Molecular characterization of Fusarium oxysporum by PCR amplification of 18S rRNA gene region

The genus Fusarium is one of the most diverse and pathologically important fungi with affinities established to Ascomycotina. Usual identification of Fusarium species based on their micro and macroscopic features and morphological characters alone may lead to incorrect species designation. In order to identify the correct species, we amplified the 18S rRNA gene region by PCR, sequenced and analyzed for sequence similarity among the NCBI data. Fusarium oxysporum, a soil borne plant pathogen was isolated from the Allium cepa. The isolate was cultured in fresh potato dextrose agar (PDA) plates for 7 days. Morphological identification was done by observing under microscope after lacto-phenol cotton blue staining. Genomic DNA isolation was carried on the grown Fusarium oxysporum. Further, PCR amplification of ITS regions was performed using Universal ITS primers. The amplified product of 18S rRNA gene was sequenced and submitted to NCBI database. The soil borne fungal pathogen of onion was identified as Fusarium oxysporum based on its morphological and molecular characteristics

Antimicrobial Activity of Protein Hydrolysate from Marine Molluscs Babylonia spirata (Linnaeus, 1758).

The present study is to investigate the antimicrobial activity of protein hydrolysate of marine water mollusks Babylonia spirata (Linnaeus, 1758). Protein hydrolysate was prepared from tissue of Babylonia spirata by enzymatic hydrolysis. Enzyme digestion were carried out with the enzyme Trypsin. The protein concentration was estimated by Bradford’s method and the protein quantification was done by using SDS PAGE analysis. Antibacterial assay was carried out against four bacterial pathogens by agar well diffusion method and antifungal activity was performed against three human pathogenic fungal strains. 2.6mg/ml protein concentration was estimated by Bradford’s method and 40 to 200 kDa protein bands were resulted in SDS PAGE analysis. In antimicrobial activity, the maximum zone of inhibition was observed against Staphylococcus aureus22.16 +1.04mm at 1000μg/ml concentration and the maximum zone of inhibition was observed in Aspergillus fumigatus13.5+0.5 in 1000μg/ml concentration. These results are signify that the protein hydrolysate of marine molluscs Babylonia spirata express remarkable antimicrobial activity.

Optimization of cholesterol oxidase production and 16S rRNA partial sequence of Bacillus cereus strain KAVK4 isolated from butter.

The aim of the present research work was to optimize cholesterol oxidase production and partial sequencing of 16S rRNA gene region of Bacillus cereus strain KAVK4 by PCR. Bacillus cereus is a gram positive bacterium isolated from butter, Tamil Nadu, India and was cultured in a nutrient medium containing cholesterol at 37°C for 24 hours. Partial sequence of 16S rRNA gene was amplified by PCR and sequenced, the sequence data was submitted to NCBI. The production of cholesterol oxidase of Bacillus cereus strain KAVK4 was analyzed by qualitative methods such as Colony staining method & Cholesterol Oxidase indicator plate method and quantitative method. The maximum production of cholesterol oxidase was checked in media with different carbon source, nitrogen source, and metal ions source. The different physical parameters like pH, temperature and time were checked. Bacillus cereus strain KAVK4 partial sequence of 16S rRNA gene region resulted 1366bp. The sequence data was submitted to NCBI and the accession number is KP792775. Bacillus cereus strain KAVK4 was optimized at media containing Fructose, Beef Extract, Ammonium Nitrate and Magnesium sulphate at pH 7.5 at Room temperature and incubation period was about 32 hrs. Bacillus cereus strain KAVK4 was found to produce maximum level of cholesterol oxidase (1.67 U/ml). The result obtained in this work was clearly indicated that Bacillus cereus strain KAVK4 was capable of producing maximum level of cholesterol oxidase at pH 7.5 and incubation was 32 hrs.