Exposure of zebrafish (Danio rerio) to trans-2-hexenal induces oxidative stress and protein degeneration of the gill.

Trans-2-hexenal (T2H) has great commercial value for development as a biopesticide, but its toxicity risk to nontarget organisms is unknown. Here, the toxicity and underlying mechanism of T2H on zebrafish (Danio rerio) were investigated. The LC50 (48 h) of T2H on zebrafish is 4.316 µg/mL, and the aldehyde group is essential to its toxicity. In 14-day chronic toxicity tests, 0.432 µg/mL T2H resulted in a higher mortality of zebrafish than the control group. Furthermore, the sensitivity of zebrafish to different administration methods was gill administration>oral administration>transdermal administration>intravenous injection. T2H induced significant cell death and ROS generation in zebrafish gill cells in a concentration-dependent manner. After treatment with 4.316 µg/mL T2H, the expression of oxidative stress-related genes (nrf2, gstp1, keap1b, sod1 and sod2) and the content of malondialdehyde (MDA) were up-regulated. Incubation with T2H caused an immediate denaturation of gill protein, which was aggravated with increasing dose of T2H. We also found that T2H at 21.225 mg/mL significantly reduced the in vitro activity of succinate dehydrogenase (SDH). Among the three amino acids tested, T2H was only found to react with methionine and glycine to form adducts, which may be the basis of the protein denaturation. This study confirmed that T2H could induce oxidative stress and protein denaturation in zebrafish gills, providing important information for risk assessment of T2H exposure.

SDH mutations confer complex cross-resistance patterns to SDHIs in Corynespora cassiicola.

Succinate dehydrogenase inhibitors (SDHIs) are one of the most frequently used fungicides in cucumber fields in China. Our previous studies indicated that the sensitivity profile of Corynespora cassiicola, the causal agent of Corynespora leaf spot, to different SDHIs varied greatly; however, the underlying mechanism remains unclear. The 50% effective concentration (EC50) values of boscalid, fluopyram, fluxapyroxad and isopyrazam in C. cassiicola collected from 2017 to 2020 shifted, with resistance frequencies of 79.83%, 78.43%, 83.19% and 49.86%, respectively. The sequence alignment of sdhB/C/D of resistant strains revealed that eight single amino acid mutations (B-H278Y/L, B-I280V, C-S73P, C-N75S, C-H134R, D-D95E and D-G109V), and three dual-mutations (B-I280V&C-S73P, B-I280V&C-N75S and C-S73P&C-N75S) conferred various SDHI resistance levels and cross-resistance profiles. The expression level of the sdhB/C/D gene and succinate dehydrogenase (SDH) activity in the mutants were significantly altered by the presence of SDHIs, compared with the wild type strain. Additionally, molecular docking results suggested that the missense mutation influenced the crystal structure of SDH and subsequently interfered with the interaction bonds and bond distances among the target protein and chemicals. In brief, amino acid mutations altered the fungicide response of target gene expression, SDH activity and the binding features of SDH-ligand complexes and subsequently conferred multiple resistance levels and complex cross-resistance patterns to SDHIs in C. cassiicola. The evaluation of C. cassiicola resistance to SDHIs provided a significant foundation for efficient chemical development and integrated CLS management strategies.

Green Leaf Volatile Trans-2-Hexenal Inhibits the Growth of Fusarium graminearum by Inducing Membrane Damage, ROS Accumulation, and Cell Dysfunction.

Fusarium graminearum, the main agent of Fusarium head blight (FHB), can cause serious yield loss and secrete mycotoxins to contaminate grain. Here, the biological activity of trans-2-hexenal (T2H) against F. graminearum was determined and its mode of action (MOA) was investigated. Furthermore, surface plasmon resonance with liquid chromatography-tandem mass spectrometry (SPR-LC-MS/MS), bioinformatic analysis, and gene knockout technique were combined to identify the binding proteins of T2H in F. graminearum cells. T2H exhibited satisfactory inhibitory activity against F. graminearum in vitro. Good lipophilicity greatly enhanced the affinity of T2H to F. graminearum mycelia and further caused membrane damage. The FgTRR (thioredoxin reductase) gene negatively regulates the sensitivity of F. graminearum to T2H by reducing the generation of reactive oxygen species (ROS) induced by T2H. Two mutant strains with FgSLX1 (structure-specific endonuclease subunit) and FgCOPB (coatomer subunit ß) genes knockout showed decreased sensitivity to T2H, suggesting that these two genes may be involved in the antimicrobial activity of T2H. Taken together, T2H can inhibit F. graminearum growth by multiple MOAs and can be used as a biofumigant to control the occurrence of FHB in the field.

Toxicity, residue and risk assessment of tetraniliprole in soil-earthworm microcosms.

Maize seed treatment with chemicals to control underground pests is a common agricultural practice, but inappropriate use of insecticides poses a considerable threat to plant development and soil nontarget organisms. In this study, the availability of tetraniliprole seed dressing to control the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae) in the maize seeding stage and its safety to earthworms (Eisenia fetida) were investigated. The selective toxicity (ST) of tetraniliprole between E. fetida and A. ipsilon was greater than 4000. No significant adverse effect of tetraniliprole seed treatment on the germination of maize seeds was observed at concentrations of 2.4-9.6 g a.i. /kg seed. Compared with the untreated control, seed treatment with tetraniliprole at 9.6 g a.i. /kg seed greatly reduced the percentage of damaged plants from 88.73% to 26.67%, and achieved the highest control effect of 69.91%. Tetraniliprole of 2.4 g a.i. /kg seed can effectively inhibit A. ipsilon until 14 days after seed germination, with the lowest mortality rate of 44.44%. During the entire exposure period, the maximum residual concentration of tetraniliprole detected in the soil (5.86 mg/kg) was considerably lower than the LC50 value of tetraniliprole to E. fetida (>4000 mg/kg). According to the low-tier risk assessment, the highest risk quotient (RQ) of tetraniliprole seed treatment to earthworms at test concentrations was 2.8 × 10-3, which was evaluated as acceptable. This study provided data support for tetraniliprole seed treatment to control underground pests in maize fields.

Evaluation of Sensitivity and Resistance Risk of Corynespora cassiicola to Isopyrazam and Mefentrifluconazole.

Necrotic lesions on leaves caused by Corynespora leaf spot (CLS) seriously threaten the quality and yield of cucumber in China. Corynespora cassiicola has developed different degrees of sensitivity to various fungicides due to its long-term and extensive application. In our work, the effect of isopyrazam and mefentrifluconazole on different life stages of C. cassiicola was examined. To determine the optimal effect of binary mixtures of isopyrazam and mefentrifluconazole, the two fungicides were mixed at different proportions. Furthermore, the disease suppression of isopyrazam, mefentrifluconazole, and their compound mixture against CLS was evaluated in greenhouse experiments. Ultraviolet (UV) mutagenesis and fungicide-selection methods were performed to assess the risk of resistance development. Among the three life stages tested, isopyrazam showed the weakest inhibition on mycelial growth, and mefentrifluconazole showed the strongest inhibition of germ tube elongation. According to Wadley's and cotoxicity coefficient methods, the optimal proportion of the two-component mixture of isopyrazam and mefentrifluconazole was 1:1. Isopyrazam, mefentrifluconazole, and their binary mixture at 1:1 reduced the disease severity of CLS on potted cucumber plants, with protective effects of 31.11, 24.65, and 42.12% and curative effect of 33.90, 37.48, and 42.84%, respectively. Compared with isopyrazam or mefentrifluconazole alone, the binary mixture of the two fungicides at 1:1 did not exert significant influence on the change of C. cassiicola sensitivity. Undoubtedly, such data will greatly facilitate the screening of new fungicides for CLS and resistance management.

A bioactivity and biochemical analysis of iminoctadine tris (albesilate) as a fungicide against Corynespora cassiicola.

Iminoctadine tris (albesilate) is a bis-guazatine fungicide, and its specific modes of action and efficacy against C. cassiicola are not yet clear. In this study, baseline sensitivity data for mycelial growth showed that the frequency distribution curve of iminoctadine tris (albesilate) EC50 values is unimodal. The EC50 values ranged from 0.1151 to 1.2101 µg/mL, with a mean of 0.5775 ±â€¯0.2677 µg/mL. Iminoctadine tris (albesilate) affected the morphological development of C. cassiicola, causing increased branching of the mycelium. The significant increase in membrane permeability and malondialdehyde content after iminoctadine tris (albesilate) treatment indicated that this fungicide caused severe damage to the membrane structure. Furthermore, 0.4 µg/mL iminoctadine tris (albesilate) could decrease the spore density of C. cassiicola from 2.6200 × 104 to 1.4967 × 104/cm2 on average in vitro, indicating that the fungicide had great potential to reduce secondary infection with C. cassiicola in the field. Additionally, 120 µg/mL iminoctadine tris (albesilate) provided over 95% curative efficacy and 81.17% preventative efficacy on detached cucumber leaves inoculated with C. cassiicola. In field trials, iminoctadine tris (albesilate) at a dose of 120 g a.i./ha exhibited 72.92% and 80.92% control efficacy in 2017 and 2018, respectively. However, the efficacy supplied by the reference fungicide azoxystrobin at 250 g a.i./ha was only approximately 50% due to the development of fungicide resistance in C. cassiicola. Taken together, the findings above provide a solid foundation for the exploration of the action mechanisms of iminoctadine tris (albesilate) against C. cassiicola and provide overwhelming evidence for the use of iminoctadine tris (albesilate) as an excellent potential alternative fungicide in the management of cucumber Corynespora leaf spot.

Evaluation of bioactivity and control efficacy of tetramycin against Corynespora cassiicola.

Tetramycin is a novel polyene antibiotic that has exhibited excellent inhibitory activity against many plant pathogens. In this study, the sensitivity of Corynespora cassiicola to tetramycin was assessed in vitro using a series of 91 isolates, and its effects on hyphae and conidia were evaluated. Preventive and curative efficacies of tetramycin against Corynespora leaf spot were evaluated using detached cucumber leaves and potted cucumber plants. The control efficacy of tetramycin against the pathogen was also determined under field conditions. Measurement of baseline sensitivity suggested that the frequency distribution of tetramycin fits a unimodal curve. Among several life stages, spore germination was most severely inhibited by tetramycin, whereas mycelial growth was found to be the least sensitive. Microscopy observation revealed that tetramycin treatment leads to abnormal morphological development of C. cassiicola. Overall, tetramycin exhibited excellent curative and preventive activities against C. cassiicola on both cucumber leaves and potted cucumber plants. Furthermore, tetramycin significantly reduced the disease severity of Corynespora leaf spot in the field. The results from this study showed that tetramycin may play an important role in Corynespora leaf spot disease management and promote its introduction into fungicide-application programs.

Detection and Characterization of QoI-Resistant Phytophthora capsici Causing Pepper Phytophthora Blight in China.

Phytophthora capsici is a highly destructive plant pathogen that has spread worldwide. To date, the quinone outside inhibitor (QoI) azoxystrobin has been the choice of farmers for managing this oomycete. In this study, the sensitivity of 90 P. capsici isolates collected from Yunnan, Fujian, Jiangxi, Zhejiang, and Guangdong in southern China to azoxystrobin was assessed based on mycelial growth, sporangia formation, and zoospore discharge. Furthermore, the mitochondrial cytochrome b (cytb) gene from azoxystrobin-sensitive and -resistant P. capsici isolates was compared to investigate the mechanism of QoI resistance. The high values for effective concentration to inhibit 50% of mycelial growth and large variation factor obtained provide strong support for the existence of azoxystrobin-resistant subpopulations in wild populations. The resistance frequency of P. capsici to azoxystrobin was greater than 40%. Sensitive P. capsici isolates were strongly suppressed on V8 medium plates containing azoxystrobin supplemented with salicylhydroxamic acid at 50 µg ml-1, whereas resistant isolates grew well under these conditions. Multiple alignment analysis revealed a missense mutation in the cytb gene that alters codon 137 (GGA to AGA), causing an amino acid substitution of glycine to arginine (G137R). The fitness of the azoxystrobin-sensitive isolate is similar to that of the G137R mutant. Additionally, the P. capsici isolates used in this study exhibited decreased sensitivity to two other QoI fungicides (pyraclostrobin and famoxadone). Necessary measures should be taken to control this trend of resistance to QoI that has developed in P. capsici in southern China.

Favorable compatibility of nitenpyram with the aphid predator, Coccinella septempunctata L. (Coleoptera: Coccinellidae).

The increasing demand for lessening the chemical input in agricultural ecosystems requires an efficient combination of pesticides and biological controls. Thus, fully understanding the compatibility of pesticides and beneficial arthropod predators is helpful and essential. In this study, we evaluated the influence of nitenpyram on both larvae and adults of Coccinella septempunctata using exposure doses of 10, 25, 50, 100, and 150% of the maximum recommended field rate (MRFR) (3, 7.5, 15, 30, and 45 g a.i. ha-1, respectively) and a blank control based on a preliminary acute 72-h toxicity experiment. In the long-term test, the LR50 (application rate causing the mortality of 50% of the individuals) of nitenpyram for C. septempunctata decreased from 73.43 to 63.0 g a.i. ha-1, while the HQ (hazard quotient) values remained below the threshold value of 2. Nitenpyram did not significantly influence the survival rate, fecundity, pupation, or adult emergence at 150% of the label rate (lowest LR50 = 63.0 g a.i. ha-1), and its demonstrated NOER (No Observed Effect application Rates) values are all above 45 g a.i. ha-1. Likewise, the total developmental time and egg hatchability were not significantly affected at 100% of the label rate (NOER = 30 g a.i. ha-1). The assessment of the total effect (E) suggested that nitenpyram could be classified as harmless to C. septempunctata below/at a dose of 30 g a.i. ha-1. The lowest LR50 and NOER values were both above the maximum recommended field application rate for nitenpyram (30 g a.i. ha-1) for controlling aphids in China. All results indicated that the on-label use of nitenpyram is compatible with the natural enemy C. septempunctata in agricultural ecosystems.

Influence of lethal and sublethal exposure to clothianidin on the seven-spotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae).

The seven-spotted ladybird beetle, Coccinella septempunctata L., as a dominant predator of aphids, has played a crucial role in integrated pest management (IPM) strategies in agricultural ecosystems. To study the risk of insecticides to C. septempunctata, the neonicotinoid clothianidin was selected for evaluation of its influence on C. septempunctata at lethal and sublethal doses. The LR50 (application rate causing 50% mortality) in the exposed larvae decreased from 19.94 to 5.91 g a.i. ha-1, and the daily HQ (hazard quotient) values increased from 3.00 to 10.15, indicating potential intoxication risks. We also determined NOERs (No Observed Effect application Rates) of clothianidin on the total developmental time (10 g a.i. ha-1), survival (2.5 g a.i. ha-1) and pupation (5 g a.i. ha-1). Moreover, clothianidin at a NOER of 2.5 g a.i. ha-1 did not profoundly affect adult emergence, fecundity or egg hatchability. The total effect (E) assessment also showed that clothianidin at 2.5 g a.i. ha-1 was slightly harmful to C. septempunctata. These results suggested that clothianidin would impair C. septempunctata when applied at over 2.5 g a.i. ha-1 in the field. Conservation of this biological control agent in agricultural ecosystems thus requires further measures to decrease the applied dosages of clothianidin.

Concentrations of imidacloprid and thiamethoxam in pollen, nectar and leaves from seed-dressed cotton crops and their potential risk to honeybees (Apis mellifera L.).

Neonicotinoid insecticides (NIs) have recently been recognized as co-factors in the decline of honeybee colonies because most neonicotinoids are systemic and can transfer into the pollen and nectar of many pollinated crops. In this study, we collected pollen, nectar and leaves from a cotton crop treated with imidacloprid and thiamethoxam to measure the residue levels of these two NIs at different application doses during the flowering period. Then, the residual data were used to assess the risk posed by the systemic insecticides to honeybees following mandated methods published by the European Food Safety Authority (EFSA), and a highly toxic risk to honeybees was highlighted. Imidacloprid was found in both pollen and nectar samples, whereas thiamethoxam was found in 90% of pollen samples and over 60% of nectar samples. Analysis of the pollen and nectar revealed residual amounts of imidacloprid ranging from 1.61 to 64.58 ng g-1 in the pollen and from not detected (ND) to 1.769 ng g-1 in the nectar. By comparison, the thiamethoxam concentrations in pollen and nectar ranged from ND to 14.521 ng g-1 and from ND to 4.285 ng g-1, respectively. The results of this study provide information on the transfer of two NIs from seed treatment to areas of the plant and provides an understanding of the potential exposure of the bee and other pollinators to systemic insecticides.

Baseline Sensitivity and Control Efficacy of Tetramycin Against Phytophthora capsici Isolates in China.

Tetramycin is a new biopesticide that combines high-level and broad-spectrum fungicidal activity, low toxicity, and environmental safety. In this study, 90 Phytophthora capsici isolates obtained from various regions in southern China were characterized for their baseline sensitivity to tetramycin. The protective and curative activities of tetramycin against P. capsici were determined on leaves of pepper, and the control efficacy of tetramycin in greenhouse experiments was also determined. Compared with mycelial growth, the formation of sporangia and the discharge of zoospores were inhibited by lower concentrations of tetramycin, approximately 5 µg ml-1 on V8 media. The frequency distribution curves for the tetramycin sensitivity were unimodal, with mean values for the fungicide concentration that reduced mycelial growth, sporangia formation, and zoospore discharge by 50% compared with the control of 1.18 ± 0.91, 0.64 ± 0.42, and 0.63 ± 0.30 µg ml-1, respectively. In addition, no correlation was observed between tetramycin and other fungicides tested, including mandipropamid, azoxystrobin, mefenoxam, fluazinam, fluopicolide, and famoxadone. Tetramycin exhibited both protective and curative effects against P. capsici in vitro, and its protective activity was better than its curative activity. In greenhouse experiments, tetramycin concentration of 60 and 90 µg ml-1 provided a protective control efficacy of 47.1 to 56.4% and curative efficacy of 43.3 to 52.7%. These results demonstrated that tetramycin could serve as an excellent alternative fungicide to control Phytophthora blight of pepper.

Activity, Translocation, and Persistence of Isopyrazam for Controlling Cucumber Powdery Mildew.

A cotyledon bioassay was conducted to assess the activity of isopyrazam against Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff, causal agent of cucumber powdery mildew. Results showed that isopyrazam has protective and curative activity against P. xanthii, with EC50 values of 0.04 and 0.05 mg liter-1, respectively. These activities are higher than those for hexaconazole, difenoconazole, pyraclostrobin, kresoxim-methyl, and azoxystrobin, fungicides currently used against cucumber powdery mildew. Isopyrazam at 0.5 mg liter-1 damaged conidiophores. Results of inoculation tests in greenhouse pots indicate that isopyrazam demonstrates a level of systemic movement in cucumber plants, especially regarding translaminar and transverse translocation. Efficacy following translaminar and transverse translocations on cotyledons and leaves treated with 60 mg liter-1 was 94.40% and 88.96%, and 95.26% and 82.83%, respectively. In addition, isopyrazam at 60 mg liter-1 exhibited a long duration of efficacy against cucumber powdery mildew, almost 2 to 3 weeks longer than that of triazoles and strobilurins. Similar trends in residual durations were observed during 2014 and 2015 greenhouse trials. Isopyrazam at 30 and 60 a.i. g ha-1 provided efficacy ranging from 83.27 to 90.83% 20 days following treatment. In conclusion, isopyrazam has translaminar and transverse translocation in cucumber leaves, and long duration of activity against cucumber powdery mildew.