A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

This protocol can help those involved in the watermelon community evaluate the fusarium wilt pathogen, and this will help determine the pathogen populations that are present and help manage it effectively. Race-typing fusarium wilt is complex and time consuming to complete, and having three viable race-typing bioassay options will allow users to choose a method best suited for their experimental conditions. All three methods help plant health practitioners diagnose the potential severity of fusarium wilt infections related to a specific isolate.

This information enables informed and effective integrated disease management decisions. To begin the preparation for planting, fill 8x16 cell starting flats with planting medium while tapping down to compress the soil. Then, sow seeds with their apex pointing upwards to a depth equal to their length.

After seeds are sown, cover the medium containing the buried seeds with Fuller's earth to a depth of 0.3175 to 0.635 centimeters, and then mist the flats to dampen the medium without creating standing or pooling water. Afterward, keep the medium moist by misting for 20 seconds every 180 minutes for five days until seed germination. After germination, water the flats once per day.

Five days post-planting, place infiltrated paper discs of 1 to 1.25 centimeter diameter containing the preferred fusarium oxysporum isolate onto one clarified V8 juice medium and 1/4 strength potato dextrose agar, or QPDA media plate, to store them in an incubator at 28 degrees Celsius for eight days. On the eighth day, transfer the V8 and QPDA plates to a biosafety cabinet before dislodging conidia by scraping a sterile cell spreader across the medium surface and pool the liquid conidia suspension to a sterile 50-milliliter culture tube. Next, quantify the spore counts and prepare the final inoculum solution by transferring the calculated volume of around 1 million spores to the fresh sterile culture tube and bringing the total volume to 30 milliliters with sterile deionized water.

To prepare for the inoculation, place 6x12 cell styrofoam flats previously sanitized with 10%bleach and well rinsed to receive the inoculated plants. Several hours prior to inoculation, thoroughly water the plants. Two hours after watering, remove the plantlets from the 8x16 cell styrofoam flats and rinse their roots.

Then, temporarily store the rinsed plants in clean containers with tap water until use, keeping the cultivars separate from one another. Later, separate the plantlets into groups of six individuals and keep the seedling groups wrapped in wet paper towels on a lab tray. Place 25 to 30 cubic centimeters of soil in the bottom of each cell of the 6x12 array styrofoam tray and use a squirt bottle to wet the soil until it is visibly damp.

Beginning with the healthy control, place a group of the undamaged six seedlings of the same cultivar into the 50-milliliter tubes containing the inoculum. To inoculate, vortex the tubes of plantlets with roots submerged for 30 seconds. Next, place a single plantlet per cell in the 6x12 styrofoam flats with the plants of the same cultivar in one column of the tray.

Cover the plantlets with planting medium and set gently. Use a syringe to wet the plantlet with 20 milliliters of water while avoiding splashing. When all plantlets have been replanted, place the plantlets overnight in an enclosed dark environment with an average temperature of 27 degrees Celsius.

To infest the kernels, prepare inoculum by isolating and culturing a fusarium oxysporum niveum strain on a plate of QPDA to the point that its growth covers half the plate. Prepare the kernels by adding sterile tap water to the one-liter glass Erlenmeyer flasks containing 200 grams of kernels to completely cover the grains up to at least five centimeters. After soaking the kernels in the flasks at 24 degrees Celsius for two hours, drain the water from the flask and plug the flask's opening with a piece of cotton roll wrapped in cheese cloth before covering the opening with aluminum foil wrap.

Sterilize the flasks in the autoclave on a gravity cycle for one hour with five minutes drying time, then allow the flasks to cool to room temperature. Transfer the grains to a mushroom growing bag with a filter. Remove the air from the bag and then fold the excess plastic around it.

Place the bag in a plastic autoclave-safe bin and cover the bin with aluminum foil wrap, then autoclave again using the same settings as before. After autoclaving, allow the fungus to incubate on the kernels in the bag for three weeks. Use a sterile cork bore of number four size to cut agar discs six millimeter in diameter from the zone of active growth on the culture plate.

Unfold the bag to place five agar discs with a strict sterile technique. Use a sterile 50-milliliter graduated cylinder to measure and add 35 milliliters of sterile tap water to the bag. Fill a plastic pot with a potting mix and then add the measured quantity of potting mix into a large plastic bag containing 14 grains of infested kernels.

Create an air cushion in the bag and seal it closed before mixing the kernels and soil by inverting the bag several times. Once done, fill a surface-sterilized pot with the infested soil mixture. Repeat the process for each remaining watermelon variety for a total of four full pots of infested soil per isolate being tested.

Next, sow six watermelon seeds in the pot with the apex end of the seed facing up. Using a spray bottle, wet the upper 0.3 to 0.6 centimeters of soil with water and then place a clear plastic dish under and over each pot. Fill 48 cell inserts with steam-pasteurized sand, peat, vermiculite in the ratio 4:1:1 and tap down the inserts to compress the soil.

Then, place the inserts in plastic trays and sow the seeds as explained earlier. Seven days before planting, inoculate five QPDA plates with infiltrated paper to store them in an incubated area for eight days on a 14 hours/10 hours dark cycle. On the seventh day, transfer two one-square-centimeter agar plugs into each 250-milliliter Erlenmeyer flask containing 100 milliliters of potato dextrose and place the Erlenmeyer flasks on a benchtop shaker.

On the inoculation day, harvest the spores by filtering the inoculum through four layers of sterile cheese cloth to determine the micro-conidial concentration of the flask. 14 days after sowing, transfer the cell inserts with the seedlings into the webbed tray, then place the webbed tray with the seedlings into a plastic tub containing the seven liters of inoculum suspension. After 15 minutes of keeping the inoculum undisturbed, transfer the cell inserts containing the inoculated seedlings into holeless trays and place the holeless trays on the greenhouse bench, followed by watering as needed.

Maintain the lighting and environmental conditions as described earlier for the root dip bioassay. Depending on the method used, start the rating by observing and calculating the incidence of wilt and plant death and take digital images to document the disease progress. In the modified tray dip method, the Black Diamond and Charleston Gray cultivars infected with a race-2 isolate showed severe symptoms and died.

In contrast, the Citrullus amarus PI cultivar showed resistance. In the root dip method, the seedlings infected with a race-0 isolate mostly survived, while nearly all seedlings infected with a race-3 isolate died. In the infested kernel method, Sugar Baby and Charleston Gray cultivars grown in race-2-infested soil have begun to wilt and die, while the Citrullus amarus PI plants still appear healthy.

Remember to sow and maintain plants appropriately, check the pathogen isolate for viability, understand the unique techniques of each inoculation method, and be consistent when assessing disease symptoms. With the results of these methods, researchers can investigate the underlying genetic determinants governing differential variance expression between the isolates through sequencing and comparative genomics analysis. Using these methods, researchers have been able to evaluate the prevalence of different virulence phenotypes across different locations and compare these phenotypes with observable genetic elements.