We are attempting to discover new types of chemistry to combat agricultural pathogens. Using iChip technology, we have isolated novel organisms, which we hope will lead to new chemistry. Testing these organisms and/or their natural products against agricultural pathogens potentially lead to finding new biopesticide candidate organisms and/or compounds.
The iChip method allows us to isolate microorganisms that are unculturable under conventional conditions by providing a period of domestication and contact with their natural environment. Our modified iChip build is simple, inexpensive, and reduces the risk of microbial contamination, making it easily accessible for a variety of research goals. Our iChip results open questions like what mechanisms drive pathogen suppression by novel microbes?
Can these bioactive compounds perform reliably in real-world trials? How do environmental factors influence their efficacy? Addressing these questions will guide sustainable biopesticide development, transforming plant disease management, and reducing reliance on synthetic fungicides.
The lab will apply iChip technology to isolate novel bacteria from disease suppressive soils produced through cover cropping, identifying novel microbes and bioactive compounds. These will be tested in controlled plant disease studies to develop innovative, sustainable alternatives to synthetic fungicides for managing agricultural diseases. To construct a modified iChip using a 5 millimeter agar punch tool, remove the bottoms of the wells from a 96-well plate.
Cut 0.05 micrometer polycarbonate membranes into rectangles, ensuring the dimensions match the bottom area of a 96-well plate. Apply a silicone sealant to adhere the polycarbonate membrane to the bottom of the 96-well plate. Ensure the adhesive seals the wells but does not entirely cover the openings of the wells.
Allow the plates to dry for 24 hours. Add 4.5 milliliters of sterile water to each of the 15 milliliter centrifuge tubes labeled A to D, and to each of the 50 milliliter tubes labeled E to H.Add 1 gram of soil into a 50 milliliter centrifuge tube. Then add 10 milliliters of water to the tube and vortex the mixture for 10 minutes.
Allow the soil suspension to settle for 10 minutes. Next, pipette 0.5 milliliters of the soil supernatant into tube A and mix thoroughly. Now, transfer 0.5 milliliters of the cell suspension from tube A to tube B and mix thoroughly.
Repeat for all remaining centrifuge tubes, completing a series of tenfold dilutions across the eight tubes. To begin, submerge the iChips completely in 95%ethanol. After 15 minutes, remove the plates from the ethanol and place them on a sterile paper towel in the laminar flow hood.
Allow the ethanol to evaporate while turning on the ultraviolet sterilizer in the laminar flow hood for 15 minutes. Pipette 360 microliters of sterile succinate minimal salts or SMS media into the first column of the plate to serve as a control. Add 45 milliliters of SMS to the cell suspension in tube E.Mix thoroughly to combine the cell suspension with the agar.
Pipette 360 microliters of the agar cell mixture into all remaining wells of the 96-well plate. Once the media is set, seal the top of the plate with a PCR plate cover. Fill the plates using tubes F, G and H to prepare a total of four plates with tenfold differences in concentrations.
Place the plates in a container containing approximately 1 inch of soil with the membrane side facing down. Incubate the plates inside the covered container in the dark at 25 degrees Celsius. After six weeks, examine the iChips for microbial growth.
Rinse the modified iChip three times with sterile water to remove all soil particulates. Wipe the top and sides of the plate with 95%ethanol, avoiding the side with the semipermeable membrane. Using a sterile blade, cut through the plate cover around a well containing a colony, and remove the square with sterile tweezers.
Using a sterile streaking tool, pierce the colony and streak onto the SMS agar plate with the four quadrant method. Incubate the plates at 25 degrees Celsius for one week or until colony growth is observed. After incubation, examine the colonies to confirm the isolation of cultures.
