Guohua Zhong

Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01

A newly isolated bacterium DG-S-01 from activated sludge utilized beta-cypermethrin (beta-CP) and its major metabolite 3-phenoxybenzoic acid (3-PBA) as sole carbon and energy source for growth in mineral salt medium (MSM). Based on the morphology, physio-biochemical characteristics, and 16S rDNA sequence analysis, DG-S-01 was identified as Ochrobactrum lupini. DG-S-01 effectively degraded beta-CP with total inocula biomass A(590 nm) = 0.1-0.8, at 20-40 °C, pH 5-9, initial beta-CP 50-400 mg L(-1) and metabolized to yield 3-PBA leading to complete degradation. Andrews equation was used to describe the special degradation rate at different initial concentrations. Degradation rate parameters q(max), K(s) and K(i) were determined to be 1.14 d(-1), 52.06 mg L(-1) and 142.80 mg L(-1), respectively. Maximum degradation was observed at 30 °C and pH 7.0. Degradation of beta-CP was accelerated when MSM was supplemented with glucose, beef extract and yeast extract. Studies on biodegradation in liquid medium showed that over 90% of the initial dose of beta-CP (50 mg L(-1)) was degraded under the optimal conditions within 5d. Moreover, the strain also degraded beta-cyfluthrin, fenpropathrin, cyhalothrin and deltamethrin. These results reveal that DG-S-01 may possess potential to be used in bioremediation of pyrethroid-contaminated environment.

Biodegradation of Chlorpyrifos and Its Hydrolysis Product 3,5,6-Trichloro-2-Pyridinol by a New Fungal Strain Cladosporium cladosporioides Hu-01

Intensive use of chlorpyrifos has resulted in its ubiquitous presence as a contaminant in surface streams and soils. It is thus critically essential to develop bioremediation methods to degrade and eliminate this pollutant from environments. We present here that a new fungal strain Hu-01 with high chlorpyrifos-degradation activity was isolated and identified as Cladosporium cladosporioides based on the morphology and 5.8S rDNA gene analysis. Strain Hu-01 utilized 50 mg·L−1 of chlorpyrifos as the sole carbon of source, and tolerated high concentration of chlorpyrifos up to 500 mg·L−1. The optimum degradation conditions were determined to be 26.8°C and pH 6.5 based on the response surface methodology (RSM). Under these conditions, strain Hu-01 completely metabolized the supplemented chlorpyrifos (50 mg·L−1) within 5 d. During the biodegradation process, transient accumulation of 3,5,6-trichloro-2-pyridinol (TCP) was observed. However, this intermediate product did not accumulate in the medium and disappeared quickly. No persistent accumulative metabolite was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis at the end of experiment. Furthermore, degradation kinetics of chlorpyrifos and TCP followed the first-order model. Compared to the non-inoculated controls, the half-lives (t 1/2) of chlorpyrifos and TCP significantly reduced by 688.0 and 986.9 h with the inoculum, respectively. The isolate harbors the metabolic pathway for the complete detoxification of chlorpyrifos and its hydrolysis product TCP, thus suggesting the fungus may be a promising candidate for bioremediation of chlorpyrifos-contaminated water, soil or crop.

Characterization of a novel cyfluthrin-degrading bacterial strain Brevibacterium aureum and its biochemical degradation pathway

Brevibacterium aureum DG-12, a new bacterial strain isolated from active sludge, was able to degrade and utilize cyfluthrin as a growth substrate in the mineral medium. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 88.6% degradation of cyfluthrin (50mgL(-1)) within 5days. The bacterium degraded cyfluthrin by cleavage of both the carboxylester linkage and diaryl bond to form 2,2,3,3-tetramethyl-cyclopropanemethanol, 4-fluoro-3-phenexy-benzoic acid, 3,5-dimethoxy phenol, and phenol, and subsequently transformed these compounds with a maximum specific degradation rate, half-saturation constant and inhibition constant of 1.0384day(-1), 20.4967mgL(-1), and 141.9013mgL(-1), respectively. A novel degradation pathway for cyfluthrin was proposed based on analysis of these metabolites. In addition, this strain was found capable of degrading a wide range of synthetic pyrethroid insecticides. Our results suggest that B. aureum DG-12 may be an ideal microorganism for bioremediation of the pyrethroid-contaminated environments.

Silencing of Rieske iron–sulfur protein using chemically synthesised siRNA as a potential biopesticide against Plutella xylostella

BACKGROUND Extensive applications and frequent long-term use of pesticides can affect behavioural mechanisms and physiological and biochemical aspects of insects, leading to resistance. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. In this context, the development of RNA interference technology has brought a turning point in the creation of new biopesticides. RESULTS Full-length cDNA of Rieske iron-sulfur protein (RISP) was cloned and characterised from Plutella xylostella L. Three siRNAs specific to RISP sequences were designed and chemically synthesised, and fed to P. xylostella larvae by coating cabbage leaves. This resulted in specific gene silencing of RISP, and consequently brought significant mortality of P. xylostella larvae compared with the control treatment. Silencing of RISP leads to significantly lower transcript levels of RISP compared with the control. In addition, the amount of ATP in the surviving larvae was lower than in the control. However, surviving larvae gradually recovered to normal transcript and protein levels. CONCLUSION This is the first demonstration of the potential use of chemically synthesised siRNA in the development of new biopesticides as a mitochondrial electron transport inhibitor.

The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

Fenpropathrin Biodegradation Pathway in Bacillus sp. DG-02 and Its Potential for Bioremediation of Pyrethroid-Contaminated Soils

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L(-1) fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h(-1), 9.0 mg L(-1), and 694.8 mg L(-1), respectively. Analysis of the degradation products by gas chromatography-mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.

Cytotoxic and Insecticidal Activities of Derivatives of Harmine, a Natural Insecticidal Component Isolated from Peganum harmala

In a continuing effort to develop novel β-carbolines endowed with better insecticidal activity, a simple high-yielding method for the synthesis of harmine compounds starting from L-tryptophan has been developed and a series of 1,3-substituted β-carboline derivatives have been synthesized and evaluated for their cytotoxicity against insect cultured Sf9 cell line in vitro and insecticidal activities against 4th instar larvae of mosquitos, Culex pipiens quinquefasciatus and mustard aphid, Lipaphis erysimi. The results demonstrated that 1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (compound 2) and methyl 1-phenyl-β-carboline-3-carboxylate (compound 13) represented the best potential compounds, with Sf9 cells inhibition rates of 71.55% and 60.21% after 24 h treatment at concentrations of 50–200 mg/L, respectively. Both compounds 2 and 13 also showed strong insecticidal activity towards 4th instar larvae of mosquitos with LC50 values of 20.82 mg/L and 23.98 mg/L, and their LC90 values were 88.29 mg/L and 295.13 mg/L, respectively. Furthermore, the LC50 values of compounds 2 and 13 against mustard aphids were 53.16 mg/L and 68.05 mg/L, and their LC90 values were 240.10 mg/L and 418.63 mg/L after 48 h treatment. The in vitro cytotoxicity of these compounds was consistent with the insecticidal activity in vivo. The results indicated that the 1- and 3-positions of the β-carboline ring deserve further investigation to develop biorational insecticides based on the natural compound harmine as a lead compound.

Laboratory and field evaluations of extracts from Rhododendron molle flowers as insect growth regulator to imported cabbage worm, Pieris rapae L. (Lepidoptera:Pieridae)

The insect growth regulating properties of rhodojaponin‐III (Abbr. R‐III) and ethyl acetate (EtOAc) extract from Rhododendron molle G. Don flowers against imported cabbage worm, Pieris rapae L., were determined under laboratory and field conditions. The values of IC50 (the median of concentration for inhibiting weight increase) for R‐III and EtOAc extract were 6.78 p.p.m. and 70.29 p.p.m. against 3rd instar larvae and 13.72 p.p.m. and 346.00 p.p.m. against 5th instar larvae, respectively. R‐III and EtOAc extract also could reduce pupating rate, pupal weight, emergence rate and extend the duration of development. Thus development of insects was inhibited significantly and development index decreased. R‐III would be superior to toosendanin as insect growth regulator. EtOAc extract 1000 p.p.m. mixture with molosultap 500 p.p.m. could achieve an effective control rate of 87.34% against P. rapae in field trials when adjusted cumulative insect‐days were used as index.

Cross-resistance and baseline susceptibility of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) to cyantraniliprole in the south of China

BACKGROUND The oriental leafworm moth, Spodoptera litura Fab. (Lepidoptera: Noctuidae), is a widely distributed polyphagous insect pest in Asia that has been shown to be resistant to various types of insecticide. The newly registered anthranilic diamide cyantraniliprole provided novel insight and great opportunities to control S. litura. RESULTS In this study, the susceptibilities of S. litura collected from South China to cyantraniliprole were measured by standard leaf-disc bioassay, and obvious variation in susceptibility was observed among the 17 field populations, with LC50 values varying from 0.206 to 1.336 mg AI L(-1). Significant correlations were detected between the LC50 values of cyantraniliprole and chlorantraniliprole (P < 0.05). However, no significant correlation (P > 0.05) was observed between the two anthranilic diamides and other insecticides with different action mechanisms (delcamethrin, chlorpyrifos, indoxacarb and emamectin benzoate). Piperonyl butoxide showed obvious synergism in Lab-Sus, ZC14 and cyantraniliprole-resistant strains, while diethyl maleate and S,S,S-tributylphorotrithioate had no obvious synergistic effects in any of the strains tested. CONCLUSION These results revealed obvious regional variation in cyantraniliprole susceptibilities among populations of S. litura from different areas, and potential cross-resistance to chlorantraniliprole, which suggested that S. litura could develop resistance to cyantraniliprole. Detoxification enzymes might not be involved in the observed tolerance in field-collected populations and the cyantraniliprole-resistant strain.

Bdor⧹⧹Orco is important for oviposition-deterring behavior induced by both the volatile and non-volatile repellents in Bactrocera dorsalis (Diptera: Tephritidae)

Several studies have shown that the selections of gravid females to potential oviposition sites from a distance were mediated by volatile signals, however, the means by which the sensory cues from non-volatile chemicals affected the insect behavior were still a controversial subject. Chemosensory in insect is a complex process, which is mediated by multigene families of chemoreceptors, including olfactory receptors, olfactory co-receptors, and odorant-binding proteins. To elucidate the chemoreception mechanism of volatile and non-volatile chemicals, the roles of Orco and OBP in oviposition-deterrent activities induced by citronellal and Rhodojaponin-III were investigated. Our results suggested that RNAi-mediated expression inhibition was successfully achieved by feeding dsRNA in Bactrocera dorsalis. High levels of Bdor\Orco expression were essential for recognizing two chemicals of different physical properties, whereas the expression of Bdor\OBP was only imperative in perception of volatile chemical. The results suggested that volatile and non-volatile chemicals may evoke distinct molecular basis for chemosensory in the flies, while Orco was essential in the perception of both chemicals. The study highlighted that the central role of Orco in chemical recognition, which enabled it to be the universally applied target of designing new botanical pesticide.