16S rDNA Phylogenetic Analysis Research Articles

Isolation and identification of a polyester-polyurethane degrading bacterium Bacillus altitudinis YX8-1

Although polyurethane (PUR) plastics play important roles in daily life, its wastes bring serious environmental pollutions. Biological (enzymatic) degradation is considered as an environmentally friendly and low-cost method for PUR waste recycling, in which the efficient PUR-degrading strains or enzymes are crucial. In this work, a polyester PUR-degrading strain YX8-1 was isolated from the surface of PUR waste collected from a landfill. Based on colony morphology and micromorphology observation, phylogenetic analysis of 16S rDNA and gyrA gene, as well as genome sequence comparison, strain YX8-1 was identified as Bacillus altitudinis. The results of high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed that strain YX8-1 was able to depolymerize self-synthesized polyester PUR oligomer (PBA-PU) to produce a monomeric compound 4, 4'-methylene diphenylamine. Furthermore, strain YX8-1 was able to degrade 32% of the commercialized polyester PUR sponges within 30 days. This study thus provides a strain capable of biodegradation of PUR waste, which may facilitate the mining of related degrading enzymes.

Soft rot disease of Belamcanda chinensis caused by Dickeya fangzhongdai in China

The rhizome of Belamcanda chinensis (belamcanda sinensis/Shegan) is an important traditional Chinese medicine, which is often used to treat lung diseases. However, soft rot disease seriously threatens the production of B. chinensis, causing its rhizomes to rot and the entire plant to die. Based on morphological characteristic and multigenic phylogenetic analyses of 16S rDNA, pel (pectate lyases encoding genes) and mdh (malic dehydrogenase encoding genes), the pathogenic isolates were identified as Dickeya fangzhongdai. One representative isolate SG-17 was selected for biological characteristics and pathogenicity tests analysis. The results showed that the optimum temperature and pH for SG-17 growth were 28 °C and pH 6, respectively. SG-17 caused typical soft rot symptoms on healthy B. chinensis plants, and produced symptoms of similar severity on Iris japonica (fringed iris) and Iris tectorum (iris), the other two species in Iridaceae family of economic and ornamental importance. Moreover, we also evaluated the impact of SG-17 on the contents of bioactive ingredients in B. chinensis rhizomes. The results show that the contents of tectorigenin and irigenin of B. chinensis rhizomes were increased significantly after infected with SG-17, while the contents of irisflorentin was decreased. As revealed by bactericides screen experiments, Ethylicin and Carbendazim · tetramethylthiuram disulfide exhibited good antibacterial activities, which could be used as the preferred agents for the control of soft rot of B. chinensis. As far as we know, this is the first report of Dickeya fangzhongdai causing soft rot on B. chinensis in the world.

Siamcapillus rubidus gen. et sp. nov. (Oculatellaceae), a novel filamentous cyanobacterium from Thailand based on molecular and morphological analyses

Cyanobacterial diversity has been extensively studied; however, scant knowledge exists concerning genetic data and higher accuracy for tropical cyanobacterial diversity. In particular, the existence of unnamed monophyletic genera within the Oculatellaceae requires further investigation. Here, two novel strains (NUACC13 and NUACC14) were isolated from a cyanobacterial mat covering border soil of a small freshwater pond in Phitsanulok Province, Thailand. A polyphasic approach combining genetic (16S rDNA and rpoC1 gene phylogenies, evolutionary distance and ITS secondary structures) and morphological information was applied to evaluate the taxonomic status of these novel strains. All phylogenetic analyses of 16S rDNA and rpoC1 gene sequences placed our strains within the Oculatellaceae, strongly supporting a novel monophyletic clade that did not belong to other known Oculatellaceae genera. The 16S rDNA sequence similarity (<96.2%) values among the closest described genera also supported the novelty of our strains. The hypothetical secondary structures (D1–D1′, Box-B, V2 and V3 helices) of the ITS region confirmed the uniqueness of our strains, indicating differences from the related genera. Morphological features of the novel strains differed from the phylogenetically closest taxa by size and color of filament and vegetative cell and the presence of a frayed sheath. Considering all the results, we herein described our strains as the novel genus Siamcapillus gen. nov. in the Oculatellaceae with the type species S. rubidus sp. nov.

Epiphytic Yeasts and Bacteria as Candidate Biocontrol Agents of Green and Blue Molds of Citrus Fruits.

Overall, 180 yeasts and bacteria isolated from the peel of citrus fruits were screened for their in vitro antagonistic activity against Penicillium digitatum and P. italicum, causative agents of green and blue mold of citrus fruits, respectively. Two yeast and three bacterial isolates were selected for their inhibitory activity on mycelium growth. Based on the phylogenetic analysis of 16S rDNA and ITS rDNA sequences, the yeast isolates were identified as Candida oleophila and Debaryomyces hansenii while the bacterial isolates were identified as Bacillus amyloliquefaciens, B. pumilus and B. subtilis. All five selected isolates significantly reduced the incidence of decay incited by P. digitatum and P. italicum on ‘Valencia’ orange and ‘Eureka’ lemon fruits. Moreover, they were effective in preventing natural infections of green and blue mold of fruits stored at 4 °C. Treatments with antagonistic yeasts and bacteria did not negatively affect the quality and shelf life of fruits. The antagonistic efficacy of the five isolates depended on multiple modes of action, including the ability to form biofilms and produce antifungal lipopeptides, lytic enzymes and volatile compounds. The selected isolates are promising as biocontrol agents of postharvest green and blue molds of citrus fruits.

Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8.

Fusarium pseudograminearum causes crown rot in wheat. This study aimed to assess the effects of the bacterial strain QTH8 isolated from Cotinus coggygria rhizosphere soil against F. pseudograminearum. Bacterial strain QTH8 was identified as Bacillus halotolerans in accordance with the phenotypic traits and the phylogenetic analysis of 16S rDNA and gyrB gene sequence. Culture filtrates of bacterial strain QTH8 inhibited the mycelial growth of F. pseudograminearum and resulted in mycelial malformation such as tumor formation, protoplast condensation, and mycelial fracture. In addition, bacterial strain QTH8 also inhibited the mycelial growth of Hainesia lythri, Pestalotiopsis sp., Botrytis cinerea, Curvularia lunata, Phyllosticta theaefolia, Fusarium graminearum, Phytophthora nicotianae, and Sclerotinia sclerotiorum. The active compounds produced by bacterial strain QTH8 were resistant to pH, ultraviolet irradiation, and low temperature, and were relatively sensitive to high temperature. After 4 h exposure, culture filtrates of bacterial strain QTH8—when applied at 5%, 10%, 15%, 20%, 25%, and 30%—significantly reduced conidial germination of F. pseudograminearum. The coleoptile infection assay proved that bacterial strain QTH8 reduced the disease index of wheat crown rot. In vivo application of QTH8 to wheat seedlings decreased the disease index of wheat crown rot and increased root length, plant height, and fresh weight. Iturin, surfactin, and fengycin were detected in the culture extract of bacterial strain QTH8 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Bacterial strain QTH8 was identified for the presence of the ituC, bacA, bmyB, spaS, srfAB, fend, and srfAA genes using the specific polymerase chain reaction primers. B. halotolerans QTH8 has a vital potential for the sustainable biocontrol of wheat crown rot.

Isolation and Molecular Characterization of Plant-Growth-Promoting Bacteria and Their Effect on Eggplant (Solanum melongena) Growth

In recent years, the optimum development of land resources has become an important task for ensuring the security of food production in China. Soil microorganisms have been considered to play an important role in conferring soil fertility and productivity. In order to obtain the plant-growth-promoting bacteria in newly reclaimed land, a total of 988 bacterial strains were isolated from nine soil samples collected from different sites in wastelands in Hangzhou (Zhejiang Province, China), a rural mountainous area. Among them, five strains exhibited substantial potential of phosphate solubilization, nitrogen fixation, siderophore production and indole acetic acid production at both pH 5.0 and pH 7.0, and also promoted eggplant growth in immature soil from newly reclaimed land. Furthermore, bacterial strains ZJ62 and ZJ3-12 were identified as Pantoeadispersa and Pantoea ananatis, respectively, while strains ZJ5, ZJ9 and ZJ174 were identified as Burkholderiaarboris, Burkholderia pyrrocinia and Burkholderia pyrrocinia, respectively, based on colony morphology observation and phylogenetic analysis of 16S rDNA and the housekeeping genes sequences. Overall, the result of this study showed that the 5 obtained bacterial strains have a great potential in promoting plant growth in immature soil from newly reclaimed land.

Isolation and characterization of a thermostable alkaline chitinase-producing Aeromonas strain and its potential in biodegradation of shrimp shell wastes

Chitinases are employed to the conversion of chitin and are produced by a wide range of bacteria. The objective of this study was to isolate chitinase-producing microorganisms with high chitinolytic activity. A thermostable alkaline chitinase producing isolate strain CQNU6-2 was obtained from soil samples and showed potential in biodegradation of shrimp shell wastes. The optimal culturing conditions of isolate CQNU6-2 is at 25°C and pH 7 for 24 h. The chitinase produced by strain CQNU6-2 exhibited maximum activity at pH 6.0 and 40°C and it could tolerate the treatment of high temperature (up to 80°C) and high pH (over 10). Taxonomic study, based on biochemical and morphological analysis and phylogenetic analysis of 16S rDNA, showed that strain CQNU6-2 was belongs to the genus Aeromonas sp. The isolate can effectively hydrolyze colloidal chitin with degradation rate of 100% and also can directly degrade the shrimp shells. Ammonium sulfate precipitation method can be used to preliminary purify the chitinase. In conclusion, strain CQNU6-2 had a promising potential for biodegradation of chitin under harsh pH or temperature conditions and could be employed to the comprehensive utilization of shrimp shell wastes.

Screening, identification and antagonistic effect of antagonistic bacteria JTFM1001 against aflatoxin contamination in corn

Aflatoxin is a strong carcinogenic and toxic fungal toxin produced by Aspergillus flavus and other Aspergillus species, and can seriously threaten the health of consumers, thus becoming a global concern. Corn, as an important oil and economic crop, is highly susceptible contaminated by aflatoxin. In this study, antagonistic bacteria with strong inhibitory effect on aflatoxin were screened to provide support for the treatment aflatoxin contamination control in corn. Ten strains which have strong antagonistic effects against A. flavus were isolated from healthy corn from different corn producing areas in China. Among them, the antagonistic bacteria JTFM1001 through corn kernels in vivo and field experiment, the inhibition effect of aflatoxin contamination reached above 70% and 55%, respectively. And the strain was identified as Bacillus subtilis based on its morphological, physiological and biochemical characteristics and phylogenetic analysis of 16S rDNA. In addition, our data showed that it can colonize in the rhizosphere and survive for a long time, forming the dominant flora, with broad application prospect. Finally, we were surprised to find that the antibacterial metabolites secreted by the antagonistic bacteria was one of the mechanisms of its inhibition of A. flavus and aflatoxin. This will provide us with new ideas and perspectives on the effective prevention and control of aflatoxin contamination in corn and corn oil.

Biological characteristics and classification of thermophilic actinomycetes showed extracellular hydrolytic enzymes producing ability isolated from compost

Compost is a highly humified organic fertilizer, rich in nutrients and a source of a variety of aerobic microorganisms, including actinomycetes, which develop in response to different levels of temperature, humidity, oxygen and pH. Microbes growing on the compost are believed to have the ability to produce extracellular hydrolytic enzymes. The purpose of this study was to determine the ability of thermophilic actinomycetes XM21 isolated from compost in producing hydrolytic enzymes, namely cellulase, amylase, protease, and lipase. The confirmation tests of hydrolytic enzymes-producing ability were conducted by inoculating the microbes into media containing cellulose, starch, gelatin and tween 80, using the method of disc diffusion. The results showed that strain XM21 capable of extracellular enzymes producing, such as cellulase, protease, amylase, lipase. Strain XM21 can grow well with high cellulase activity in a wide range of temperature between 30-55oC, optimum at 45oC. The strain can grow well on different media, utilized carbon sources with pH 5-10, and salinity of 0-5%. On the agar plate, the strain has white aerial mycelia, the mature spore chains appeared spirals, moderately long, bearing 10 to 35 spores each. Based on the biological characteristics and phylogenetic analysis of 16S rDNA, it can be concluded that strain XM21 is close to Streptomyces flavovariabilis (98, 12%), hence identified as Streptomyces flavovariabilis XM21.

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize (“Rojo Criollo”) and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and β-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.

First Report of Pectobacterium aroidearum Causing Soft Rot of Amorphophallus konjac in China

Konjac (Amorphophallus konjac) is an important cash crop in southwest China, grown as a source of glucomannan for industrial use. In July 2019, symptoms of stem soft rot were observed on A. konjac in Kunming (24.97697941°N, 102.79377675°E), Yunnan province (southwestern China). Symptoms included stem soft rot with a foul smell, inner part of plant disintegration, and wilting of leaves. Twenty-five to 30% of the plants in the field were damaged. Bacteria were isolated on LB (tryptone/yeast extract/NaCl) agar medium and incubated at 28 to 30°C for 48 to 78 h. The isolated colonies were streaked on LB medium for plates to obtain single colonies. The morphology of the bacterium was short, rod-shaped, with flagellum, with a slightly obtuse circle at both ends. DNA of bacterial isolates was extracted, and polymerase chain reaction (PCR) amplifications were performed using 16S rDNA primers (27F, 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R, 5′-GGTTACCTTGTTACGACTT-3′) and gapA primers (gapA-7-F and gapA-938-R) (Cigna et al. 2017) followed by sequencing. The 16S rDNA sequence of the 1,442-bp product (strain Ecc-K1, accession no. MN161137) had 100% similarity to the 16S rDNA sequences of Pectobacterium aroidearum (MK875007 and NR159926) by using BLAST (Altschul et al. 1990). Phylogenetic analysis of 16S rDNA performed by the CLUSTAL Omega tool (Sievers et al. 2011) showed that the new sequence (accession no. MN161137) and the P. aroidearum NR159926 type strain sequences comprise the same cluster. BLAST analysis (NCBI) repeated with the sequence MN161137 showed the most similar accession P. aroidearum strain Ecc15b MK961266.1 (99.93% similarity) and grouped MN161137 with Ec15b and Pcc M1 DQ205331.1 distant from other P. aroidearum. The P. aroidearum accessions MK875007 and NR159926 had 99.86 and 99.99.65% similarity to MN161137, respectively, and grouped with other P. aroidearum. Similarly, the gapA sequence of the 870-bp product (accession no. MN478003) had 95.84% similarity to the gapA sequences of CP024956 by using BLAST. Moreover, biochemical reactions using the Biolog GEN III System (Biolog, Haywood, CA) identified that the new isolated strain has a similarity value between the strain and P. aroidearum of 0.660 (24 h), greater than the critical value of 0.50, and the genetic distance was 5.389; the identification result has the highest similarity index to P. aroidearum. Therefore, combined results from morphological, molecular, and biochemical reaction results confirmed this species as P. aroidearum (Nabhan et al. 2013). Pathogenicity tests were confirmed on 1-year-old A. konjac seedlings was injected into stem and petiole with a bacterial suspension containing 10⁸ CFU/ml (10 μl/inoculation site). Control seedlings received sterile water. Inoculated and control seedlings (six each) were kept in a greenhouse and watered as needed. Stem soft rot, inner medulla disintegration, and wilt of leaves symptoms developed on inoculated seedlings within 3 to 10 days. The bacterium was reisolated from inoculated seedlings and was identified by above PCR and DNA sequencing methods, thus fulfilling Koch’s postulates. No symptoms were visible in the control seedlings. P. aroidearum has been reported as a pathogen of Chinese cabbage (Xie et al. 2018) and Cucurbita pepo (Moraes et al. 2017). To our knowledge, this is the first report of soft rot caused by P. aroidearum on A. konjac in China, which expanded the known host range for this pathogen.

Peach witches’-broom, an emerging disease associated with ‘Candidatus Phytoplasma phoenicium’ and ‘Candidatus Phytoplasma aurantifolia’ in Iran

During field surveys carried out from 2013 to 2017 in the eight main peach producing provinces of Iran, symptoms of a phytoplasma-like peach witches'-broom disease (PWIB), inducing severe yellowing, little leaf, internode shortening, crown and stem witches’-broom, decline, and death, were observed. The aim of this work was to identify and characterize the agent(s) associated with PWIB by biological assays and molecular analyses. PWIB agents were successfully transmitted under controlled conditions from scions of in field-affected peach trees, exhibiting severe or mild symptoms, to peach and bitter almond seedlings inducing phytoplasma-like symptoms. A 16S rDNA fragment of 1250 bp was amplified by nested-PCR from all PWIB-affected trees and grafted seedlings. Nucleotide sequence identity, presence of species-specific signature sequences, in silico RFLP, single nucleotide polymorphisms, and phylogenetic analyses of 16S rDNA allowed the assignation of the phytoplasma strains identified in seven Iranian provinces in peach trees with severe PWIB symptoms to four SNP genetic lineages of ‘Ca. P. phoenicium’ (subgroup 16SrIX-B and its variant). PWIB phytoplasma strains identified in Abarkooh (Yazd province) in peach trees with mild symptoms were attributed to the species ‘Ca. P. aurantifolia’ (subgroup 16SrII-C). This report of a wide spread of ‘Ca. P. phoenicium’ in association with PWIB in Iran supported its capability of adaptation to a broad range of fruit tree species, such as peach, nectarine, and apricot. As ‘Ca. P. phoenicium’ and ‘Ca. P. aurantifolia’ are the etiological agents of other important plant diseases in Iran and neighbouring countries, further investigations are needed to determine the role played by peach in their epidemiological pathways.

Apricot yellows associated with ‘ Candidatus Phytoplasma phoenicium’ in Iran

Almond witches’ broom associated with ‘ Candidatus Phytoplasma phoenicium’ is an economically important disease of almond in Iran and Lebanon. During surveys of almond witches’ broom in 2012–2015, an apricot yellows disease was observed in Fars Province of Iran. The characteristic symptoms of the disease were leaf yellowing, inward leaf curl, scorch of leaf margins, shortened internodes, production of rosettes at the tips of the branches, and decline, stunting, and death of affected trees. Healthy bitter almond and apricot seedlings, grafted with shoots from symptomatic trees, exhibited phytoplasma-type symptoms. A 16S rDNA fragment of 1,250 bp was amplified by nested-PCR from affected trees and grafted seedlings. Nucleotide sequence identity, presence of species-specific signature sequences, and phylogenetic analysis of 16S rDNA allowed the assignation of the phytoplasma strains identified to the ‘ Ca . P. phoenicium’. In vitro and in silico RFLP analyses of the amplified fragment allowed affiliation of the apricot yellows phytoplasma to a molecular variant in the subgroup 16SrIX-B. Within the population strains identified in this and previous studies, 16 genetic lineages were determined within 16S rDNA nucleotide sequences by the combination of 19 single nucleotide polymorphisms. The apricot yellows phytoplasma strains belong to a unique genetic lineage distinguished by the presence of three lineage-specific SNPs. This first report of ‘ Ca . P. phoenicium’ in association with apricot yellows in Iran opens new perspectives on the epidemiology of almond witches’ broom, suggesting possible adaptation of the phytoplasma to other fruit tree species.

Identification and Phylogenetic Analysis of Pear Psyllids (Hemiptera: Psyllidae) in Chinese Pear Orchards

Pear psyllids are among the most damaging pests in pear orchards, but little knowledge exists of psyllid species in cultivated pear orchards in China. In this study, DNA sequence analyses of the 16S rDNA and cytochrome oxidase I (COI) DNA regions were performed to identify pear psyllids from 28 regions of 20 provinces in China and to classify their genetic relationships to understand the origin of the species. The results showed that Cacopsylla chinensis Yang et Li (Hemiptera: Psyllidae) was found in most pear orchards in China, but Cacopsylla qianli (Hemiptera: Psyllidae) was found in only the cities of Guiyang (Guizhou province) and Xiangyang (Hubei province). The results for the 16S rDNA and COI regions were similar. Based on the nucleotide sequences and phylogenetic analyses of 16S rDNA and COI, C. chinensis could be divided into three groups: lineages I, II, and III. Based on 16S rDNA and COI, lineage II showed approximately 4% and 3% difference from lineage I, and lineage III showed approximately 12% and 9% difference from lineage I, respectively. C. chinensis lineage I was found in most provinces of China, while C. chinensis lineage II samples were mainly found in the Bohai rim region of China, and lineage III samples were found in Northeast China. The results of this study will provide information to pear producers regarding effective control measures to prevent further damage from pear psyllids.

Photobacterium carnosum sp. nov., isolated from spoiled modified atmosphere packaged poultry meat

Analysis of spoilage-associated microbiota of modified-atmosphere packaged poultry meat revealed four different bacterial isolates that could not be assigned to known species. They showed a Gram-negative staining behavior, were facultatively aerobic, non-motile with variable cell morphology. Phylogenetic analysis of 16S rDNA and gyrB, rpoD and recA revealed a distinct lineage within the genus Photobacterium with Photobacterium (P.) iliopiscarium DSM 9896T, P. phosphoreum DSM 15556T, P. kishitanii DSM 19954T, P. piscicola LMG 27681T and P. aquimaris DSM 23343T as closest relatives.The designated type strain TMW 2.2021T is non-luminous and grew at 0–20°C (optimum 10–15°C), within pH 5.0–8.5 (optimum 6–8) and in the presence of 0.5–3% (w/v) NaCl (optimum 1%). Major cellular fatty acids of TMW 2.2021T were summed feature 3 (C16:1ω7c/iso-C15 3-OH), C16:0, C18:1ω7c and summed feature 2 (C12:0 aldehyde and C10.928 unknown). Quinone analysis revealed Q-8 as sole respiratory ubiquinone. The genome of TMW 2.2021T has a size of 4.56Mb and a G+C content of 38.49mol%. The ANI value between TMW 2.2021T and the type strain of closest relative P. iliopiscarium DSM 9896T was 91.43%. Fingerprinting on the base of M13-RAPD-PCR band pattern and MALDI-TOF MS profiles allowed intraspecies differentiation between our isolates but also supported their distinct lineage to a novel species. Based on phylogenetic, genomic, phenotypic and chemotaxonomic data, strain TMW 2.2021T and further strains represent a novel species of the genus Photobacterium, for which the name Photobacterium carnosum sp. nov. is proposed. The type strain is TMW 2.2021T (=DSM 105454T=CECT 9394T).

Đặc điểm sinh học của xạ khuẩn nội sinh trên cây nghệ ở Nam Định và tiềm năng sinh tổng hợp chất kháng khuẩn

Endophytic actinomycetes associated with medicinal plants have widely reported to inhibit microbial pathogens and shown the potential applications. In traditional medicine, Curcuma longa has been used for thousand years. This is a precious medicinal plant, with the desire to find endophytic Actinomycetes strains with many precious properties on medicinal plants. In the paper, roots, branches and leaves of Curcuma longa were collected at Giao Thuy district, Nam Dinh province, Vietnam. The results showed that 8 endophytic Actinomycetes strains were isolated, in which, there are two strains named NVGR3 and NVGC3 could inhibit all tested strains (18.5%) and especially, NVGR3 strain has the best antibacterial activity. The isolated NVGR3 was selected for studying on biological characteristics. This strain reached high biomass yields as grow in several cultivation media at pH 5÷10, salinity up to 7% and growth temperature of 15÷37oC. Based on the results of morphological, physiological and biochemical characterizations and phylogenetic analysis of 16S rDNA, the isolated NVGR3 was identified as Streptomyces collinus species named Streptomyces collinus NVGR3.

African origin of Bradyrhizobium populations nodulating Bambara groundnut (Vigna subterranea L. Verdc) in Ghanaian and South African soils.

Flavonoids secreted by legumes play a major role as signal molecules for attracting compatible rhizobia. The aim of this study was to assess and understand the diversity of microsymbionts nodulating Bambara groundnut (Vigna subterranea L. Verdc.) landraces of different seedcoat colours using restriction fragment length polymorphism and phylogenetic analysis. Seedcoat pigmentation of landraces had effect on the diversity of microsymbionts of Bambara groundnut. Even when planted together in one hole, nodulating bradyrhizobia clustered differently. For example, 16S rDNA-RFLP typing of rhizobial samples TUTVSBLM.I, TUTVSCRM.I and TUTVSRDM.I originating respectively from Black, Cream and Red landraces that were co-planted in the same hole at Manga in the Sudano-sahelian savanna, as well as TUTVSCRK.I and TUTVSRDK.I respectively from Cream and Red landraces co-planted at Kpalisogu in the Guinea savanna, revealed different 16S rDNA- RFLP types. Phylogenetic analysis of 16S rDNA, glnII, recA and atpD sequences showed that Vigna subterranea was nodulated specifically by a diverse group of Bradyrhizobium species (e.g. Bradyrhizobium vignae, and a novel group of Bradyrhizobium spp.) in soils from Ghana and South Africa. The recA gene phylogeny showed incongruency with the other housekeeping genes, indicating the possibility of lateral gene transfer and/or recombination events. The grouping of isolates according to symbiotic gene (nifH and nodD) phylogenies revealed inter- and intra-specific symbiotic plasmid transfer and different evolutionary history. The results also showed that a cropping history and physico-chemical environment of soils increased bradyrhizobial diversity in Ghana and South Africa.

Characterization of Uncultured Genome Fragment from Soil Metagenomic Library Exposed Rare Mismatch of Internal Tetranucleotide Frequency.

Exploring the genomic information of a specific uncultured soil bacterium is vital to understand its function in the ecosystem but is still a challenge due to the lack of culture techniques. To examine the genomes of uncultured bacteria, a metagenomic bacterial artificial chromosome library derived from a soil sample was screened for 16S rDNA-containing clones. Five clones (4C6, 5E7, 5G4, 5G12, and 5H7) containing uncultured soil bacteria genome fragment (with low 16S rDNA similarity to isolated bacteria) were selected for sequencing. Clone 5E7 and 5G4 showed only 82 and 83% of 16S rDNA identity to known sequences. Phylogenetic analysis of 16S rDNA indicated that 5E7 and 5G4 were potentially from new class of Chloroflexi. Only one-third of the 5G4 open reading frames have significant hits against HMMER. Internal tetranucleotide frequency analysis indicated that the unknown region of 5G4 was poorly correlated with other parts of the clone, indicating that this section might be obtained through lateral transfer. It was suggested that this region rich for unknown genes is under fast evolution.

Isolation and genetic characterization of Nocardia seriolae from snubnose pompano Trachinotus blochii in Vietnam.

A total of 480 cage-cultured fish were collected from 4 coastal provinces in central Vietnam to investigate the causative agent of nocardiosis. Fish displayed unique characteristics such as paleness and lethargy and exhibited haemorrhages and ulcers on the skin. Prominent white nodules varying in size were observed in the spleen, kidney, and liver. Furthermore, histopathological sections showed typical granulomatous lesions in these organs. Using the Ziehl-Neelsen staining method, isolated bacteria exhibited acid-fast, bead-like filament morphology when cultured in brain-heart infusion medium or Ogawa medium. Phylogenetic analysis of 16S rDNA confirmed that the isolated bacterium was Nocardia seriolae. This study demonstrates for the first time an outbreak of N. seriolae in snubnose pompano in central Vietnam.

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23–25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don’t show homology to each other, but all of them are built up from 3–6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70–75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9–1.7mM of KM and 80–120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.