In the agricultural industry, research concerning the discovery of alternatives to synthetic pesticides has been growing significantly over the past two decades. By examining the natural products that bioactive soil microbes produce, our team envisions discovering new bio pesticides that already exist in the environment. Thin-layer chromatography-direct bioautography, or TLC-DB has primarily been used to determine the bio-activity of plant extracts against pathogenic bacteria and fungi.
In this video, bacterial extracts are assayed without the need to induce sporulation of the pathogen, which allows for the examination of a wider range of pathogens. TLC-DB will continue to be one of the tools that we use to focus on the discovery of novel natural products for bio-control. This will benefit the agriculture and agrifood sectors and help mitigate the resistance that pathogens have been developing to commercial pesticides.
To begin, using a pipette, spot five microliters of each extract two centimeters apart on the dots marked on the bottom line of the thin-layer chromatography, or TLC plate. Let the spots dry on the TLC plate and repeat until about five milligrams of each extract are loaded onto the plate. Prepare a one to two solution of dichloromethane and methanol in a TLC developing tank.
Put the TLC plate into the developing tank and develop it until the solvent reaches the line marked two centimeters from the top of the plate. Once developed, remove the TLC plate from the tank Immediately, spot the positive controls on the dots above the solvent line. Place the plate in an ethanol sterilized TLC plate box before all solvent evaporates.
Scrape the mycelial mat of the five pathogen plates using a sterile metal spatula, then transfer it into a 50 milliliter centrifuge tube. After adding 25 milliliters of potato dextrose broth amended with agar, add sterile glass beads to the tube and vortex for five minutes to break up the mycelial mat. After assembling the chromatography sprayer in the hood, attach the tubing, then attach the flow meter to the setup.
Transfer the mycelial suspension to the chromatography sprayer using a sterile syringe with a needle tip to ensure large pieces of mycelia do not clog the sprayer. Place the previously developed TLC plates on sterile paper towels in the laminar flow hood to reduce hand contact with the plate while applying the media suspension. Connect the pathogen sample to the assembled sprayer and apply three coats of the suspension to the TLC plate, allowing the plate to dry completely between applications.
Next, prepare the incubation setup by adding 50 milliliters of sterile water into a sterilized plastic plate box. Place four Petri plates in it for the TLC plate to sit on. Also, place sterilized folded cellulose sheets or filter paper on each side of the box to hold moisture.
Place the completed assay plate onto four empty Petri dishes in the box. Incubate the assay for three days to one week or until an even layer of mycelia grows across the plate, except for around the positive controls and zone of inhibition. Then, using a metal spoon, scrape the silica off the inhibition zones and place it into micro centrifuge tubes.
Add 500 microliters of methanol to each tube and vortex to extract the metabolites from the silica. Centrifuge the tubes to pellet the silica, and transfer the supernatant to liquid chromatography vials for analysis. Imaging under ultraviolet light showed the separation of metabolites on the TLC plate.
After the incubation, the pathogen appeared to grow evenly across the entire plate, except over the positive controls and the inhibition zones.
