16S rRNA Nucleotide Sequence Research Articles

Isolation and Characterization of Antagonistic Bacterial Strains for Biocontrol of Collar Rot (Aspergillus niger) in Groundnut (Arachis hypogaea L.)

Aspergillus niger is a pathogenic fungus causing collar rot disease in groundnut (Arachis hypogaea L.). The species is currently recognized as one of the most severe threats to groundnut worldwide, negatively impacting on productivity and quality of groundnut. This study was conducted to isolate bacterial strains capable of inhibiting the pathogenic A. niger QT1 strain as well as the production of some extracellular cell wall hydrolytic enzymes. A preliminary screening resulted in a total of 138 bacterial isolates exhibiting endo-β-1,3-glucanase activity. The second screening resulted in 10 isolates showing strong antagonistic activities against A. niger QT1 of 41.05 to 61.6% inhibitory ability. Among these, five isolates produced high cell wall hydrolytic enzymes, including endo β-1,3-glucanase, chitinase, and cellulase. Using the nucleotide sequence of 16s rRNA as a marker, it was revealed that these isolates were identified as Bacillus velezensis (2 strains), and Bacillus siamensis (3 strains). The in vivo evaluation of the potential application for treatment of A. niger QT1 demonstrated that all five strains effectively controlled collar rot disease of groundnuts. The highest efficacy was observerd in the treatment supplemented with B. velezensis 137 with no groundnut death. The present results strongly supported for the application of bacteria as biocontrol agents in order to replace the use of chemical plant fungal pesticides.

Genetic similarities and phylogenetic analysis of Muntjac (Muntiacus spp.) by comparing the nucleotide sequence of 16S rRNA and cytochrome B genome.

This study aimed to identify the phylogenetic similarities among the muntjac (Muntiacus spp.). The phylogenetic similarities among seven major muntjac species were studied by comparing the nucleotide sequence of 16s rRNA and cytochrome b genome. Nucleotide sequences, retrieved from NCBI databases were aligned by using DNASTAR software. A phylogenetic tree was created for the selected species of muntjac by using the maximum likelihood method on MEGA7 software. The results of nucleotide sequences (16s rRNA) showed phylogenetic similarities between, the M. truongsonensis and M. rooseveltorum had the highest (99.2%) while the lowest similarities (96.8%) found between M. crinifrons and M. putaoensi. While the results of nucleotide sequences (Cty b) showed the highest similarity (100%) between M. muntjak and M. truongsonensis and the lowest s (91.5%) among M. putaoensis and M. crinifrons. The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including M. putaoensis, M. truongsonensis, M. rooseveltorum, and M. muntjak, and the second one including M. crinifrons and M. vuquangensis. The M. reevesi exists separately in the phylogenetic tree. The phylogenetic tree of muntjac species using cytochrome b genes shows that the M. muntjak and M. truongsonensis are clustered in the same group.

Characterization and Antagonistic Effect of Culturable Apple-Phyllosphere Endophytic Bacteria from the Cold Plateau in Yunnan, China

The endophytic bacteria in apple leaves from apple-producing areas of Yunnan, China were isolated and identified on the basis of bacterial colony morphology and nucleotide sequences of 16S rRNA and rpoB genes. The endophytic bacterial isolates with nitrogen, phosphorus, and potassium utilization abilities were screened by culturing on functional media. A total of 5709 isolates of culturable endophytic bacteria (CEB) were isolated from 30 apple leaf samples collected from different regions. A total of 39 CEB representative isolates were identified as Bacillus velezensis, B. subtilis, B. licheniformis, B. safensis, B. pumilus, and Priestia megaterium. Among them, B. velezensis and B. subtilis were the main CEB, accounting for 55.00% and 34.37%, respectively, which exhibited potential inhibition on not only the main apple disease pathogens of Alternaria alternata, Valsa mali, Fusarium oxysporum, and Rosellinia necatrix, but also some important and uncontrollable phytopathogens, including F. oxysporum f. sp. cubense that causes banana Fusarium wilt, and Phytophthora nicotianae that causes tobacco black shank. Among these isolated endophytic bacteria species, a total of 10 strains, including b3, b4, b16, b17, b20, and b23 of B. subtilis, b7, b24, and b28 of B. licheniformis, and b38 of B. velezensis, can fix nitrogen; 8 strains, including b7 and b28 of B. licheniformis, b5, b10, and b23 of B. subtilis, b8 of B. safensis, and b6 of Priestia megaterium, could dissolve inorganic phosphorus; 11 strains, including b9, b12, b14, b30, b34, and b43 of B. velezensis, b6 of Priestia megaterium, and b17, b18, b20, and b26 of B. subtilis, could degrade organic phosphorus; and 5 strains, including b4, b5, and b26 of B. subtilis, and b7 and b28 of B. licheniformis could dissolve potassium. These strains are valuable resources of endophytic bacteria that have adapted to the ecological environment of the Cold Plateau apple-production area and could be used as plant disease biocontrol agents and biofertilizers of crops. The culturable phyllosphere endophytes in apple leaves relate to geographic locations, apple varieties, and environment, providing the basis to explain the mechanisms underlying the establishment of apple endophyte diversity and may help to devise apple disease management strategies.

Molecular detection and genetic characterization of Coxiella-like endosymbionts in dogs and ticks infesting dogs in Northeast India

An epidemiological study was performed to determine the role of dogs and ticks infesting dogs in the transmission of Q fever in humans and animals from April 2019 to March 2020 in the northeastern hill states of India. In total, 245 pet and stray dogs irrespective of age or sex were sampled, without specific inclusion or exclusion criteria. In total, 478 ticks belonging to three species were detected, namely Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum. The DNA extracted from blood and tick samples was assayed for molecular characterization of Coxiella burnetii targeting the 16S rRNA and superoxide dismutase (SOD) genes. Amplified PCR products were purified, cloned and custom sequenced. PCR assay showed 3.3% (8/245) of the dogs were positive for Coxiella-like bacteria. Coxiella-like bacterial DNA was detected in adult fully engorged females of R. sanguineus (7.7%, 13/168), R. (B.) microplus (3.3%, 4/123) and H. anatolicum (1.9%, 1/54). Coxiella-like bacterial DNA lacked in adult male or nymphal stage. The infection rate did not vary significantly between seasons, nor according to sex or age of the host. Six nucleotide sequences of 16S rRNA and SOD genes are discussed.

Occurrence of Leaf Spot Disease on Watermelon Caused by Pseudomonas syringae pv. syringae

Typical bacterial symptoms, water-soaking brown and black leaf spots with yellow halo, were observed on watermelon seedlings in nursery and field of Gyeongnam and Jeonnam provinces. Bacterial isolates from the lesion showed strong pathogenicity on watermelon and zucchini. One of them was rod-shaped with 4 polar flagella by observation of transmission electron microscopy. They belonged to LOPAT group 1. The phylogenical trees with nucleotide sequences of 16S rRNA and multi-locus sequencing typing with the 4 house-keeping genes (gapA, gltA, gyrB, and rpoD) of the isolates showed they were highly homologous to Pseudomonas syringae pv. syringae and grouped together with them, indicating that they were appeared as P. syringae genomospecies group 1. Morphological, physiological, and genetical characteristics of the isolates suggested they are P. syringae pv. syringae. We believe this is the first report that P. syringae pv. syringae caused leaf spot disease on watermelon in the Republic of Korea.

Revision and phylogeny of the genus Loxoneptera Hampson, 1896 (Lepidoptera, Crambidae, Pyraustinae), based on morphology and molecular data.

The genus Loxoneptera Hampson, 1896 is revised based on external appearance and genitalia. It is comprised of eleven species, of which three are described as new species from China: L. crassiuncata Chen & Zhang, sp. nov., L. triangularis Chen & Zhang, sp. nov., and L. rectacerosa Chen & Zhang, sp. nov.; six species are proposed as new combinations: L. carnealis (Swinhoe, 1895), comb. nov., L. medialis (Caradja, 1925), comb. nov., L. pentasaris (Meyrick, 1932), comb. nov., L. bipunctalis (Hampson, 1912), comb. nov., L. brevipalpis (Snellen, 1890), comb. nov., and L. dichroma (Moore, 1888), comb. nov. A new replacement name, L. hampsoni Chen & Zhang, nom. nov., is proposed for L. carnealis Hampson, 1896, the type species of the genus, because it is a secondary homonym of L. carnealis (Swinhoe, 1895), comb. nov. External characters and genitalia morphology of all species are figured. Nucleotide sequences of COI, 16S rRNA, 28S rRNA, and EF-1α were used for the molecular analysis and phylogeny of Loxoneptera species.

Morphological, biochemical and molecular identification of rhizobacteria isolates with potential for biocontrol of fungal plant pathogens

Rhizobacteria have huge potential for biocontrol activity against many plant pathogens. Isolation and identification are therefore crucial to understand their microbial diversity and ecological importance. The study sought to identify eight yam (Dioscorea sp.) rhizobacterial isolates found to be inhibitory in a previous study to several fungal pathogens, using polyphasic approach (morphological, biochemical, and molecular approaches). Morphological and biochemical characterizations were carried out at the Noguchi Memorial Institute for Medical Research (NMIMR), Legon-Accra, Ghana while the Molecular characterization was done at Functional Biosciences Inc., Wisconsin, USA. Morphological and biochemical characters determined were texture, consistency, the morphology of cell growth on nutrient agar, citrate utilization, catalase reaction, Gram reaction, and endospore formation. Molecular identification was achieved through the amplification and sequencing of the 16SrRNA and gyrB regions of the eight rhizobacterial genomic DNA followed by BLAST analysis. Sequencing data were also deposited at GenBank to obtain accession numbers for the rhizobacteria. All the eight bacterial isolates were shown to possess rod-shaped bacilli cells, Gram-positive, and were endospore producers. The amplification of the 16SrRNA and the gyrB regions of the bacterial DNA and subsequent BLAST of these two regions revealed that they were all members of the Bacillus subtilis group at percentage similarity between 99-100%. Four out of the eight isolates were, Bacillus subtilis, three were B. amyloliquefaciens and the last, B. velezensis. Sequence data of both the 16SrRNA and gyrB deposited at the GenBank provided GenBank accession numbers for nucleotide sequence of 16SrRNA as GenBank MT535846-MT535853 and gyrB as GenBank MT908225-MT908232 respectively. This is the first attempt to identify these rhizobacteria, which have huge potential as fungal antagonists of many plant pathogens in Ghana.

Phylogenetic Relationships of Flounders from the Family Pleuronectidae (Ostichties: Pleuronectiformes) Based on 16S rRNA Gene

The systematics and phylogeny of flatfish is investigated on the basis of an incomplete nucleotide sequence of 16S rRNA and the complete Co-1 and Cyt-b sequences. In total 62 specimens of 14 species of our own collections were submitted to the GenBank/BOLD repositories and studied. Four types of gene trees were reconstructed: Bayesian (BA), maximum likelihood (ML), minimum evolution (ME), and neighbor joining (NJ). These trees showed basically similar topology. Two separate branches on the trees support the previously identified subfamilies Hippoglossoidinae and Pleuronectinae with the monophyletic status of these taxa. The subfamily Pleuronectinae can be considered monophyletic if the tribe Microstomini is excluded from it and genus Lepidopsetta is moved into the tribe Pleuronectini. Three sets of nucleotide sequences were formed and independently studied. One set included all the obtained 16S rRNA gene sequences (291 bp), the second set included a sample of longer 16S rRNA sequences (617 bp), and the third set consisted of three gene sequences: 16S rRNA, Co-1, and Cyt-b (2926 bp). All three data sets gave a similar phylogenetic signal, which is consistent with the traditional concept of the taxonomy of the Pleuronectiformes order; however, the second and third sets provided better resolution of topology.

Detection of Rhodococcus fascians, the Causative Agent of Lily Fasciation in South Korea.

Rhodococcus fascians is an important pathogen that infects various herbaceous perennials and reduces their economic value. In this study, we examined R. fascians isolates carrying a virulence gene from symptomatic lily plants grown in South Korea. Phylogenetic analysis using the nucleotide sequences of 16S rRNA, vicA, and fasD led to the classification of the isolates into four different strains of R. fascians. Inoculation of Nicotiana benthamiana with these isolates slowed root growth and resulted in symptoms of leafy gall. These findings elucidate the diversification of domestic pathogenic R. fascians and may lead to an accurate causal diagnosis to help reduce economic losses in the bulb market.

Detection of Ehrlichia sp. in Amblyomma sculptum parasitizing horses from Brazilian Pantanal wetland

Bacteria of the genus Ehrlichia are transmitted by ticks and also are an important cause of infection in wild and domestic mammals. Infection with Ehrlichia spp. has been reported in horses, especially in the USA, Nicaragua and Brazil. In this study, we report the parasitism by Amblyomma sculptum, Rhipicephalus microplus and Dermacentor nitens ticks in horses from a ranch located in south Pantanal wetland. Molecular and serological analyzes to determine infection by Ehrlichia spp. in horses and their respective ticks were carried out. A total of 12 horses were submitted to blood collection to investigate antibodies by indirect immunofluorescence assay (IFA) using Ehrlichia canis crude antigens and to be tested by polymerase chain reaction (PCR) in order to amplify fragments of the 16S rRNA, dsb, groEL and sodB gene of Ehrlichia spp. A total of 164 tick specimens were removed from horses, stored in isopropanol and later identified as D. nitens, A. sculptum and R. microplus. DNA from ticks were extracted and subjected to the same PCR assays to detect Ehrlichia spp. Anti-Ehrlichia spp. antibodies were detected in five/12 (41.7 %) horses by IFA, with antibody titers ranging from 40 to 160. All horse DNA samples were negative for the 16S rRNA, dsb, groEL and sodB of Ehrlichia spp. One A. sculptum female was positive to all target genes of Ehrlichia. This tick was parasitizing an Ehrlichia-seropositive horse with antibody titer of 80. Nucleotide sequences of 16S rRNA, dsb, groEL and sodB genes showed close relationship with different strains of Ehrlichia detected in wild mammals, Amblyomma ticks and horses from Brazil and Argentina. Detection of anti-Ehrlichia sp. antibodies suggests that horses have been exposed to an ehrlichial agent in the Pantanal. Future studies on Ehrlichia infection should be carried out to better elucidate and to bring new information about equine ehrlichiosis, since these animals are important hosts of ticks in the Brazilian Pantanal wetlands.

The morphological characteristic strain KPABG LID-610023 of Chroococcus s.l. group (Cyanoprokaryota)

The Chroococcus-like strain was collected from anthropogenic meadow on the Barents Sea coast (Murmansk Region, Russia). It was studied by light microscope, the nucleotide sequences of 16S rRNA and ITS were obtained. Based on 16S rRNA phylogenetic analysis, the strain KPABG LID-610023 was placed in the clade of Inacoccus in spite of morphological and molecular differences from one already established species: Inacoccus carmineus. Morphologically the tested strain did not show colored hyaline envelopes, and nanocytes were absent. The nucleotide sequences of 16S rRNA gene clearly demonstrated a genetic difference, separating it from Inacoccus carmineus. In addition, we compared ITS sequences of KPABG LID-610023 strain and Inacoccus carmineus and found that nucleotide composition, length, and secondary structures of D1–D1' and Box-B were different. These results suggest that the strain KPABG LID-610023 appear to be a new species of the genus Inacoccus. Possibly this new species is a cryptic taxon due to its morphological similarity with Chroococcus pallidus Näg.

Molecular phylogenetic, population genetic and demographic studies of Nodularia douglasiae and Nodularia breviconcha based on CO1 and 16S rRNA

Freshwater mussels belonging to the genus Nodularia (Family Unionidae) are known to be widely distributed in East Asia. Although phylogenetic and population genetic studies have been performed for these species, there still remain unresolved questions in their taxonomic status and biogeographic distribution pathways. Here, the nucleotide sequences of CO1 and 16S rRNA were newly determined from 86 N. douglasiae and 83 N. breviconcha individuals collected on the Korean Peninsula. Based on these data, we revealed the following results: (1) N. douglasiae can be divided into the three genetic clades of A (only found in Korean Peninsula), B (widely distributed in East Asia), and C (only found in the west of China and Russia), (2) the clade A is not an independent species but a concrete member of N. douglasiae given the lack of genetic differences between the clades A and B, and (3) N. breviconcha is not a subspecies of N. douglasiae but an independent species apart from N. douglasiae. In addition, we suggested the plausible scenarios of biogeographic distribution events and demographic history of Nodularia species.

A novel process of anodic water denitrification in an upflow tubular microbial fuel cell: effect of C/N ratio

AbstractBACKGROUNDA high nitrate concentration in groundwater in some parts of Iran has been detected due to untreated wastewater discharge and agriculture activities. Therefore, the development of treatment technology in this area using a new biological method was carried out.RESULTSActivated sludge originating from slaughterhouse wastewater treatment plants and immobilized on graphite granules exhibited noticeable denitrification activity in a double chamber upflow tubular microbial fuel cell (UTMFC). The effects of three C/N values at different initial nitrate concentrations and external resistances on nitrate and chemical oxygen demand (COD) removal efficiencies as well as cell performance were studied at a fixed hydraulic retention time (HRT) of 12 h. Based on nucleotide sequence of 16S rRNA, four bacterial strains from anodic biofilm were also identified. The UTMFC operated in a continuous mode with C/N values of 20 and 10 was able to denitrify up to 150 mg L–1 nitrate without more accumulation of nitrite and ammonium ions than is permitted for drinking water. The maximum nitrate removal capacity of 0.25 ± 0.01 kg NO3−‐N/ (m3 net anodic compartment ‘NAC’.day) was achieved at 400 mg L–1 initial nitrate concentration and at C/N = 10. Additionally, the maximum current density and power generation at this ratio were found to be 46.04 A m–3 NAC and 16.96 W/m3 NAC, respectively. No efficient denitrification was observed at C/N = 5 while nitrite/ammonium formation increased.CONCLUSIONThe experimental data documented the feasibility of bioelectricity generation and substrate removal utilizing acetate containing water under the adopted operating conditions of UTMFC, and the proposed system allowed the simultaneous heterotrophic denitrification/electricity generation. © 2020 Society of Chemical Industry

Hydrocarbon-Oxidizing Bacteria from Urban Lake Beloye (Moscow): Identification and Phylogenetic Analysis

Cultured hydrocarbon-oxidizing bacteria have been isolated for the first time from bacterioplankton in urban Lake Beloye (Moscow). The taxonomic positions of two bacterial strains (2012B and 2012C) isolated from this lake have been determined. Lipids of the strain 2012B comprise C14:0–C19:0 fatty acids, the most abundant of them being C15:0 (54%), C16:0 (17%), C17:0 (10%), and 10-methyl С18:0 (3.5%). Lipids of the strain 2012C comprise C14:0–C19:0 fatty acids, the most abundant of them being C15:0 (45%), C16:0 (32%), and C17:0 (9%). A phylogenetic analysis of strain 2012B is performed using the nucleotide sequences of the 16S rRNA (KP779654.1) and alkB (KR422620.1) genes and the strain is identified as a typical member of the genus Rhodococcus spp. (Actinobacteria, Nocardiaceae). The combination of molecular identification and analysis of biochemical and physiological properties makes it possible to identify strain 2012B as Rhodococcusqingshengii 2012B. A phylogenetic analysis of strain 2012C is performed using the nucleotide sequences of 16S rRNA (MG966152) and shows the highest identity (99.57%) of strain 2012С with Pseudomonas psychrotolerans and Pseudomonas oryzihabitans

Изучение свойств штаммов бактерий рода Рaenibacillus, выделенных с семян нута

A necessary condition for the creation of effective biological preparations with a complex effect is the presence of bacterial strains that have several mechanisms of a positive effect on the plant. Preference is given to those microorganisms that are able to survive in adverse environmental conditions. The aim of the study was to search for new bacterial strains characterized by antagonism towards phytopathogenic microflora and exhibiting tolerance to soil contamination by lead ions and chloride ions. As a result of the screening, bacterial strains were isolated from the surface of chickpea seeds that were identified as Paenibacillus peoriae ANT 13 and Paenibacillus jamilae ANT 14 based on cultural-morphological and physiological-biochemical properties, as well as a comparative analysis of the nucleotide sequences of the gene 16S rRNA. The strains P. peoriae ANT 13 and P. jamilae ANT 14 exhibit antagonistic activity against phytopathogenic micromycetes. The strain P. jamilae ANT 14 is resistant to NaCl (content in the medium is up to 7 %), the strain P. peoriae ANT 13 is able to grow in the presence of Pb2 + ions in an amount up to 3150 mg/l. The use of isolated isolates for presowing treatment of wheat seeds (Triticum aestivum L.) helped to reduce the spread of root rot of seedlings and increase the length of shoots. Inoculation of seeds of alfalfa changeable (Medicago varia Mart.) of P. peoriae ANT 13 and P. jamilae ANT 14 increased the activity of nodulation and contributed to the growth of the root system of the plant, bacterization of chickpea seeds (Cicer arietinum L.) affected the increase in the mass of shoots and roots of this culture. The research results allow us to consider strains of P. peoriae ANT 13 and P. jamilae ANT 14 as promising objects of agricultural biotechnology.

Notes on the sinistral helicoid snail Bertia cambojiensis (Reeve, 1860) from Vietnam (Eupulmonata, Dyakiidae).

Since the time of the original description there have been no precise locality records in Cambodia of Bertia cambojiensis (Reeve, 1860) and it was believed to be extinct. In 2012, a joint Natural History Museum survey with Vietnamese colleagues rediscovered living populations of this huge sinistral helicoid snail in a protected area of southern Vietnam. The genitalia and radula morphology are re-assessed and type specimens of all recognised congeners are figured herein. The unique morphological characters of this species are a small and simple penis, well-developed amatorial organ complex that incorporates four amatorial organ ducts, a short gametolytic organ complex and spiked papilla, and radula morphology with unicuspid teeth. The type locality of B. cambojiensis, which has been contentious, is determined here to be in the vicinity of ‘Brelum’, Vietnam, near the border with Cambodia. In addition, the nucleotide sequences of barcoding genes COI, 16SrRNA and 28S fragments were provided for further comparison.

First report of ‘Candidatus Phytoplasma aurantifolia’(16SrII-A) associated with strawflower phyllody

Four strawflower plant samples showing phyllody disease symptoms were collected from the experimental plot at the College of Horticulture, Bangalore, Karnataka (India). The phytoplasma infection in strawflower was confirmed through PCR using 16Sr RNA and SecY gene specific primers. All four strawflower plant samples gave positive amplification 16Sr RNA (1.8 kb) and SecY gene (1.6 kb) for the phytoplasma. The amplified products were cloned and sequenced. The sequences of four clones obtained were identical. Therefore, a representative isolate (SWF1) nucleotide (nt) sequence of 16Sr RNA and SecY gene comparisons were made with the available phytoplasmas 16Sr RNA and SecY gene nt sequences from NCBI database. The analysis showed that strawflower phyllody phytoplasma shared the highest identity of 97.3 to 98.5% (16S rRNA) and 99.4 to 99.5% (SecY gene) with ‘Candidatus Phytoplasma aurantifolia’ group (16SrII) isolates reported from different parts of the world. This was well supported by close clustering of strawflower phytoplasma in the current study with ‘Ca. P. aurantifolia’ 16SrII groups in the phylogenetic analysis. The virtual RFLP pattern generated for the phytoplasma from strawflower phyllody was similar (similarity coefficient 0.97) to the reference pattern of 16Sr group II and subgroup A (‘Ca.P. aurantifolia’, 16SrII). This is the first report of phytoplasma associated with the phyllody disease of strawflower in India.

Identification and Phylogenetic Analysis of Xanthomonas albilineans (Ashby) Dowson Based on Multiple Gene Sequences in Yunnan Province, China

Sugarcane leaf scald caused by Xanthomonas albilineans (Ashby) Dowson is an extremely destructive disease. In 2017, suspected symptoms of sugarcane leaf scald was observed, in Baoshan, Mengding and Jinping, Yunnan Province, China. The bacteria was isolated from the symptomatic stalks and was subsequently identified as the causal agent using Koch’s postulates. BLASTN analysis showed that the amplified nucleotide sequences of 16S rRNA, gyrB and rpoD genes from all isolates shared 100% sequence identity with the nucleotide sequences of corresponding genes of X. albilineans strain GPE PC73 (Acc no. FP565176). Based on symptom diagnosis in the field, colony morphology, validation of Koch’s postulates and molecular identification of 16S rRNA, gyrB and rpoD genes, the disease was confirmed as sugarcane leaf scald caused by X. albilineans. Partial 16S rRNA, gyrB and rpoD gene sequence analysis of 36 X. albilineans strains from Yunnan and Guangxi provinces, China, was determined. The results revealed that the gyrB gene was the most sensitive in differentiating X. albilineans strains from Yunnan and Guangxi provinces.

Identification of geosmin biosynthetic gene in geosmin-producing colonial cyanobacteria Coelosphaerium sp. and isolation of geosmin non-producing Coelosphaerium sp. from brackish Lake Shinji in Japan

Geosmin is an earthy-muddy smelling compound produced in aquatic ecosystems by microorganisms including cyanobacteria. An increase in geosmin levels affecting the local fishery occurred in May 2007 in Lake Shinji, Japan, and geosmin-producing colonial cyanobacterium, Coelosphaerium sp. G2, was isolated from a water sample from the lake and identified. Cyanobacteria Coelosphaerium sp. is commonly found in Lake Shinji; however, prior to 2007, earthy-muddy odors were not a frequent issue. Further, there was no information regarding the geosmin biosynthetic gene in colonial cyanobacteria. Here, the geosmin biosynthetic gene (geoA) in strain G2 was identified and its nucleotide sequence was determined. It was found that geoA had 79% and 78% identity with geoA from filamentous geosmin-producing cyanobacteria Fischerella sp. PCC 9431 and geoA2 from Phormidium sp. P2r, respectively. The deduced amino acid sequence of GeoA consisted of two domains that were annotated as terpene cyclase. In 2015, geosmin non-producing Coelosphaerium sp. S3C5 was isolated from Lake Shinji and identified by morphological and genetic analyses. There was no difference in morphology or nucleotide sequences of 16S rRNA and 16S-23S internal transcribed spacer (ITS) between geosmin-producing and non-producing strains, which are therefore closely related and can exist in Lake Shinji. Distinguishing the two strains by observation under a microscope and sequencing of 16S rRNA and 16S-23S ITS have proven difficult. Inconsistency between the appearance of Coelosphaerium cells and the detection of the odor in water samples could therefore be attributed to dominance by the geosmin-producing strain or the non-producing strain. The increase in earthy smell is assumed to be caused by an increase in the geosmin-producing strain in Lake Shinji. Genetic analysis of geoA in Coelosphaerium sp. and the relative abundances of geosmin-producing and non-producing Coelosphaerium strains in Lake Shinji can be used to mitigate the economic damages caused by geosmin. Development of a molecular method to monitor the geosmin-producing strain in water ecosystems is equally important to alleviate the earthy smell caused by this particular strain.

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n=17) and green Texas cichlid (Herichthys cyanoguttatus) (n=1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed-field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%-100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 103 CFU/fish and 2.08 × 102 CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.