Denitrification performance and microbial communities of solid-phase denitrifying reactors using poly (butylene succinate)/bamboo powder composite.

This study explored the denitrification performance of solid-phase denitrification (SPD) systems packed with poly (butylene succinate)/bamboo powder composite to treat synthetic aquaculture wastewater under different salinity conditions (0‰ Vs. 25‰). The results showed composite could achieve the maximum denitrification rates of 0.22 kg (salinity, 0‰) and 0.34 kg NO3--N m-3 d-1 (salinity, 25‰) over 200-day operation. No significant nitrite accumulation and less dissolved organic carbon (DOC) release (<15 mg/L) were found. The morphological and spectroscopic analyses demonstrated the mixture composites degradation. Microbial community analysis showed that Acidovorax, Simplicispira, Denitromonas, SM1A02, Marinicella and Formosa were the dominant genera for denitrifying bacteria, while Aspergillus was the major genus for denitrifying fungus. The co-network analysis also indicated the interactions between bacterial and fungal community played an important role in composite degradation and denitrification. The outcomes provided a potential strategy of DOC control and cost reduction for aquaculture nitrate removal by SPD.

Aerobic denitrification performance and nitrate removal pathway analysis of a novel fungus Fusarium solani RADF-77.

Increasing nitrogenous contaminants have caused immense challenges to the environment and human health. As compared to physical and chemical methods, biological denitrification is considered to be an effective solution due to its environmental friendliness, high efficiency, and low cost. In the present work, a novel fungal strain identified as Fusarium solani (RADF-77) was isolated from cellulose material-supported denitrification reactor; this strain is capable of removing nitrogen under aerobic conditions. The average NO3--N removal rate for RADF-77 were 4.43 mg/(L·h) and 4.50 mg/(L·d), when using glucose and tea residue as carbon source, respectively. The nitrogen balance revealed that 53.66% of N vanished via gaseous products. Transcriptional results revealed that respiratory and assimilative nitrate reductases may work together for nitrate removal. Our results indicate that RADF-77 could be used as a potential means of enhancing nitrate-removal performance, as well as recycling tea residue, which is the main byproduct of the manufacture of tea extracts.

Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb.

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain (V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5'- and 3'-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5'-UTR) and -UUUACUACU (in the 3'-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).

Microarray-based identification of tomato microRNAs and time course analysis of their response to Cucumber mosaic virus infection.

A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with Cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

A gyrB-targeted PCR for rapid identification of Paenibacillus mucilaginosus.

Paenibacillus mucilaginosus, one of the typical silicate bacteria, has long been used as a biofertilizer in agriculture and has recently shown potential in bioleaching and wastewater engineering. There has been considerable research involving the isolation of P. mucilaginosus for various utilizations; therefore, rapid identification of this species is of great interest. Herein, we describe a specific polymerase chain reaction (PCR) method developed for a rapid identification of P. mucilaginosus, which might provide potential utilization in the investigation of populations, detection of biofertilizers, and identification of novel isolates on a large scale. A gyrB-targeted species-specific primer pair, F2 (5'-ACG GAT ATC TCC CAG ACG TTC AT-3') and R5 (5'-ACG GGC ACG CTG CGC CTG TAC G-3'), was successfully designed to selectively amplify a 519-bp amplicon from P. mucilaginosus. Good specificity was demonstrated by both reference strains and total soil deoxyribonucleic acid, from which only the gyrB gene of P. mucilaginosus was amplified. The detection limit was 4-10 cells per assay. Using the culture-PCR method, 20 of 26 soil isolates on a nitrogen-free medium were rapidly identified as P. mucilaginosus, which was confirmed by sequencing of the gyrB gene.

Transfer of Bacillus mucilaginosus and Bacillus edaphicus to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov. and Paenibacillus edaphicus comb. nov.

Bacillus mucilaginosus and Bacillus edaphicus were reclassified based on their 16S rRNA and gyrB gene sequences, DNA-DNA hybridization, fatty acid methyl esters and other taxonomic characteristics. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences indicated that strains of B. mucilaginosus and B. edaphicus were members of the genus Paenibacillus, with over 90.4 % and 70.3 % sequence similarity, respectively. Their DNA G+C contents were 54.5-56.8 mol%. The DNA-DNA relatedness values of B. edaphicus VKPM B-7517(T) with B. mucilaginosus KNP414 and B. mucilaginosus CGMCC 1.236 were 89.2 % and 88.7 %, respectively. The major isoprenoid quinone of B. mucilaginosus and B. edaphicus was MK-7 (94.1-95.7 %). The peptidoglycan type was A1gamma (meso-diaminopimelic acid) and the major polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were anteiso-C(15 : 0), C(16 : 1)omega11c and C(16 : 0). Phenotypic features and fatty acid profiles supported the similarity of B. mucilaginosus and B. edaphicus to Paenibacillus validus CCTCC 95016(T) and confirmed their relationship with members of the genus Paenibacillus. Therefore, it is proposed that Bacillus mucilaginosus and Bacillus edaphicus be transferred to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov. (type strain HSCC 1605(T)=VKPM B-7519(T)=VKM B-1480D(T)=CIP 105815(T)=KCTC 3870(T)) and Paenibacillus edaphicus comb. nov. (type strain VKPM B-7517(T)=DSM 12974(T)=CIP 105814(T)), respectively.

Purification and characterization of keratinase from a new Bacillus subtilis strain.

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.

Type-dependent differential expression of neuropeptide Y in chicken hypothalamus (Gallus domesticus).

Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY between layer-type and meat-type of chickens, which are two divergent kinds of the domestic chickens in feeding behavior and body weight, we detected mRNA levels of NPY in hypothalamic infundibular nucleus (IN), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of these two types of chickens using one-step real time RT-PCR. The meat-type chicken had more food daily (about 1.7 folds) and greater body weights (about 1.5 folds) and brain weights than the layer-type chicken at the age of 14 d. In the meat-type of chicken, NPY mRNA levels of the IN and PVN were significantly greater than those of the LHA, and were not significantly different between the IN and PVN. However, in the layer-type of chicken, NPY mRNA levels were significantly greater in the IN than those in the LHA and PVN, and were not significantly different between the PVN and LHA. In all these hypothalamic regions, the layer-type of chicken had significantly higher NPY mRNA levels than the meat-type chicken did. These results suggest the expression of NPY in the hypothalamus has a type-dependent pattern in domestic chickens.

2b ORFs encoded by subgroup IB strains of cucumber mosaic virus induce differential virulence on Nicotiana species.

Cucumber mosaic virus (CMV)-encoded 2b protein from subgroup IA or subgroup II was shown to be a determinant of virulence in many solanaceous hosts. In this study, the virulence of 2b proteins from subgroup IB strains was analysed using four intraspecies hybrid viruses, which were generated by precise replacement of the 2b open reading frame (ORF) in subgroup IA strain Fny-CMV with the 2b ORFs of four subgroup IB strains, Cb7-CMV, PGs-CMV, Rad35-CMV and Na-CMV, generating FCb7(2b)-CMV, FPGs(2b)-CMV, FRad35(2b)-CMV and FNa(2b)-CMV, respectively. FCb7(2b)-CMV was more virulent than Fny-CMV, and was similar in phenotype to its parental virus Cb7-CMV on the three Nicotiana species tested. FNa(2b)-CMV also was virulent on these host species, equivalent to Fny-CMV or Na-CMV. However, FRad35(2b)-CMV only caused mild mosaic or undetectable symptoms on all the host species tested, and was less virulent than Fny-CMV or Rad35-CMV. FPGs(2b)-CMV infected all the host species systemically, and induced either mosaic or barely visible symptoms, demonstrating that the inability of PGs-CMV to infect these three Nicotiana species was not due to its 2b protein. The diverse virulence was shown to be mediated by the 2b proteins rather than the C-terminal overlapping parts of the 2a proteins, and was associated with the level of viral progeny RNA accumulation in systemically infected leaves, but not with the rate of long-distance viral movement in host plants. Through analysis of encapsidation of viral RNAs, there was an apparent correlation between the virulence and the high level of encapsidated RNA 2 in virions of Fny-CMV, FCb7(2b)-CMV and FNa(2b)-CMV.

[Detection of two viruses infecting Pinellia ternata in China].

OBJECTIVE: To study viruses infecting Pinellia ternata in China. METHOD: Symptom observation, DAS-ELISA and RT-PCR detection were applied. RESULT AND CONCLUSION: During a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.

Quantitative determination of cucumber mosaic virus genome RNAs in virions by real-time reverse transcription-polymerase chain reaction.

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct) values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2, RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about 1.00:1.17:3.58:5.81. These results were confirmed by Lab-on-a-chip and northern blot hybridization assays. Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.

[Isolation and identification of a newbacterial pathogen infecting larvae of honeybee (Apismellifera ) Perish].

A bacteroidal disease of honeybee (Apis mellifera ) larvae was found in some regions of Zhejiang Province, China , in early spring 2005. The diseased larvae lost its shine, became yellow and rotted when serious. This symptom was different to any bacteroidal disease of honeybee larval been reported. So, it is considered to be a new bacteroidal disease of honeybee larval. Five pure cultures of bacteria were separated from ten collections of diseased honeybee larvae, named as L1, L2, L3, IA and L5. Among these five pure cultures, only L2 could make the healthy honeybee larvae become diseased in both field and lab test. The symptom caused by L2 was similar to the natural-infection. From the diseased larvae caused by L2 could isolate bacteria the same as L2. Thus 12 was determined as the causing agent of this bacteroidal disease of honeybee larval. L2 was identified according to the characteristics of morphology, physiological biochemical characteristics and 16S rRNA gene sequence. As a result, the morphology and physiological biochemical characteristics of L2 were similar to E. faecium. And its 16S rRNA sequences highly matched to E. faecium, the similarities between them were higher than 99%. The overall similarity values between L2 and the published 16S rRNA sequences of 41 typical species of Enterococcus were 93.9% - 99.5% , the top value was between 12 and E. faecium. In the phylogenetic tree, L2 and E. faecium were assembled in the same ramification. So 12 was identified as E. faecium. Although Enterococcus faecium was known as pathogen to many post, account for 12% of all nosocomial infections, only second to E. coli, but there is no report about this bacteria infects honeybee up to now. So it is a new pathogen to honeybee. The isolation and identification of pathogen of this new bacteroidal larvae disease, afford a good feasibility for available prevention and cure to this new disease.

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

[Coexistence and competition of two satellite RNAs of Cucumber mosaic virus in systemic hosts].

Two Cucumber mosaic virus (CMV) satellite RNAs, namely Yns and Yi and of 385nt and 369nt respectively, were introduced to systemic host plants with a satellite RNA-free isolate of CMV (CNa), after in vitro transcription of satellite RNAs from cDNA clones and by co-inoculation with CMV genomic RNAs. The competition and coexistence of the two satellite RNAs were studied by RT-PCR detection, double-stranded RNA analysis and sequencing comparison. The results showed that, in the inoculated leaves of Nicotiana glutinosa, both satellite RNA presented together after inoculated 5 days after pseudo-recombination. In the systemic leaves of N. glutinosa, both satellite RNAs were detected at 5 days and 10 days post first inoculation, however, only the 369nt satellite (Yi) was recovered at 15 days post first inoculation and second inoculation transferred from the pseudo-recombination plants. Comparison of full sequences of the satellite RNAs obtained by RT-PCR from pseudo-recombinant virus showed that no mutation and mutual exchange of the satellite genome has been found. The results indicate that both 369nt satellite RNA and 385nt satellite RNA can be introduced to CMV-CNa with in vitro transcription products and both replicated to a high level depending on the helper virus. But after transferring to new systemic plants, only one satellite of 369nt can be kept as co-infection for long with the helper CMV. Co-existence and competition are found between different satellite RNAs in the same plant.

[Distribution of Rubisco and RCA in Brassica chinensis chloroplasts and effect of TuMV-infection on their cellular localization].

The cellular localizations of Rubisco and Rubisco activase (RCA) in Chinese cabbage (Brassica chinensis L. cv. Suzhou) leaves were investigated by immunogold-labeling electron microscopy. The results showed that Rubisco and RCA were mainly located in chloroplasts of mesophyll, guard cell of stomatal apparatus and parenchyma of vascular bundle. A high density of gold particles were localized preferentially to the chloroplast stroma. In contrast, there were no specific binding of gold particles detected in the cytoplasm, vacuole, mitochondria and other organelles. As infected by turnip mosaic virus, the density of gold particles decreased lightly in the abnormal chloroplasts and dropped by much lower (58.44% and 64.67% as that in the health chloroplasts respectively) in swollen chloroplasts. This indicated that virus infection caused the decreases of Rubisco and RCA in host plant, which then affected plant photosynthesis.

[Accumulation of coat protein of Turnip mosaic virus in host chloroplasts and its effect on PS II activity].

Intact chloroplasts were isolated from Chinese cabbage (Brassica chinensis L. cv. Suzhou) and mustard (B. juncea L. cv. Wenzhou) leaves inoculated with Turnip mosaic virus (TuMV). The proteins attached to the surface of chloroplasts were digested, then the total proteins of chloroplasts were extracted. By analysis of the SDS-PAGE pattern, one extra protein band with the same mobility and molecular weight as TuMV-coat protein was found in the chloroplasts of infected leaves. Results of Western blotting showed that this band was highly reactive to the antiserum against TuMV-CP. The detection of immunogold-particles also showed the presence of TuMV-CP in chloroplasts of both host plant species as compared with the healthy control plant. Electron microscopic photograph showed some virus particles and cylindrical inclusions in the cytoplasm of host plants. Some chloroplasts showed abnormalities, including inhibited lamellar development, swollen and rounded, membrane vesiculation and disintegration. On the tenth day after TuMV inoculation, F(v) /F(o), F(v)/ F(m), Phi(PS II) and q(P) values all became lower, but the q(N) values were higher, as compared with the healthy control plants. The results provided the evidence for the presence of TuMV-CP in the diseased chloroplasts. It suggests that the accumulation of TuMV-CP inside chloroplasts affect photosynthesis in virus-infected plants by inhibiting the PS II activity.