Ridge planting increases the rhizosphere microbiome diversity and improves the yield of Pinellia ternata (Thunb.) Breit in North China.

Pinellia ternata (Thunb.) Breit is an important traditional Chinese medicine. In North China, conventional flat planting of P. ternate is prone to root rot during the rainy season, leading to severe yield loss. Variations in planting patterns (e.g., ridge planting) can effectively alleviate this situation. However, the relationship between planting patterns and the changes induced by rhizosphere microbiome still needs to be determined. In this study, we clarified the effect of ridge planting on the yield of P. ternata and rhizosphere microbial community using high-throughput amplicon sequencing of 16S rRNA. Field experiments showed that ridge planting could increase the yield of P. ternata by 72.69% compared with flat planting. The high-throughput sequencing results demonstrated that fungal and bacterial communities in rhizosphere siols of flat and ridge planting showed obvious difference in diversity, structure, relative abundance, and community composition. The fungal phyla Zygomycota, Basidiomycota, Glomeromycota, and the bacterial phyla Chlamydiae, Tenericutes, and Hydrogenedentes were present in a higher relative abundance in the rhizosphere of ridge planting. Adonis multivariate analysis of variance results showed that 29 bacterial genera were significantly up/down-regulated, and only 4 fungal genera were changed considerably in ridge planting soil, indicating that the bacterial community composition varied significantly between the two treatments. Correlation analysis revealed that the yield of P. ternata was positively correlated with fungal genera Emericellopsis while negatively correlated with bacterial genera Acetobacter, Iamia, and fungal genera Thielavia. Overall, this study showed that ridge cropping significantly impacts the diversity and composition of the rhizosphere microbiome. It creates an environment favorable for crop growth and can be an effective planting strategy for P. ternata in areas with irrigation and high monsoon rainfall in North China.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae).

Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (RPL32, RPS23, SDHA, UBQ, UCCR, GST, TATA-box, HSP70, GAPDH, RPL7A, SOD, RPS3, Actin, α-tubulin, AK, and EF1) were carefully chosen in R. pedestris, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in R. pedestris is contingent upon the specific experimental conditions. RPL7A and EF1 are optimal reference genes for developmental stages. Furthermore, α-tubulin and EF1 exhibit the most stable expression across various adult tissues. RPL32 and RPL7A exhibit the most stable expression for adult age. For nymph age, RPL32 and SOD display the most stable expression. For temperature conditions, RPS23 and RPL7A were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (RpedOBP37) and cytochrome P450 6a2 (RpedCYP6) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on R. pedestris, serving as a fundamental groundwork for future investigations on stable reference genes in R. pedestris as well as other organisms.

The Lethal and Sublethal Effects of Lambda-Cyhalothrin and Emamectin Benzoate on the Soybean Pest Riptortus pedestris (Fabricius).

Riptortus pedestris (Fabricius, 1775) (Hemiptera: Alydidae) is a major soybean pest in East Asia that can cause soybean staygreen syndrome. To date, no insecticides have been registered for the control of R. pedestris in China, and these insects are primarily controlled in the field through the application of broad-spectrum insecticides including lambda-cyhalothrin (LCT) and emamectin benzoate (EMB). Here, the lethal and sublethal effects of LCT and EMB on R. pedestris were comprehensively evaluated. LCT and EMB were both found to exhibit high levels of toxicity and concentration-dependent repellent effects for R. pedestris. The exposure of third instar nymphs from the F0 generation to LC30 concentrations of LCT and EMB resulted in a significant increase in the duration of nymph development and adult pre-oviposition period (APOP), together with reductions in fifth instar nymph and adult body weight, longevity, oviposition days, fecundity, vitellarium length, lateral oviduct diameter, and vitellogenin (Vg) gene expression as compared to control treatment. Strikingly, these suppressive effects were transmitted to the F1 generation, which similarly experienced the prolongation of preadult development and the preoviposition period (TPOP). Relative to control-treated populations, the F1 generation for these insecticide-treated groups also exhibited significant decreases in population parameter values. Overall, these data offer new insight into the impact that LCT and EMB treatment can have on R. pedestris, providing a valuable foundation for the application of these pesticides in the context of integrated pest management strategies aimed at soybean crop preservation.

Identification of highly active compounds from insecticidal plant Oroxylum indicum L. (Vent.) and the induction of apoptosis by lapachol on Sf9 cells.

The extraction of biopesticides from plants has become a promising field for agricultural development. To explore a high-efficiency and viable method for the screening of plant compounds with insecticidal activity, we screened for active ingredients in the insecticidal plant, Oroxylum indicum L. Vent, using Sf9 cells. A CCK-8 cytotoxicity assay kit was used for high-throughput screening of 34 compounds contained in O. indicum. The apoptosis-inducing effect of the highly cytotoxic compound on Sf9 cells was investigated by morphological characterization using inverted microscopy, caspase-3 activity assay, and DNA gel electrophoresis. Finally, the biological activity of compounds against aphids was evaluated using the leaf-pest dipping methods and leaf dipping methods. Results showed that among the main compounds identified, lapachol, chrysin, and baicalein had good proliferation inhibitory effects on Sf9 cells, with their recorded IC50 being 11.53 mg/L, 38.39 mg/L, and 42.10 mg/L, respectively. Moreover, the IC50 value of lapachol was lower than the control insecticides rotenone (18.03 mg/L) and fipronil (21.04 mg/L). Apoptosis assay further showed that lapachol promoted the production of caspase-3 and led to DNA fragmentation in Sf9 cells. Lapachol showed high biological activity against Aphis gossypii, Sitobion avenae, and Semiaphis heraclei, with its recorded LC50 being 104.40, 101.80, and 110.29 mg/L, respectively, which were comparable to the activity of the control insecticide rotenone. High-throughput screening of active ingredients in the insecticidal plant O. indicum using Sf9 cells is feasible, and the identification of lapachol as the main aphidicidal active substance is valuable for further study.

Detoxification enzyme is involved in the temperature effect on the toxicity of tetrachlorantraniliprole to Plutella xylostella.

The efficacy of insecticides is usually influenced by temperature. Insecticides can be divided into "positive", "negative" and "non-effect" temperature coefficient insecticides (TCI). To assess the temperature-dependent effect of tetrachlorantraniliprole (TET) on Plutella xylostella Linnaeus and to elucidate the mechanism of temperature affects TET toxicity, we determined the toxicity of TET against P. xylostella from 15 °C to 35 °C by leaf dipping method. Moreover, we compared the transcriptome data of the third-instar larvae treated by TET, chlorfenapyr (CHL, non-effect TCI), and the control group at 15, 25, 35 °C, respectively. The results showed that the toxicity of TET against P. xylostella increased with increasing temperature from 15 °C to 35 °C. A total of 21 differential expressed genes (DEGs) of detoxification enzymes were screened by RNA-seq, in which 10 up-regulated genes (3 UGTs, 2 GSTs, 5 P450s) may involve the positive temperature effect of TET, and their expression patterns were consistent with qPCR results. Furthermore, the enzyme activities of GSTs and UGTs significantly increased after TET was treated at 15 °C. Especially, the temperature coefficient (TC) of TET was significantly reduced mixed with UGTs enzyme inhibitor 5-NI. Overall, TET showed higher insecticidal activity with increasing temperature, in which detoxifying enzymes associated with regulation of the positive temperature effect of TET on P. xylostella, such as UGTs, GSTs and P450s, are strongly involved. The transcriptome data provide in-depth information to understand the TET mechanism against diamondback moth. Most importantly, we identified detoxification enzymes that might be involved in regulating TET's positive temperature effect process, and contributed to efficient pest management.

Genome-wide identification of candidate chemosensory receptors in the bean bug Riptortus pedestris (Hemiptera: Alydidae) and the functional verification of its odorant receptor co-receptor (Orco) in recognizing aggregation pheromone.

A sophisticated and sensitive olfactory system plays a vital role in the survival and reproduction of insects. Chemosensory receptors are indispensable for the molecular recognition and discrimination of semiochemicals. Riptortus pedestris is a notorious pest of legume plants, resulting in yield losses and quality decreases in soybeans. It is well accepted that R. pedestris highly relies on its olfactory system in detecting aggregation pheromones, host volatiles, and pesticides; however, little research focused on its chemosensory receptors. In the present study, we identified 237 odorant receptors (ORs), 42 gustatory receptors (GRs), and 31 ionotropic receptors (IRs) from the reported genome of R. pedestris, and analyzed their phylogenetic relationship with other hemipteran species. Through the results of RNA-seq and real-time quantitative PCR (qRT-PCR), we found that RpedORs displayed different expression levels in the antennae of R. pedestris at different development stages. To further verify the function of odorant receptor co-receptor (Orco), an obligate and unique insect OR, we silenced RpedOrco by RNA interference (RNAi) method. The results showed that silencing RpedOrco could significantly impair the response to aggregation pheromone in R. pedestris, indicating that RpedOrco plays an essential role in odorant detection. Our results can provide the theoretical foundations for revealing the olfactory recognition mechanism of R. pedestris and help explore and develop novel olfactory-based agents against this pest.

Influence of pre-exposure time on the toxicities of different temperature effect insecticides to Apolygus lucorum (Hemiptera: Miridae).

Temperature can have influences on the toxicities and efficacies of insecticides. Therefore, it is important to accurately evaluate the temperature effect (TE) on the toxicities of insecticides to insects. Previous studies have shown that the pre-exposure of insects to temperatures before their contact with insecticides, caused variations in their toxicities. However, most of these studies focused on the TE of the insecticides post-treatment. In this study we hypothesized that pre-exposure time of insect at different temperature can influence the toxicities of insecticides. We then evaluated the influence of different pre-exposure time (0, 2, 4, 8, 12 and 24 h) on toxicities of three different temperature effect insecticides (TEIs) to Apolygus lucorum at 15, 25 and 35°C respectively. We found that all toxicities of three TEIs to A. lucorum did not vary with pre-exposure time at 25°C. The LC50 of hexaflumuron (positive TEI) only decreased (from 1800.06 to 237.40 mg/L) at 15°C, with an increase in the pre-exposure time. Whereas the LC50 of ß-cypermethrin (negative TEI) decreased from 225.43 to 60.79 mg/L at 35°C. These results also showed that the temperature coefficients (TCs) of the toxicities were influenced by pre-exposure time at different temperatures. For hexaflumuron, all the TCs at 25°C and 35°C decreased, as the pre-exposure time increased. For ß-cypermethrin, the TCs decreased significantly only at 35°C. The toxicity and TCs of phoxim (non-effect TEI) showed no obvious fluctuation at the tested temperatures. These results showed that when the pre-exposure times were extended, the toxicities of the positive / negative TEI showed an increase at the temperature where the pest was less sensitive to the insecticides. These results can be applied to determine the toxicities / bioactivities of different insecticides accurately at different temperatures.

Temperature-dependent variations in toxicity of diamide insecticides against three lepidopteran insects.

The effect of temperature on the toxicities of four diamide insecticides (chlorantraniliprole, cyantraniliprole, flubendiamide, tetraniliprole) against three lepidopteran insects (Helicoverpa armigera, Plutella xylostella, Athetis lepigone) were determined from 15 to 35 °C by exposing third-instar larvae to dip-treated cabbage leaf. The results indicated that increase in temperature led to an increase significantly and regularly in the toxicities of the four diamide insecticides against P. xylostella and H. armigera, but not for A. lepigone. The temperature coefficients (TCs) of the four diamide insecticides increased from 15 to 35 °C. Tetraniliprole for H. armigera (+825.83), chlorantraniliprole for P. xylostella (+315.65) and cyantraniliprole for H. armigera (+225.77) exhibited high positive TCs. For A. lepigone, temperature had a positively weak or no effect on the toxicities of most of the diamide insecticides from 20 to 30 °C, but a higher effect from 30 to 35 °C. In addition, the toxicities of chlorantraniliprole, cyantraniliprole and tetraniliprole all decreased from 15 to 20 °C. This study can guide pest managers in choosing suitable ambient field temperature when spraying diamide insecticides against lepidopteran insects.

Analysis of Differentially Expressed Transcripts in Apolygus lucorum (Meyer-Dür) Exposed to Different Temperature Coefficient Insecticides.

The existence of a temperature effect of insecticides frustrated the control of the green plant bug Apolygus lucorum (Meyer-Dür). Previous studies mostly focused on the application of insecticides, but the underlying mechanism remains incompletely understood. Here, we report a transcriptome profiling of A. lucorum treated by three kinds of temperature coefficient insecticides (TCIs) (positive TCI: imidacloprid, negative TCI: b-cypermethrin and non-effect TCI: phoxim) at 15 °C, 25 °C and 35 °C by using next- and third-generation RNA-Seq methods. A total of 34,739 transcripts were annotated from 277.74 Gb of clean data. There were more up-regulated transcripts than down-regulated transcripts in all three kinds of TCI treatments. Further Venn diagrams indicate the regulatory transcripts and regulatory modes were different at the three temperatures. The responses to imidacloprid involved more detox and stress response transcripts such as cytochrome P450 (CYP450), carboxylesterase (CarE) and catalase (CAT) at 35 °C, which was the case for beta-cypermethrin at 15 °C. UDP-glucuronyltransferase (UGT) and heat shock protein (HSP) transcripts were heavily involved, and thus deserve particular note in the temperature effect of insecticides. This high-confidence transcriptome atlas provides improved gene information for further study on the insecticide temperature effect related physiological and biochemical processes of A. lucorum.

Treating wheat seeds with neonicotinoid insecticides does not harm the rhizosphere microbial community.

Wheat aphids damage wheat plants directly by feeding on them and indirectly by transmitting plant pathogenic viruses, both of which result in low yield and plant death. Due to their high root absorption and systemic characteristics, neonicotinoid insecticidal seed treatments are increasingly applied to control wheat aphids throughout the growing season in China. Ecological concerns are raised in some research, because neonicotinoids can persist and accumulate in soils. They are prone to leach into waterways, and are found in crop nectars and pollens, where they may be harmful to pollinators. Less information is available about the effect of neonicotinoid seed treatments on soil microorganisms. Here, we posed the hypothesis that neonicotinoids are not harmful to soil microbial communities. We tested our hypothesis by evaluating the effects of two neonicotinoids, imidacloprid and clothianidin, on soil microbiomes using high-throughput sequencing during three points in the wheat growth season. Except for the imidacloprid-treated soil in the seedling stage, the community richness and diversity were not affected according to Chao1, ACE and the Shannon indices, and species distribution histogram at the phylum level. However, Beta diversity indices showed that the species richness of the bacterial and fungal community was suppressed by neonicotinoids in seedling stage (high neonicotinoids concentrations), whereas by the reviving period, the changes reverted into stimulation of the soil microorganisms (low neonicotinoids concentrations). Overall, the general microbiome recovered at the end of the wheat planting season. Generally, wheat seed dressing with neonicotinoid insecticides control aphids during the entire growth period, and have no lasting adverse effects on the soil microbiome. This study provides an understanding of the influence of neonicotinoids on crop land ecology at the level of soil microbe communities.

The Joint Toxicity of Different Temperature Coefficient Insecticides on Apolygus lucorum (Hemiptera: Miridae).

The effect of temperature on the cotoxicity coefficient (CTC) value was used to evaluate mixture efficacy of different temperature coefficient chemicals from 15 to 35°C by exposing third-instar Apolygus lucorum (Meyer-Dür) to dip-treated asparagus bean pods. The results indicated the joint toxicity of same temperature coefficient insecticide (TCI) types were unaffected by temperature. This means that even when temperatures change, the mixture ratios of the highest CTC values remained the same, and the effect of temperature on the joint toxicity of same TCI types was only on the CTC values. However, the effect of temperature was variable when considering the joint toxicity of different TCI types. The effect of temperature on the joint toxicity of both strong positive and strong negative TCI types was clear, and the highest CTC values of mixture ratios changed with temperature regularly. When comparing the influence of temperature between strong/slight positive/negative insecticides, the results indicated a greater influence of the strong TCI. Paradoxically, the highest CTC value of the imidacloprid and methomyl mixture did not change with temperature changes consistently, even with the variance of imidacloprid ratios, a strong TCI. These results will guide pest managers in choosing the most effective insecticide mixtures for A. lucorum control under given environmental conditions.

Synthesis and biological activity of novel benzimidazole derivatives as potential antifungal agents.

In the present study, a series of novel benzimidazole derivatives containing chrysanthemum acid moieties was designed and synthesized. Preliminary investigation of biological activity indicated that all of the compounds exhibited lower activity than that of beta-cypermethrin against Plutella xylostella and Lipaphis erysimi; meanwhile, they showed good inhibitory activity against Botrytis cinerea and Sclerotinia sclerotiorum in vitro. The fungicidal activity of compound 8a against B. cinerea was approximately equal to that of thiabendazole and was twice as active against S. sclerotiorum as was thiabendazole. In addition, compound 9e displayed the most potent inhibitory activity against both fungi and was almost twice as potent as thiabendazole.