Isolation and characterization of the unicellular diazotrophic cyanobacterium Group C TW3 from the tropical western Pacific Ocean

A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5-3.0 μm in width and 4.0-6.0 μm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h(-1) when cultured at 30°C and 140 μmol m(-2) s(-1) of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light-dark cycle, was 7.31 × 10(-15) mol N cell(-1) day(-1) . Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.     

Genome sequence of strain TW25, a novel member of the genus Ornithinibacillus in the family Bacillaceae

Ornithinibacillus sp. strain TW25, belonging to the family Bacillaceae, was isolated from a dead ark clam during a mass mortality event. Here, the draft genome sequence of strain TW25 (3,843,870 bp, with a G+C content of 36.7%) is reported. This is the first Ornithinibacillus genome to be sequenced.     

Genome sequence of Ruegeria sp. strain KLH11, an N-acylhomoserine lactone-producing bacterium isolated from the marine sponge Mycale laxissima

Ruegeria sp. strain KLH11, isolated from the marine sponge Mycale laxissima, produces a complex profile of N-acylhomoserine lactone quorum-sensing (QS) molecules. The genome sequence provides insights into the genetic potential of KLH11 to maintain complex QS systems, and this is the first genome report of a cultivated symbiont from a marine sponge.     

Efficient bioremediation of tannery wastewater by monostrains and consortium of marine Chlorella sp. and Phormidium sp.

This study evaluated the bioremediation potential of two marine microalgae Chlorella sp. and Phormidium sp., both individually and in consortium, to reduce various pollutants in tannery wastewater (TW). The microalgae were grown in hazardous 100% TW for 20 days, and the reductions in biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), total phosphorous (TP), chromium (Cr) and total dissolved solids (TDS) of the wastewater monitored periodically. Both marine isolates reduced the BOD and COD by ≥90% in the consortium and by over 80% individually. Concentrations of TN and TP were reduced by 91.16% and 88%, respectively, by the consortium. Removal/biosorption efficiencies for chromium ranged from 90.17–94.45%. Notably, the TDS, the most difficult to deal with, were reduced by >50% within 20 days by the consortium. The novel consortium developed in this study reduced most of the ecologically harmful components in the TW to within the permissible limits of discharge in about 5 to 15 days of treatment. Thus, both the tested marine strains of Chlorella and Phormidium sp. are promising for bioremediating/detoxifying TW and adequately improve the water quality for safe discharge into open water bodies, in particular when used as a consortium.     

Degradation and induction specificity in actinomycetes that degrade p-nitrophenol.

We have isolated two soil bacteria (identified as Arthrobacter aurescens TW17 and Nocardia sp. strain TW2) capable of degrading p-nitrophenol (PNP) and numerous other phenolic compounds. A. aurescens TW17 contains a large plasmid which correlated with the PNP degradation phenotype. Degradation of PNP by A. aurescens TW17 was induced by preexposure to PNP, 4-nitrocatechol, 3-methyl-4-nitrophenol, or m-nitrophenol, whereas PNP degradation by Nocardia sp. strain TW2 was induced by PNP, 4-nitrocatechol, phenol, p-cresol, or m-nitrophenol. A. aurescens TW17 initially degraded PNP to hydroquinone and nitrite. Nocardia sp. strain TW2 initially converted PNP to hydroquinone or 4-nitrocatechol, depending upon the inducing compound.     

Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga

Host-pathogen interactions have been widely studied in humans and terrestrial plants, but are much less well explored in marine systems. Here we show that a marine macroalga, Delisea pulchra, utilizes a chemical defence - furanones - to inhibit colonization and infection by a novel bacterial pathogen, Ruegeria sp. R11, and that infection by R11 is temperature dependent. Ruegeria sp. R11 formed biofilms, invaded and bleached furanone-free, but not furanone-producing D. pulchra thalli, at high (24°C) but not low (19°C) temperatures. Bleaching is commonly observed in natural populations of D. pulchra near Sydney, Australia, during the austral summer when ocean temperatures are at their peak and the chemical defences of the alga are reduced. Furanones, produced by D. pulchra as a chemical defence, inhibit quorum sensing (QS) in bacteria, and this may play a role in furanone inhibition of R11 infection of furanone-free thalli as R11 produces QS signals. This interplay between temperature, an algal chemical defence mechanism and bacterial virulence demonstrates the complex impact environmental change can have on an ecosystem.     

Nicotine degradation by two novel bacterial isolates of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. TW and <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. TY and their responses in the presence of neonicotinoid insecticides

Two novel nicotine-degrading bacterial strains were isolated from tobacco waste and identified as Acinetobacter sp. TW and Sphingomonas sp. TY based on morphology, physiological and biochemical tests, Biolog analysis and 16S rDNA sequencing. The 16S rDNA sequences have been deposited in GenBank under the accession numbers FJ753401 for TW and FJ754274 for TY. The best culture conditions for nicotine degradation were 25–37°C and pH 7.0–8.0 for strain TW and 25–30°C and pH 6.0–7.0 for strain TY. Under the best conditions, the cell growth and nicotine-degradation kinetics of the two isolates were assessed, and 1.0 g/l nicotine was completely degraded within 12 and 18 h for TW and TY, respectively. Moreover, the presence of four widely-used commercial neonicotinoid insecticides in the medium had no effects on nicotine degradation by TW; among the four tested neonicotinoids, only thiamethoxam significantly delayed nicotine degradation by TY. TW and TY were also able to degrade selected neonicotinoids. This is the first report of nicotine degradation by Acinetobacter sp. and Sphingomonas sp. This study showed that these two newly isolated bacteria may be suitable for the disposal of tobacco waste and the reduction of nicotine in tobacco leaves.     

Paenibacillus xylaniclasticus sp. nov., a xylanolytic-cellulolytic bacterium isolated from sludge in an anaerobic digester

A mesophilic, facultative, anaerobic, xylanolytic-cellulolytic bacterium, TW1(T), was isolated from sludge in an anaerobic digester fed with pineapple waste. Cells stained Gram-positive, were spore-forming, and had the morphology of straight to slightly curved rods. Growth was observed in the temperature range of 30 to 50°C (optimum 37°C) and the pH range of 6.0 to 7.5 (optimum pH 7.0) under aerobic and anaerobic conditions. The strain contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone was menaquinone with seven isoprene units (MK-7). Anteiso-C(15:0), iso-C(16:0), anteiso-C(17:0), and C(16:0) were the predominant cellular fatty acids. The G+C content of the DNA was 49.5 mol%. A phylogenetic analysis based on 16S rRNA showed that strain TW1(T) belonged within the genus Paenibacillus and was closely related to Paenibacillus cellulosilyticus LMG 22232(T), P. curdlanolyticus KCTC 3759(T), and P. kobensis KCTC 3761(T) with 97.7, 97.5, and 97.3% sequence similarity, respectively. The DNA-DNA hybridization values between the isolate and type strains of P. cellulosilyticus LMG 22232(T), P. curdlanolyticus KCTC 3759(T), and P. kobensis KCTC 3761(T) were found to be 18.6, 18.3, and 18.0%, respectively. The protein and xylanase patterns of strain TW1(T) were quite different from those of the type strains of closely related Paenibacillus species. On the basis of DNA-DNA relatedness and phenotypic analyses, phylogenetic data and the enzymatic pattern presented in this study, strain TW1(T) should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus xylaniclasticus sp. nov. is proposed. The type strain is TW1(T) (=NBRC 106381(T) =KCTC 13719(T) =TISTR 1914(T)).     

A novel lipopeptide produced by a Pacific Ocean deep-sea bacterium, Rhodococcus sp. TW53

Aims: Our goal was to find a novel, biosurfactant‐producing bacterium from Pacific Ocean deep‐sea sediments. Methods and Results: An oil‐degrading biosurfactant‐producing bacterium TW53 was obtained from deep‐sea sediment, and was identified through 16S rDNA analysis as belonging to the genus Rhodococcus. It lowered the surface tension of its culture to 34·4 mN m^?1. Thin layer chromatography (TLC) showed that the crude biosurfactants of TW53 were composed of lipopeptides and free fatty acids (FA). The lipopeptides were purified with column chromatography and then hydrolysed with 6 mol l^?1 HCl. Gas chromatography‐mass spectrometry analysis showed that the hydrolyte in the hydrophobic fraction contained five kinds of FA with chain lengths of C₁₄–C₁₉, and C₁₆H₃₂O₂ was a major component making up 59·18% of the total. However, 3‐hydroxyl FA was not found, although it is usually found in lipopeptides. Silica gel TLC revealed that the hydrolyte in the hydrophilic fraction was composed of five kinds of amino acids; consistently, ESI‐Q‐TOF‐MS analysis confirmed the composition results and provided their sequence tentatively as Ala‐Ile‐Asp‐Met‐Pro. Furthermore, the yield and CMC (critical micelle concentrations) of purified lipopeptides were examined. The purified product reduced the surface tension of water to 30·7 mN m^?1 with a CMC value of 23·7 mg l^?1. These results suggest that Rhodococcus sp. TW53 produces a novel lipopeptide that we have named rhodofactin. Conclusion: The deep‐sea isolate Rhodococcus sp. TW53 was the first reported lipopeptide‐producing bacterium of this genus. The lipopeptides had novel chemical compositions. Significance and Impact of the Study: Rhodococcus sp. TW53 has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution.     

Ornithinibacillus scapharcae sp. nov., isolated from a dead ark clam

A novel Gram-positive, aerobic, motile, hemolytic, endospore-forming and rod-shaped bacterium TW25^T was isolated from a dead ark clam during a mass mortality event on the South coast of Korea. The strain grew optimally at 30°C, at pH 8–9, and with 1% (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain TW25^T was associated with the genus Ornithinibacillus and that it was most closely related to the type strain of Ornithinibacillus californiensis (98.5% similarity). The dominant cellular fatty acids were iso-C15:0, anteiso-C15:0 and C16:0. The peptidoglycan amino acid type was A4β, containing l-ornithine and d-aspartic acid. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, four unidentified phospholipids, two unidentified aminolipids and two unidentified lipids. The major respiratory quinone was menaquinone-7 (MK-7). The G + C content of genomic DNA was 36.7 mol%. DNA–DNA hybridization experiments with related strains revealed lower than 11 ± 3% relatedness. Based on this polyphasic taxonomic study, strain TW25^T represents a novel species in the genus Ornithinibacillus, for which the name Ornithinibacillus scapharcae sp. nov. is proposed. The type strain is TW25^T (=KACC 15116^T = JCM 17314^T).     

The Recovery of Repeated-Sprint Exercise Is Associated with PCr Resynthesis,while Muscle pH and EMG Amplitude Remain Depressed

The physiological equivalents of power output maintenance and recovery during repeated-sprint exercise (RSE) remain to be fully elucidated. In an attempt to improve our understanding of the determinants of RSE performance we therefore aimed to determine its recovery following exhaustive exercise (which affected intramuscular and neural factors) concomitantly with those of intramuscular concentrations of adenosine triphosphate [ATP], phosphocreatine [PCr] and pH values and electromyography (EMG) activity (a proxy for net motor unit activity) changes. Eight young men performed 10, 6-s all-out sprints on a cycle ergometer, interspersed with 30 s of recovery, followed, after 6 min of passive recovery, by five 6-s sprints, again interspersed by 30 s of passive recovery. Biopsies of the vastus lateralis were obtained at rest, immediately after the first 10 sprints and after 6 min of recovery. EMG activity of the vastus lateralis was obtained from surface electrodes throughout exercise. Total work (TW), [ATP], [PCr], pH and EMG amplitude decreased significantly throughout the first ten sprints (P<0.05). After 6 min of recovery, TW during sprint 11 recovered to 86.3±7.7% of sprint 1. ATP and PCr were resynthesized to 92.6±6.0% and 85.3±10.3% of the resting value, respectively, but muscle pH and EMG amplitude remained depressed. PCr resynthesis was correlated with TW done in sprint 11 (r = 0.79, P<0.05) and TW done during sprints 11 to 15 (r = 0.67, P<0.05). There was a ∼2-fold greater decrease in the TW/EMG ratio in the last five sprints (sprint 11 to 15) than in the first five sprints (sprint 1 to 5) resulting in a disproportionate decrease in mechanical power (i.e., TW) in relation to EMG. Thus, we conclude that the inability to produce power output during repeated sprints is mostly mediated by intramuscular fatigue signals probably related with the control of PCr metabolism.     

具ACC脱氨酶活性的碱蓬内生细菌的分离、鉴定及其生物学特性

【目的】具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的盐生植物碱蓬内生细菌的分离及生物学特性的研究有助于探索内生细菌与宿主植物耐盐性的关系。【方法】采用研磨法从健康碱蓬植株的根、茎、叶中分离具有ACC脱氨酶活性的碱蓬内生细菌,根据形态特征、生理生化、API鉴定系统和16S rRNA对菌种进行鉴定,并分别测定了菌株产ACC脱氨酶、铁载体、吲哚乙酸、赤霉素、脱落酸、蛋白酶及溶磷、固氮和拮抗病原菌的特性。【结果】将分离得到的内生细菌LP11、SS12、TW1和TW2分别鉴定为栖稻假单胞菌(Pseudomonas oryzihabitans)、假单胞菌(Pseudomonas sp.)、成团泛菌(Pantoea agglomerans)和恶臭假单胞菌(Pseudomonas putida),4株菌除具有较高的ACC脱氨酶活力之外,还可不同程度地产生铁载体、吲哚乙酸、赤霉素和脱落酸,且均有溶磷作用,但无固氮能力及蛋白酶活力,唯有菌株SS12对萝卜枯萎病菌(Fusariumoxysporum f.sp.conglutinans)和黄瓜枯萎病菌(F.oxysporum f.sp.cucumerinum)具有拮抗作用。【结论】从盐生植物碱蓬中分离到的假单胞菌属和泛菌属内生细菌,具有丰富多样的生物学特性。     

A novel multienzyme complex from a newly isolated facultative anaerobic bacterium, Paenibacillus sp. TW1

A multienzyme complex from newly isolated Paenibacillus sp. TW1 was purified from pellet-bound enzyme preparations by elution with 0.25% sucrose and 1.0% triethylamine (TEA), ultrafiltration and Sephacryl S-400 gel filtration chromatography. The purified multienzyme complex showed a single protein band on non-denaturing polyacrylamide gel electrophoresis (native-PAGE). The high molecular mass of the purified multienzyme complex was approximately 1,950 kDa. The complex consisted of xylanase and cellulase activities as the major and minor enzyme subunits, respectively. The complex appeared as at least 18 protein bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and as 15 xylanases and 6 cellulases on zymograms. The purified multienzyme complex contained xylanase, α-L-arabinofuranosidase, carboxymethyl cellulase (CMCase), avicelase and cellobiohydrolase. The complex could effectively hydrolyze corn hulls, corncobs and sugarcane bagasse. These results indicate that the multienzyme complex that is produced by this bacterium is a large, novel xylanolytic-cellulolytic enzyme complex.     

Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish

After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.     

Ruegeria arenilitoris sp. nov., isolated from the seashore sand around a seaweed farm

A Gram-negative, motile and rod-shaped bacterial strain, G-M8^T, which was isolated from seashore sand around a seaweed farm at Geoje island in South Korea, was characterized taxonomically. It grew optimally at 30–37 °C, at pH 7.0–8.0 and in presence of 2 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain G-M8^T joined the cluster comprising the type strains of Ruegeria atlantica and Ruegeria lacuscaerulensis, showing 97.5 % sequence similarity, by a bootstrap resampling value of 85.8 %. It exhibited 16S rRNA gene sequence similarity values of 95.4–96.7 % to the type strains of the other Ruegeria species. Strain G-M8^T exhibited the highest gyrB sequence similarity value (88.5 %) to the type strain of R. lacuscaerulensis. Strain G-M8^T contained Q-10 as the predominant ubiquinone and C_18:1 ω7c as the predominant fatty acid. The polar lipid profile of strain G-M8^T was similar to that of R. atlantica KCTC 12424^T. The DNA G+C content of strain G-M8^T was 64.6 mol% and its mean DNA–DNA relatedness values with R. atlantica KCTC 12424^T and R. lacuscaerulensis KCTC 2953^T were 18 ± 5.3 and 10 ± 3.6 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, demonstrated that strain G-M8^T is distinguished from other Ruegeria species. On the basis of the data presented, strain G-M8^T (=KCTC 23960^T = CCUG 62412^T) represents a novel species of the genus Ruegeria, for which the name Ruegeria arenilitoris sp. nov. is proposed.     

Column Flushing of Phenanthrene and Copper (II) Co-Contaminants from Sandy Soil Using Tween 80 and Citric Acid

The clean-up of soils co-contaminated with heavy metals and organic compounds is a contemporary issue of remediation efforts. Column flushing was conducted to investigate the performance of nonionic surfactant and/or organic acid solutions, 4000 mg/L Tween 80 (TW80), and/or 0.04 mol/L citric acid (CA), to enhance the simultaneous removal of phenanthrene and copper (II) from the co-contaminated sandy soil. The flushing effects were compared when TW80, CA, TW80 after CA (CA/TW80), CA after TW80 (TW80/CA), and a mixture of TW80 and CA (TW80-CA) were used as flushing agents. The maximum concentrations of phenanthrene in effluent solutions occurred at 3.3, 4.7, 5.3, and 15.3 h during TW80, TW80/CA, TW80-CA, and CA/TW80 flushing and those of copper (II) at 2.7, 3.3, 3.3, and 14.0 h during CA, CA/TW80, TW80-CA, and TW80/CA flushing, respectively. Phenanthrene was mainly desorbed through partitioning into TW80 micelles in aqueous phase while copper (II) was effectively removed through complexation with CA. The removal efficiencies were up to 81.5%, 5.9%, 99.9%, 91.6%, and 99.8% for phenanthrene, and 0.1%, 76.7%, 85.7%, 78.1%, and 84.4% for copper (II) by TW80, CA, TW80/CA, TW80-CA, and CA/TW80. However, it took a long time to use TW80/CA and CA/TW80 to clean phenanthrene and copper (II) efficiently. The overall removal efficiencies of contaminants in the soil column increased with flushing time in the Sigmoidal Model. The results indicated that a combination of TW80 and CA has potential for in situ clean-up of heavy metals and polycyclic aromatic hydrocarbons (PAHs) from co-contaminated soils.     

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin

The terminal web of the intestinal brush border contains a spectrin- like protein, TW 260/240 (Glenney, J. R., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854.) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N., R. E. Cheng, and M. Willard, 1983, Cell, 32:953-965; Glenney J. R., P. Glenney, and K. Weber, 1983, J. Cell Biol., 96:1491-1496). We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin. Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross- linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength. However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules. At salt concentrations at (150 mM KCl) and above (300 mM KCl) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0 degree and 37 degrees C. We also observed (in the presence of 75 mM KCl) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates. We have studied the interaction of TW 260/240 with actin using techniques of co-sedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt- and pH- dependent. Under optimum conditions (25-75 mM KCl, at pH 7.0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered. At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (greater than 150 mM KCl), the binding of TW 260/240 to actin was inhibited. The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F- actin.     

Tea waste as a supplement for the cultivation of <Emphasis Type="Italic">Ganoderma</Emphasis> <Emphasis Type="Italic">lucidum</Emphasis>

Tea waste (TW) was investigated as a new supplement for substrate mixtures in Ganoderma lucidum cultivation. The effects of sawdust (S) based substrates supplemented with TW at the various levels (75S:25TW, 80S:20TW, 85S:15TW, and 90S:10TW) and Ganoderma lucidum strains on yield, biological efficiency (BE) and the chemical composition of fruiting bodies were determined in solid-state fermentation. Significant differences were found among substrates regarding yield and BE, while yield and BE of the strains were not different. The substrate formulations producing highest yield and BE were 80S:20TW (87.98 g/kg substrate and 34.90%) and 75S:25TW (82.30 g/kg substrate and 31%). Yield and BE of substrates containing TW were generally higher than that of the control (80sawdust:18wheat bran:1sucrose:1 CaCO₃). Nitrogen, potassium, iron, and manganese contents and C:N ratios of substrates were strongly correlated with yield. BE showed positive and significant correlations with potassium, iron and manganese. Moisture content, potassium, magnesium, calcium, iron, and zinc contents of the fruiting bodies were affected by both strain and substrate. It was concluded that TW can be used as a supplement for substrate preparation in G. lucidum cultivation.     

Isolation and identification of bacterial strains with insecticidal activities from Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

Several pathogenic bacteria strains were isolated from dead larvae of the weevil, Rhynchophorus ferrugineus Oliver, collected on palm trees in Taiwan, China. Strains of pure cultures were temporarily named TW‐1, TW‐2, TW‐3, TW‐4, TW‐5, TW‐6, TW‐7, TW‐8, TW‐9, TW‐10, TW‐11, TW‐12, TW‐13 and TW‐14. These strains were identified by synthetically using colony morphological characters, physiological and biochemical tests and molecular biological analysis. Our results showed that these 14 isolated strains belonged to four genera and five species. The expected approximate 1400‐bp bands were obtained through 16S rDNA universal primer amplification. The sequencing results obtained from a commercial sequencing company were initially compared with a corresponding database from NCBI using BLAST, and a phylogenetic tree was constructed using mega 5.1 software. The phylogenetic results indicated that these isolated strains had more than 99% homology with type strains. To further confirm these strains, numerous physiological and biochemical indexes were confirmed. TW‐1, TW‐2, TW‐3, TW‐9, TW‐12, TW‐13 and TW‐14 were identified as Serratia marcescens; TW‐4, TW‐5, TW‐8 and TW‐10 as Staphylococcus sciuri; TW‐6 as Klebsiella pneumonia subsp. pneumonia; TW‐7 as Proteus vulgaris and TW‐11 as Proteus mirabilis. The preliminary single‐dose bioassay on fourth instar larvae showed that S. marcescens had a higher virulence at a density of 1.89 OD₆₀₀ (56.37% mortality) than the other strains. The corrected mortalities of the other strains were all less than 50%. The lowest toxicities were found for P. vulgaris and P. mirabilis where the corrected mortalities were only 28.18% and 25.57%, respectively. LC₅₀ of S. marcescens was 1.2 × 10⁷ CFU/ml inoculums. Our results indicated that S. marcescens from facultative cultivable bacterial flora isolated from R. ferrugineus could potentially be used as a microbial control agent for this widespread pest.