A constitutive serine protease inhibitor suppresses herbivore performance in tea (Camellia sinensis).

Protease inhibitors promote herbivore resistance in diverse plant species. Although many inducible protease inhibitors have been identified, there are limited reports available on the biological relevance and molecular basis of constitutive protease inhibitors in herbivore resistance. Here, we identified a serine protease inhibitor, CsSERPIN1, from the tea plant (Camellia sinensis). Expression of CsSERPIN1 was not strongly affected by the assessed biotic and abiotic stresses. In vitro and in vivo experiments showed that CsSERPIN1 strongly inhibited the activities of digestive protease activities of trypsin and chymotrypsin. Transient or heterologous expression of CsSERPIN1 significantly reduced herbivory by two destructive herbivores, the tea geometrid and fall armyworm, in tea and Arabidopsis plants, respectively. The expression of CsSERPIN1 in Arabidopsis did not negatively influence the growth of the plants under the measured parameters. Our findings suggest that CsSERPIN1 can inactivate gut digestive proteases and suppress the growth and development of herbivores, making it a promising candidate for pest prevention in agriculture.

Mixture toxicity of pyraclostrobine and metiram to the zebrafish (Danio rerio) and its potential mechanism.

The interplay between pesticides plays a critical role in ecotoxicology since these chemicals rarely emerge as single substances but rather in mixtures with other chemicals. In the present work, we purposed to clarify the combined toxic impacts of pyraclostrobine (PYR) and metiram (MET) on the zebrafish by using numerous indicators. Results exhibited that the 4-day LC50 value of MET to fish embryos was 0.0025 mg a.i. L-1, which was lower compared with PYR (0.019 mg a.i. L-1). Combinations of PYR and MET presented a synergetic impact on fish embryos. Contents of POD, CYP450, and VTG were drastically increased in the plurality of the single and joint treatments relative to the baseline value. Three genes, including vtg1, crh, and il-8, related to the endocrine and immune systems, were also surprisingly up-regulated when fish were challenged by the individual and mixture pesticides compared with the baseline value. These results afforded valuable information on the latent toxicity mechanisms of co-exposure for PYR and MET in the early growth stage of fish. Moreover, our data also revealed that frequent application of these two pesticides might exert a potentially ecotoxicological hazard on aquatic ecosystems. Collectively, the present study provided valuable guidance for the risk evaluation of chemical combinations.

Simultaneous determination of isoxaflutole and its two metabolites in corn under field conditions by LC-MS/MS.

OBJECTIVE: An improved QuEChERS method was established and verified for simultaneous determination of isoxaflutole and its metabolites diketonitrile and benzoic acid analogue residues in corn and plants. This method was mainly used to study the digestion rule and final residue level of isoxaflutole and its metabolites in corn and plants. It was hoped that the safe use of isoxaflutole in corn can be achieved eventually. METHOD: The method was completed by means of ultra-performance liquid chromatography with triple quadrupole mass spectrometry. The extraction of the target substance was through acetonitrile solution containing 1% acetic acid, and the purification was through primary secondary amine, octadecylsilane and graphitized carbon black sorbent. RESULTS: In the method, the quantitative limits and detection limits of the three analytes were 0.005-0.01 and 0.001-0.003 mg kg-1 respectively. The half-life of isoxaflutole in the plants in Shandong and Anhui was 36.4 and 42.1 days respectively, and the digestion dynamics all conformed to the first-order kinetics. The final residues of isoxaflutole in green corn and mature corn were less than 0.02 mg kg-1 of the maximum residue limit set by the Codex Alimentarius Commission. CONCLUSIONS: The residual amount of isoxaflutole in corn and plants at harvest time was acceptable when isoxaflutole was applied once at a dose of 121.5 g a.i. ha-1 . © 2021 Society of Chemical Industry.

[Determination of cyanamide residue in grapes and cherries by liquid chromatography-tandem mass spectrometry coupled with precolumn derivatization].

A method was developed for the quantitative determination of cyanamide in grapes and cherries by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with dansyl chloride (DNS) precolumn derivatization. First, the samples were homogenized, and then extracted with ethyl acetate under ultrasonication. The water was removed using anhydrous sodium sulfate, and the extract was concentrated and derivatized with dansyl chloride under alkaline condition. The separation was performed on a Shimadzu Shim-pack XR-ODS column (75 mm×2.0 mm, 1.6 µm) with the mobile phases of methanol and 2 mmol/L ammonium acetate aqueous solution (containing 0.05% (v/v) formic acid) in a gradient elution mode. The identification and quantification of cyanamide were carried out by MS/MS in positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) mode. The calibration curves showed good linearities in the range of 0.01-1.0 mg/L with the correlation coefficients not less than 0.9990. The average recoveries of cyanamide spiked at 0.01, 0.05 and 1.0 mg/kg in cherries and grapes were between 75% and 81%, and the relative standard deviations (RSDs) were between 6.5% and 9.8%. Both the limits of quantification (LOQs) of the analytes were 0.01 mg/kg. The method is simple, rapid, accurate and suitable for the confirmation and quantification of cyanamide in cherries and grapes.