Bio‐preparation of (R)‐DMPM using whole cells of Pseudochrobactrum asaccharolyticum WZZ003 and its application on kilogram‐scale synthesis of fungicide (R)‐metalaxyl

Methyl (R)‐N‐(2,6‐dimethylphenyl)alaninate ((R)‐DMPM) is a key chiral intermediate for the production of (R)‐metalaxyl, which is one of the best‐selling fungicides. A new strain, Pseudochrobactrum asaccharolyticum WZZ003, was identified as a biocatalyst for the enantioselective hydrolysis of (R,S)‐DMPM. The key parameters including pH, temperature, rotation speed and substrate concentrations were optimized in the enantioselective hydrolysis of (R,S)‐DMPM. After the 48 h hydrolysis of 256 mM (R,S)‐DMPM under the optimized reaction conditions, the enantiomeric excess of product (e.e._p) was up to 99% and the conversion was nearly 50%. Subsequently, the unhydrolyzed (S)‐DMPM was converted to (R,S)‐DMPM through the n‐butanal‐catalyzed racemization. Furthermore, stereoselective hydrolysis of (R,S)‐DMPM catalyzed by whole cells of P. asaccharolyticum WZZ003 was scaled up to kilogram‐scale, offering (R)‐MAP‐acid with 98.6% e.e._p and 48.0% yield. Moreover, (R)‐metalaxyl was prepared at kilogram scale after subsequent esterification and coupling reactions. Therefore, a practical production process of (R)‐DMPM and (R)‐metalaxyl with the prospect of industrialization was developed in this study. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:921–928, 2018     

强力安对食用菌生产中杂菌的毒力测定

试验利用滤纸片、管碟抑菌圈法测定了强力安杀菌剂对木霉TrichodermaviridePers.exFr、链孢霉MoniliasitophilaMont等食用菌生产上的杂菌的毒力,结果表明强力安具有明显的抑菌、杀菌作用。试验还测定了强力安对食用菌的作用,结果表明强力安对平菇Pleurotusostreatus(JacexFr)Quel、金针菇Flammulinavelutipes（Fr）Sing具抑制力,而对黑木耳Auriculariaauricula(l.exHook)Underw、香菇Lentinulaedodes(Berk)Pegler则未发现抑制作用。根据强力安对供试菌的最低抑制浓度,作者提出了生产实际上的用药浓度,以供参考。     

Development and application of a simple,rapid and sensitive method for detecting moderately carbendazim‐resistant isolates in Botrytis cinerea

Sustainable disease management depends on the ability to monitor the development of fungicide resistance in pathogen populations. A point mutation resulting in an alteration (F200Y) at codon 200 of the target protein β‐tubulin leads to a moderate level of resistance to carbendazim in Botrytis cinerea. Although traditional methods remain a cornerstone in detection of fungicide resistance, molecular methods that do not require the isolation of pathogens, can detect the presence of resistance alleles at low frequencies, and require less time and labour than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting the moderately carbendazim‐resistant isolates in B. cinerea based on loop‐mediated isothermal amplification (LAMP). By using specific LAMP primers, we detected the resistance‐conferring mutation underlying β‐tubulin F200Y. The concentrations of LAMP components and LAMP parameters were optimised, resulting in reaction temperatures and times of 61–65°C and 45 min, respectively. The feasibility of the LAMP assay was verified by assaying the diseased samples with artificial inoculation in the different hosts. The LAMP assay developed in the current study was specific, stable, repeatable and sensitive, and was successfully applied for detection of moderately carbendazim‐resistant isolates of B. cinerea in plant samples.     

Carbendazim resistance and dimethachlone sensitivity of field isolates of Sclerotinia sclerotiorum from oilseed rape in Henan Province,China

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases in oilseed rape‐growing areas of China. To determine the frequency of resistance of field isolates of S. sclerotiorum to carbendazim and dimethachlone, a total of 556 isolates from 10 different regions of Henan Province were obtained between 2015 and 2016. The frequency of isolates with a high‐resistance phenotype and a moderate‐resistance phenotype to carbendazim was 69.2% and 10.8%, respectively. However, S. sclerotiorum isolates resistant to dimethachlone were not detected. The baseline sensitivity of S. sclerotiorum to dimethachlone was distributed as a unimodal curve with a mean EC₅₀ value of 0.39 ± 0.09 μg ml^?1 for the inhibition of mycelial growth. Four dimethachlone‐resistant mutants were obtained from 20 wild‐type isolates induced by exposure to increasing concentrations of the fungicide in vitro. The mutants showed high levels of resistance to dimethachlone, with resistance factors that ranged from 179 to 323. Positive cross‐resistance occurred between dimethachlone and procymidone, iprodione, and fludioxonil; however, no cross‐resistance was observed for carbendazim and boscalid. The fitness of the dimethachlone‐resistant mutants was significantly lower than that of the wild‐type isolates, as measured by mycelial growth, hyphal dry weight, sclerotium number and dry weight, and pathogenicity. Additionally, based on osmotic tests, the inhibition of mycelial growth caused by NaCl applied at different concentrations was significantly higher for the dimethachlone‐resistant mutants than for their wild‐type parents.     

Novel synthesis scheme and in vitro antimicrobial evaluation of a panel of (E)-2-aryl-1-cyano-1-nitroethenes

Drug resistance has become a major concern in the field of infection management, therefore searching for new antibacterial agents is getting more challenging. Our study presents an optimized and eco-friendly synthesis scheme for a panel of nitroalkenes bearing various functional groups in the aromatic moiety and bromine or cyano substituents in 1 position of nitrovinyl moiety. The presence of nitrolefine group outside the ring minimalizes genotoxic properties while conjugation of aryl group with nitrovinyl moiety increases stability of the compounds. Then our research focused on evaluation of biological properties of such obtained (E)-2-aryl-1-cyano-1-nitroethenes. As they exhibit strong bacteriostatic and bactericidal activities against reference bacteria and yeast species with no detectable cytotoxicity towards cultured human HepG2 and HaCaT cells, they could be promising candidates for the replacement of traditional nitrofurane-containing antibacterial drugs. Nevertheless, validation of the obtained data in an in vivo model and additional safety studies on mutagenicity are still required.     

Characterization of adaptability components of Brazilian isolates of Macrophomina pseudophaseolina

Macrophomina pseudophaseolina is a new Macrophomina species reported on different crop and weed species in Brazil, India and Senegal, but to date there are no studies about its adaptability components. In this work, a collection of 62 M. pseudophaseolina isolates obtained from roots of the weed species Trianthema portulacastrum and Boerhavia diffusa collected in Northeastern Brazil, was used to: (a) study the effect of temperature and salinity on mycelial growth, (b) to determine their sensitivity to the fungicide carbendazim and (c) to assess their aggressiveness on melon and watermelon seedlings. Results showed variability among M. pseudophaseolina isolates. The optimum temperature for mycelial growth ranged between 26.4 and 38.1ºC. NaCl reduced the in vitro growth of all isolates, which were also highly sensitive to the fungicide carbendazim, exhibiting EC₅₀ values ranging from 0.013 to 0.089 mg/L a.i. Disease severity values on melon and watermelon seedlings showed that M. pseudophaseolina isolates were more aggressive in melon than in watermelon. Information about adaptability components of M. pseudophaseolina obtained in this study could be incorporated on breeding programs for melon and watermelon crops.     

Discovery of oxathiapiprolin,a new oomycete fungicide that targets an oxysterol binding protein

Oxathiapiprolin is the first member of a new class of piperidinyl thiazole isoxazoline fungicides with exceptional activity against plant diseases caused by oomycete pathogens. It acts via inhibition of a novel fungal target—an oxysterol binding protein—resulting in excellent preventative, curative and residual efficacy against key diseases of grapes, potatoes and vegetables. Oxathiapiprolin is being developed globally as DuPont™ Zorvec™ disease control with first registration and sales anticipated in 2015. The discovery, synthesis, optimization and biological efficacy are presented.     

Synthesis,cytotoxicity, antimicrobial and anti-biofilm activities of novel pyrazolo[3,4-b]pyridine and pyrimidine functionalized 1,2,3-triazole derivatives

A series of novel pyrazolo[3,4-b]pyridine and pyrimidine functionalized 1,2,3-triazole derivatives 8a–g and 9a–g were prepared starting from 6-trifluoromethylpyridine-2(1H)one 2 via selective O-alkylation, followed by cyclisation using hydrazine hydrate to obtain 6-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 4. Compound 4 was diazotized followed by reaction with sodium azide, resulted in 3-azido-6-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridine 5. Compound 5 was further cyclized with N-/O-propargylated pyrimidine derivatives under Sharpless conditions and obtained compounds 6 and 7, respectively. Each set of compounds 6 and 7 were alkylated with different alkyl halides and obtained respective products 8 and 9. All the products were screened for cytotoxicity against four human cancer cell lines such as A549-Lung (CCL-185), MCF7-Breast (HTB-22), DU145-Prostate (HTB-81) and HeLa-Cervical (CCL-2), compounds 9d, 9e and 9f which showed promising activity have been identified. The products were also screened for antimicrobial, anti bio-film and MBC activities. Promising compounds in each case have been identified.