Optimization of green silver nanoparticles as nanofungicides for management of rice bakanae disease.

Rice bakanae, a devastating seed-borne disease caused by Fusarium species requires a more attractive and eco-friendly management strategy. The optimization of plant-mediated silver nanoparticles (AgNPs) as nanofungicides by targeting Fusarium species may be a rational approach. In this study, Azadirachta indica leaf aqueous extract-based AgNPs (AiLAE-AgNPs) were synthesized through the optimization of three reaction parameters: A. indica leaf amount, plant extract-to-AgNO3 ratio (reactant ratio), and incubation time. The optimized green AgNPs were characterized using ultraviolet-visible light (UV-Vis) spectroscopy, field emission scanning electron microscopy (FESEM) with energy dispersive X-ray (EDX) spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and powder X-ray diffraction (XRD) techniques. The optimal conditions for producing spherical, unique, and diminutive-sized AgNPs ranging from 4 to 27 nm, with an average size of 15 nm, were 2 g AiLAE at a 1:19 ratio (extract-to-AgNO3) and incubated for 4 h. Fusarium isolates collected from infected soils and identified as F. fujikuroi (40) and F. proliferatum (58 and 65) by PCR were used for seed infestation. The AgNPs exhibited concentration-dependent mycelial growth inhibition with EC50 values ranging from 2.95 to 5.50 µg/mL. The AgNPs displayed exposure time-dependent seed disinfectant potential (complete CFU reduction in F. fujikuroi (40) and F. proliferatum (58) was observed at a concentration of 17.24 µg/mL). The optimized green AgNPs were non-toxic to germinating seeds, and completely cured bakanae under net-house conditions, suggesting their great nano-fungicidal potency for food security and sustainable agriculture.

Cold adaptation: structural and functional characterizations of psychrophilic and mesophilic acetate kinase.

Acetate kinase catalyzes the reversible magnesium-dependent phosphoryl transfer from ATP to acetate to form acetyl phosphate and ADP. Here, we report functional and some structural properties of cold-adapted psychrotrophic enzyme; acetate kinase with those from mesophilic counterpart in Escherichia coli K-12. Recombinant acetate kinase from Shewanella sp. AS-11 (SAK) and E. coli K-12 (EAK) were purified to homogeneity following affinity chromatography and followed by Super Q column chromatography as reported before [44]. Both purified enzymes are shared some of the common properties such as (similar molecular mass, amino acid sequence and similar optimum pH), but characterized shift in the apparent optimum temperature of specific activity to lower temperature as well as by a lower thermal stability compared with EAK. The functional comparisons reveal that SAK is a cold adapted enzyme, having a higher affinity to acetate than EAK. In the acetyl phosphate and ADP-forming direction, the catalytic efficiency (k(cat)/K(m)) for acetate was 8.0 times higher for SAK than EAK at 10 °C. The activity ratio of SAK to EAK was increased with decreasing temperature in both of the forward and backward reactions. Furthermore, the activation energy, enthalpy and entropy in both reaction directions that catalyzed by SAK were lower than those catalyzed by EAK. The model structure of SAK showed the significantly reduced numbers of salt bridges and cation-pi interactions as compared with EAK. These results suggest that weakening of intramolecular electrostatic interactions of SAK is involved in a more flexible structure which is likely to be responsible for its cold adaptation.

Fluorescence studies on the stability, flexibility and substrate-induced conformational changes of acetate kinases from psychrophilic and mesophilic bacteria.

The acetate kinase from the Antarctic psychrophilic Shewanella sp. AS-11 (SAK) has a significantly higher catalytic efficiency at low temperatures when compared with that from mesophilic Escherichia coli K-12 (EAK). To examine the stability and conformational flexibility of SAK and EAK, steady state intrinsic fluorescence studies were performed. EAK contains only one Trp at a position 46, while SAK contains two Trps at positions 46 and 388. From the fluorescence emission spectra, quenching with acrylamide, Cs(+) and I(-) at different temperatures and denaturation with guanidine-HCl, it was revealed that the SAK bears more flexible and unstable structure than that of EAK. Substrate-induced conformational changes reflect that SAK reached transition state through more conformational changes than EAK. In combination of our thermodynamic studies on the enzymatic reaction and present research findings, it can be concluded that these structural features of SAK may contribute to its high catalytic efficiency at low temperatures.