Although Spodoptera frugiperda populations with the ryanodine receptor (RyR) I4734M mutation occurred in their native regions, this mutation has not been detected in the Eastern Hemisphere. In order to clarify the functional role of this mutation, the first HDR-mediated RyRI4734M mutation was introduced into S. frugiperda using CRISPR/Cas9 technology and a homozygous SfRyRI4734M strain was successfully established. Meanwhile, a novel, non-invasive genotyping method based on insect fecal DNA to efficiently identify genome-edited individuals was developed. Compared with the wild type, the genome-edited SfRyRI4734M strain showing high-level resistance to chlorantraniliprole (396.7-fold) and tetraniliprole (149.1-fold), and moderate resistance to cyantraniliprole (32.3-fold) and flubendiamide (29.5-fold). Reciprocal crossing experiments indicated that resistance to chlorantraniliprole was inherited in an autosomally incompletely recessive mode. Furthermore, the SfRyRI4734M substitution adversely reduced the fitness and flight ability of S. frugiperda. The mutant strain exhibited significantly decreased fecundity and severely impaired flight distance and velocity. These findings provided in vivo genetic validation of SfRyRI4734M mutation in diamide resistance, demonstrating this mutation conferred subtle differences on the binding affinities of four diamides. Moreover, our results firstly demonstrated that SfRyRI4734M mutation could affect muscle function, thereby reducing the flight ability of S. frugiperda.

Myzus persicae is a worldwide insect pest with high resistance to many traditional insecticides. Cycloxaprid, a novel cis-configuration neonicotinoid insecticide, is effective in controlling neonicotinoid-resistant insect pests. Lethal and sublethal effects of cycloxaprid on M. persicae were conducted in this study. Results showed that cycloxaprid had higher toxicity to the laboratory and field resistant M. persicae than imidacloprid. Because of the resistance, imidacloprid showed lower control efficacy (<60%) against M. persicae, which falls short of the efficacy required for practical agricultural management. However, cycloxaprid exhibited higher control efficacies (>84.79%) against M. persicae in the field. In addition, in order to quantify the sublethal impacts of cycloxaprid, we conducted a life table analysis on M. persicae. When resistant M. persicae was treated with LC25 of cycloxaprid or imidacloprid, the longevity and fecundity of F1 adults were significantly decreased. Meanwhile, the intrinsic rate of increase (rm), finite rate of increase (λ) and net reproduction rate (Ri) of F1 generation M. persicae were reduced in cycloxaprid and imidacloprid treatments. Therefore, cycloxaprid shows high potential as a candidate insecticide for managing imidacloprid-resistant M. persicae. Importantly, our laboratory data indicate that exposure to its low sublethal concentration (LC25) inhibits population growth parameters, suggesting a low risk of inducing pest resurgence under such conditions.

In recent years, honeybee populations have declined dramatically, raising serious concern due to their vital role as pollinators in maintaining global ecosystems and biodiversity. Among the various potential threats, neonicotinoid pesticides have attracted increasing attention for their sublethal toxicity to bees and the differing toxicological profiles of various neonicotinoid compounds toward non-target organisms. In this study, we investigated selective toxicity at the molecular level by examining the binding interactions between a key chemosensory protein in Apis mellifera, AmelCSP3, and three representative neonicotinoid compounds developed across different decades. The analysis was conducted using spectroscopic techniques, surface plasmon resonance, and molecular modeling. The findings reveal that clothianidin binds CSP3 most strongly, followed by thiamethoxam and then paichongding. Thermodynamic parameters derived from fluorescence analysis indicated that the binding process was spontaneous and primarily driven by hydrophobic interactions. Notably, clothianidin-a primary metabolite of thiamethoxam-demonstrated slightly stronger binding than its parent, suggesting that metabolic transformation can exacerbate non-target risk. Paichongding, a third-generation neonicotinoid, showed the highest dissociation constant (KD) and a temperature-dependent decrease in association constant (Ka), indicating it may exert weaker olfactory disruption in Apis mellifera at elevated temperatures. This study offers mechanistic insight into how specific structural features of neonicotinoids influence their binding behavior with Apis mellifera chemosensory proteins, providing molecular-level evidence for their differential non-target effects and informing the rational design of pollinator-friendly pesticides.

RNA pesticides have emerged as a promising alternative to conventional chemical pesticides due to their high specificity and minimal environmental impact. However, the instability of RNA molecules in the environment and the challenges associated with their effective delivery to target pests limit their broader application. This study addresses these challenges by developing a dual delivery system using chitosan (CS) and Metal-Organic Frameworks (MOFs) to enhance the delivery and efficacy of double-stranded RNA (dsRNA) and cycloxaprid against Diaphorina citri, a vector of citrus greening disease. The CS-MOF nanoparticles were synthesized and characterized using scanning electron microscopy (SEM) and dynamic light scattering (DLS). Insect bioassays demonstrated that the codelivery system significantly improved insecticidal activity, achieving over 80% mortality in D. citri within 2 days. The results indicate that the encapsulation of dsRNA within MOFs enhances its stability, while the controlled release properties of the nanoparticles improve the efficacy of cycloxaprid. This novel approach shows great potential in overcoming the limitations of RNA pesticides and offers a sustainable solution for pest management in agriculture. Future research should optimize the delivery system, conduct field trials, and explore its applicability to other agricultural pests.

In mountainous citrus orchards, the application of conventional ground sprayers for the control of citrus red mite (Panonychus citri) is often constrained by complex terrain and low operational efficiency. The Unmanned Aerial Spraying System (UASS), due to its low-altitude, low-volume, and high-maneuverability characteristics, has emerged as a promising alternative for pest management in such challenging environments. To evaluate the spray performance and field efficacy of different UASS types in controlling P. citri, five representative UASS models (JX25, DP, T1000, E-A2021, and T20), four mainstream pesticide formulations, and four novel tank-mix adjuvants were systematically assessed in a field experiment conducted in a typical hilly citrus orchard. The results showed that T20 delivered the best overall spray deposition, with upper canopy coverage reaching 10.63%, a deposition of 3.01 μg/cm2, and the highest pesticide utilization (43.2%). E-A2021, equipped with a centrifugal nozzle, produced the finest droplets and highest droplet density (120.3–151.4 deposits/cm2), but its deposition and coverage were lowest due to drift. Nonetheless, it exhibited superior penetration (dIPR 72.3%, dDPR 73.5%), facilitating internal canopy coverage. T1000, operating at higher flight parameters, had the weakest deposition. Formulation type had a limited impact, with microemulsions (MEs) outperforming emulsifiable concentrates (ECs) and suspension concentrates (SCs). All adjuvants improved spray metrics, especially Yimanchu and Silwet, which enhanced pesticide utilization to 46.8% and 46.4% for E-A2021 and DP, respectively. Adjuvant use increased utilization by 4.6–11.9%, but also raised ground losses by 1.5–4.2%, except for Yimanchu, which reduced ground loss by 2.3%. In terms of control effect, the rapid efficacy (1–7 days after application, DAA) of UASS spraying was slightly lower than that of ground sprayers — electric spray gun (ESG), while its residual efficacy (14–25 DAA) was slightly higher. The addition of adjuvants improved both rapid and residual efficacy, making it comparable to or even better than ESG. E-A2021 with 5% abamectin·etoxazole ME (5A·E) and Yimanchu achieved 97.4% efficacy at 25 DAA. Among UASSs, T20 showed the rapid control, while E-A2021 outperformed JX25 and T1000 due to finer droplets effectively targeting P. citri. In residual control (14–25 DAA), JX25 with 45% bifenazate·etoxazole SC (45B·E) was most effective, followed by T20. 5A·E and 45B·E showed better residual efficacy than abamectin-based formulations, which declined more rapidly. Adjuvants significantly extended control duration, with Yimanchu performing best. This study demonstrates that with optimized spraying parameters, nozzle types, and adjuvants, UASSs can match or surpass ground spraying in P. citri control in hilly citrus orchards, providing valuable guidance for precision pesticide application in complex terrain.

Honeybees, essential pollinators for maintaining biodiversity, are experiencing a sharp population decline, which has become a pressing environmental concern. Among the factors implicated in this decline, neonicotinoid pesticides, particularly those belonging to the fourth generation, have been the focus of extensive scrutiny due to their potential risks to honeybees. This study investigates the molecular basis of these risks by examining the binding interactions between Apis mellifera L. chemosensory protein 3 (AmelCSP3) and neonicotinoids with a cis-oxygen bridge heterocyclic structure. Employing surface plasmon resonance (SPR) in conjunction with multispectral techniques and molecular modeling, this study meticulously analyzed the binding affinity, specificity, and kinetics under conditions that simulate real-world exposure scenarios. Key parameters such as the number of binding sites (n), binding constants (Ka), dissociation constants (KD), and binding distances (r) were quantitatively assessed. The findings revealed that hydrogen bonding and hydrophobic interactions serve as the primary forces driving the binding process, with fluorescence quenching mechanisms involving both dynamic and static interactions. Molecular docking and dynamics simulations further illustrated the stability of these interactions within the active site of the protein. Of particular interest, cis-structured neonicotinoids demonstrated distinct binding characteristics compared to their trans-structured counterparts, including an inverse relationship between the binding constant and temperature. These findings offer critical insights for the design of cis-structured neonicotinoid compounds that are safer for pollinators, thus reducing the impact on non-target organisms such as bees. Furthermore, this research enhances the understanding of the interaction mechanisms between cis-structured neonicotinoid substances and honeybee proteins, providing a foundation for future studies on the environmental safety of these compounds.

Honeybees are vital for biodiversity and agricultural productivity, yet their populations are declining globally, partly due to exposure to neonicotinoid pesticides. Odorant-binding protein 14 (OBP14) plays an important role in honeybee chemosensation, but its involvement in neonicotinoid toxicity remains underexplored due to limitations in traditional fluorescence spectroscopy techniques. This gap hampers our understanding of neonicotinoid risks to honeybee health. Here, we explored the molecular interactions between OBP14 from Apis mellifera and three widely used neonicotinoids (imidacloprid, thiamethoxam, and clothianidin) using molecular modeling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and multispectroscopy. SPR and ITC characterized the binding affinity, specificity, and thermodynamic parameters of AmelOBP14 interacting with three neonicotinoid compounds, revealing that the binding process is spontaneous and primarily driven by hydrophobic and electrostatic interactions. Molecular modeling highlighted that phenylalanine residue Phe54, near the binding site, plays a critical role in these interactions. UV-vis absorption spectroscopy and synchronous fluorescence spectroscopy (SFS) support slight changes in the microenvironment around the aromatic amino acids of OBP14. Fourier Transform Infrared Spectroscopy (FTIR) and circular dichroism spectroscopy (CD) indicate a decrease in the α-helix content of OBP14, suggesting a change in its secondary structure, while three-dimensional (3D) fluorescence spectroscopy confirms the non-fluorescent nature of the OBP14 polypeptide backbone. The study results revealed its potential as a biomarker for pesticide risk assessment, providing important insights into the molecular mechanisms by which neonicotinoids may impair bee chemosensory function, and offering guidance for the design of safer pesticides to minimize harm to these important pollinators.

The sustained application of insecticides has led to the development of resistance. Carboxylesterases (CarEs) play important roles in the resistance and synergism of chlorantraniliprole and carbaryl. In this study, the key Spodoptera frugiperda CarEs gene, SfCarE, was identified. SfCarE was highly expressed at the 4th instar and in the midgut of the 6th instar larvae. Knockdown of SfCarE resulted in significant decrease in CarE activity and an increase in susceptibility to chlorantraniliprole, as well as to the mixture of chlorantraniliprole and carbaryl. Metabolic assays demonstrated that the SfCarE protein can metabolize both carbaryl and chlorantraniliprole, while carbaryl can effectively slow down the metabolism of chlorantraniliprole, thereby increasing its retention and insecticidal effect. Molecular docking and molecular dynamics simulation indicated that the predominant modes of interaction between SfCarE and the compounds were hydrogen bonds and van der Waals forces. Additionally, carbaryl exhibited a higher degree of stability in binding with SfCarE than chlorantraniliprole. Therefore, carbaryl was preferentially metabolized by SfCarE, which protecting chlorantraniliprole from metabolism. In summary, this study preliminarily reveals a synergistic mechanism in which the metabolic rate of chlorantraniliprole is reduced when combined with carbaryl. This research demonstrated that the SfCarE-mediated metabolic and synergistic interactions between chlorantraniliprole and carbaryl may provide valuable insights for the development of novel insecticidal combinations for integrated resistance management (IRM).

The ryanodine receptors (RyRs) represent an optimal target for insecticide development. To explore novel structures of RyRs agonists, a series of compounds containing 1,2,4-oxadiazole were designed and synthesized based on the RyRs structure. The compounds were confirmed by 1H and 13C nuclear magnetic resonance as well as high-resolution mass spectrometry. Additionally, bioassays demonstrated that the majority of the newly synthesized compounds exhibited complete mortality (100%) against Plutella xylostella and Mythimna separata at a concentration of 50 mg/L. Furthermore, compound II5 showed a slightly higher LC50 value (LC50 = 0.20 mg/L) compared to the control chlorantraniliprole (LC50 = 0.06 mg/L) against M. separata. Through the toxicity study of the target compounds on Spodoptera frugiperda carrying the resistant mutant RyR (I4790M), it was confirmed that these target compounds have a similar mode of action to chlorantraniliprole. Moreover, the binding modes of these compounds were investigated, revealing that 1,2,4-oxadiazole serves as an effective substitute structure for amide bonds. These findings provide valuable insights for further structural optimization.

In the intricate web of ecological relationships, pollinators such as the Italian honeybee (Apis mellifera) play a crucial role in maintaining biodiversity and agricultural productivity. This study focuses on the interactions between three neonicotinoid compounds and the honeybee's chemosensory protein 3 (CSP3), a key player in their olfactory system. Employing advanced spectroscopic techniques and molecular modeling, we explore the binding dynamics and conformational changes in CSP3 upon exposure to these pesticides. The research reveals that all three neonicotinoids considerably quench CSP3's fluorescence through a dynamic and static mixing mechanism, indicating a strong binding affinity, predominantly driven by hydrophobic interactions. UV-visible absorption, synchronous fluorescence, and 3D fluorescence spectra support slight changes in the microenvironment around the aromatic amino acids of CSP3. Circular dichroism spectra indicate a reduction in CSP3's α-helix content, suggesting structural alterations. Molecular docking and dynamics simulations further elucidate the binding modes and stability of these interactions, highlighting the role of specific amino acids in CSP3's binding cavity. Findings provide critical insights into molecular mechanisms by which neonicotinoids may impair honeybee chemosensory function, offering implications for designing safer pesticides and understanding the broader ecological impact of these chemicals on pollinator health.

INTRODUCTION:Diaphorina citri is the most serious pest of citrus worldwide because it is the natural insect vector of huanglongbing. Cycloxaprid (Cyc) was highly toxic to D. citri. However, the poor solubility and stability had limited its development. OBJECTIVES:In order to improve the insecticidal effect and stability to harsh climatic conditions of Cyc. METHODS:Cyc was chosen as the representative pesticide, 4,4'-methylenebis (phenyl isocyanate), PEG-600 and n-butanol were used to prepare sustained-release nano-gelation particles (Cyc@NGs). RESULTS:Cyc@NGs enhance the toxicity of Cyc more than 3 folds. Furthermore, Cyc@NGs showed excellent anti-rain and anti-UV capacity. After being exposed to ultraviolet light for 12 h, Cyc decreased by 100 %, while the insecticide content of Cyc@NGs only decreased by 25 %. Additionally, Cyc@NGs possessed better wettability on citrus leaves, mainly benefitting from its lower contact angle on citrus leaves. Moreover, FITC-labeled nano-gelation particles (FITC-NGs) exhibited high capability to penetrate and enrich in citrus leaf tissue and D. citri midgut. Consequently, NGs promoted the translocation and durability of insecticides, thereby, increasing the insecticidal activity. The results suggested that nano-gelation particle is a promising platform to deliver insecticides and Cyc@NGs would be the suitable candidate for the effective management of D. citri.

BACKGROUND:Citrus huanglongbing (HLB) is a devastating disease in citrus, caused by Candidatus Liberibacter asiaticus (CLas), which primarily resides in the phloem where chemicals cannot effectively reach, posing a significant challenge in controlling HLB. To address these challenges, plant essential oils (EOs), widely used as transdermal enhancers and known for their benefits for plant tissues, were investigated for their potential to enhance chemical permeation. RESULTS:In this study, seven EOs - eugenol, carvacrol, eucalyptol, geraniol, linalool, cinnamaldehyde, and d-limonene - were evaluated for their potential to enhance chemical penetration into citrus leaves. Preliminary screening with tracer methods revealed that geraniol provided the greatest enhancement of permeation, achieving the deepest and broadest distribution into the spongy tissue of the citrus leaf. The permeation levels of rhodamine B and crystal violet through the cuticular layer were elevated from 0.55 mg L-1 and 0.11 mg L-1 to 20.04 mg L-1 and 7.14 mg L-1, respectively, by the addition of geraniol. Further validation through high-performance liquid chromatography-mass spectrometry (HPLC-MS) confirmed that the penetration of fludioxonil and tetracycline into citrus leaf tissues was enhanced by 15.07-fold and 20.43-fold, respectively. In planta experiments confirmed that adding geraniol to tetracycline and oxytetracycline via foliar application eliminated CLas bacteria in HLB-affected citrus more efficiently than controls. CONCLUSION:Our study underscores the potential of geraniol as an effective EO-based permeation enhancer for combating HLB. By significantly improving the delivery and efficacy of bactericidal treatments, geraniol offers a promising strategy for managing this catastrophic citrus disease, potentially transforming the approach to controlling HLB. © 2024 Society of Chemical Industry.

Angelica sinensis (Oliv.) Diels belongs to the Apiaceae family. The root of A. sinensis, is used in traditional Chinese medicine for its antioxidant and immune regulation properties. The main active compounds in A. sinensis include organic acids, phthalides and coumarins, and their biosynthetic pathways are the focus of international attention. A. sinensis is prone to early flowering and bolting, which negatively impacts production for several reasons, including germplasm degradation and quality instability in artificial cultivation. The identification of top-geoherbalism of A. sinensis has also become the focus of recent research, as it would allow selection for breeds with excellent medicinal quality and remarkable curative effects. Advances in sequencing technology and bioinformatic methodologies have enabled extensive molecular and genetic studies in A. sinensis. In this review, we summarize the latest molecular research advances related to A. sinensis, including biosynthetic pathways and regulation of active compounds, and molecular underpinnings of early bolting and flowering and top-geoherbalism. We discuss limitations of the current research and propose prospective topics in need of further exploration.

Amomi Fructus (Sharen, AF) is a traditional Chinese medicine (TCM) from three source species (or varieties), including Wurfbainia villosa var. villosa (WVV), W. villosa var. xanthioides (WVX), or W. longiligularis (WL). Among them, WVV has been transplanted from its top-geoherb region, Guangdong, to its current main production area, Yunnan, for >50 years in China. However, the genetic and transcriptomic differentiation among multiple AF source species (or varieties) and between the origin and transplanted populations of WVV is unknown. In our study, the observed overall higher expression of terpenoid biosynthesis genes in WVV than in WVX provided possible evidence for the better pharmacological effect of WVV. We also screened six candidate borneol dehydrogenases (BDHs) that potentially catalyzed borneol into camphor in WVV and functionally verified them. Highly expressed genes at the P2 stage of WVV, Wv05G1424 and Wv05G1438, were capable of catalyzing the formation of camphor from (+)-borneol, (-)-borneol and DL-isoborneol. Moreover, the BDH genes may experience independent evolution after acquiring the ancestral copies, and the following tandem duplications might account for the abundant camphor content in WVV. Furthermore, four populations of WVV, WVX, and WL are genetically differentiated, and the gene flow from WVX to WVV in Yunnan contributed to the greater genetic diversity in the introduced population (WVV-JH) than in its top-geoherb region (WVV-YC), which showed the lowest genetic diversity and might undergo genetic degradation. In addition, terpene synthesis (TPS) and BDH genes were selected among populations of multiple AF source species (or varieties) and between the top- and non-top-geoherb regions, which might explain the difference in metabolites between these populations. Our findings provide important guidance for the conservation, genetic improvement, and industrial development of the three source species (or varieties) and for identifying top-geoherbalism with molecular markers, and proper clinical application of AF.

为有效防控新入侵的迁飞性害虫草地贪夜蛾,指导科学用药,本研究采用饲料表面涂药法测定了 13类20种杀虫剂对云南省玉溪市红塔区(红塔种群)、广东省广州市花都区(花都种群)、广西壮族自治区钦州市钦南区(钦南种群)、云南省玉溪市华宁县(华宁种群)、海南省海口市秀英区(秀英种群)5个种群草地贪夜蛾3龄幼虫的毒力;进而,以红塔种群为相对敏感种群,以华宁种群为抗性种群,测定甲维盐、溴虫氟苯双酰胺、虱螨脲、氯虫苯甲酰胺、杀铃脲、虫螨腈、高效氯氟氰菊酯、茚虫威和氟苯虫酰胺9种杀虫剂对2个种群草地贪夜蛾5龄幼虫的毒力.结果表明,20种杀虫剂对5个种群草地贪夜蛾3龄幼虫的毒力大小顺序依次为:甲维盐、溴虫氟苯双酰胺＞虱螨脲、杀铃脲、氟铃脲、氯虫苯甲酰胺＞高效氯氟氰菊酯、虫螨腈、多杀霉素＞溴氰虫酰胺、茚虫威、唑虫酰胺＞氟苯虫酰胺、联苯菊酯、甲氧虫酰肼＞毒死蜱、乙酰甲胺磷＞噻虫嗪＞啶虫脒、甲萘威;9种杀虫剂对2个种群草地贪夜蛾5龄幼虫的毒力大小顺序依次为:甲维盐、溴虫氟苯双酰胺＞氯虫苯甲酰胺＞虱螨脲＞杀铃脲＞虫螨腈＞高效氯氟氰菊酯、茚虫威＞氟苯虫酰胺,研究结果表明,甲维盐、溴虫氟苯双酰胺、虱螨脲、氯虫苯甲酰胺4种杀虫剂对草地贪夜蛾3龄和5龄幼虫具有很高的活性,是防治草地贪夜蛾的理想药剂,该研究为合理选择有效杀虫剂进行田间防治提供了科学依据.

Prunus quanzhouensis is a new species of Rosaceae discovered in Guangxi, China, which is here described and illustrated. Despite its morphological similarities to P. campanulata, P. conradinae and P. xueluoensis, it can be distinguished from them by being shrubs or small trees, with 1.5–3.0 m tall, leaves with 10–13 pairs of lateral veins, abaxially glabrous involucral bracts, adaxially appressed villous bracts, flowers measuring 1–1.2 cm in diameter, tubular hypanthium with about 9×3 mm, oblique sepals, 30–35 stamens, and a style that is villous and longer than the stamens. Less than six populations of this new species have been found in the type locality, with approximately 130 mature individuals, which accounted for more than 95% of the total population. Based on direct observation in the field and the IUCN Red List Categories and Criteria, P. quanzhouensis is classified as Endangered (EN).

Sulfoxaflor is a widely used sulfoximine insecticide that has been regarded as an important alternative insecticide for IPM strategies, but a comprehensive study of its potential ecological toxicity is still lacking. In the present work, the growth, longevity, predation and reproduction toxicity of Coccinella septempunctata caused by sulfoxaflor were evaluated. In addition, the potential mechanisms of decreased fecundity in C. septempunctata were investigated by analyzing the transcriptional and protein levels of reproduction-related gene vitellogenin (Vg). In a 20-day acute contact toxicity test, decreased survival proportion, pupation rate, adult emergence ratio, and increased hazard quotient (HQ) values were observed. Moreover, sublethal dosages of sulfoxaflor significantly inhibited the predation, longevity, fecundity and net reproduction rate of progeny. In addition, LR30 of sulfoxaflor dramatically down-regulate the mRNA-expression (F0: 65.38-fold, F1: 2.24-fold) and protein content (F0: 1.35-fold, F1: 1.36-fold) of Vg in the F0 and F1 generations. These results suggested that sulfoxaflor could inhibit the gene and protein content of Vg, thereby reducing the fecundity of C. septempunctata. Our study indicated that sulfoxaflor has potential risks to parent and progeny generations of C. septempunctata. These results provide valuable reference for optimal usage of sulfoxaflor in IPM systems.

In this study, we sequenced and assembled the complete chloroplast genome of Chloranthus nervosus Collett ex Hemsl. 1890. The total length of the complete chloroplast sequence was found to be 158,002 bp. It consisted of a large single-copy (LSC) region of 87,127 bp, a small single-copy (SSC) region of 18,541 bp, and a pair of inverted repeat (IR) regions, each with a length of 26,167 bp. The overall GC content of the complete chloroplast genome was 38.9%, with the LSC region, SSC region, and IR regions exhibiting GC contents of 37.4%, 34.1%, and 43.1%, respectively. The annotation of the chloroplast genome revealed a total of 131 genes, comprising 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis revealed that the seven sampled species of Chloranthus were divided into two clades. Within the clade characterized by long filamentous anther connectives, C. nervosus showed the closest relation to C. japonicus. These findings validated the previous preliminary results on the phylogenetic relationships of the seven species of Chloranthus with strong support.

On the basis of the structures of natural methylxanthines and chalcone, a series of novel chalcone analogues containing a methylxanthine moiety, Ia-Ig, and their N-acyl pyrazoline derivatives IIa-IIz and IIaa-IIaf were synthesized and identified through melting points, 1H NMR, 13C NMR, and HRMS. The single crystal of compound IId was obtained, which further illustrated the structural characteristics of the methylxanthine-acylpyrazoline compounds. The biological tests showed that some of them displayed favorable insecticidal activities toward Plutella xylostella L. and were superior to the natural methylxanthine compound caffeine while being comparable with the insecticide triflumuron (e.g., compound Ic: LC50 = 16.8508 mg/L, IIf: LC50 = 1.5721 mg/L, against P. xylostella). Of these compounds, Ic, IIf, and IIu could serve as novel insecticidal leading structures for further study. Some of the compounds showed good fungicidal activities (e.g., compound Ig: EC50 = 14.74 μg/mL, against Rhizoctonia cerealis; IIf: EC50 = 7.06 μg/mL, against Physalospora piricola; IIac: EC50 = 5.37 and 8.19 μg/mL, against Phytophthora capsici and Sclerotinia sclerotiorum, respectively); Ic, Ig, IIa, IIf, IIr, IIs, IIv, IIac, and IIaf could be novel fungicidal leading compounds for further exploration. Furthermore, most of the tested compounds exhibited apparent herbicidal activities against Brassica campestris at a concentration of 100 μg/mL; among others, compound IIa was the best one both toward Brassica campestris and Echinochloa crusgalli and deserves further investigation. The structure-activity relationships of these compounds were also summarized and discussed in detail. The contrast experiment results of compounds C-1 and C-2 showed a positive effect on the biological activity enhancement from the combination of the methylxanthine moiety with the N-dichloroacetyl phenylpyrazoline skeleton. In addition, two 3D-QSAR models with predictive capability were constructed based on the insecticidal and fungicidal activities to afford deep insight into the bioactivity profiles of these compounds. This research provides useful guidance and reference for the discovery and development of novel xanthine natural product-based pesticides.

Deoxynivalenol (DON) and zearalenone (ZEN) pose a serious threat to human health, and have been frequently detected in the aqueous environment. To protect consumers from the harm of mycotoxins, a nanozyme-mediated multiplexed lateral flow immunoassay (LFIA) integrated with a smartphone was developed for rapid, highly sensitive and simultaneous quantitative detection of DON and ZEN in the aqueous environment. Highly efficient peroxidase mimicking core-shell Au@Pt nanozymes were synthesized by one-pot method, and then used as signal amplification to highly improve sensitivity of the detection, while a smartphone-based quantitative detection device could rapidly quantify results to improve the detection efficiency of the LIFA for on-site detection. After optimization, the detection time of the assay was 10 min, and the detection limits of the LIFA for DON/ZEN were 0.24/0.04 ng/mL, which were improved 416 and 150 folds compared to the conventional gold nanoparticles (GNPs)-based LFIA. Moreover, there was no obvious cross-reaction with other related mycotoxins, indicating that LFIA had a high specificity. The average recoveries of DON and ZEN from corn, wheat and three water samples were obtained from 94.3 % to 107.9 % with relative standard deviations of 0.2-7.6 %. Furthermore, the accuracy and reliability of the LIFA were evaluated with three spiked water samples, and the results presented good correlations with analytic results from the enzyme-linked immunosorbent assay (R2 =0.988 for DON, and 0.983 for ZEN). The results indicate the proposed LIFA was potentially a rapid, on-site simultaneous and highly sensitive method for DON and ZEN detection in the aqueous environment.