Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus is a migratory endoparasitic nematode known to cause severe environmental damage and economic losses in pine forest ecosystems. This present study investigated the nematicidal metabolites of actinomycetes in vitro and evaluated the disease control efficacy of the active compound and metabolites under greenhouse and field conditions. RESULTS: Five thousand types of actinobacteria from Korean forest soil samples were screened to identify novel nematicidal agents against the pine wood nematode. Streptomyces sp. AN091965 showed the strongest nematicidal activity. One active compound, spectinabilin, was obtained by nematicidal asssy-directed fractionation, and it showed significant nematicidal activity against B. xylophilus, with an LC50 value of 0.84 µg mL-1 . Spectinabilin effectively suppressed the development of pine wilt disease in 5-year-old Pinus densiflora trees, even at 0.9 mg per tree under greenhouse conditions. Moreover, the acetone extract of the active strain's mycelia efficiently suppressed the development of pine wilt disease under field conditions. CONCLUSION: To the best of our knowledge, this the first report to describe the nematicidal activity of spectinabilin against B. xylophilus. The cell extracts described herein merit further field studies as potential nematicides against the pine wood nematode. © 2018 Society of Chemical Industry.

Combination therapy with polymyxin B and netropsin against clinical isolates of multidrug-resistant Acinetobacter baumannii.

Polymyxins are last-resort antibiotics for treating infections of Gram-negative bacteria. The recent emergence of polymyxin-resistant bacteria, however, urgently demands clinical optimisation of polymyxin use to minimise further evolution of resistance. In this study we developed a novel combination therapy using minimal concentrations of polymyxin B. After large-scale screening of Streptomyces secondary metabolites, we identified a reliable polymixin synergist and confirmed as netropsin using high-pressure liquid chromatography, nuclear magnetic resonance, and mass spectrometry followed by in vitro assays using various Gram-negative pathogenic bacteria. To evaluate the effectiveness of combining polymixin B and netropsin in vivo, we performed survival analysis on greater wax moth Galleria mellonella infected with colistin-resistant clinical Acinetobacter baumannii isolates as well as Escherichia coli, Shigella flexineri, Salmonella typhimuruim, and Pseudomonas aeruginosa. The survival of infected G. mellonella was significantly higher when treated with polymyxin B and netropsin in combination than when treated with polymyxin B or netropsin alone. We propose a netropsin combination therapy that minimises the use of polymyxin B when treating infections with multidrug resistant Gram-negative bacteria.

Pseudonocardia endophytica sp. nov., isolated from the pharmaceutical plant Lobelia clavata.

An endophytic actinomycete strain, designated YIM 56035(T), was isolated from the inner tissue of the traditional Chinese medicinal plant Lobelia clavata. The strain stained Gram-positive, was aerobic and exhibited branching, white aerial mycelium and yellowish-brown substrate mycelium. The strain formed spore chains on aerial hyphae. The G+C content of the genomic DNA was 70.3 mol%. On the basis of morphological, physiological, chemotaxonomic and phylogenetic characteristics, strain YIM 56035(T) was assigned to the genus Pseudonocardia. Analysis of 16S rRNA gene sequences showed 98.5, 97.3, 97.3 and 97.1 % similarity to the closely related type strains Pseudonocardia kongjuensis LM 157(T), Pseudonocardia autotrophica IMSNU 20050(T), Pseudonocardia ammonioxydans H9(T) and Pseudonocardia compacta IMSNU 20111(T), respectively. The results of DNA-DNA hybridizations and comparison of some phenotypic characteristics revealed that the strain represents a novel species of the genus Pseudonocardia. The name Pseudonocardia endophytica sp. nov. is proposed, with the type strain YIM 56035(T) (=DSM 44969(T) =CCTCC AA 206026(T) =KCTC 19150(T)).

Streptomyces mayteni sp. nov., a novel actinomycete isolated from a Chinese medicinal plant.

An actinomycete strain, designated YIM 60475(T), was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained LL-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA-DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475(T) is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475(T) (=CCTCC AA 207005(T) = KCTC 19383(T)).

Differentiation of Streptomyces spp. which cause potato scab disease on the basis of partial rpoB gene sequences.

In a previous study, we found that the phylogenetic analysis of partial rpoB sequences can be used effectively to phylogenetically differentiate Streptomyces spp. [B.J. Kim, C.J. Kim, J. Chun, Y.H. Koh, S.H. Lee, J.W. Hyun, C.Y. Cha, Y.H. Kook, Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences, Int. J. Syst. Evol. Microbiol. 54 (2004) 593-598]. In the present study, we analyzed the partial rpoB gene sequences of 19 reference Streptomyces strains associated with potato scab. Furthermore, to empirically confirm the usefulness of rpoB gene analysis for the phylogenetic differentiation of Streptomyces spp., we applied the proposed system to 27 potato scab isolates obtained from the Korean provinces of Jeju-do and Kangwon-do. Phylogenetic relationships among these isolates using the devised rpoB gene-based methods were generally similar to those reported for 16S rRNA gene-based analysis. Isolates from potato scab lesion in Korea were also clearly differentiated into their phylogenetic groups by this method. In addition, the deduced RpoB amino acid sequences were also found to be useful for differentiating these strains. Our data demonstrate that the rpoB gene-based method can be used as a means of complementing other genetic methods such as 16S rRNA gene analysis or DNA-DNA hybridization to phylogenetically differentiate potato scab related Streptomyces spp.

Runella limosa sp. nov., isolated from activated sludge.

A Gram-negative bacterium, designated strain EMB111(T), was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. Cells were long and rod-shaped. The isolate was strictly aerobic and non-motile. The strain grew optimally at 25-30 degrees C and pH 7.5-8.0. The predominant fatty acids of strain EMB111(T) were iso-C(15 : 0), C(16 : 1)omega5c, iso-C(17 : 0) 3-OH, iso-C(15 : 0) 3-OH, C(16 : 0) 3-OH, C(16 : 0) and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH). The strain contained a large amount of phosphatidylglycerol and small amounts of two unknown phospholipids (PL1, PL2) as the polar lipids. The major isoprenoid quinone was menaquinone-7. The G+C content of the genomic DNA was 42.7 mol%. Phylogenetic analysis showed that strain EMB111(T) formed a phyletic cluster with members of the genus Runella within the family Flexibacteraceae and was most closely related to Runella slithyformis ATCC 29530(T) with a 16S rRNA gene sequence similarity of 94.8 %. On the basis of chemotaxonomic data and molecular properties, strain EMB111(T) represents a novel species within the genus Runella, for which the name Runella limosa sp. nov. is proposed. The type strain is EMB111(T) (=KCTC 12615(T)=DSM 17973(T)).

Flavobacterium croceum sp. nov., isolated from activated sludge.

A Gram-negative, non-motile, rod-shaped bacterium, designated strain EMB47(T), was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. Growth was observed between 10 and 40 degrees C (optimum, 25-35 degrees C) and between pH 5.0 and 8.5 (optimum, pH 7.5-8.0). The predominant fatty acids of strain EMB47(T) were iso-C(16 : 0) 3-OH, iso-C(15 : 1) G, C(15 : 0), iso-C(15 : 0), iso-C(14 : 0) and iso-C(16 : 0) and it contained phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylcholine as polar lipids. The G+C content of the genomic DNA was 40.8 mol% and the major quinone was MK-6. Comparative 16S rRNA gene sequence analyses showed that strain EMB47(T) formed a distinct phyletic line within the genus Flavobacterium. The levels of 16S rRNA gene sequence similarity with respect to Flavobacterium species were below 94.7 %. On the basis of the phenotypic, chemotaxonomic and molecular data, strain EMB47(T) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium croceum sp. nov. is proposed. The type strain is EMB47(T) (=KCTC 12611(T)=DSM 17960(T)).

Inhibitory effect on proliferation of vascular smooth muscle cells and protective effect on CCl(4)-induced hepatic damage of HEAI extract.

The effects of methanol extract from Hericium erinaceus cultivated with Artemisia iwayomogi (HEAI) on proliferation of vascular smooth muscle cells and CCl(4)-induced hepatic damage were evaluated. HEAI was shown to have a potent inhibitory effect on the proliferation of vascular smooth muscle cells (VSMCs). Interestingly, a methanol extract of Hericium erinaceus showed no inhibitory effect on the proliferation of VSMCs, while a methanol extract of Artemisia iwayomogi possessed strong inhibitory effects on the proliferation of VSMCs. Therefore, the inhibitory effects of HEAI may be caused by the changes of chemical components in the culture broth after the addition of Artemisia iwayomogi in the HEAI growth media. HEAI also had a strong protective effect on CCl(4)-induced hepatic damage in rats. The activity was evaluated using biochemical parameters such as glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and alkaline phosphatase (ALP). HEAI treatment caused a significant reduction in the activity of GOT but not of GPT and ALP in comparison with CCl(4) treatment alone. Histopathological studies showed that liver samples treated with HEAI were significantly different when compared to non-treated animals after CCl(4) exposure.