Design and Synthesis of Novel Oxathiapiprolin Derivatives as Oxysterol Binding Protein Inhibitors and Their Application in Phytopathogenic Oomycetes.

Oomycetes, particularly those from the genus Phytophthora, are significant threats to global food security and natural ecosystems. Oxathiapiprolin (OXA) is an effective oomycete fungicide that targets an oxysterol binding protein (OSBP), while the binding mechanism of OXA is still unclear, which limits the pesticide design, induced by the low sequence identity of Phytophthora and template models. Herein, we generated the OSBP model of the well-reported Phytophthora capsici using AlphaFold 2 and studied the binding mechanism of OXA. Based on it, a series of OXA analogues were designed. Then, compound 2l, the most potent candidate, was successfully designed and synthesized, showing a control efficiency comparable to that of OXA. Moreover, field trial experiments showed that 2l exhibited nearly the same activity (72.4%) as OXA against cucumber downy mildew at 25 g/ha. The present work indicated that 2l could be used as a leading compound for the discovery of new OSBP fungicides.

Two single mutations in carboxylesterase 001C improve fenvalerate hydrolase activity in Helicoverpa armigera.

Carboxylesterases (CarEs) usually play critical roles in the detoxification of toxic chemicals and therefore may be involved in insecticide resistance in agricultural pests. Previous work has shown that CarE 001C from Helicoverpa armigera was able to metabolize the isomers of cypermethrin and fenvalerate. In this study, seven mutants of CarE 001C with single amino acid substitution were produced and expressed in the Escherichia coli. Enzyme kinetic analysis indicated that all seven mutations dramatically reduced enzymatic activities toward the generic substrate α-naphthyl acetate, but in vitro metabolism assay showed that two of the mutations, H423I and R322L, significantly improved hydrolase activities toward fenvalerate, with their recorded specific activities being 3.5 and 5.1 nM·s-1·mg -1 proteins, respectively. Further, thermostability assay showed that the stability of one mutant enzyme was enhanced. This study will help us better understand the potential of CarEs in insecticide detoxification and resistance in H. armigera.

Enhanced hydrolysis of ß-cypermethrin caused by deletions in the glycin-rich region of carboxylesterase 001G from Helicoverpa armigera.

BACKGROUND: Carboxylesterase (CarE) is a major class of enzyme involved in the detoxification of toxic xenobiotics in various insect species. Previous work has shown that the carboxylesterase gene CarE001G found in Helicoverpa armigera is more active and can metabolize synthesized pyrethroids, such as ß-cypermethrin, one of the commonly used commercial insecticides for lepidopteran pest control. In addition, CarE001G is very special as it has a very specific glycine-rich region located adjacent to its C-terminal. But whether mutations in this unique sequence can change the biochemistry and function of CarE001G are unknown. RESULTS: In this study, four variants of CarE001G with different deletions in the glycine-rich region were obtained and functionally expressed in Escherichia coli. The recombinant proteins were purified and confirmed by Western blot and mass spectrometry analyses. These mutant enzymes showed high catalytic efficiency toward the model substrate α-naphthyl acetate. Inhibition study showed that ß-cypermethrin had relatively strong inhibition on CarE activities. In vitro metabolism assay showed that the mutant enzymes significantly enhanced their metabolic activities toward ß-cypermethrin with specific activities between 4.0 and 5.6 nmol L-1 min-1 mg-1 protein. Molecular docking analyses consistently demonstrated that deletion mutations in the glycine-rich region may facilitate the anchoring of the ß-cypermethrin molecule in the active binding pocket of the mutant enzymes. CONCLUSION: The data show that deletion mutations can cause qualitative change in the capacity of CarEs in the detoxification of ß-cypermethrin. This indicates that deletion mutations in the glycine-rich region may have the potential to cause synthesized pyrethroid (SP) resistance in H. armigera in the future. © 2020 Society of Chemical Industry.

Design, synthesis, and fungicidal evaluation of novel oxysterol binding protein inhibitors for combatting resistance associated with oxathiapiprolin.

Oxathiapiprolin, the first successful oxysterol binding protein (OSBP) inhibitor for oomycete control, is regarded as an important milestone in the history of fungicide discovery. However, its interaction with OSBP remain unclear. Moreover, some plant pathogenic oomycetes have developed medium to high resistance to oxathiapiprolin. In this paper, the three-dimensional (3D) structure of OSBP from Phytophthora capsici (pcOSBP) was built, and its interaction with oxathiapiprolin was systematically investigated by integrating molecular docking, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations. The computational results showed that oxathiapiprolin bound to pcOSBP forms H-bonds with Leu73, Lys74, Ser69, and water molecules. Then, based on its interaction with pcOSBP, oxathiapiprolin was structurally modified to discover new analogs with high fungicidal activity and a low risk of resistance. Fortunately, compound 1e was successfully designed and synthesized as the most potent candidate, and it showed a much lower resistance risk (RF < 1) against LP3-M and LP3-H in P. capsici. The present work indicated that the piperidinyl-thiazole-isoxazoline moiety is useful for further optimization. Furthermore, compound 1e could be used as a lead compound for the discovery of new OSBP inhibitors.

Functional Characterization of Two Carboxylesterase Genes Involved in Pyrethroid Detoxification in Helicoverpa armigera.

Insect carboxylesterases are major enzymes involved in metabolism of xenobiotics including insecticides. Two carboxylesterase genes, CarE001A and CarE001H, were cloned from the destructive agricultural pest Helicoverpa armigera. Quantitative real-time polymerase chain reaction showed that CarE001A and CarE001H were predominantly expressed in fat body and midgut, respectively; developmental expression analyses found that the expression levels of both CarEs were significantly higher in fifth-instar larvae than in other life stages. Recombinant CarE001A and CarE001H expressed in the Escherichia coli exhibited high enzymatic activity toward α-naphthyl acetate. Inhibition assays showed that organophosphates had strong inhibition on CarEs activity compared to pyrethroids. Metabolism assays indicated that CarE001A and CarE001H were able to metabolize ß-cypermethrin and λ-cyhalothrin. Homology modeling and molecular docking analyses demonstrated that ß-cypermethrin could fit nicely into the active pocket of both carboxylesterases. These results suggested that CarE001A and CarE001H could play important roles in the detoxification of pyrehtroids in H. armigera.

M233I Mutation in the ß-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide.

Three phenotypes were detected in 161 Botrytis cinerea field isolates, including Zox(S)Car(S) (sensitive to zoxamide and carbendazim), Zox(S)Car(R) (sensitive to zoxamide and resistant to carbendazim), and Zox(R)Car(R) (resistant to zoxamide and carbendazim), but not Zox(R)Car(S) (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 µg/ml. Two stable Zox(R)Car(S) isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the ß-tubulin of Zox(S)Car(R), Zox(R)Car(R), Zox(R)Car(S), respectively. Molecular docking indicated that position 198 in ß-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between ß-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of ß tubulin.

Wild Type Sensitivity and Mutation Analysis for Resistance Risk to Fluopicolide in Phytophthora capsici.

Crown, root, and fruit rot caused by Phytophthora capsici is an increasing problem for vegetable growers in Michigan and the United States. The newly registered fungicide fluopicolide is effective to limit crop loss but the potential for P. capsici to develop resistance is not well known. A laboratory study assessed the risk of P. capsici developing resistance to fluopicolide. Baseline sensitivity to fluopicolide was determined using 126 P. capsici Michigan isolates. Values of effective concentrations for 50% inhibition of mycelial growth ranged from 0.08 to 0.24 µg/ml and were distributed as a unimodal curve, indicating that all isolates were sensitive to fluopicolide. Mutants resistant to fluopicolide were obtained from five isolates by screening zoospores on fluopicolide-amended (5 µg/ml) media at a mutation frequency above 1.0 × 10-7. The mutant isolates were clustered with either intermediate (resistance factor [RF] = 3.53 to 77.91) or high (RF = 2481.40 to 7034.79) resistance. Resistance was stable through 10 mycelial transfers on fungicide-free medium. All resistant mutants showed similar fitness in zoospore production, cyst germination, and virulence compared with their sensitive parents, with few exceptions. No cross-resistance was detected between fluopicolide and five other fungicides. There could be a moderately high risk of field populations of P. capsici developing resistance to fluopicolide, and populations should be monitored.

Baseline sensitivity and resistance-risk assessment of Phytophthora capsici to iprovalicarb.

Iprovalicarb has been used to control Phytophthora capsici, a devastating pathogen of many economically important crops. To evaluate the risk of fungicide resistance, 158 isolates of P. capsici were examined for sensitivity to iprovalicarb by measuring mycelial growth. Values of effective concentrations for 50% mycelial growth inhibition varied from 0.2042 to 0.5540 µg/ml and averaged 0.3923 (±0.0552) µg/ml, with a unimodal distribution. This is the first report of P. capsici isolates highly resistant to iprovalicarb (resistance factor >100). Resistance of the isolates was stable through 10 transfers on iprovalicarb-free medium, and most resistant isolates had the same level of fitness (mycelial growth, zoospore production, and virulence) as their corresponding parents, indicating that iprovalicarb resistance was independent from other general growth characters. There was cross-resistance among all tested carboxylic acid amide (CAA) fungicides, including iprovalicarb, flumorph, dimethomorph, and mandipropamid, but not with non-CAA fungicides, including azoxystrobin, chlorothalonil, cymoxanil, etridiazole, metalaxyl, and zoxamide. Based on the present results, resistance risk of P. capsici to CAAs could be moderate and resistance management should be considered.

Fluence-response dynamics of the UV-induced SOS response in Escherichia coli.

Bacteria in nature often suffer sudden stresses, such as ultraviolet (UV) irradiation, nutrient deprivation, and chemotoxins that would cause DNA damage and DNA replication failure, which in turn trigger SOS response. According to the strength and duration of the stress, the SOS system not only repairs DNA damage but also induces mutagenesis, so as to adapt to the changing environment. The key proteins in charge of mutagenesis are UmuD and UmuD'. In this paper, we quantitatively measure the growth rate and cellular levels of proteins UmuD and UmuD' in Escherichia coli after various fluences of UV irradiation. To compare with the experimental observations, an ordinary differential equation model is built to describe the SOS response. Considering the fact that the DNA lesions affect cellular protein production and replication origination, the simulation results fit well with the experimental data. Our results show how the fluence of UV irradiation determines the dynamics of the inducing signal and the mutation frequency of the cell.

[Nonbiologic risk factors of pulmonary tuberculosis among adults in Henan: a case-control study].

OBJECTIVE: To study the nonbiologic risk factors of tuberculosis (TB) among adults. METHODS: 1:2 matched case-control study was conducted. 158 new smear positive pulmonary TB patients with 25 - 60 years of age were notified and registered from three county anti-TB institutions in Henan from October 2002-March 2004, were selected as cases. Two healthy persons were selected to match each case, with the same sex and age group, from the nearest neighbors of the case as controls. Interview was carried out with a uniform designed questionnaire at the residence of the object. Univariate and multivariate condition logistic regression models were used. RESULTS: The results of univariate analysis showed that marital status, education, self-employed occupation, smoking, out-migration for work away from hometown, household annual income level and household property were significantly associated with TB prevalence (P < 0.05); multivariate analysis showed that marital status, self-employed occupation, smoking, household economic condition and out-migration for work, away from home were also associated with TB (P < 0.05), and OR values were 2.826, 2.350, 1.536, 0.707, 2.096, respectively. CONCLUSION: Being single, smoking, self-employed occupation and out-migration for work were nonbiologic risk factors of TB while good household economic condition was a protective factor.

[Biological effect of seed-coating in Carthamus tinctorins].

OBJECTIVE: To study the Biological effect of seed-coating in Carthamus tinctorins. METHOD: Two kinds of seedcoating chemicals SCF1 and SCF2 were used in this experiment, the seed YM-99 and 27981-99 were coated by three kinds of ratio of seedcoating chemicals to seed. It was investigated that the germination energy and germination percentage in the room and the emergence rate, seedling stage growing, pest in the field. RESULT: Seedoating can improve the emergence rate and seedling stage growing, it also can effectively control aphid, rust and virosis during the growing period in C. tinctorins. CONCLUSION: Seedcoating has significant biological effect in C. tinctorins.

Cost of malaria control in China: Henan's consolidation programme from community and government perspectives.

OBJECTIVE: To assist with strategic planning for the eradication of malaria in Henan Province, China, which reached the consolidation phase of malaria control in 1992, when only 318 malaria cases were reported. METHODS: We conducted a prospective two-year study of the costs for Henan's malaria control programme. We used a cost model that could also be applied to other malaria programmes in mainland China, and analysed the cost of the three components of Henan's malaria programme: suspected malaria case management, vector surveillance, and population blood surveys. Primary cost data were collected from the government, and data on suspected malaria patients were collected in two malaria counties (population 2 093 100). We enlisted the help of 260 village doctors in six townships or former communes (population 247 762), and studied all 12 325 reported cases of suspected malaria in their catchment areas in 1994 and 1995. FINDINGS: The average annual government investment in malaria control was estimated to be US$ 111 516 (case-management 59%; active blood surveys 25%; vector surveillance 12%; and contingencies and special projects 4%). The average cost (direct and indirect) for patients seeking treatment for suspected malaria was US$ 3.48, equivalent to 10 days' income for rural residents. Each suspected malaria case cost the government an average of US$ 0.78. CONCLUSION: Further cuts in government funding will increase future costs when epidemic malaria returns; investment in malaria control should therefore continue at least at current levels of US$ 0.03 per person at risk.