The diamondback moth (Plutella xylostella (Linn.), DBM) is the most destructive insect pest of cruciferous vegetables throughout the world and has become highly resistant to pyrethroid insecticides. To furtherly elucidate the occurrence characteristics of knockdown resistance in DBM, by means of the nucleotide sequencing method, the knockdown resistance allele frequencies in both the laboratory and field strains were detected. Besides, the genotypes of the dead individuals after fenvalerate treatment were also determined. Only L1014F and T929I mutations were found. Same resistance allele frequencies of 98.75%~100% were found in the 2 mutation sites in the of 2nd to 4th instar larvae of laboratory strain. The resistance allele frequencies for L1014F and T929I mutations in field strains, collected from Fujian, Zhejiang, Inner Mongolia and Beijing, were of 90.00%~100% and 88.75%~100%, respectively. And that were both of 98.75%~100% in the 2 sites in the field strain collected from vegetable fields in Pudang, Fuzhou of during November, 2010 and November, 2011. The dead insects after fenvalerate treatment at different concentrations were all resistant homozygotes in 2 mutation sites. The results indicated that genotypes at the mutation sites were mainly resistant homozygous in DBM and knockdown resistance allele frequencies varied little with larval instar, occurrence seasons in the same locality and different collection localities in the same season. T929I mutation occurred only together with L1014F mutation, whereas, when the genotype was heterozygously resistant in L1014F site, then that would be homozygously susceptible or heterozygously resistant in T929I site. Genotype at mutation sites was not the sole factor that determined the sensitivity of DBM to fenvalerate. This study will help to betterly understand the evolution characteristics of knockdown resistance in DBM.

Vegetative insecticidal protein 3A (Vip3A) has a broad specturm toxicity torwards Lepidoptera pests. The 2.4 kb entire coding region of vip3Aa gene (GenBank No. KT307982) was amplified by PCR with total DNA extracted from Bacillus thuringienesis (Bt) NBIC-380 which had high insecticidal activity against corn borer (Ostrinia nubilalis) as template. The vip3Aa was inserted into multiple cloning sites of the Escherichia coli expression vector pGEX-6p-1 and pET-28a, Bacillus expression vector pBMB1A to generate the recombinant expression vector pGV3, pEV3 and pBV3. The pGV3 and pEV3 were transformed into E. coli BL21 (DE3), respectively, and pBV3 was electro- transformed into Bt BMB171 and Bacillus subtilis (Bs) 168 strains. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) showed one protein of 88 kD. The expressed protein in E. coli was purified by GST affinity chromatography and Ni affinity chromatography, which demonstrated no toxicity towards Helicoverpa armigera second instar larvae. The proteins expressed in BtBMB171 and Bs168 had activities against H. armigera second instar larvae with medium lethal concentration (LC50) of 6.185 6 and 2.984 6 mg/mL, respectively. Bioassay indicated that expression of vip3Aa gene from Bt NBIC-380 strain had certain toxicity torwards H. armigera. This work enriches the fundamental research of Vip and provides basic data for construction of high toxicity and broad spectrum engineering strains.

To study the effects of Bacillus thuringiensis on rice (Oryza sativa) seedling under cadmium stress, the plant growth parameters of germination rates of rice seeds, the yellow and brown rates of rice seedlings and chlorophyll content were determined by direct measurement method, apparent observation and ultraviolet spectrophotometry. Atomic force microscopy was applied to observe the surface changes of B. thuringiensis before and after cadmium treatment, and some granular dots were found on the bacterial surface, which implied there was surface adsorption of cadmium on B. thuringiensis, indicated that B. thuringiensis could be a potential bacterium in the practical applications of Cd2+ contaminated bioremediation on rice soil. Additionally, the growth of rice seedling was significantly inhibited under the cadmium stress, and the inhibition activity was enhanced with the increasing concentration of cadmium. However, the seedlings had no significant difference of shoot height and root length in 50 and 400 mg/L Cd2+ treatment groups between Cd and Cd+Bt groups (P＞0.05), which indicated that there was no significant changes in the shoot height and root length of the seedlings before and after Bt treatment under the cadmium stress. The significant difference of chlorophyll a and b was found in 100 and 400 mg/L Cd2+ treatment between Cd and Cd+Bt groups (P＜0.05). The rice seed germination rate had significant difference in 200 mg/L Cd2+ treatment group between Cd and Cd+Bt groups (P＜0.05). Meanwhile, the yellow rate and brown spot rate of Cd+Bt and Cd treatments were also monitored. It was found that the yellow rates had significant difference in 50 and 400 mg/L Cd2+ treatment between Cd and Cd+Bt groups (P＜0.05). In addition, the brown spot did not appear in the treatment group of 50 mg/L Cd2+, while the brown spot rate had significant difference in 400 mg/L Cd2+ treatment between Cd and Cd+Bt groups (P＜0.05), which indicated that both the yellow rate and brown spot rate of Cd+Bt were lower than the Cd treatment. The above results demonstrated that B. thuringiensis could reduce the concentration of free cadmium in rice soil by adsorption process, thereby relieve the cadmium stress and increase the chlorophyll content of rice seedlings. The present work provides a theoretical basis for the application of the Bt strain in the field of cadmium contamination.

This study was based on Bacillus thuringiensis(Bt) strain FH21 with high toxic of Plutella xylostella larvae. Using an improved polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method, cry1Bb3, cry1Da4, cry1Ja3 and cry1Id3 genes were cloned from strain FH21 with the GenBank No. KJ619659, KJ619660, JQ228425 and KJ619661, respectively. The 4 genes were inserted into expression vector pEB, and transformed into Escherichia coli strain Rosetta (DE3), which were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) at low temperature. The total protein of FH21 and the 4 proteins were used for bioassay on P. xylostella, Helicoverpa armigera and Ostrinia furnacalis. The total protein of FH21 had high insecticidal activity against 3 insects with lethal concentration 50 (LC50) of 0.801, 0.502 and 2.858 mg/L, respectively. Cry1Bb3, Cry1Ja3 and Cry1Id3 had high insecticidal activity against P. xylostella and O. furnacalis with LC50 of 1.860, 1.697, 1.538, 1.800, 2.270 and 0.840 mg/L, respectively. This study showed that Bt FH21strain against agricultural pests with a high insecticidal activity, insecticidal genes were assayed and insecticidal activity were determined simultaneously. The cry1 genes provide more choices for important agricultural pest.

Rice dwarf virus (RDV) is mainly transmitted by leafhopper (Nephotettix cincticeps) in a persistent-propagative manner. The genome of RDV consists of 12 dsRNA segments, encoding at least 7 structural proteins (P1, P2, P3, P5, P7, P8 and P9) and 5 non-structural proteins (Pns4, Pns6, Pns10, Pns11 and Pns12). Pns11 of RDV is known to be a gene silencing suppressor in plant, and participates in the formation of viroplasm for viral replication and assembly of progeny virions in the cultural cells of N. cincticeps. However, the definite function of Pns11 in viroplasm in insect vector is still unknown yet. In the present study, RNA interference (RNAi) was firstly applied via delivering the synthesized dsRNAs in vitro from Pns11 gene into cultural cells of N. cincticeps. Immuno-fluorescence assay showed that when Pns11 was knocked down, the antigens of Pns11 were significantly reduced, and RDV was restricted within 1~2 cultural cells. The infectivity evaluated with the fluorescent antibody focus counting method showed the level of the viral infection decreased about 5 times. These results indicated that RNAi targeting on Pns11 gene significantly reduced the expression of Pns11, leading the RDV accumulation and infection, was blocked in the cultural cells. Thus, Pns11 was assumed to associate with viral replication. Then, microinjection of dsRNAs from Pns11 into the body of N. cincticeps was performed. qRT-PCR test showed the knockdown of Pns11 significantly decreased the viruliferous rate of insect more than 60%. And the RNAi effect steadily lasted for more than 12 d, which was nearly a circulative transmission period. qRT-PCR assay showed that knockdown of Pns11 caused more than 80% reduction in the relative expression levels of P8 and Pns11 genes. It was suggested that RNAi targeting on Pns11 gene inhibited the viral accumulation in body of N. cincticeps, and decreased the number of viruliferous insect. Immuno-fluorescence assay displayed that at 6 d after microinjection, RDV and Pns11 were detected in the epithelia cells of filter chamber and midgut of N. cincticeps in control, while blocked in 1~2 epithelia cells of filter chamber in treatment of Pns11 knockdown. At 12 d after microinjection, RDV and Pns11 were detected in the whole of intestines and salivary gland in control, while still blocked in 2~3 epithelia cells of filter chamber in the treatment of Pns11 knockdown. These observations indicated that knockdown of Pns11 inhibited the spread of RDV in insect vector via blocking viral replication. Finally, capability of viral transmission by insect was tested. The result showed that when Pns11 was knocked down, insect vectors were unable to transmit RDV in 14 d after microinjection. These results confirmed that Pns11 was a viral replication factor, which was necessary for RDV infection and multiplication. It also verified Pns11 was an effective target to block viral replication and transmission. This study theoretically bases for virus disease control via blocking RDV multiplication and circulation in insect vector.

Bacteriocins are peptides or proteins with antimicrobial activities, and produced by some bacteria through the ribosome way. Because of their harmless and low drug resistance, they are welcomed in food and health safety. In this study, a putative bacteriocin gene AOI-3 was predicted from a whole-genome sequenced Bacillus thuringiensis (Bt) BRC-ZYR2 and amplified by PCR with total DNA. Then the 231 bp PCR product was purified and ligated to pET32a expression vector by seamless cloning technology, and transformed into Escherichia coli JM109. The selected clones were digested and sequenced. Sequence alignment showed that nucleotide homology between the gene AOI-3 and a gene (GenBank No. CP003763.1) in Bt HD-789 whole-genome was 99%, while the amino acid sequence homology with a putative Bt bacteriocin biosynthesis protein(GenBank No. WP033699510.1) was 100%. Furthermore, the conserved domain of AOI-3 bacteriocin belonged to DUF2762 (domain of unknown function, DUF) superfamily, which were annotated as holin-like proteins BhlA. Amino acid composition analysis showed that the gene sequence encoded 76 amino acids, the molecular weight was 8 813.62 Da, the isoelectric point was 4.82, without signal peptide, containing a transmembrane domain. A recombinant plasmid was transformed into BL21 (DE3) and induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular weight of the expressed peptide was about 8.8 kD in supernatants, which was completely coincided with the forecasted result. The expressed peptide was furtherly purified by Ni-NTA affinity chromatography. Herein, the cloning, expression and purification of the putative bacteriocin gene AOI-3 provide basic data for evaluation of its anti-microbial activities, and elucidation of its struture and mechanism.

Rice ragged stunt virus (RRSV) is transmitted by Nilaparvata lugens in a persistent propagative manner. The nonstructural protein 6 (Pns6) encoded by RRSV is a constituent of viroplasm, but the functional role of Pns6 in viral infection of insect vector is still unkown. In this study, RNA interference (RNAi) induced by synthesized dsRNAs was used to investigate the function of Pns6 in N. lugens. Second-instar nymphs of N. lugens were fed with dsRNAs targeting the Pns6 gene (dsPns6), green fluorescent protein (GFP) gene (dsGFP) or 10% sucrose diet control through a membrane-feeding method, then fed on diseased rice (Oryza sativa) plants for 2 d. At 9 d post-first access to diseased plants, immunofluorescence assay showed that the viruliferous rates of insects treated by dsGFP and 10% sucrose diet control were 27% and 30%, respectively, while the viruliferous rate of insects treated by dsPns6 was 10%. The qRT-PCR results displayed that inhibiting the expression of Pns6 could also significantly reduced (P＜0.05) the expression levels of viral structural proteins P8 and P9. In addition, the transmission rate of insects treated with dsGFP and diet control were 20% and 22%, respectively. In contrast, the transmission rate of insects treated with dsPns6 was 6%. These results revealed that Pns6 was an important protein essential for RRSV multiplication in N. lugens. Inhibition of Pns6 expression could significantly reduce (P＜0.05) the viruliferous rate and block viral multiplication, finally prevent the transmitting ability of insects. The results also confirmed that Pns6 of RRSV could act as a good target for blocking the multiplication of RRSV in its insect vector.

Bacillusthuringiensis (Bt) is a gram-positive and spore-forming bacteria. It can form parasporal crystal during spore-forming, which is specific toxic to many insects. Bt formulation is a microbial bioinsecticide, which is the largest produced and the most widely used. But the Bt formulation is sensitive to UV in the application process, thus shortens the duration of active ingredient, which the disadvantage would limit the application of Bt formulation. Improving the biological insecticidal activity and UV-resistant ability of Bt insecticidal crystal protein by loading with nanomaterialsis is regarded as a promising technology, due to its advantages of small particle size, large specific surface area, high efficiency and low toxicity, environmental-friendly properties and improving the stability of Bt preparations. In the present study, Bt insecticidal crystal protein was loaded with nano-Mg(OH)2 to understand the compound biological insecticidal activity and resistance to UV. The nano-Mg(OH)2 was prepared by co-precipitation of magnesium chloride hexahydrate and sodium hydroxide, and the X-ray powder diffraction (XRD) was applied to analyze the size of synthesized nano-Mg(OH)2, and the size of nano-Mg(OH)2 at (101) direction was 12 nm according to the scherrer equation. Scanning electron microscope (SEM) analysis showed that the sample contained a lot of small nano-flakes structure with uniform dispersion, and the morphology of Mg(OH)2 particles loading with Cry toxin had no obvious changes. Bt LLP29 was selected as a target strain because it had high insecticidal activity to mosquitoes (Culicidae), and the Bt insecticidal crystal protein was obtained by alkali soluble method. Results of sodium dodecyl sulfonate - polyacrylamide gelelectrophoresis (SDS-PAGE) and bioassay indicated that the loaded nano-Mg(OH)2 could effectively improve the insecticidal activity of protein, protect the protein structure from destructing, and enhance the ability of resistance to ultraviolet radiation. The present work provides the fundamental information to gain biocompatibility and environmental-friendly nano-formulation of Bt bioinsecticide.

Galectins are a family of β-galactoside-binding proteins that widely distribute among insect species which play an important role in insect immune regulation. Thus the research of Aedes aegypti galectins is of great significance. In this study, primers were designed based on the A. aegypti galectin12 gene sequence, and the entire coding region of galectin12 gene was amplified by qRT-PCR with extracted total RNA. PCR product was purified and ligated to pMD-18T cloning vector. The selected positive clone was used for digested analysis and nucleotide sequencing. After endonucleases digestion, fragments were connected to pET-32a vector and obtained 23 kD protein. In addition, the expressed protein was purified by Ni-NTA affinity chromatography, and obtained purified protein bioassay result indicated that galectin12 could supporess mosquito Bt toxies. Cloning, expression and purification of Galectin 12 would help to furtherly understand the mechanism of galectin against Cry toxin for A. aegypti.

Empoasca vitis (Gothe), one of the most serious tea plant (Camellia sinensis) insects, occurs throughout the Chinese tea growing areas and causes significant losses in both production and quality of tea. It is of important significance to solve pest problems by biological pesticide and initially explore the intoxication mechanisms of Bacillus thuringiensis Cry toxins to E. vitis midgut cells. Using specific primers, peptide sequence bound to midgut receptors by screening phage library was amplified. The 750 bp PCR product was purified and cloned into the cloning vector pMD-18T, then the recombinant plamid DNA was used for EcoRⅠ/XhoⅠdigested identification and gene sequencing. The targeted fragment was subcloned into expression vector, and then the strain with recombinant plasmid of pT-egfp-32a was induced and expressed. Moreover, the fusion protein was purified by His affinity chromatography and its binding activity to E. vitis midgut was verified by Western blot. Results indicated that the relative molecular weight of T-EGFP was about 46 kD in supernatant after purification step. After that, it also achieved good effect that Western blot had been identified its binding activity to E. vitis midgut brash border membrane vesicle (BBMV). Successful projects for expression, purification and verification of T-EGFP bound to midgut receptors of E. vitis would provide basic data for further research on understanding the interaction mechanism between Cry toxins and E. vitis midgut cells, and also contribute to further study of directed modification for Bt Cry toxin against E. vitis.

DEAD-box RNA helicase (RH) ubiquitously exists in most organisms and participates, if not all, steps of cellular RNA metabolism. Generally, containing 9 highly conserved motifs is an important standard to identify a DEAD-box RNA helicase. In the present study, the CDS of a rice (Oryza sativa L. japonica. cv. Nipponbare) gene OsRH37, encoding a putative DEAD-box RNA helicase, was cloned. The amino acid sequence of OsRH37 was analyzed by bioinformatics method and it was indicated that the amino acid sequence identity between OsRH37 and 3 other Arabidopsis thaliana DEAD-box RNA helicases including AtRH17, 37 and 52 was＞70%, whereas the amino acid sequence identity between OsRH37 and DEAD-box RNA helicases from different animals was generally＜60%. The overexpression vector and RNAi vector of OsRH37 were constructed, and by using rice genetic transformation system the overexpression and RNAi transgenic rice plants of OsRH37 were obtained. Six lines (66.7%) among 9 lines of OsRH37 overexpressed transgenic rice and 13 lines (92.9%) among 14 lines of OsRH37 RNAi transgenic rice were identified to be positive, respectively. The relative expression level of OsRH37 among wild type, overexpression lines, and RNAi lines were detected by sqRT-PCR. The results indicated that compared with the wild type (WT), the expression level of OsRH37 in overexpression lines was obviously up-regulated while that in RNAi lines was significantly down-regulated. To observe the subcellular localization pattern of OsRH37, the stop codon TAG deleted CDS was constructed into the transient expression vector pEarleyGate102, harboring a cyan fluorescence protein (CFP), by Gateway technology and the leaves of Nicotiana benthamiana agroinfiltrated with CFP-OsRH37 were observed under confocal microscope at 48 h post infiltrated. The results of subcellular localization analysis suggested that OsRH37 fused with CFP was mostly localized at nucleus, karyosome and cytoplasmic granular structures. The bioinformatics analysis, obtained transgenic rice plants and subcellular localization analysis of OsRH37 can provide foundational insights for the important roles of OsRH37 in the processes of the rice growth, development and stress responses.

Spinosad, produced by Saccharopolyspora spinosa, is a kind of high-efficiency and low-toxicity bio-insecticide wildly used as pesticide, veterinary drug, hygienic insecticide, etc. The ability of S. spinosa to utilize starch and maltose was low, making a huge obstacle for the industrial fermentation of spinosad. In order to improve the ability of S. spinosa to use starch, the maltose transport related genes malEFG from Streptomyces avermitilis were expressed in a spinosad high-yield strain S. spinosa ASAGF73. The integration expression vector pSET152::malEFG was transformed into ASAGF73 through conjugal transfer, and the malEFG genes were successfully expressed in the resulting transformant. ASAGF73 and the transformant were cultured in single carbon source medium to study the effects of malEFG, showing that the transformant consumed dextrin and maltose betterly than ASAGF73. In the media with dextrin or maltose as the sole carbon source, expression of malEFG genes promoted obviously the growth of S. spinosa, resulting in improvement of spinosad yields. This study provides theoretical significance and application value for spinosad fermentation process using starch as main carbon source.

Rice stripe virus (RSV) is a type member of Tenuivirus, inducing rice (Oryza sativa) stripe disease in East Asia such as Korea, Japan and China, causing large yield loss in rice production. In the present study, yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) technique were used to detect the self-interaction sites of the RSV coat protein (CP). A series of CP deleted mutants were constructed and transformed into yeast strain Y2H Gold, observing the growth of yeast transformants on quadruple drop out SD/-Leu-Trp-His-Ade/X-α-gal agar plates. It was revealed that C terminal domain from residue 307~318 played an essential role in the interaction, deleting or damaging of which abolished the self-interaction. Site-directed mutants were generated to investigate the specific sites of CP that involved in the interaction. It revealed that the L308, V309, F312 and F313 amino acids were critical for CP self-interaction, which were highly conserved in Tenuivirus. BiFC assay was performed to confirm these results. Agrobacterium tumefaciens that carried the CP deleted mutants or site-directed mutants were infiltrated into the epidermal leaves of Nicotiana benthamiana. Confocal microscope was used to observe the yellow fluorescent protein (YFP) 48 h post inoculation (hpi). The CPΔ307~322, CPL308A, CPV309A, CPF312A and CPF313A had no signal, indicating there was no self-interaction, while CPΔ319~322 showed YFP similar to the full-length CP mainly aggregated in cytoplasm, consistent with results in Y2H. The cellular localization of these CP mutants was different from that of the full length wild-type CP. The site-directed mutants that had lost the ability of self-interaction showed diffused localization in the plasma membrane, while a substantial proportion of the full length CP gathered in the cytoplasm, forming numerous discrete aggregates, suggesting that the CP self-interaction and the subcellular localization in plant were interdependent processes. It was presumed that the forms of dimmer or higher multimer of CP might involve in different processes from the individual protein. These results may be useful in understanding the important role of CP in the process of RSV infection.

Plant parasitic nematode can parasitize various plant species and cause the yield loss of plants, which has been a serious problem in agricultural system around the world. Effectively integrated pest management (IPM) practices are needed to control these nematodes. One of these IPM strategies is the use of resistance inducers or triggers. Resistance of plant induced by special triggers can enhance defense ability of plant against different nematodes. This paper reviewed the criterions and types of induced resistance, main triggers and their physiological and biochemical mechanism in inducing plant to defense against nematodes. To determine whether plant resistance was induced by triggers, the inducing time, virulence factors and gene type of host should be taken into consideration. In recent years, research on induced resistance was focused on the microscopic observation of the interaction of nematode and host, especially on the feeding site of nematode. Reactive oxygen species (ROS) are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. During times of environmental stress, ROS levels can increase dramatically and be generated by exogenous sources such as microorganisms and chemicals. Effects of ROS on cell metabolism are well documented in a variety of species. These include not only roles in programmed cell death but also positive effects such as the induction of host defense genes. When a plant recognizes an attacking pathogen, one of the first induced reactions is to rapidly produce superoxide (O2-) or hydrogen peroxide (H2O2) to strengthen the cell wall. This prevents the spread of the pathogen to other parts of the plant, essentially forming a net around the pathogen to restrict movement and reproduction. Thereafter, the obseravtion of ROS has been another research area in the interaction of nematode and plants. Serveral compounds have been found to trigger resistance of various plants to defense against different pathogens. Chemicals such as acibenzolar s-methyl (ASM) and β-aminobutyric acid (BABA) have been verified to be effective in increasing the resistance of tomato (Lycopersicon esculentum) and decrease the number of root knots and eggs of Meloidogyne javanica. Potassium silicate can decrease the number of M. incognita, but increase the activity of peroxidase. Exogenous hormones such as abscisic acid (ABA), jamonic acid (JA) and ethylene (ET) have been found to be effective in decreasing infection and propagation of root-knot nematode (RKN). Some beneficial microorganisms such as Pseudomonas fluorescens and P. aeruginosa are found to effectively reduce the infection of Heterodera schachtii and M. javanica. Bacillus sphaericus can induce potato (Solanum tuberosum L.) to defense against the Globodera pallida. Arbuscular mycorrhizal fungi (AMF) can reduce the infection of M. incognita and increase the transcriptional expression level of defense-related genes and protein-synthesisrelated genes. Plant extracts from Foeniculum vulgare or Ocimum basilicum was found to decrease the population of M. javanica and M. incognita of tomato. In addition, biochar additions to soil have been shown to not only significantly improve soil tilth, nutrient retention and availability to plants, but also induce resiatance of some plants to defense against fungi, bacteria and nematodes. Research of induced resistance will be vital to formulate the integrated pest management strategy of nematodes, reduce the application of chemical nematicides and protect the ecological condition of agricultural system.

In order to select atrazine-degrading consortium and strain resources, in the present research, by enrichment culture, a stable 4-member bacterial consortium NC1 was isolated from long-term use of atrazine surface corn-planted soil (Nancha area, Yichun City, Heilongjiang Province, China). The consortium NC1 was attenuated and plated on mineral salts medium agar plates, strains were repeatedly crossed vaccination on mineral salts medium agar plates for separation of pure culture, 4 bacterial isolates, named strain YJY1, YJY2, YJY3 and YJY5. Based on physiological and biochemical tests, and 16S rDNA gene sequence analysis, 4 bacterial strains were shown to belong to the Gram-negative species Enterobacter sp., Achromobacter xylosoxidans, Pseudomonas aeruginosa and Klebsiella pneumonia, respectively. These species were not stated previously as being capable of atrazine-degrading. By growth capacity measuring, consortium NC1, strain YJY5 could use atrazine as single nitrogen source for growth, and strain YJY1, YJY2 and YJY3 could not. Consortium NC1 and 4 strains could hardly grow in MSM with cyanuric acid as the sole nitrogen source. Consortium NC1 and strain YJY5 had a short lag period (8~10 h), then was logarithmic phase. Strain YJY5 was stagnate phase of 28~36 h, and consortium NC1 was still growing rapidly at 36 h and capable of degrading 100 mg/L atrazine to nontoxic cyanuric acid. Altogether this was a new combination of isolates in an atrazine- degrading consortium, and its degradation rate and the growth were both faster than that of the sole strain. The high atrazine degradation ability of the consortium NC1 showed good potential for atrazine biodegradation. This research will contribute toward a better understanding of the metabolic activities of atrazine-degrading consortium, which are generally considered to be responsible for atrazine mineralization in the natural environment. The results provide information and reference for the study on biodegradation of atrazine work.

Serratia sp., one of the opportunistic pathogen, possesses certain insecticidal activity against insect. In this study, Serratia sp. strain BRC-CXG2 (KT366770) was isolated from the corpse of Monochamus alternatus Hope which was suffered from sepsis and was identified through morphological identification, 16S rDNA phylogenetic analysis, physiological and biochemical reactions, drug sensitive test and biometric identification. The results showed that the strain BRC-CXG2 belonged to one of new species of Serratia sp. and possessed different levels of sensitivity to a number of common medicines, such as cefoperazone, cefotaxime and ceftazidime. In addition, the prodigiosin isolated from this strain increased significantly the toxicity of spore and crystal mixture produced by Bacillus thuringiensis israelensis (Bti) LLP29 to Aedes aegypti. The research provides a theoretical basis for the effective development of Bt synergists (cotoxicity coefficient: 128.06), and construction of new genetically engineered bacteria.

Brevibacillus brevis FJAT-0809-GLX is a biocontrol agent with broad-spectrum antifungal activities. Ethylparaben has been confirmed to be one of its main functional components, but isolation and purification of this component from the fermentation broth is very complicated and difficult. Thus, it is urgent to develope a rapid, simple and effective method for quantitative determination of ethylparabens in the fermentation broth of B. brevis FJAT-0809-GLX. An automated sysmtem using high performance liquid chromatography (HPLC) with ultraviolet detection was developed for the determination of ethylparaben in fermentation broth in the present study. The mobile phase consisted of methanol/distilled water (65∶35 V/V) using a gradient program at a flow rate of 1.0 mL/min with 254 nm detection and an injection volume of 10 μL. Good linearity was obtained over the concentration range of 1.0~20 μg/mL with the coefficient of determination of 0.998 7. For the ethylparaben, the limit of HPLC detection was 1.0×10-15 μg/mL. Subsequently, the ethylparabens in fermentation broth were detected at different time points. The results indicated that the peak times and peak areas of ethylparabens were all stable within 48 h. The average recovery rate of the additive ethylparabens from fermentation broth was 96.2%~105.3% and the relative standard deviation (RSD) was 0.8%. Finally, the ethylparaben concentration in an optimizated fermentation culture medium was detected using this HPLC determination system. The results showed that its retention time was at 3.12 min and the peak area was 136. Therefore, its concentration reached approximately 2.111 μg/mL according to the standard curve. In conclusion, a simple, sensitive and quick HPLC method was established for determination of ethylparabens in fermentation broth of B. brevis FJAT-0809-GLX. The results will lay a foundation for tracking detection of functional components from fermentation broth of B. brevis.