Risk assessment for resistance to fludioxonil in Corynespora cassiicola in Liaoning China.

Cucumber corynespora leaf spot, caused by Corynespora cassiicola, is the primary disease of cucumber leaves in greenhouses in China. Fludioxonil is a phenylpyrrole fungicide that inhibits C. cassiicola growth. We studied the sensitivity of 170 isolates of C. cassiicola to fludioxonil and evaluated resistance risk. All of the isolates were sensitive to fludioxonil. The EC50 values ranged from 0.082 to 0.539 µg/mL with a mean of 0.207 ± 0.0053 µg/mL. Laboratory-created mutants with a high resistance factor to fludioxonil were genetically stable after 10 transfers and showed positive cross-resistance to iprodione and procymidone but not to azoxystrobin, carbendazim, pydiflumetofen, and prochloraz. There was no significant difference in mycelial growth and temperature adaptation between the mutant s and the sensitive isolates, except for pathogenicity and sporulation. The resistant isolates accumulated less glycerol than their parental isolates and were more sensitive to osmotic stress. The histidine kinase activity of the sensitive isolates was significantly inhibited compared to that of the resistant mutants. Sequence alignment of the histidine kinase gene CCos revealed that the mutants RTL4, RXM5, and RFS102 had point mutations at different sites that resulted in amino acid changes at G934E, S739F, and A825P in the CCos protein. The mutant RFS102 had an alanine deletion at site 824. After fludioxonil treatment, CCos expression by RFS20 was significantly lower than that of the parental isolate. Our findings demonstrate that C. cassiicola exhibits moderate resistance to fludioxonil.

Enhanced metabolic ability enabled wild panicgrass (Panicum miliaceum L. var. ruderale kit.) resistance to ALS inhibitor herbicide.

Wild panicgrass (Panicum miliaceum L. var. ruderale kit.) is an annual grass weed that primarily occurs in maize fields. Nicosulfuron is a widely used selective herbicide that effectively controls gramineous weeds in maize fields. However, owing to its long-term and extensive application, the control of P. miliaceum has been substantially reduced. The objective of this study was to determine the resistance pattern to ALS inhibitors in P. miliaceum and investigate the underlying resistance mechanisms. These are important for guiding the prevention and eradication of resistant weeds. Whole plant bioassays showed P. miliaceum had evolved high levels of resistance to nicosulfuron and multiple resistance to atrazine and mesotrione. The ALS gene sequence results indicated the absence of mutations in the resistant population. Additionally, there was no significant difference found in the inhibition rate of the ALS enzyme activity (I50) between the resistant and sensitive populations. Following the application of malathion the resistant P. miliaceum population became more sensitive to nicosulfuron. At 96 h after application of nicosulfuron, glutathione-S-transferase activity in the resistant population was significantly higher than that in the susceptible population. The study reveals that the main cause of resistance to ALS inhibitor herbicide in P. miliaceum is likely increased metabolism of herbicides. These findings may assist in devising effective strategies for preventing and eliminating resistant P. miliaceum.

Investigation of resistance mechanisms to fomesafen in Ipomoea nil from China.

Recently, the herbicide fomesafen has frequently failed to control the troublesome weed Ipomoea nil in soybean fields in Liaoning Province, China. Hence, we collected 10 suspected resistant populations and evaluated their sensitivity to fomesafen. The results revealed various degrees of Ipomoea nil resistance to fomesafen, with a resistance index of 2.88 to 22.43; the highest value occurred in the LN3 population. Therefore, the mechanisms of the resistance in LN3 to fomesafen were explored. After fomesafen treatment, the expression levels of InPPX1 and InPPX2 genes were 4.19- and 9.29-fold higher, respectively, in LN3 than those in the susceptible (LN1) population. However, mutations and copy number variations were not detected between the two populations. Additionally, malathion pretreatment reduced the dose necessary to halve the growth rate of LN3 by 58%. Liquid chromatography with tandem mass spectrometry demonstrated that metabolism of fomesafen was significantly suppressed by malathion. Moreover, LN3 displayed increased reactive oxygen species scavenging capacity, which was represented by higher superoxide dismutase and peroxidase activities after fomesafen application than those in LN1. An orthogonal partial least squares-discriminant analysis revealed that the high resistance in LN3 could be attributed mainly to enhanced metabolism. Fortunately, the fomesafen-resistant I. nil remained sensitive to 2,4-D-ethylhexylester and bentazon, providing methods for its control.

Sensitivity to 12 Fungicides and Resistance Mechanism to Trifloxystrobin, Carbendazim, and Succinate Dehydrogenase Inhibitors in Cucumber Corynespora Leaf Spot (Corynespora cassiicola).

Corynespora cassiicola is the causal agent of cucumber Corynespora leaf spot, which affects many economically important plant species. Chemical control of this disease is hampered by the common development of fungicide resistance. In this study, 100 isolates from Liaoning Province were collected, and their sensitivity to 12 fungicides was determined. All the isolates (100%) were resistant to trifloxystrobin and carbendazim, and 98% were resistant to fluopyram, boscalid, pydiflumetofen, isopyrazam, and fluxapyroxad. However, none were resistant to propiconazole, prochloraz, tebuconazole, difenoconazole, and fludioxonil. The Cytb gene of trifloxystrobin-resistant isolates encoded the G143A mutation, whereas the ß-tubulin gene of carbendazim-resistant isolates encoded the E198A and E198A and M163I mutations. Mutations in SdhB-I280V, SdhC-S73P, SdhC-H134R, SdhD-D95E, and SdhD-G109V were associated with resistance to the succinate dehydrogenase inhibitors (SDHIs). Trifloxystrobin, carbendazim, and fluopyram were barely effective on the resistant isolates, whereas fludioxonil and prochloraz were effective on the isolates that were resistant to the quinone outside inhibitors (QoIs), SDHIs, and benzimidazoles. Ultimately, this study demonstrates that fungicide resistance seriously threatens the effective control of Corynespora leaf spot.

Multiple resistance of Echinochloa phyllopogon to synthetic auxin, ALS-, and ACCase-inhibiting herbicides in Northeast China.

Echinochloa phyllopogon is a self-pollinating allotetraploid weed and a serious threat to global rice production. One sensitive and three multiple-resistant populations collected from two provinces of Northeast China were used to analyze the mechanism of multiple resistance of E. phyllopogon to penoxsulam, metamifop, and quinclorac. Compared with the sensitive population LN12, LN1 showed higher resistance to these three herbicides; LN24 showed medium resistance to penoxsulam and metamifop and higher resistance to quinclorac (274-fold); HLJ4 showed low resistance to penoxsulam and high resistance to metamifop and quinclorac. Target sequence analysis showed no mutations in acetolactate synthase or acetyl-CoA carboxylase genes. In-vitro enzyme activity analysis showed that the activity of the target enzyme of multiple herbicide-resistant populations was similar to that of the sensitive population. The P450 inhibitor, malathion, noticeably increased the sensitivity of LN1, LN24, and HLJ4 to penoxsulam, LN1 to metamifop, and HLJ4 to quinclorac. Under all four treatments, the GSTs activities of resistant and sensitive populations showed an increasing trend from day 1 to day 5, but the sensitivity and activity of GSTs were higher in the multiple-resistant population than that in the sensitive population LN12. This study identified the development of multiple-resistant E. phyllopogon populations that pose a serious threat to rice production in rice fields in Northeast China, preliminarily confirming that multiple-resistance was likely due to non-target-site resistance mechanisms. These populations of E. phyllopogon are likely to be more difficult to control.

Mutation at the 197 site and P450-mediated metabolic resistance are involved in bensulfuron-methyl resistance in Sagittaria trifolia.

Sagittaria trifolia control is threatened by the emergence of resistance to acetolactate synthase (ALS)-inhibiting herbicides. Hence, we systematically uncovered the molecular mechanism of resistance to the main herbicide (bensulfuron-methyl) in Liaoning Province from target-site and non-target-site resistance perspectives. The suspected resistant population (TR-1) exhibited high-level resistance. A new amino acid substitution (Pro-197-Ala) in resistant Sagittaria trifolia for ALS was detected, and the molecular docking results showed that the spatial structure of ALS changed significantly after the substitution, manifested by an increase in the number of contacted amino acid residues and the disappearance of hydrogen bonds. Dose-response test of transgenic Arabidopsis thaliana further demonstrated that the Pro-197-Ala substitution conferred bensulfuron-methyl resistance. The assays found that the sensitivity of the ALS enzyme in TR-1 to this herbicide was decreased in vitro; and this population had developed resistance to other types of ALS-inhibiting herbicides. Furthermore, the resistance of TR-1 to bensulfuron-methyl was significantly alleviated after co-treatment with a P450-inhibitor (malathion). TR-1 metabolized bensulfuron-methyl significantly faster than sensitive population (TS-1) did, but this gap was narrowed after malathion treatment. Overall, the resistance of Sagittaria trifolia to bensulfuron-methyl was derived from the mutation of the target-site gene and the enhancement of the P450s-mediated detoxification metabolism.

Evaluation of efficacy and mechanism of Bacillus velezensis CB13 for controlling peanut stem rot caused by Sclerotium rolfsii.

Peanut stem rot, caused by Sclerotium rolfsii, considerably affects crop productivity. Application of chemical fungicides harms the environment and induces drug resistance. Biological agents are valid and eco-friendly alternatives to chemical fungicides. Bacillus spp. are important biocontrol agents that are now widely used against several plant diseases. This study aimed to evaluate the efficacy and mechanism of a potential biocontrol agent Bacillus sp. for controlling peanut stem rot caused by S. rolfsii. Here, we isolated a strain of Bacillus from pig biogas slurry that considerably inhibits the radial growth of S. rolfsii. The strain CB13 was identified as Bacillus velezensis on the basis of morphological, physiological, biochemical characteristics and phylogenetic trees based on the 16S rDNA and gyrA, gyrB, and rpoB gene sequences. The biocontrol efficacy of CB13 was evaluated on the basis of colonization ability, induction of defense enzyme activity, and soil microbial diversity. The control efficiencies of B. velezensis CB13-impregnated seeds in four pot experiments were 65.44, 73.33, 85.13, and 94.92%. Root colonization was confirmed through green fluorescent protein (GFP)-tagging experiments. The CB13-GFP strain was detected in peanut root and rhizosphere soil, at 104 and 108 CFU/g, respectively, after 50 days. Furthermore, B. velezensis CB13 enhanced the defense response against S. rolfsii infection by inducing defense enzyme activity. MiSeq sequencing revealed a shift in the rhizosphere bacterial and fungal communities in peanuts treated with B. velezensis CB13. Specifically, the treatment enhanced disease resistance by increasing the diversity of soil bacterial communities in peanut roots, increasing the abundance of beneficial communities, and promoting soil fertility. Additionally, real-time quantitative polymerase chain reaction results showed that B. velezensis CB13 stably colonized or increased the content of Bacillus spp. in the soil and effectively inhibited S. rolfsii proliferation in soil. These findings indicate that B. velezensis CB13 is a promising agent for the biocontrol of peanut stem rot.

Occurrence and mechanism of target-site resistance to bensulfuron-methyl in Monochoria korsakowii from China.

Monochoria korsakowii is an increasingly significant threat to rice production across China, particularly in Liaoning province. Few studies have reported herbicide resistance in M. korsakowii, and resistance status and mechanisms are poorly understood. Here, thirty field populations of M. korsakowii were collected from 11 rice-growing regions of Liaoning, and 97% of populations had evolved resistance to bensulfuron-methyl (BM), with majority (24 of 28) showing high resistance levels (RI > 10). The first in-depth analysis of molecular features of AHAS1 and AHAS2 in BM-resistant populations showed that four Pro197 mutations (Pro197 to His, Ala, Leu or Ser) in AHAS1 and one mutation (Pro197Ser) in AHAS2 were identified. Notably, novel double Pro197Ser mutations co-occurred in both AHAS1 and AHAS2 in the most resistant line LN-20. Furthermore, resistant mutants were used to investigate the effect of Pro197 mutations on AHAS functionality, binding modes, gene expression and cross-resistance in M. korsakowii. All the detected Pro197 mutations considerably reduced in vitro AHAS sensitivity to BM by weakening hydrogen bonds and hydrophobic interactions in the predicted BM-AHAS complexes, especially the double Pro197Ser mutations. This novel resistance mutation combination slightly impacted the extractable AHAS activity, and increased the affinity and catalytic rate of pyruvate. Also, the AHAS expression level was significantly up-regulated. Moreover, all mutations provided resistance only to other sulfonylureas herbicides but not triazolopyrimidine or pyrimidinyl-benzoates herbicides. In conclusion, bensulfuron-methyl resistance in M. korsakowii was grim in Liaoning, China, and amino acid mutations on AHAS isozymes were the primary resistance mechanism. Double Pro197Ser mutations in both AHAS1 and AHAS2 confer higher herbicide resistance than single mutations in AHAS1. Thus, this work deepens our understanding of resistance status and mechanisms of M. korsakowii.

Increase in glutathione S-transferase activity and antioxidant damage ability drive resistance to bensulfuron-methyl in Sagittaria trifolia.

Weeds tend to develop resistance to herbicides with time. Understanding the resistance mechanisms evolved by weeds would help manage weed infestation. Sagittaria trifolia, a paddy weed found in the rice fields of Liaoning, China, has developed resistance to bensulfuron-methyl, causing severe yield losses in rice. This study deciphers the underlying mechanisms in terms of non-target-site resistance toward bensulfuron-methyl. We compared the ability of glutathione S-transferase (GST) mediated detoxification metabolism and reactive oxygen species (ROS) scavenging between sensitive (NHS) and resistant (NHR) populations of S. trifolia. The resistance ratio of NHR was 210; but the ratio was significantly decreased after GST-inhibitor treatment (44.9). This indicated that a GST-mediated enhancement of detoxification metabolism stimulated the development of resistance. Similarly, higher GST activity was observed in NHR; but the activity equaled that of NHS after GST-inhibitor treatment. However, treatment with the GST-inhibitor did not completely reverse bensulfuron-methyl resistance in NHR, indicating that additional factors contributed to herbicide resistance in these plants. We observed a rapid increase in H2O2 and malondialdehyde accumulation in the case of NHS after bensulfuron-methyl application, whereas those of NHR remained relatively stable, implying that NHR exhibited higher ROS-scavenging capacity under herbicide stress. Further, NHR showed higher glutathione and ascorbic acid contents and higher activities of glutathione reductase and dehydrogenase reductase, all of which contribute towards herbicide resistance in these plants. Our results indicate that GST-mediated detoxification metabolism of bensulfuron-methyl and ROS scavenging capacity contributed to the development of resistance in S. trifolia.

P450s mediated enhanced herbicide metabolism involved in the thifensulfuron-methyl resistance in Ipomoea purpurea L.

Ipomea purpurea (L.) Roth. reduces dry land crop yield and quality in Northeast China, especially in Liaoning Province. Frequent use of thifensulfuron-methyl in recent years has resulted in herbicide resistance in I. purpurea. We evaluated resistance levels of I. purpurea to thifensulfuron-methyl, an acetolactate synthase (ALS) inhibitor, in Liaoning Province and further investigated the resistance mechanisms. The results showed that 15 populations of I. purpurea have evolved up to 5.81-34.44-fold resistance to thifensulfuron-methyl, compared to the susceptible population (S), among which LN3 was the most resistant. DNA sequencing of the ALS gene in susceptible and resistant populations did not reveal any target site mutations that could be associated with resistance to thifensulfuron-methyl in I. purpurea. Additionally, no significant difference was detected between the in vitro ALS activity of LN3 and S. The GR50 of LN3 decreased sharply by 47% when malathion (a P450 inhibitor) was applied with thifensulfuron-methyl. Absorption of thifensulfuron-methyl by LN3 was equal to that of S; however, LN3 metabolized the herbicide significantly faster. This was repressed after the inhibition of P450s activity. Collectively, our results confirmed that I. purpurea in Liaoning Province has developed resistance to thifensulfuron-methyl and implied that the resistance was conferred by the increase in detoxification mediated by P450s. Furthermore, LN3 was sensitive to fluroxypyr, which can be used as an alternative to control I. purpurea.

Comparative Assessment of Phytoconstituents, Antioxidant Activity and Chemical Analysis of Different Parts of Milk Thistle Silybum marianum L.

Silybum marianum L. is a therapeutic plant belonging to the family Asteraceae, which has exhibited silymarin, a principal component used to cure various physiochemical disorders. The study appraised the phytochemical analysis, antioxidant activity and chemical analysis of an extract from the seed, stem and leaves. Qualitative and quantitative phytochemical analysis was evaluated by the Folin-Ciocalteu reagent method and aluminum chloride colorimetric method, respectively. While the antioxidant activity was determined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and acetate buffer in ferric chloride (FRAP) assay, respectively, the chemical profile was evaluated by Gas Chromatography-Mass Spectrometry (GC-MS) assay. The study outcomes identified that alkaloids, glycosides, flavonoids, terpenoids, steroids and catcholic tannins were present in seed, stem and leaves extracts except for saponins and Gallic tannins. Whereas, phenols were absent only in seed extract. Quantitative analysis revealed the presence of phenols and flavonoids in appreciable amounts of 21.79 (GAE/g), 129.66 (QE/g) and 17.29 (GAE/g), 114.29 (QE/g) from the leaves and stem extract, respectively. Similarly, all extracts expressed reasonable DPPH inhibition (IC50) and FRAP reducing power such as 75.98, 72.39 and 63.21% and 46.60, 51.40 and 41.30 mmol/g from the seeds, stem and leaves extract, respectively. Additionally, chemical analysis revealed the existence of 6, 8 and 9 chemical compounds from the seeds, stem and leaves extract, respectively, corresponding to 99.95, 99.96 and 98.89% of the whole extract. The chemical compound, Dibutyl phthalate was reported from all extracts while, Hexadecanoic acid, methyl ester and Silane, (1,1-dimethylethyl), dimethyl (phenylmethoxy) were reported only from the seed and leaves extract. Moreover, Methyl stearate was also a major compound reported from all extracts except for seed extract. It is demonstrable that extracts from different parts of S. marianum possess significant antioxidant activity, as well as valuable chemical compounds accountable for therapeutic effects that might be incorporated as an alternative to synthetic chemical agents.

Antioxidant, antifungal, and aphicidal activity of the triterpenoids spinasterol and 22,23-dihydrospinasterol from leaves of Citrullus colocynthis L.

Terpenoids from natural plant sources are valuable for their diverse biological activities that have important roles in the medical and agrochemical industries. In this study, we assessed the antioxidant, antifungal, and aphicidal activities of a mixture of spinasterol and 22,23-dihydrospinasterol from the leaves of Citrullus colocynthis. We used 1,1-diphenyl-2-picrylhydrazyl (DPPH) to assess antioxidant activity, and we measured antifungal activity using mycelium growth inhibition assays with three pathogenic fungi, Magnaporthe grisea, Rhizoctonia solani, and Phytophthora infestans. Aphicidal activity against adults of Myzus persicae was determined using in vitro and in vivo assays. Spinasterol and 22,23-dihydrospinasterol exhibited moderate antioxidant activity, even at lower concentrations: 19.98% at 0.78 µg mL-1, 31.52% at 3.0 µg mL-1, 36.61% at 12.5 µg mL-1, and 49.76% at 50 µg mL-1. Spinasterol and 22,23-dihydrospinasterol showed reasonable levels of fungicidal activity toward R. solani and M. grisea, with EC50 values of 129.5 and 206.1 µg mL-1, respectively. The positive controls boscalid and carbendazim were highly effective against all fungi except boscalid for M. grisea (EC50 = 868 µg mL-1) and carbendazim for P. infestans (EC50 = 8721 µg mL-1). Significant insecticidal activity was observed in both residual and greenhouse assays, with LC50 values of 42.46, 54.86, and 180.9 µg mL-1 and 32.71, 42.46, and 173.8 µg mL-1 at 72, 48, and 24 h, respectively. The antioxidant activity of spinasterol and 22,23-dihydrospinasterol was strongly positively correlated with their antifungal and insecticidal activity. Spinasterol and 22,23-dihydrospinasterol therefore show good antioxidant and aphicidal activity with moderate fungicidal activity, making them suitable candidates for an alternative to synthetic agents.

Molecular basis of cross-resistance to acetohydroxy acid synthase-inhibiting herbicides in Sagittaria trifolia L.

Acetohydroxy acid synthase (AHAS)-inhibiting herbicides are one of the most commonly used herbicides for controlling the growth of Sagittaria trifolia L. in paddy fields in Northeastern China. In this study, we collected five suspected resistant populations of S. trifolia (R1-R5) from three different provinces of Northeastern China. The results of whole-plant bioassays revealed that those populations showed high level of resistance to bensulfuron-methyl with resistance index (GR50 R/S) ranging from 39.90 to 88.50. The results of AHAS-activity assays were consistent with the results of the whole-plant bioassays. The AHAS gene analysis showed that R2 and R3 populations contained Pro-197-Leu mutations that were highly resistant to penoxsulam; R1 and R4 populations contained Pro-197-Ser mutations that were highly resistant to bispyribac­sodium; R5 population contained Trp-574-Leu mutation that showed high resistance to IMI, PT, PTB and SU herbicides. The AHAS with resistance mutations showed less sensitivity to feedback inhibition by BCAAs and R genotypes had increased free BCAAs.

Spinasterol, 22,23-Dihydrospinasterol and Fernenol from Citrullus Colocynthis L. with Aphicidal Activity against Cabbage Aphid Brevicoryne Brassicae L.

Brevicoryne brassicae is a problematic pest in cabbage and other field crops. Synthetic pesticides are used to control this pest, but they are injurious for human health and the environment. The present study aimed to purify and identify the active compounds from Citrullus colocynthis leaves with an appraisal of their efficacy against B. brassicae. Separation and purification were performed via different chromatographic techniques. Molecular analysis and chemical structures were recognized by mass spectrum (MS) and nuclear magnetic resonance (NMR), respectively. Moreover, in vitro and in vivo aphicidal activity was assessed using various concentrations, i.e., 6.25, 12.5, 25 and 50 µg/mL at 12, 24, 48 and 72 h exposure. The outcome shows that mass spectrum analyses of the purified compounds suggested the molecular formulae are C30H50O and C29H50O, C29H48O. The compounds were characterized as fernenol and a mixture of spinasterol, 22,23-dihydrospinasterol by 1H-NMR and 13C-NMR spectrum analysis. The toxicity results showed that the mixture of spinasterol and 22,23-dihydrospinasterol showed LC50 values of 32.36, 44.49 and 37.50 µg/mL by contact, residual and greenhouse assay at 72 h exposure, respectively. In contrast, fernenol recorded LC50 values as 47.99, 57.46 and 58.67 µg/mL, respectively. On the other hand, spinasterol, 22,23-dihydrospinasterol showed the highest mortality, i.e., 66.67%, 53.33% and 60% while, 30%, 23.33% and 25% mortality was recorded by fernenol after 72 h at 50 µg/mL by contact, residual and greenhouse assay, respectively. This study suggests that spinasterol, 22,23-dihydrospinasterol are more effective against B. brassicae which may be introduced as an effective and suitable substitute of synthetic chemical pesticides.

Novel sulfonamides against Botrytis cinerea with no positive cross-resistance to commercial fungicides: Design, synthesis and SAR study.

Thirty-four novel compounds were synthesized using chesulfamide (N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide), a high-profile fungicide, as the lead compound, and their structures were characterized by 1H NMR, 13C NMR, MS and elemental analysis. Additionally, the structure of (1S,2R)-2-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl)cyclohexane-1-sulfonamide (IV-9) was confirmed by X-ray single crystal diffraction. The mycelium inhibition tests, spore germination inhibition tests, tomato pot tests and field trials were performed against strains of B. cinerea. Bioassay results showed that most of target compounds had good fungicidal activity against B. cinerea, in particular, IV-9 exhibited similar or superior effects to procymidone, boscalid and pyrisoxazole in all in vitro and in vivo tests. Moreover, there was no positive cross-resistance found between the compound IV-9 and eight commercial fungicides (azoxystrobin, boscalid, chlorothalonil, diethofencarb, fludioxonil, procymidone, pyrimethanil and pyrisoxazole) in the cross-resistance validation test performed by an innovative method.

Insecticidal activity and biochemical composition of Citrullus colocynthis, Cannabis indica and Artemisia argyi extracts against cabbage aphid (Brevicoryne brassicae L.).

Plant extracts contain many active compounds, which are tremendously fruitful for plant defence against several insect pests. The prime objectives of the present study were to calculate the extraction yield and to evaluate the leaf extracts of Citrullus colocynthis (L.), Cannabis indica (L.) and Artemisia argyi (L.) against Brevicoryne brassicae and to conduct biochemical analysis by gas chromatography-mass spectrometry (GC-MS). The results suggested that when using ethanol, C. colocynthis produced a high dry yield (12.45%), followed by that of C. indica and A. argyi, which were 12.37% and 10.95%, respectively. The toxicity results showed that A. argyi was toxic to B. brassicae with an LC50 of 3.91 mg mL-1, followed by the toxicity of C. colocynthis and C. indica, exhibiting LC50 values of 6.26 and 10.04 mg mL-1, respectively, which were obtained via a residual assay; with a contact assay, the LC50 values of C. colocynthis, C. indica and A. argyi were 0.22 mg mL-1, 1.96 and 2.87 mg mL-1, respectively. The interaction of plant extracts, concentration and time revealed that the maximum mortality based on a concentration of 20 mg L-1 was 55.50%, the time-based mortality was 55% at 72 h of exposure, and the treatment-based mortality was 44.13% for A. argyi via the residual assay. On the other hand, the maximum concentration-based mortality was 74.44% at 20 mg mL-1, the time-based mortality was 66.38% after 72 h of exposure, and 57.30% treatment-based mortality was afforded by A. argyi via the contact assay. The biochemical analysis presented ten constituents in both the A. argyi and C. colocynthis extracts and twenty in that of C. indica, corresponding to 99.80%, 99.99% and 97% of the total extracts, respectively. Moreover, the detected caryophylleneonides (sesquiterpenes), α-bisabolol and dronabinol (Δ9-THC) from C. indica and erucylamide and octasiloxane hexamethyl from C. colocynthis exhibited insecticidal properties, which might be responsible for aphid mortality. However, A. argyi was evaluated for the first time against B. brassicae. It was concluded that all the plant extracts possessed significant insecticidal properties and could be introduced as botanical insecticides after field evaluations.

Phytochemical Analysis, Biochemical and Mineral Composition and GC-MS Profiling of Methanolic Extract of Chinese Arrowhead Sagittaria trifolia L. from Northeast China.

Sagittaria trifolia is a medicinal foodstuff of China and East Asia belonging to the family Alismataceae. Samples of S. trifolia tubers were collected from Meihekow, Siping, Jilin, Harbin and Wuchang from Northeast China. The current study was aimed to evaluate the qualitative and quantitative analysis, antioxidant activity, biochemical analysis and chemical composition of different populations of S. trifolia. By using Folin-Ciocalteu, aluminium chloride colourimetric and 1,1-diphenyl-1-picrylhydrazyl (DPPH), total phenol and flavonoids content and antioxidant activity was analysed. Furthermore, chemical composition, biochemical analysis and mineral substances were also determined. The results showed the presence of flavonoids, phenols, saponins, tannins, glycosides and steroids except for alkaloids and terpenoids by qualitative analysis. Quantitative analysis revealed that highest total phenol, flavonoids content and antioxidant potential identified from Meihekow, i.e., 2.307 mg GAE/g, 12.263 mg QE/g and 77.373%, respectively. Gas chromatography-mass spectrometry results showed the presence of 40 chemical compounds corresponding to 99.44% of total extract that might be responsible for antioxidant properties. Mineral and biochemical analysis revealed the presence of calcium, magnesium, potassium, sodium, iron, copper, zinc and, carbohydrate, protein, fibre and fat contents, respectively. Interestingly, all S. trifolia populations collected from different locations possess similar composition. The dietary values, phytoconstituents, antioxidant activities and nutritional and curative chemical compounds of S. trifolia are beneficial for the nutritherapy of human beings.

Synthesis, Fungicidal Activity and SAR of 2-Thiazolamide/Pyrazolamide-Cyclohexylsulfonamides against Botrytis cinerea.

In order to explore more efficient sulfonamides against Botrytis cinereal, 36 novel cyclohexylsulfonamides were synthesized by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt) condensation reaction using chesulfamide as a lead compound, introducing thiazole and pyrazole active groups. Their structures were characterized by 1H-NMR, 13C-NMR, mass spectrum (MS), and elemental analysis. Compound III -31 was further confirmed by X-ray single crystal diffraction. The in vitro and in vivo fungicidal activities against B. cinerea were evaluated by three bioassay methods. The results of mycelial growth demonstrated that median effective concentration (EC50) values of nine compounds were close to boscalid (EC50 = 1.72 µg/mL) and procymidone (EC50 = 1.79 µg/mL) against B. cinerea (KZ-9). In the spore germination experiment, it was found that compounds III-19 and III-31 inhibited germination 93.89 and 98.00%, respectively; at 10 µg/mL, they approached boscalid (95.97%). In the tomato pot experiment, the control effects of two compounds (III-21 and III-27) were 89.80 and 87.90%, respectively, at 200 µg/mL which were significantly higher than boscalid (81.99%). The structure-activity relationship (SAR) was also discussed, which provided a valuable idea for developing new fungicides.

In vitro and in vivo studies of the metabolic activation of chelidonine.

Chelidonium majus L. is a herbal medicine widely employed in Europe and Western Asia. Chelidonine (CHE) is a major constituent of the herb and has been reported to be an inhibitor of the cytochrome P450 enzymes (CYP). The major objective of the present study was to study the metabolic pathways of CHE in order to identify potential reactive metabolites responsible for the enzyme inhibition. Three oxidative metabolites (M1-M3) were detected in human liver microsomal incubations after exposure to CHE. M1 and M2 were two isomers of catechol derivatives, and M3 was a dicatechol compound. The M1-M3 metabolites were also observed in bile of rats given CHE. A total of five glutathione (GSH) conjugates (M4-M8) were detected in microsomes containing CHE, GSH, and NADPH. Moreover, M4 and M6 originated from M1, M5 and M7 resulted from M2, and M8 was a M3-derived GSH conjugate. Three biliary CHE-derived GSH conjugates (M4, M5 and M8) were found in CHE-treated rats. This indicates that CHE was bioactivated to ortho-quinone derivatives both in vitro and in vivo. Recombinant P450 enzyme incubations demonstrated that the CYPs3A4, 1A2, 2C19 and 2D6 were mainly involved in metabolic activation of CHE. This study generated data that may be useful in understanding possible mechanisms of CHE-induced P450 inhibition.

Synthesis, fungicidal activity and SAR of 3,4-dichloroisothiazole-based cycloalkylsulfonamides.

To develop more valuable and effective fungicide candidates, a novel series of 3,4-dichloroisothioxazole-based cycloalkylsulfonamides were synthesized and their structures were identified by 1H NMR, 13C NMR, MS and elemental analysis. Compound 3k was further confirmed by X-ray single crystal diffraction. The in vitro bioassay results demonstrated that the target compounds showed significant fungicidal activity on mycelial growth and spore germination of Botrytis cinerea. Especially, compound 3j, with prominent inhibition effect on mycelial with EC50 and EC80 values of 1.4 and 23.7 µg/mL respectively, was comparable to the selected commercial fungicide. Moreover, at 50 µg/mL, the inhibition rate of compound 3j on spore germination was recorded up to 89.7%. The further in vivo bioassay results indicated compound 3j continued to show high control effect on tomato leaves, flowers and fruit at 200 µg/mL, with control efficiencies of 94.3%, 89.3% and 91.9%, respectively. The structure-activity relationship showed that the compound with a five-membered ring possessed the best activity after the introduction of the active fragment of the 3,4-dichloroisothioxazole, provided a valuable idea for further creation of new fungicides.