PCR amplification of the hrcV gene through specific primers for detecting Pseudomonas syringae pathovars.

Pseudomonas syringae pathovars are important pathogens among phytopathogenic bacteria causing a variety of diseases in plants. These pathogens can rapidly disseminate in a large area leading to infection and destruction of plants. To prevent the incidence of the bacteria, appropriate detection methods should be employed. Routinely serological tests, being time-consuming and costly, are exploited to detect these pathogens in plants, soil, water and other resources. Over the recent years, DNA-based detection approaches which are stable, rapid, specific and reliable have been developed and sequence analysis of various genes are widely utilized to identify different strains of P. syringe. However, the greatest limitation of these genes is inability to detect numerous pathovars of P. syringae. Herein, by using bioinformatic analysis, we found the hrcV gene located at pathogenicity islands of bacterial genome with the potential of being used as a new marker for phylogenetic detection of numerous pathovars of P. syringae. Following design of specific primers to hrcV, we amplified a 440 bp fragment. Of 13 assayed pathovars, 11 were detected. Also, through experimental procedures and bioinformatic analysis it was revealed that the designed primers have the capacity to detect 19 pathovars. Our findings suggest that hrcV could be used as a gene with the merit of detecting more pathovars of P. syringae in comparison with other genes used frequently for detection purposes.

Encapsulation of propolis extracted with methylal in the chitosan nanoparticles and its antibacterial and cell cytotoxicity studies.

In this study we develop novel type of antibacterial chitosan-propolis NPs to improve theantimicrobial activity against various pathogens. To this aim, we primarily extracted propolis with methylal and ethanol as green solvents and its encapsulation with chitosan NPs. The developed propolis loaded chitosan NPs indicated antimicrobial and anti-biofilm properties against various gram positive and negative. FTIR revealed the successful encapsulation of the propolis extract with Ethanol (PE) and Methylal (PM) into the chitosan nano career matrix. HPLC and GC-MASS also confirmed the presence of flavonoids and phenols compounds of propolis extracted with both solvents. In addition, we confirmed the total phenolic and flavonoid compounds in propolis by calorimetric method of Folin-Ciocalteu and aluminum trichloride complex formation assays, respectively. PE-CH and PM-CH were optimized regarding physicochemical properties such as particle size, zeta potential, and poly dispersity index (PDI) index. DLS and SEM micrographs confirmed a spherical morphology in a range of 360-420 nm with Z potential values of 30-48 mV and PDI of 0.105-0.166 for PE-CH and PM-CH, respectively. The encapsulation efficiency was evaluated using colorimetric analysis, with median values ranging from 90 to 92%. The MIC values within the range of 2 to 230 µg/ml and MBC values between 3 to 346 µg/ml against both gram-positive and negative bacteria. While both PE and PM showed a significant reduction in the number of E. coli, S. aureus, and S. epidermidis, the use of PE-CH and PM-CH led to a statistically significant and greater reduction in number of E. coli, S. aureus, and S. epidermidis strains on the biofilm, pre-formed biofilm and planktonic phases. Besides, the DPPH assay showed significant antioxidant activity for these NPs within the range of 36 to 92%. MTT assay for MHFB-1, HFF, L929, MDF, and MCF-7 cells exhibited statistically significant differences in each other that show the IC50 between 60-160 µg/ml for normal cells and 20 for cancer cells. Finally the present study indicated that both PM and PM-CH greater than PE and PE-CH in which contain high flavonoid and phenolic contents with a high antioxidation potential antioxidant properties, which could be beneficial for cell proliferation and antibiotic and anticancer applications.