Evaluation of Phytochemical, Antimicrobial, Antioxidant Activity and Cytotoxic Potentials of Agave americana

Ethnomedicinal plants are being used as a source of medicine from ancient time but they lack the proof of modern scientific evidence for their effectiveness. This study focuses on the evaluation of phytochemical, antimicrobial, antioxidant properties of one of the ethnomedicinal plant Agave americana from Dhulikhel region of Nepal. The plant extract was prepared using solvent-based warm soxhlet extraction from the leaves of the plant and antimicrobial activity against six different non-resistant clinical isolates of bacteria (Staphylococcus aureus, Shigella, Klebsiella pneumoniae, Escherichia coli, Bacillus thuringiensis, and Salmonella paratyphi) was evaluated using agar disc diffusion method along with qualitative analysis for presence/absence of phytochemicals. Antioxidant activity was measured by DPPH assay and the cytotoxicity was evaluated using MCF-7 (human breast adenocarcinoma) cancer cell. Presence of phytochemicals like alkaloids, flavonoids, reducing sugars and saponins were detected in the plant extract. The extract was found to show some level of antimicrobial activity against Staphylococcus aureus, Escherichia coli and Bacillus thuringiensis at 50, 100 and 200 mg/ml. The IC50 value of the extract was found to be 7.68 μg/ml. The extracts of Agave americana showed 50 % cell-death of MCF-7 in 12 h at 5 μg/ml. Although this study provided some scientific evidence for the medicinal value of Agave americana, further studies are still needed for the detailed evaluations of every molecule present in this plant along with screening in larger geographical area of Nepal

Phytochemical screening, antimicrobial activity and cytotoxicity of Nepalese medicinal plants Swertia chirayita and Dendrobium amoenum

Research on medicinal plants are important to Nepal because most of its rural population relies on it as mode of medicine. Medicinal plants namely Swertia chirayita and Dendrobium amoenum were collected from mid hills of Nepal. The present study was undertaken to find the antimicrobial activity, phytochemical presence and their cytotoxicity in different extraction medium. The percentage yield from the plants were highest in warm methanol extraction with 12.6%, followed by ethyl acetate and lowest was for cold methanol. Plant extract showed the presence of antioxidants like alkaloid, terpenoids, flavonoids, tannin, glycosides. The Brine Shrimp Bioassay of methanol and ethyl acetate extract showed cytotoxicity. Chiraito extract showed LC50 of 199 ppm for Dhunche sample, 128.82 ppm for Daman sample and 131.82 ppm of Illam sample. The antibacterial activity of methanol extract of Chiraito and Dendrobium amoenum showed significant bioactivity by inhibiting growth of microbial species selected for the test. The zone of inhibition shown by the extracts was comparable to the standard antibiotics. Similarly, methanol extract of Chiraito also showed significant antifungal activity with the zone of inhibition comparable to amphotericin.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 48-5

Evaluation of Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis for biological control of Ralstonia wilt of tomato [version 3; referees: 2 approved]

Background: Ralstonia spp. is a major pathogenic microbe for tomato, which invades the roots of diverse plant hosts and colonizes xylem vessels causing wilt, especially in tropical, subtropical and warm-temperate regions. Ralstonia spp. produces several virulence factors helping it to invade the plant’s natural defense mechanism. Native isolates of Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis can be used as biocontrol agents to control the bacterial wilt and combined application of these beneficial microbes can give better results. Methods: Bacterial wilt infection in the field was identified by field experts and the infected plant part was used to isolate Ralstonia spp. in CPG media and was positively identified. Subsequently, the efficacy of the biocontrol agents was tested and documented using agar well diffusion technique and digital microscopy. 2ml of the microbial concentrate (109 cells/ml) was mixed in one liter of water and was applied in the plant root at the rate of 100 ml per plant as a treatment method. Results: It was observed that the isolated Trichoderma spp. AA2 and Pseudomonas fluorescens PFS were most potent in inhibiting the growth of Ralstonia spp., showing ZOI 20.67 mm and 22.33 mm, respectively. Digital microscopy showed distinct inhibitory effect on the growth and survival of Ralstonia spp. The results from the field data indicated that Trichoderma spp. and Pseudomonas fluorescens alone were able to prevent 92% and 96% of the infection and combination of both were more effective, preventing 97% of infection. Chemical control methods prevented 94% of infection. Bacillus subtilis could only prevent 84 % of the infection.   Conclusions: Antagonistic effect against Ralstonia spp. shown by native isolates of Trichoderma spp. and P. fluorescens manifested the promising potential as biocontrol agents. Combined application gave better results. Results shown by Bacillus subtilis were not significant

Evaluation of Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis for biological control of Ralstonia wilt of tomato [version 2; referees: 2 approved]

Background: Ralstonia spp. is a major pathogenic microbe for tomato, which invades the roots of diverse plant hosts and colonizes xylem vessels causing wilt, especially in tropical, subtropical and warm-temperate regions. Ralstonia spp. produces several virulence factors helping it to invade the plant’s natural defense mechanism. Native isolates of Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis can be used as biocontrol agents to control the bacterial wilt and combined application of these beneficial microbes can give better results. Methods: Bacterial wilt infection in the field was identified by field experts and the infected plant part was used to isolate Ralstonia spp. in CPG media and was positively identified. Subsequently, the efficacy of the biocontrol agents was tested and documented using agar well diffusion technique and digital microscopy. 2ml of the microbial concentrate (109 cells/ml) was mixed in one liter of water and was applied in the plant root at the rate of 100 ml per plant as a treatment method. Results: It was observed that the isolated Trichoderma spp. AA2 and Pseudomonas fluorescens PFS were most potent in inhibiting the growth of Ralstonia spp., showing ZOI 20.67 mm and 22.33 mm, respectively. Digital microscopy showed distinct inhibitory effect on the growth and survival of Ralstonia spp. The results from the field data indicated that Trichoderma spp. and Pseudomonas fluorescens alone were able to prevent 92% and 96% of the infection and combination of both were more effective, preventing 97% of infection. Chemical control methods prevented 94% of infection. Bacillus subtilis could only prevent 84 % of the infection.   Conclusions: Antagonistic effect against Ralstonia spp. shown by native isolates of Trichoderma spp. and P. fluorescens manifested the promising potential as biocontrol agents. Combined application gave better results. Results shown by Bacillus subtilis were not significant

Field Images of plot design showing pictures of before the treatment and effects after the treatment.

There is significant decrease in the occurrence of disease for the treated plots whereas the bacterial wilt has severely effected in the untreated plot. <div>We also confirm we have permission from the individuals to use the image ‘Field Counselling’’<br></div

Images of the plates showing the zone of inhibition by different antagonists against Ralstonia spp.

Clear zone of inhibition obtained by agar well diffusion technique indicates the bioefficacy of the selected bioantagonists against R. solanacearum