Glutathione S-transferase genes in the epsilon group were reported to function in insecticide resistance. SlGSTE12 was validated to be overexpressed in pyrethroid-and organophosphate-resistant populations of Spodoptera litura compared to a susceptible population. A functional study of heterologously expressed SlGSTE12 showed that K m and V max for 1-chloro-2,4dinitrobenzene (CDNB) conjugating activity were 0.70 ± 0.18 mmol L −1 and 90.6 ± 9.4 nmol mg −1 min −1 , respectively. β-Cypermethrin and cyhalothrin showed much weaker inhibition of SlGSTE12 activity to CDNB conjugation than fenvalerate, chlorpyrifos, and phoxim. Ultrahigh-performance liquid chromatography analysis showed that SlGSTE12 had significant metabolism activity to fenvalerate and phoxim both in vitro and in Escherichia coli, especially to chlorpyrifos, and slight metabolism activity toward cyhalothrin only in vitro. Silencing of SlGSTE12 by RNAi increased the mortality to fenvalerate, cyhalothrin, and chlorpyrifos significantly. SlGSTE12 also had a significant antioxidant ability against cumene hydroperoxide. Our study suggested that SlGSTE12 could metabolize phoxim, fenvalerate, cyhalothrin, and especially chlorpyrifos. SlGSTE12 might also participate in pyrethroid and organophosphate resistance by antioxidant activity.
Citation: Liu J., Li P., Lu L., Xie L., Chen X., Zhang B. (2019): Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua. Plant Protect. Sci., 55: 61-71.Abstract: Eight commonly used candidate reference genes, 18S ribosomal RNA (rRNA) (18S), 28S rRNA (28S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1α), ribosomal protein L7 (RPL7), Alpha-tubulin (α-TUB), and TATA box binding protein-associated factor (TBP), were evaluated under various experimental conditions to assess their suitability in different developmental stages, tissues and herbicide treatments in Avena fatua. The results indicated the most suitable reference genes for the different experimental conditions. For developmental stages, 28S and EF1α were the optimal reference genes, both EF1α and 28S were suitable for experiments of different tissues, whereas for herbicide treatments, GAPDH and ACT were suitable for normalizations of expression data. In addition, GAPDH and EF1α were the suitable reference genes.
BACKGROUND: Spodoptera litura is an important agricultural pest and has developed serious resistance to multiple insecticides. The resistance level to several insecticides is reported to be unstable, but the mechanism is less reported.RESULTS: Chlorpyrifos and phoxim resistance level in a field-collected population of S. litura declined continuously from the first to the tenth generation and remained stable at the 11th and 12th generations without insecticide exposure. Synergist experiment showed that diethyl maleate and piperonyl butoxide significantly increased mortality to chlorpyrifos and phoxim in the first and sixth generations, but not in the 12th generation. The expression of 31 identified glutathione S-transferase (GST) genes in the third-instar larvae of S. litura in the first, sixth and 12th generations was determined, and eight genes were seen to decrease significantly in the sixth and 12th generations compared with the first generation. SlGSTe9 was selected for further functional study as it had higher abundance and significantly higher expression in the chlorpyrifos-resistant population than in the susceptible population. The recombinant protein of SlGSTE9 showed metabolism activity to chlorpyrifos in vitro and in Escherichia coli, but not to phoxim. Silencing of SlGSTe9 increased the cumulative mortality to chlorpyrifos significantly. SlGSTE9 also showed antioxidant activity to cumene hydroperoxide. CONCLUSION: Our results suggest that SlGSTe9 is directly involved in chlorpyrifos resistance stability, but not in phoxim. SlGSTE9 may also participate in insecticides resistance by relieving the oxidase stress induced by insecticides.
Rhyzopertha dominica (Fabricius) has developed extensive pesticide resistance in the last several decades. We have developed a supercritical fluid extraction method for Trigonella foenum-graecum L. (TFG) and studied the contact toxicities of the extracts to R. dominica. The extraction method was designed with orthogonal experiments to preserve and collect all the possible active components. Contact toxicity and efficiency of extraction were used as standard values to optimize extraction conditions, which were achieved at 55°C under 25 Mpa of pressure. The extraction efficiency for 200 g of dry sample reached 6.21% with 30 ml of 95% alcohol. Extracts loaded on filter paper showed dose and time dependent toxicities to adult R. dominica with a LC 50 value of 65.02 lg/cm 2 after 3 days post treatment. Our extensive in vivo studies indicated the extracts from Trigonella foenum-graecum seeds have high efficacy against pesticide resistant R. dominica. The active ingredient(s) from the extract shows promise as a novel pesticide candidate.
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