The overall goal of this procedure is to accurately quantify mycotoxin biosynthesis, spoilation and growth in order to study molecular seed fungal interactions. This is accomplished by first setting up a kernel bioassay. Next kedia are enumerated, then mycotoxins are quantified.
Finally, the fungal biomass is measured. Ultimately, results can be obtained that show the effects of seed physiology on the reproduction development and secondary metabolism of myco toxigenic fungi through the quantification of kedia biomass and secondary metabolites using spore enumeration techniques coupled with high performance liquid chromatography. The main advantage of propagating this technique is to standardize methods across multiple labs to results from different laboratories can be cross interpreted.
This method can help answer key questions in the field of molecular seed microbe interactions such as the mode by which pathogenic fungi hijack plant lipid metabolism as a virulent Strategy. Generally, individuals new to this method may struggle because different strains of angi exhibit different growths and developmental patterns. So it is imperative that you optimize the conditions for your favorite fungal seed interaction Two weeks prior to performing the maze.
Kernel bioassay culture. The fungal pathogens on potato dextrose agar or PDA at 28 degrees Celsius. When ready to begin select kernels similar in age, shape, and size, and place them in 50 milliliter tubes.Surface.
Sterilize the kernels by adding 70%ethanol and shaking the tubes at room temperature for five minutes. Next, shake with sterile water for one minute and with 6%sodium hypochlorite for 10 minutes. Rinse with sterile water three times with shaking for five minutes each.
Then pat the kernels dry on autoclave towels to infect with aspergillus flavus. Use an 18 gauge needle to create a small wound, 0.5 centimeters deep on the embryo side of each kernel. For a fusarium vertices infection, use a razor blade to make a 0.5 millimeter deep cut.
Prepare the inoculum suspension by adding five milliliters of autoclave, 0.1%tween 20 solution to the cultured plates, and use cell spreader to scrape the spores. Remove the mycelium by gravity, filtering the solution through at least four layers of autoclave cheesecloth. Use a hemo cytometer to calculate the spore concentration and adjust to tend to the six spores per milliliter.
For both fungal species. Create a humidity chamber in a plastic container by lining it with five sheets of paper towels and adding 100 milliliters of sterile water. Next place four kernels into each autoclave.
20 milliliter glass in installation vial. Weigh them and record their mass. Add 200 microliters of spore suspension to each vial cap and vortex to evenly coat the kernels with the suspension.
Then loosen the caps to freely allow air exchange and place the vials into the humidity chamber. Incubate the kernels under a 12 hour light 12 hour dark photo period at 28 degrees Celsius for seven days or until desired After the infection period, add five millimeters of autoclave 0.01%between 20 to the simulation vials containing infected kernels and vortex thoroughly for one minute. Using a wide VRE pipette tip, immediately dilute the spore suspension by transferring two separate 200 microliter aliquots into two milliliter einor tubes containing 1.8 milliliters of 0.01%Tween 20.
Use a hemo cytometer to count each aliquot twice. To quantify aflatoxin, add the kernels from a single sample into a 50 milliliter blender cup with 20 milliliters of 80%methanol and 0.05 grams of sodium chloride. Cover and blend at high speed for one minute.
Place a fluided filter paper over a clean collection vessel and pour the extract into the filter. Transfer 10 milliliters of filtrate into a clean vessel. Add 20 milliliters of distilled water and mix well.
Filter the sample through a 1.5 micron glass microfiber filter into a clean cup. Apply one milliliter of filtered extract to an ALE test column and using compressed air force the filtered extract through the column at a rate of one to two drops per second. Wash the column twice with one milliliter of distilled water by passing it through the column at one to two drops per second until air comes through.
Elute the column with one milliliter of 100%HPLC grade methanol at a one to two drop per second rate collected into a Glass Q Vet. Add one milliliter of ale test developer to the EIT and mix well. Place the Q VET into a calibrated fluorimeter and read at 60 seconds For acin B one or FB one analysis, add 10 milliliters of a 50 50 solution of acetonide trial in water to each kernel bioassay sample and extract overnight at a room temperature without agitation.
Mix the extract with six milliliters of deionized water and apply it to a C 18 solid phase column that has been preconditioned with two milliliters of acetyl nitrile, followed by two milliliters of water. After loading the sample, wash the column with two milliliters of water, followed by two milliliters of acetyl nitrile in water. Elute the FB one containing sample for HPLC analysis with two milliliters of acetyl nitrile in water.
Derivatize the FB one by transferring one milliliter of the column EIT to a vial containing 0.1 milliliters of borate buffer and 0.1 milliliters of aldehyde and incubate for 10 minutes. Stop the reaction by adding 0.5 milliliters of an acetyl nitrile and 0.01 molar boric acid solution. Analyze FB one by HPLC using a linear gradient of acetyl nitrile 0.1 molar sodium phosphate.
Compare the samples to an FB one standard curve to analyze sterol culture, the fungus on corn for seven to 14 days. After the incubation period, add 10 milliliters of chloroform methanol to each vial. Shake the samples well and then incubate the vials in the dark at room temperature for 24 hours.
Centrifuge the samples at 5, 000 RPM for three minutes. Then filter the supernatant through a 0.45 micron nylon membrane. Inject the sample directly into an HBLC column and elude with 100%methanol at a flow rate of 1.5 milliliters per minute.
Quantify the samples by comparing peak areas to a standard curve generated from HPLC grade or goole. Following inoculation and incubation in a humidity chamber for two to three days. Fungal growth should begin to appear on the kernels as shown here.
Seventies post-treatment vegetative growth on inoculated kernels should be clearly visible while mock controls should be uninfected, periods of longer incubation are conducive to more copious vegetative growth. This figure shows that a flavus wild type NR RL 3 3 5 7 displayed maximum values of colonization, aflatoxin, accumulation, and kedia production at four, six, and eight days respectively. However, when mycotoxin contamination and tion were compared per unit of rol, the greatest levels were observed at four and six days respectively.
Shown here are the representative for fumonisin and rol from HPLC chromatograms using methods demonstrated in this video. Fumonisin levels range from 3, 500 to 8, 000 nanograms per gram of kernel and rol levels range from 5, 000 to 10, 000 nanograms per gram of kernel. Once the kernel bioassays are done, ation, mycotoxins and fungal biomass can all be measured within two to four days, depending on the number of experimental samples.
While attempting this procedure, it is important to remember to carry off the methods in as sterile field as possible Following this procedure. Other methods like phyto, harmonics, lipidomics and proteomics, transcriptomics or other inter atomics can be performed in order to answer additional questions like which genes, proteins, and hormone pathways are involved in this important cross kingdom interactions.
