetes from the neuston population may be an interesting source for discovery of new antimicrobial agents.

Thermus thermophilus TMY isolated from silica scale taken from a geothermal power plant

To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains. The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting. Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus. The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered. The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.

Occurrence and distribution of ‘Candidatus Phytoplasma trifolii’ associated with diseases of solanaceous crops in Lebanon

A survey for phytoplasma diseases in tomato and pepper fields in Lebanon was conducted during 2003 and 2004. Tomato plants with stunting, yellowing or purplish leaves, proliferation of laterals buds, hypertrophic calyxes and virescent flowers were found in 25% of the tomato fields surveyed, where they represented 2–8% of the plants. Pepper plants displaying stunting and yellowing of leaves, were found in 27% of the fields and 1–4% of the plants were affected. Phytoplasmas infecting tomato and pepper had identical 16S-rDNA RFLP profiles and sequences. A phytoplasma isolate named PTL was transmitted by dodder from a diseased tomato plant to a periwinkle (Catharanthus roseus) plant in which it induced leaf yellowing, virescence and phyllody. 16S-rDNA phylogenetic analysis classified PTL as a strain of ‘Candidatus Phytoplasma trifolii’.

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus Phormidium (Oscillatoriales) from different habitats and geographical sites

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.

‘ Candidatus Rhabdochlamydia crassificans’, an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea)

The genus Rickettsiella comprises various intracellular bacterial pathogens of arthropods, exhibiting a chlamydia-like developmental cycle. Species may be divided into two main groups, the R. popilliae– R. grylli group and the R. chironomi group. Previous phylogenetic studies based on the 16S ribosomal RNA encoding gene showed that two Rickettsiella species, one from each group, belong in reality to two distantly related lineages, the γ- Proteobacteria ( R. grylli) and the Chlamydiales (‘ Candidatus Rhabdochlamydia porcellionis’, a pathogen of terrestrial isopods). In the present work, the 16S rDNA sequence of another Rickettsiella-like species, causing abdominal swelling to its cockroach host Blatta orientalis, was determined and phylogenetic analysis performed. Identical 16S rDNA sequences of 1495 nucleotides were obtained from fat body and ovary tissues of both healthy and diseased cockroach individuals. The sequence shared only 73% of similarity with R. grylli, but 82–87% with most Chlamydiales, and even 96.3% with ‘ Candidatus Rhabdochlamydia porcellionis’. Phylogenetic analyses confirmed the affiliation of the cockroach pathogen within the order Chlamydiales, and based on ultrastructural characteristics and genetic analyses, we propose its inclusion in the ‘ Candidatus Rhabdochlamydia’ as a distinct taxon, ‘ Candidatus Rhabdochlamydia crassificans’. These results extend our knowledge of the phylogenetic diversity of the Chlamydiales.

Isolation and characterization of Pseudoalteromonas strains with divergent polycyclic aromatic hydrocarbon catabolic properties

Fifteen strains of polycyclic aromatic hydrocarbon (PAH)-catabolizing bacteria, identified as Pseudoalteromonas spp. were isolated from Eagle Harbor, Puget Sound, USA, using a most probable number procedure in which naphthalene or phenanthrene was the sole carbon and energy source. Despite having identical 16S rDNA sequences, some catabolized many PAHs, whereas others oxidized only naphthalenes. A putative naphthalene 1,2-dioxygenase gene fragment was polymerase chain reaction-amplified from the naphthalene-degrading strains and shown to be almost identical to a gene present in Neptunomonas naphthovorans, suggesting horizontal transfer.

Isolation of Sulfate-Reducing Bacteria from the Terrestrial Deep Subsurface and Description of Desulfovibrio cavernae sp. nov.

Deep subsurface sandstones in the area of Berlin (Germany) located 600 to 1060 m below the surface were examined for the presence of viable microorganisms. The in situ temperatures at the sampling sites ranged from 37 to 45 degrees C. Investigations focussed on sulfate-reducing bacteria able to grow on methanol and triethylene glycol, which are added as chemicals to facilitate the long-term underground storage of natural gas. Seven strains were isolated from porewater brines in the porous sandstone. Three of them were obtained with methanol (strains H1M, H3M, and B1M), three strains with triethylene glycol (strains H1T, B1T, and B2T) and one strain with a mixture of lactate, acetate and butyrate (strain H1-13). Due to phenotypic properties six isolates could be identified as members of the genus Desulfovibrio, and strain B2T as a Desulfotomaculum. The salt tolerance and temperature range for growth indicated that the isolates originated from the indigenous deep subsurface sandstones. They grew in mineral media reflecting the in situ ionic composition of the different brines, which contained 1.5 to 190 g NaCl x l(-1) and high calcium and magnesium concentrations. The Desulfovibrio strains grew at temperatures between 20 and 50 degrees C, while the Desulfotomaculum strain was thermophilic and grew between 30 and 65 degrees C. The strains utilized a broad spectrum of electron donors and acceptors. They grew with carbon compounds like lactate, pyruvate, formate, n-alcohols (C1-C5), glycerol, ethylene glycol, malate, succinate, and fumarate. Some strains even utilized glucose as electron donor and carbon source. All strains were able to use sulfate, sulfite and nitrate as electron acceptors. Additionally, three Desulfovibrio strains reduced manganese oxide, the Desulfotomaculum strain reduced manganese oxide, iron oxide, and elemental sulfur. The 16S rRNA analysis revealed that the isolates belong to three different species. The strains H1T, H3M and B1M could be identified as Desulfovibrio indonesiensis, and strain B2T as Desulfotomaculum geothermicum. The other Desulfovibrio strains (H1M, H1-13, and B1T) showed identical 16S rDNA sequences and similarities as low as 93% to their closest relative, Desulfovibrio aminophilusT. Therefore, these isolates were assigned to a new species, Desulfovibrio cavernae sp. nov., with strain H1M as the type strain.

Phylogenetically diverse new sulfur chemolithotrophs of alpha-proteobacteria isolated from Indian soils.

Five facultative sulfur chemolithotrophs were isolated from soils to study the diversity of sulfur lithotrophy. Phenotypic characteristics, including sulfur lithotrophic properties and chemotaxonomic features of the isolates, were similar to those of the members of the colorless sulfur bacteria. 16S rDNA sequence analyses rendered placing the isolates to three distinct phylogenetic clusters of alpha-proteobacteria. Three isolates, AS001, AS002, and KCT002, were identified as members of the genus Paracoccus. The strains AS001 and AS002, having identical 16S-rDNA sequence, showed significant 16S rDNA sequence similarity (99.1%) to Paracoccus versutus. The strain KCT002 showed highest (98%) 16S rDNA sequence similarity to P. alcaliphilus and 96% similarity to the pair AS001 and AS002. Isolate KCT001 appeared to be closely related to Pseudaminobacter salicylatoxidans, although sulfur lithotrophy of P. salicylotoxidans is not known. The other isolate, TCK, showed almost identical 16S rDNA (99.9%) sequence with two recently described unclassified chemolithoautotrophic arsenite oxidizing strains. Physiological and chemotaxonomic characteristics and phylogenetic analyses of the five new strains emphasize the need of polyphasic bacterial taxonomy of sulfur lithotrophs.