From Water to Water: Insight into the Translocation of Pesticides from Plant Rhizosphere Solution to Leaf Guttation and the Associated Ecological Risks.

Plant guttation is an important source of water/nutrients for many beneficial insects, while the presence of pesticides in guttation has been considered as a new exposure route for nontarget insects. This study aimed to elucidate how 15 diverse pesticides are translocated from growth media to guttation by maize plants through a hydroponic experiment. All pesticides were effectively translocated from the growth solution to maize guttation and reached a steady state within 5 days. The strong positive correlation (R2 = 0.43-0.84) between the concentrations of pesticides in guttation and in xylem sap demonstrated that xylem sap was a major source of pesticides in guttation. The relationship between the bioaccumulation of pesticides in guttation (BCFguttation) and the chemical Kow was split into two distinct patterns: for pesticides with log Kow > 3, we identified a good negative linear correlation between log BCFguttation and log Kow (R2 = 0.71); however, for pesticides with log Kow < 3, all data fall close to a horizontal line of BCFguttation ≅ 1, indicating that hydrophilic pesticides can easily pass through the plants from rhizosphere solution to leaf guttation and reach saturation status. Besides, after feeding with pesticide-contaminated guttation, the mortality of honeybees was significantly impacted, even at very low levels (e.g., ∑600 µg/L with a mortality of 93%). Our results provide essential information for predicting the contamination of plant guttation with pesticides and associated ecological risks.

Pesticides in Greenhouse Airborne Particulate Matter: Occurrence, Distribution, Transformation Products, and Potential Human Exposure Risks.

Pesticides are frequently sprayed in greenhouses to ensure crop yields, where airborne particulate matter (PM) may serve as a carrier in depositing and transporting pesticides. However, little is known about the occurrence and fate of PM-borne pesticides in greenhouses. Herein, we examined the distribution, dissipation, and transformation of six commonly used pesticides (imidacloprid, acetamiprid, prochloraz, triadimefon, hexaconazole, and tebuconazole) in greenhouse PM (PM1, PM2.5, and PM10) after application as well as the associated human exposure risks via inhalation. During 35 days of experiment, the six pesticides were detected in all PM samples, and exhibited size- and time-dependent distribution characteristics, with the majority of them (>64.6%) accumulated in PM1. About 1.0-16.4% of initially measured pesticides in PM remained after 35 days, and a total of 12 major transformation products were elucidated, with six of them newly identified. The inhalation of PM could be an important route of human exposure to pesticides in the greenhouse, where the estimated average daily human inhalation dose (ADDinh) of the six individual pesticides was 2.1-1.2 × 104 pg/kg day-1 after application (1-35 days). Our findings highlight the occurrence of pesticides/transformation products in greenhouse PM, and their potential inhalation risks should be further concerned.

Greenhouse cultivation enhances pesticide bioaccumulation in cowpeas following repeated spraying.

Understanding pesticide residue patterns in crops is important for ensuring human health. However, data on residue accumulation and distribution in cowpeas grown in the greenhouse and open field are lacking. Our results suggest that acetamiprid, chlorantraniliprole, cyromazine, and thiamethoxam residues in greenhouse cowpeas were 1.03-15.32 times higher than those in open field cowpeas. Moreover, repeated spraying contributed to the accumulation of pesticide residues in cowpeas. Clothianidin, a thiamethoxam metabolite, was detected at 1.04-86.00 µg/kg in cowpeas. Pesticide residues in old cowpeas were higher than those in tender cowpeas, and the lower half of the plants had higher pesticide residues than did the upper half. Moreover, pesticide residues differed between the upper and lower halves of the same cowpea pod. Chronic and acute dietary risk assessments indicated that the human health risk was within acceptable levels of cowpea consumption. Given their high residue levels and potential accumulation, pesticides in cowpeas should be continuously assessed.

Optimizing Analysis Methods: Rapid and Accurate Determination of Cuaminosulfate Residues with LC-MS/MS Based on Box-Behnken Design Study.

In view of the defects in the previous detection of cuaminosulfate, which only focused on the analysis of copper ions, there is currently no analysis method available to determine the actual state of cuaminosulfate as chelated or bound. In order to investigate the dissipation and terminal residues in soil and watermelon of cuaminosulfate for food safety and environmental risk, a highly effective technique was developed to detect cuaminosulfate residues in watermelon and soil, and field experiments were conducted in China. After single-factor experiments, residual cuaminosulfate in samples was extracted by pure water, purified using a liquid-liquid approach combined with a dispersive solid-phase extraction, and detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). The Box-Behnken design (BBD) study was used to find the optimal solutions for the time of liquid-liquid purification, the amount of extraction solvent, and the amounts of cleanup sorbents for the analytical method. The average recovery of the method was in the range of 80.0% to 101.1%, the average relative standard deviation (RSD) was 5.3-9.9%, and the detection limit was lower than 0.05 mg/kg. The BBD study not only improved the extraction rate of the method, but also saved time and was operated easily. The final residues of cuaminosulfate in watermelon at different sampling intervals were all lower than 0.05 mg/kg under field conditions. The cuaminosulfate in soils dissipated following exponential kinetics, with half-life values in the range of 9.39 to 12.58 days, which varied by different locations. Based on the validated method, food safety residues and soil residues can be determined rapidly and accurately.

Dissipation dynamics and comparative dietary exposure assessment of mefentrifluconazole in rice.

A fast and sensitive analytical method based on UHPLC coupled with tandem mass spectrometry was established to investigate the dissipation and final residual amounts of mefentrifluconazole in rice, and dietary risk to consumers was evaluated. The method provided good linearity (R2 ≥ 0.9979), accuracy (recovery range, 79.0-101.5%), precision (relative standard deviation range, 1.3-13.9%), and sensitivity (limit of quantification, 0.005 mg/kg). The dissipation dynamics of mefentrifluconazole in rice followed first-order kinetics, with half-lives of 2.8-16.6 days. The final residues of mefentrifluconazole in various samples of harvested brown rice ranged from less than the limit of quantification to 0.092 mg/kg, the latter value being higher than the maximum residue limit recommended by the European Union. Comparative dietary exposure of mefentrifluconazole was assessed using field data and Chinese dietary patterns for different genders, regions, and age data. Although the results showed acceptable levels of risk for both acute exposure (the percentage of the acute reference dose ≤ 0.7483%) and chronic dietary intake (the percentage of acceptable daily intake ≤ 31.8516%), more studies of children are needed because they are at higher risk than other groups. This work provides the necessary data for registering and establishing the maximum residue limit for mefentrifluconazole in rice in China and reveals the potential risks to different groups of long-term application of mefentrifluconazole to rice and other crops.

Catalytic degradation of dimethomorph by nitrogen-doped rice husk biochar.

N-doped biochar is widely used for activating persulfate to degrade organic pollutants. Which type of N atom is the key factor for activation is still unclear and needs to be further explored and analyzed. In this study, four kinds of biochar were prepared using urea and rice husk as precursors, and tested for the catalytic degradation of dimethomorph. Increasing the nitrogen doping level caused the catalytic removal efficiency of dimethomorph in the presence of peroxymonosulfate increased from 16.6% to 86.8%. A correlation analysis showed that the ability of N-doped biochar to activate PMS is mainly related to the content of pyrrole N, graphite N and carbonyl and the degree of defects. In experiments on electron paramagnetic resonance and free radical suppression, the reactive species of SO4•-, 1O2,·OH and O2.- were detected, among which 1O2 was found to be the main agent in the nonradical pathway. The degradation pathways for dimethomorph were analyzed based on a total of 8 degradation products identified by high-performance liquid chromatography-time of flight mass spectrometry (HPLC-Q-TOFMS). The results of this study provide a fundamental basis for using agricultural waste to produce inexpensive and efficient nonmetal catalysts that are highly effective in reducing dimethomorph levels in agricultural lands.

Spontaneous In-Source Fragmentation Reaction Mechanism and Highly Sensitive Analysis of Dicofol by Electrospray Ionization Mass Spectrometry.

Although dicofol has been widely banned all over the world as a kind of organochlorine contaminant, it still exists in the environment. This study developed a high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) detection technique for dicofol, an environmental pollutant, for the first time using in-source fragmentation. The results confirmed that m/z 251 was the only precursor ion of dicofol after in-source fragmentation, and m/z 139 and m/z 111 were reasonable product ions. The main factors triggering the in-source fragmentation were the H+ content and solution conductivity when dicofol entered the mass spectrometer. Density functional theory can be used to analyze and interpret the mechanism of dicofol fragmentation reaction in ESI source. Dicofol reduced the molecular energy from 8.8 ± 0.05 kcal/mol to 1.0 ± 0.05 kcal/mol, indicating that the internal energy release from high to low was the key driving force of in-source fragmentation. A method based on HPLC-MS/MS was developed to analyze dicofol residues in environmental water. The LOQ was 0.1 µg/L, which was better than the previous GC or GC-MS methods. This study not only proposed an HPLC-MS/MS analysis method for dicofol for the first time but also explained the in-source fragmentation mechanism of compounds in ESI source, which has positive significance for the study of compounds with unconventional mass spectrometry behavior in the field of organic pollutant analysis and metabonomics.

Highly-Selective Analytical Strategy for 90 Pesticides and Metabolites Residues in Fish and Shrimp Samples.

The analysis of pesticide residues in aquatic products is challenging due to low residue levels and the complex matrix interference. In this study, we developed a simple, fast method for the trace analysis of 90 pesticides and metabolites in aquatic products. The analytes covered a wide polarity range with log Kow (log octanol-water partition coefficient) ranging from -1.2 to 6.37. Grass carp (Ctenopharyngodon idellus) and prawn (Penaeus chinensis) samples were chosen to validate the quantification method. The samples were extracted by 0.2% formic-acetonitrile, cleaned by solid-phase extraction (PRiME HLB), and analyzed by high performance liquid chromatography-tandem mass spectrometry. The results showed good linearities for the analytes and were observed in the range of 0.05-50 µg/L. The recoveries of the method were within 50.4-118.6%, with the relative standard deviations being lower than 20%. The limits of quantifications (LOQs) of the method were in the range of 0.05-5.0 µg/kg, which were superior to values compared with other research. The developed method was applied to detect pesticide residues in prawn samples from eastern coastal areas of China. Three herbicide residues of diuron, prometryn, and atrazine were detected in prawn samples. The method was sensitive and efficient, which is of significance in expanding the screening scope and improving the quantitative analysis efficiency in aquatic products.

Occurrence and impact of carbendazim and hymexazol residues on yeast growth and ochratoxin A contamination during wine production.

BACKGROUND: Grapes are highly vulnerable to infection by carbon black aspergilli, which produce ochratoxin A (OTA), a mycotoxin. Carbendazim and hymexazol are widely applied to control grape diseases. Howerver, fungicides, toxigenic fungi, and OTA can be transferred from grapes to wine causing potential safety issues. The impact of these residues on fungal populations and OTA during vinification are currently unclear. Here we investigated the effects of carbendazim and hymexazol on the viability of Aspergillus carbonarius and OTA contamination during an indoor wine-processing experiment. RESULTS: The population size of A. carbonarius substantially increased at 24 h followed by a significantly decreased at 72 h after destemming and crushing. However, carbendazim and hymexazol notably inhibited the growth of A. carbonarius in must samples. In addition, yeast growth was substantially deleyed by carbendazim, hymexazol, and OTA during the first 3 days in compared with the control. Carbendazim, hymexazol, and OTA residues declined over time, and the processing factors (PFs) for carbendazim and hymexazol throughout vinification were 0.164, 0.074, and 0.185-0.476, respectively. Carbendazim and hymexazol each reduced OTA concentrations. However, there was no significant difference after 48 h. Addition of carbendazim or hymexazol significantly reduced the level of A. carbonarius but had no significant effect on the final concentration of OTA in mature wine. CONCLUSION: The wine-making process can reduce the residues of OTA, carbendazim, and hymexazol in grapes, but it is recommended that grapes chosen to make wine should be free of A. carbonarius contamination. © 2023 Society of Chemical Industry.

Trace determination of imidacloprid and its major metabolites in wheat-soil system.

Trace analysis method is a reliable basis for studying the translocation and metabolism of imidacloprid used as an insecticide in wheat, and it clarifies whether biologically active metabolites including residual imidacloprid, have long-lasting insecticidal potency against wheat aphids under seed treatment during the entire growth period. In this study, a highly sensitive analytical method was established to determine the residues of imidacloprid and its six metabolites (5-hydroxy imidacloprid, imidacloprid olefin, imidacloprid guanidine, imidacloprid urea, 6-chloronicotinic acid, and imidacloprid nitrosimine) in wheat-soil systems, such as in wheat leaves, wheat ears, wheat grains, roots, and soil. All the compounds were extracted using an ACN:water (8:2, v/v) mixture and purified by dispersive solid-phase extraction. The average recoveries ranged from 74.4% to 109.5% for all matrices, with intra- and inter-day variations of less than 14.9%. The limit of quantitation was in the range of 0.001-0.005 mg/kg. The method is demonstrated to be sensitive and accurate for monitoring imidacloprid and its metabolites at trace levels during the entire growth period under field conditions.

First report of leaf blight disease of Livistona chinensis caused by Alternaria alternata in Pakistan.

Livistona chinensis (Jacq.) is also known as fan palm and is commonly grown in the subtropical region of the world. This plant is widely cultivated in Asia for ornamental purpose and also used in Chinese medicines (Li et al. 2019). In May 2021, severe leaf blight was observed on L. chinensis leaves in ornamental plant nurseries, located at Pattoki (30°59'41.5"N 73°48'43.8"E) District Kasur, Punjab province, Pakistan. The disease incidence was up to 50% and the initial symptoms appeared as chlorotic brown spots on the upper portion of leaves. Later, the spots expanded and changed into elliptical lesions on the leaves. The lesions with dark brown margins coalesced to cause extensive tissue necrosis of leaves and exhibited blight (Figure 1). Two to three leaves were taken from each infected plant. Infected leaves of each sample of L. chinensis were excised into small pieces (3-4 mm) with the help of sterilized scissor and surface disinfected with 1% NaClO for 20s and rinsed 3 times with sterilized distilled water. To isolate the potential causal organism, these pieces were plated on Potato Dextrose Agar (PDA) medium and incubated at 28 °C with 70 % relative humidity for 7 days. Purified cultures were obtained through single spore culture on PDA. All obtained isolates were preserved in 30% glycerol at -80°C. The fungal colony colour was olive to dark greenish and dark brown to black on the reverse side. The conidia (n=36 per isolate) were greenish to brown in colour, ellipsoid to obclavate, ovoid, irregular and measured an average range from 10.9 to 30.7 µm long x 6.3 to 12.5 µm wide with 2 to 5 transverse and 0 to 3 longitudinal septa (Figure 2). The genomic DNA was extracted from all isolates (n=40) and multi-locus sequence analysis approach was used for molecular identification. The Internal Transcribed Spacers (ITS) region, Alt a1 major allergen (ALT) gene, glyceraldehyde-3-phosphate dehydrogenase (GPD), actin (ACT) gene and histone 3 genes were amplified using ITS1/4 (White et al. 1990), Alt-4for/Alt-4rev (Lawrence et al. 2013), GPD1/GPD2 (Guerber et al. 2003), ACT512F/ACT783R (Carbone and Kohn, 1999) and H3-1a/H3-1b (Luan et al. 2007). Based on morphological and molecular characteristics, all isolates were identified as Alternaria alternata. The sequences of the representative isolate APLB-3 were submitted in the GenBank with the accession numbers (ITS: MZ663802), (ALT: MZ666883), (ACT: MZ666885), (GPD: MZ666884), and (Histone3: MZ666886) showing 100% similarity with ITS accession MK968038, ALT accession MN702781, ACT accession MT318253, GPD accession MT524743 and histone 3 accession MH824369. For pathogenicity test, potted L. chinensis plants (n=9) leaves were pin-pricked with sterilized needle (Bajwa et al. 2010) and inoculated with spore suspensions (107 spore/ml) of APLB-3 (1ml/leaf) to confirm Koch's postulates. After 14 days, the inoculated leaves showed chlorotic brown spots and leaf blight symptoms similar to those observed on infected plants in nurseries. The plants grown as the control group (n=9) were sprayed with sterilized distilled water and had no symptoms (Figure 3). The experiment was performed three times. The fungal pathogen was re-isolated from the artificial inoculated leaf tissues and identified as A. alternata based on morphological and molecular characterization. To our knowledge, this is the first record of A. alternata causing leaf blight disease of L. chinensis in Pakistan. This disease may potentially decrease the value of ornamental plants in Pakistan under favourable conditions and proper management strategies should be applied.

First record of Leaf Spot of Ficus religiosa Caused by Alternaria alternata in Pakistan.

Ficus religiosa (L.) belongs to the family Moraceae, native to India and commonly known as 'Peepal'. It has high medicinal value due to its antibacterial, antiviral and antioxidant properties (Singh et al., 2015; Kalpana et al., 2009). In August 2021, leaf spots were observed on F. religiosa trees in Pabbi forest park Kharian (32°50'01.4"N 73°50'17.7"E), District Gujrat, Pakistan. The disease incidence was recorded approximately 30%. The leaf spots were irregular in shape, brown in colour, 3-9 mm in size and encircled by yellowish halo. In severe condition, the spots coalesced and produced necrotic areas on leaf surface (Figure 1). The samples (n=21) were collected based on symptoms and infected leaf segments were excised into small pieces, surface disinfected with 1% NaClO for 20s and rinsed 3 times with sterilized distilled water. The pieces were plated on Potato Dextrose Agar (PDA) medium and incubated at 28°C for 7 days. All the pure cultures were obtained through single spore method on PDA and preserved in 30% glycerol at -80°C. The colonies were olive green to dark brown with white margin and later turned dark olive or black with enormous sporulation. Conidia (n=24) were obclavate, ovoid, brown in colour and measuring 10.2 to 34.1 µm long x 5.9 to 12.3 µm wide with 1 to 6 transverse and 1 to 3 longitudinal septa (Figure 2). Based on these characteristics, the pathogen was identified as Alternaria alternata (Gilardi, G., et al. 2019). For molecular identification, the Internal Transcribed Spacers (ITS) region, endopolygalacturonase (endoPG) gene and major allergen (Alt a1) gene were amplified using ITS1/4 (White et al. 1990), PG3/PG2b (Andrew et al. 2009) and Alt-4for/Alt-4rev (Lawrence et al. 2013) primers respectively. Based on molecular characteristics, all isolates were identified as A. alternata. The sequences of the representative isolate FLB-1 were submitted in the GenBank with the accession numbers OL514181 for ITS, OK315658 for endoPG and OK315659 for Alt al showing 100% similarity with ITS accession KP124298, and endoPG accession AY205020 and 99.7% with Alt al accession KP123847 sequences of CBS106.24 A. alternata after BLASTn queries. The Phylogenetic reconstruction based on maximum likelihood, using Mega X (Kumar et al. 2018) and FLB-1 grouped with A. alternata (Figure 3). Pathogenicity test was performed on nine months old healthy F. religiosa (L.) seedlings (n=12) to fulfil the Koch's postulates. The leaves were pinpricked and sprayed with FLB-1 conidial suspension (107 spores/ml) by using atomizer (Bajwa et al., 2010). The leaves of F. religiosa (L.) seedlings (n=12) sprayed with sterilized distilled water served as control. All the seedlings were incubated at 25 ± 3°C in the glasshouse. The experiment was performed three times under the same conditions. The typical symptoms appeared on inoculated leaves after 7-14 days that were similar to the symptoms observed on original infected F. religiosa (L.) trees. In the control treatment leaves remained asymptomatic (Figure 4). The pathogen from the artificial infected leaves was re-isolated and identified as A. alternata based on morphological and molecular characteristics. To our knowledge, this is the first report of leaf spot of F. religiosa (L.) caused by A. alternata in Pakistan. The leaves of F. religiosa (L.) are commonly used in Asia for different purposes and this leaf spot disease may represent a significant threat to F. religiosa (L.) tree health.

First Report of Bacterial Leaf Spot of Ficus benghalensis Caused by Pseudomonas cichorii in Pakistan.

Ficus benghalensis L. belongs to the family Moraceae, native to Asia and commonly known as Banyan. It has been identified as an important medicinal tree due to its antioxidant, anti-diabetic, and anti-inflammatory properties (Singh et al., 2009; Thite et al., 2014). In March 2021, leaf spots were observed on Banyan trees in the Kharian forest zone, District Gujrat, Punjab Province, Pakistan. Initial symptoms on leaves were irregular, water-soaked, and light brown lesions. The lesions turned dark brown at the centre, and the margins gradually turned yellow. The average size of lesions was 12 to 20 × 8 to 13 mm. The lesions coalesced and produced necrotic areas on the leaf (Figure 1). Samples (n=34) were collected based on symptoms and infected leaf segments were excised into small pieces (10-20 mm), surface disinfected with 1% NaClO for 10 seconds and rinsed three times with sterilized distilled water (SDW). Ten pieces/sample were mashed and soaked in 1.5 ml SDW to obtain a suspension. Later, 10 µL of the suspension was streaked on Nutrient agar (NA) and King's B medium (KBM) and incubated for 72 h at 30°C. After 72 h bacterial colonies appeared on NA and KBM medium. Each colony was re-streaked for three times to obtain the purified colonies. Morphological and biochemical characteristics of isolated bacterial cultures were performed by following the method of Schaad et al. (2001). Bacterial colonies appeared pale yellow to creamy, smooth, and circular with undulated margins on both NA and KBM medium. The colonies produced a fluorescent blue colour on KBM under the UV light. Isolated bacterial cultures were positive for oxidase, negative for levan production and arginine dihydrolase. Based on these characteristics, the pathogen was identified as Pseudomonas species. For molecular identification, 16S rRNA and rpoB genes were amplified and sequenced using the following primers: 27F/1492R (Lane, 1991) and LAPS/LAPS27 (Ait Tayeb et al. 2005), respectively. All the isolates were identified as P. cichorii after BLASTn analysis. The sequences of isolate BLS-01 obtained in this study were deposited in GenBank with accession No. OK397593 for 16S rRNA and OK423684 for rpoB exhibiting 100 % similarity with P. cichorii Accession No. MK356431 for 16S rRNA and JQ267563 for rpoB. A pathogenicity test was performed on healthy Banyan seedlings to fulfil Koch's postulates. Leaves from seedling plants were inoculated with 3 mL of BLS-01 suspension (108 CFU/ml) by spraying the inoculum on leaves using a sterilized spray bottle. The leaves sprayed with sterilized distilled water served as control (Figure 2). The experiment was performed three times following the same protocol as described above. Symptoms that appeared on inoculated leaves after 7-10 days were similar to the symptoms observed on original infected Banyan tree leaves in the forest zone. Control leaves remained asymptomatic during the whole experiment. The pathogen from the artificial infected leaves was re-isolated and identified as P. cichorii based on morphological, biochemical, and molecular characteristics. To our knowledge, this is the first report of leaf spot of F. benghalensis caused by P. cichorii in Pakistan.

Residual levels of five pesticides in peanut oil processing and chips frying.

BACKGROUND: Pesticide contamination in oil crops and processed products is an important food safety concern. The study was aimed to investigate the pesticide residue changes in press processing of peanut oil and frying of chips. RESULTS: Five pesticides - chlorpyrifos, deltamethrin, methoxyfenozide, azoxystrobin and propargite - which are often applied during growth period in peanut plants, were selected to investigate their residue changes in cold press processing of peanut oil and frying of potato chips. Results showed that the residues of the five pesticides were decreased by 3.1-42.6% during air-drying before oil pressing. The residues of chlorpyrifos, deltamethrin, methoxyfenozide and propargite in peanut oil were 2.05-3.63 times higher than that in peanut meal after cold pressing of the oil, except for azoxystrobin having a slightly lower residue in peanut oil, with 0.92 times that in peanut meal. The processing factors of the five pesticides in peanut oil ranged from 1.17 to 2.73 and were highly related to the log Kow of the pesticides. The higher the log Kow , the more easily was the pesticide partitioned in the peanut oil. Besides, as frying time increase during preparation of chips, the concentration of pesticides in peanut oil decreased gradually by 6.7-22.1% compared to the first frying. In addition, 0.47-11.06% of the pesticides were transferred to the chips through frying with contaminated oil. CONCLUSION: This is first report showing that pesticides can transfer from contaminated oil to chips. There exists a potential dietary health risk by using pesticide-contaminated oil for frying chips. This work could provide basic data for accurate dietary risk assessment of pesticide residues in peanut oil and its frying products. © 2021 Society of Chemical Industry.

Thifluzamide exposure induced neuro-endocrine disrupting effects in zebrafish (Danio rerio).

Thifluzamide is widely used fungicide and frequently detected in aquatic system. In this study, the toxicity of fungicide thifluzamide to non-targeted aquatic organisms was investigated for neuroendocrine disruption potentials. Here, zebrafish embryos were exposed to a series of concentrations of thifluzamide for 6 days. The results showed that both the development of embryos/larvae and the behavior of hatched larvae were significantly affected by thifluzamide. Importantly, the decreased activity of acetylcholinesterase (AchE) and the increased contents of neurotransmitters such as serotonin (5-HT) and norepinephrine (NE), along with transcriptional changes of nervous system related genes were observed following 4 days exposure to thifluzamide. Besides, the decreased contents of triiodothyronine (T3) and thyroxine (T4) in whole body, as well as significant expression alteration in hypothalamic-pituitary-thyroid (HPT) axis associated genes were discovered in zebrafish embryos after 4 days of exposure to thifluzamide. Our results clearly demonstrated that zebrafish embryos exposed to thifluzamide could disrupt neuroendocrine, compromise behavior and induce developmental abnormality, suggesting impact of this fungicide on developmental programming in zebrafish.

Quantitative determination of pyriproxyfen and its metabolite residues in bee products of China using a modified QuEChERS approach with UPLC-MS/MS.

Given the key role of bees as indicators for environmental assessment, residues in bees and bee products have attracted great interest. In this regard, an improved, highly sensitive method for quantifying the insecticide pyriproxyfen and its four metabolites (4'-OH-Pyr, DPH-Pyr, 2-OH-PY, 4'-OH-POP) in honeybees, larvae, and bee products (honey, pollen, royal jelly and wax) should be established. For this purpose, we used ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry for rapid quantification (≤5 min). Recoveries for various matrices ranged from 73.77% to 114.97%, with satisfactory intra-day and inter-day precision (relative standard deviations of 0.03-8.61% and 0.10-7.25%, respectively). The results demonstrated excellent linearity (R2 > 0.9903) with a limit of quantification of 1 µg/kg for six different matrices. We collected and analyzed 597 samples (honey, bees and wax) from four major beekeeping areas in China. Only 47 of these samples contained residues of pyriproxyfen and two of its metabolites (2-OH-PY, 4'-OH-Pyr), and high levels of contamination were found in bee samples (2-739 µg/kg), with substantive accumulation in wax (levels were 9.49% higher than in other samples). The result demonstrate that the method provides a reliable and convenient means of monitoring pyriproxyfen and its metabolites in bee products for better product quality, human health, and international commercial competition and also lays a foundation for risk assessment of potential pyriproxyfen contamination in China.

First Report of Postharvest Stem End Rot of mango fruit (Mangifera indica) caused by Lasiodiplodia theobromae in China.

China is the second largest producer of mango in the world, a fruit has high nutritive value and a rich source of fiber (Kuhn et al., 2017). In late June 2019, a postharvest stem-end rot disease was observed in different local fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district of Beijing, China. Black rot symptomatic lesions were observed on the fruit surface which initially started from the stem end of the mango fruit (Fig. 1). Approximately 45 % of mango fruits were affected with the disease. Symptomatic portions from collected fruit samples (n=40) were cut into small pieces (2mm2), rinsed with 1% NaClO for 20s and then washed three times with sterilized distilled water (SDW) for surface disinfection. The disinfected pieces were then placed on sterilized filter paper for drying. Later, these pieces were placed on Potato Dextrose Agar (PDA) plates and incubated at 28°C for seven days. The resulting fungal colonies were purified by the single spore isolation technique. The isolated fungal colonies were initially greenish to gray in color, later turning olive-black to black. Conidia were dark brown in color, oval-shaped, two-celled and measured 22.4 to 25.7 (24.06 ± 0.15) µm in length and 10.2 to 12.8 (11.3 ± 0.13) µm in width (n=36). Based on the symptoms, culture morphology and microscopic characters, Lasiodiplodia theobromae was suspected as the causal agent, and similar results were reported by Pavlic et al., 2004 and Burgess et al., 2006. For molecular identification, a multi-locus sequence analysis approach was used. The Internal Transcribed Spacers (ITS) region, elongation factor 1 alpha (EF1-α) and ß-tubulin genes were amplified and sequenced using ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), and Bt2a/Bt2b (Glass and Donaldson, 1995) primers respectively. The sequences of isolate MFT9 were deposited to GenBank (MW115977 (ITS), (MW118595 (EF1-α) and MW118596 (ß-tubulin). All sequences showed more than 99.5% similarity with reported sequences of Lasiodiplodia theobromae isolate IBL340 with accessions numbers KT247466 (ITS), KT247472 (EF1-α) and KT247475 (ß-tubulin). Phylogenetic reconstruction based on Maximum Likelihood, using Mega X (Kumar et al., 2018), grouped isolate MFT9 with isolates representing L. theobromae. Pathogenicity testing was performed on 18 fresh, healthy, medium-sized mango fruits for each treatment to fulfill Koch's postulate. The fruits were disinfested with 1% NaClO and punctured with a sterilized needle to create approximately 2mm2 wounds for inoculation. Fruits were inoculated with 15µL of fresh inoculum (107 spores/mL) from isolate MFT9. Control fruits were inoculated with 15µL of SDW and both the inoculated and control fruits were incubated at 28°C for seven days of post inoculation. The rot lesions appeared at the point of inoculation and gradually spread on the fruit surface. The symptoms were similar to the symptoms observed on the original fruit samples (Fig. 2). This experiment was conducted three times under the same conditions, with control fruits remaining asymptomatic each time. The re-isolated fungus was identified as L. theobromae based on symptoms and morpho-molecular analysis, described above. L. theobromae is also reported as a causal agent responsible for a postharvest stem-end rot on Coconut in China (Zhang, et al., 2019). To our knowledge, this is the first report of L. theobromae causing postharvest stem-end rot of mango fruit in China. This finding suggests that L. theobromae is a potential problem for mango fruit production in China.

First record of Colletotrichum alienum Causing postharvest Anthracnose disease of mango fruit in China.

Mango (Mangifera indica L.) is one of the world's most significant economic fruit crops, and China is the second-largest producer of mango (Kuhn et al., 2017). Postharvest mango anthracnose is caused by Colletotrichum species and reduce the self-life of mature fruit (Wu et al., 2020). Colletotrichum species also cause postharvest anthracnose and fruit rot disease of Apple, Banana and Avocado (Khodadadi et al., 2020; Vieira et al., 2017; Sharma et al., 2017). In July 2019, mango fruits cv. 'Jin-Hwang' were observed at different fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district, Beijing, China, exhibiting typical symptoms of anthracnose including brown to black lesions in different size (≤ 2 cm) with identified border on the mango fruit surface. Later, the lesions were coalesced and extensively cover the surface area of the fruit. The lesions were also restricted to peel the fruit and pathogen invaded in the fruit pulp. About 30% of mango fruits were affected by anthracnose disease. The margins of lesions from infected mango fruits (n=56) were cut into 2 × 2 mm pieces, surface disinfected with NaClO (2% v/v) for 30 s, rinsed thrice with distilled water for 60s. These pieces were placed on PDA medium and incubated at 25°C for 7 days. Pure culture of fungal isolates was obtained by single spore isolation technique. Initially, the fungal colony was off white, and colony extended with time, turning light gray at the center. The morphological examination revealed that conidia were hyaline, oblong, and unicellular. The conidia were measured from 10 days old culture and dimensions varied from 13.3 to 15.8 µm in length and 4.6 to 6.1 µm in width. For molecular identification, a multi-locus sequence analysis; the Internal Transcribed Spacers (ITS) region, partial actin (ACT) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and chitin synthase (CHS-1) gene were amplified by using the primer sets ITS1/4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), GDF1/GDR1 (Guerber et al. 2003) and CHS1-79F/CHS-1-354R (Carbone and Kohn 1999) respectively. The partial sequences of MTY21 were deposited to GenBank accessions (MT921666 (ITS), MT936119 (ACT), MT936120 (GAPDH) and MT936118 (CHS-1). All obtained sequences showed 100% similarity with reported sequences of Colletotrichum alienum ICMP.18691 with accessions numbers JX010217 (ITS), JX009580 (ACT), JX010018 (GAPDH) and JX009754 (CHS-1) which represented the isolate MTY21 identified as C. alienum by constructing Maximum Likelihood phylogenetic tree analysis using Mega X (Kumar et al., 2018). For the confirmation of Koch's postulates, the pathogenicity test was conducted on 36 fresh healthy mango fruits for each treatment. Fruits were punctured with the help of a sterilized needle to create 2mm2 wounds and inoculated with 10µL inoculum (107 spores/mL) of MTY21. Control mango fruits were inoculated with 10µL sterilized distilled water and incubated at 25 °C with 90% relative humidity. The lesions appeared at the point of inoculation and gradually spread on the fruit surface after 7 days post inoculation. The symptoms were similar to the symptoms on original fruit specimens. The re-isolated fungus was identified as C. alienum based on morphological and molecular analysis. Mango anthracnose disease caused by several Colletotrichum species has been reported previously on mango in China (Li et al., 2019). Liu et al. (2020) reported C. alienum as the causal organism of anthracnose disease on Aquilaria sinensis in China. C. alienum has been previously reported causing mango anthracnose disease in Mexico (Tovar-Pedraza et al., 2020) To our knowledge, this is the first report of C. alienum causing postharvest anthracnose of mango in China. The prevalence of C. alienum was 30% on mango fruit which reflects the importance of this pathogen as a potential problem of mango fruit in China.

Monitoring the behavior of imazalil and its metabolite in grapes, apples, and the processing of fruit wine at enantiomeric level.

BACKGROUND: Imazalil is widely used in agriculture, which may pose a threat to food safety. This study aimed to investigate the fate of imazalil and its main metabolite, R14821 (imazalil-M), in field grapes and apples, and in the processing of fruit wine at the enantiomeric level. RESULTS: Analysis method was established to determine imazalil and imazalil-M enantiomers in grape, apple, fruit wine and pomace. The method showed acceptable recoveries of 71.6-99.9% and precision with relative standard deviation of 0.3-11.7%. Processing factors (PFs) were 0.15-0.40 (for imazalil enantiomers) and <0.13-0.83 (for imazalil-M enantiomers) during the wine-making process. The PFs after individual steps including washing, peeling, fermentation, and clarification were all less than 1. No enantioselective dissipation of imazalil was found in grapes under field conditions with half-lives of 23.82-24.49 days. R-(-)-imazalil degraded slightly faster than S-(+)-imazalil in apples under field conditions with half-lives of 9.82-10.09 days. S-(+)-imazalil-M preferentially degraded in field grapes and apple. No significant enantioselectivity of imazalil and imazalil-M was observed during the wine-making process. The enantiomeric fraction (EF) values of imazalil were 0.484-0.511 and 0.509-0.522 in grape wine and cider, respectively. The EFs were 0.484-0.501(in grape wine) and 0.484-0.504 (in cider) for imazalil-M. CONCLUSION: The results showed that the wine-making process could reduce imazalil and imazalil-M residues in grapes and apples. The finding of non-enantioselectivity of imazalil during the processing of fruit wine was useful for accurate risk assessment for imazalil in raw and processing fruits. © 2021 Society of Chemical Industry.

Preparation and Biological Property Evaluation of Novel Cationic Lipid-Based Liposomes for Efficient Gene Delivery.

Novel cationic lipid-based liposomes prepared using an amphiphilic cationic lipid material, N,N-dimethyl-(N',N'-di-stearoyl-1-ethyl)1,3-diaminopropane (DMSP), have been proposed to enhance the transfection of nucleic acids. Herein, we designed and investigated liposomes prepared using DMSP, soybean phosphatidylcholine, and cholesterol. This novel gene vector has high gene loading capabilities and excellent protection against nuclease degradation. An in vitro study showed that the liposomes had lower toxicity and superior cellular uptake and transfection efficiency compared with Lipofectamine 2000. An endosomal escape study revealed that the liposomes demonstrated high endosomal escape and released their genetic payload in the cytoplasm efficiently. Mechanistic studies indicated that the liposome/nucleic acid complexes entered cells through energy-dependent endocytosis that was mediated by fossa proteins. These results suggest that such cationic lipid-based liposome vectors have potential for clinical gene delivery.