Formation Of Norcantharidin Microemulsion And Its Effect On Non-target Soil Microbes

Biopesticides are generally categorized as i)natural products of plants,micrbes or animals which possess pesticide activities and ii)living organisms which are able to effectively control pests and plant pathogens.The effect of synthetic pesticides on soil microorganisms–key organisms in geochemical cycles–has been comprehensively studied but on the other hand,little is known regarding the impact of biopesticides on soil microbes and this could be attributed to the general belief that their natural origin guarantees low non-target toxicity.The biopesticide cantharidin,a well-known defensive terpenoid compound produced by blister beetles,shows good activity controlling various pests and plant pathogens.Whereas the procedure for synthesizing cantharidin under assay conditions is complex and difficult,as its analog norcantharidin,exhibits a similar mode of action as to cantharidin:they causing death by inhibiting protein serine/threonine phosphatases（PSPs）in vivo.Norcantharidin has been widely used in controlling cancer instead of cantharidin because it is much cheaper and easier to synthesize.In this study,norcantharidin is used as research material,and we focus on following three objectives:（1）pseudo ternary phase diagrams were constructed to test the optimal composition of the 5%norcantharidin microemulsion;then the acute LC₅₀to P.xylostella and the acute EC₅₀ to pear canker were estimated as well as the mechanism of emulsion stability;（2）we explored the degradation rate of norcantharidin microemulsion and cantharidin emulsifiable concentrate in the soil at different concentrations and temperatures;and（3）in comparison with traditional synthetic pesticides,the impacts of biopesticides on soil basal SIR,soil enzymes as well as soil microbial community structure and diversity based on DGGE fingerprinting are investigated.The optimal composition of the microemulsion based on single factor study,pre-experiment and some essential evaluation indices（e.g.,physico-chemical properties of norcantharidin,pseudo ternary phase diagrams and emulsion appearance）are presented as follows:when dimethylformamide,cyclohexanone and ethyl butyrate were mixed in a ratio of1:2:1（v/v）,norcantharidin attained a great solubility of 50 g/L;Tx13 and Tw80 formed a single continuous phase with a transparent appearance when the best ratio（w/w）of the surfactants Tx13 to Tw80 was 1:1 with optimal characters;pseudo-ternary phase diagrams suggested that mixed surfactants to ethanol-1:1 was the most appropriate Km to form a stable norcantharidin microemulsion.The pharmaceutical properties of norcantharidin microemulsion were tested by conductometry,dilution stability,cloud point and thermodynamic stability.Results showed that norcantharidin microemulsion was a transparent and uniform buff liquid under natural conditions and no oil floating or delamination was observed.Its cloud point was greater than 72°C.The percolation threshold（φ_p）wasφ_w≈18.36%;the first phase inversion occurred atφ_w≈42%and the maximum value(1.25 S m^-1,φ_w≈55%)suggested the second phase inversion from reverse swollen micelles（w/o）to direct micelles（o/w）.Microemulsions remained homogeneous and transparent during the dilution process,although DSL results indicated an increase in the droplet size along with the increase of the dilution ratio.GC was used to determine the content of norcantharidin microemulsion after different treatments.The retention time was stable and the linear curve fit well when the norcantharidin concentrations range from 0 to 1.6 mg/ml;thus the results were accurate and reliable.After storage at 0 and 54°C for 14 days,the appearance of the prepared norcantharidin microemulsion remained transparent and uniform,without any precipitate or phase separation.The decomposition rates of norcantharidin were 2.13%and 2.51%,i.e.,there was no significant change in the stability of the microemulsion.Subsequently insecticidal bioassay indicated that the acute LC₅₀of norcantharidin microemulsion to P.xylostella was estimated to be 12.48 mg/L and the acute EC₅₀ of norcantharidin microemulsion to Valsa ceratosperma was estimated to be 0.17 mg/L.GC was used to determine the residues of cantharidin and norcantharidin in soil.The linear curve fit well and the results were accurate and reliable.The evolution of pesticide concentrations in soil fit more accurately with the first-order dynamical reaction equation.In cantharidin-treated soil,the half-lives were 0.78,1.25 and 2.23 days for 1.25,6.25 and 62.5mg/kg,respectively.The corresponding values of norcantharidin are 1.32,1.68 and 1.94 days for 12.5,62.5 and 625 mg/kg,respectively.The evolution of pesticide concentration in soil fits more accurately with the first-order dynamical reaction equation at different temperatures.In cantharidin-treated soil,the half-lives were 1.14 d（10°C）,1.09 d（20°C）,0.98 d（30°C）days,respectively.The corresponding values of norcantharidin are 1.37 d（10°C）,1.27 d（20°C）,1.21 d（30°C）,respectively.A general reduction in the invertase activity with pesticides application in the soil was evident at 3 and 7 days.The higher the concentration,the stronger the adverse effect.However,the detrimental effect caused by pesticide application on soil invertase gradually weakened over with time.Compared to the non-treated soil,the maximum value of invertase was 123.07±5.36 mg glucose kg^-1 DW soil 24^-1 at 12.5 mg kg^-1 DW of norcantharidin.In carbendazim-treated soils,alkaline phosphate activity was significantly stimulated during the whole incubation time,but the application of norcantharidin and cantharidin distinctly inhibited that on days 3 and 7.These changes were transient.After about two weeks,the harmful effects due to the application of pesticides phased out and eventually became comparable with non-treated samples.Neutral phosphatase activity presented an‘inhibition-recovery-stimulation’trend in carbendazim-,cantharidin-and norcantharidin-treated samples.In the case of soil microbial biomass（as measured by SIR）,conspicuously lower values of SIR were observed for both cantharidin-and norcantharidin-treated soils（2.64 mg and 2.13 mg,respectively）.By contrast,there is no significant difference between the samples treated with cantharidin and norcantharidin and the control until the end of the experiments.Carbendazim appeared to have no clear effect on soil SIR at all incubation times,except in the highest concentrations.In our study,microbial parameters were susceptible to the pesticides type and concentration.Regarding biological pesticides,the effects induced by cantharidin and norcantharidin on the entire soil fungus followed a similar pattern.In cantharidin-and norcantharidin-treated soils,pesticides induced a marked decrease in fungal population abundance at days 3 and 7;then the inhibitory effect gradually weakened along with the incubation time.Results of DGGE fingerprinting indicated that the application of pesticides inhibited some sensitive colonies and induced new colonies and had no impact on some communities,indicating that tolerant strains to these pesticides were developed under selective pressure.Our work provides insights into the potential toxicity of norcantharidin to non-target organisms,and helps pave the way for the rational utilization of norcantharidin in pest control.