We would like to acknowledge Dr. Ali and the Plant Molecular Diagnostic Laboratory as well as Dr. Pingsheng Ji at the University of Georgia, whose leadership and support helped establish our Fon program.

1. Determining race by root-dip method (RDM)
<ol>
	<li>Preparation of&#160;the experimental environment
	<ol>
		<li>Because symptom expression is highly dependent on environmental conditions, maintain plants in a controlled area. Monitor relative humidity, temperature, photoperiod, and light intensity (Figure 1).
		<ol>
			<li>Set the temperature to 26-28 &#176;C, relative humidity to 50-75%, and set a 16 h photoperiod to ensure adequate plant growth and health. 
			N...

Three methods of race typing have been presented. Each of these methods is best suited to particular questions and experimental conditions. The infested kernel inoculation method (soil infestation) is perhaps simpler and more straightforward, making it especially useful for the assessment of pathogenicity30. Using this method for simple race-typing is highly effective. However, applying the method to determine the resistance of a specific cultivar could be challenging, given that each plant may no...

The soilborne fungi that make up the Fusarium oxysporum species complex (FOSC) are impactful hemibiotrophic plant pathogens that can cause serious disease and yield loss in a diverse range of crops1. Fusarium wilt of watermelon, caused by F. oxysporum f. sp. niveum (Fon), has been increasing in scope, incidence, and severity across the world in the last several decades2,3. In seedlings, the symptoms of Fusarium wilt often resemble damping-off. In older plants, the foliage becomes gray, chlorotic, and necrotic. Eventually, wilting of the plants progresses to full plant collapse and death4. Direct yield loss occurs due to the symptoms and plant death, while indirect yield loss can occur due to sun damage caused by the elimination of the foliar canopy5. Sexual reproduction and associated reproductive structures have never been observed in F. oxysporum. However, the pathogen produces two types of asexual spores, micro- and macroconidia, as well as larger, long-term survival structures called chlamydospores, which can survive in the soil for many years6.
The FOSC is classified into formae speciales based on observed host ranges, usually limited to one or a few host species1. Although recent research has indicated that this species complex may be a composite of 15 different species, the particular species that infect watermelon are currently unknown7. F. oxysporum f. sp. niveum (Fon) is the name for the groups of strains that exclusively infect Citrullus lanatus or the domesticated watermelon8,9. F. oxysporum strains within most pathogenic formae speciales display certain levels of diversity with regard to their genetic components and virulence toward a host species. For instance, one strain may infect all cultivars of a host, whereas another may only infect the more susceptible cultivars. To account for such variation, these groups are informally classified into races based on evolutionary relationships or common phenotypic characteristics. Within Fon, four races (0, 1, 2, and 3) have been characterized based on their pathogenicity against a set of select watermelon cultivars, with the discovery of race 3 occurring recently10.
Despite this apparent diversity, the morphologies of spores or hyphae are not distinguishable between the races of Fon races, meaning that molecular or phenotypic assays are needed to identify an isolate&#39;s unique race11. Molecular research has identified some genetic differences. For example, the role of Secreted in Xylem (SIX) effectors has been studied for years in F. oxysporum, and some of these effectors have been located on the chromosomes exchanged during horizontal gene transfer12. For example, SIX6 is found in Fon races 0 and 1 but not in race 213. SIX effectors have been implicated in the pathogenicity of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense, which cause Fusarium wilt on tomato and banana, respectively14,15,16,17. The analysis of SIX effector profiles among strains of F. oxysporum f. sp. spiniciae, the Fusarium wilt pathogen on spinach, has enabled classification that accurately reflects genetic and phenotypic diversity18. However, the differences between virulence mechanisms of Fon races are currently not entirely understood, and molecular assays developed upon their use have shown inconsistent and inaccurate results19. Therefore, phenotypic results from infection assays are currently the best way to classify isolates.
F. oxysporum initially infects hosts through the roots before making its way up the xylem20. This makes direct inoculation of the roots of a given host cultivar an effective way to perform race-typing and is the basis of the root-dip and tray-dip inoculation methods21. When not infecting a host, F. oxysporum resides in the soil and can remain dormant for years. Growing susceptible watermelon cultivars in soil from a field of interest is one way to test for the presence of Fon. Expanding this method to include cultivars of different known levels of resistance in soil that is deliberately infested with Fon is also a good way to perform race-typing (Table 1) and is the basis of the infested kernel seeding method. The modified tray-dip method is a variation of the original tray-dip method that allows for a high-throughput race-typing where many plants and field isolates can be investigated rapidly22. Important factors of a quick and successful race-typing bioassay include using cultivars that have documented differences in resistance to the different pathogen races, ensuring that the inoculum is both biologically active and abundant during infection, maintaining an environment that is both conducive for the pathogen and host, and using a consistent rating system for severity or incidence of disease. This paper describes the root-dip23,24, infested kernel seeding25,26, and modified tray-dip22 methods for phenotypic race-typing based on the principles described above.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100% Fuller&#8217;s Earth</td>
			<td>Sigma-Aldrich</td>
			<td>F200-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L glass Erlenmeyer Flask</td>
			<td>PYREX</td>
			<td>4980-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL graduated cylinder</td>
			<td>Lab Safety Supply</td>
			<td>41121805</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Eppendorf Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-413-921</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil wrap</td>
			<td>Reynolds Wrap</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Walmart</td>
			<td>587192290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Fisher Scientific</td>
			<td>03-391-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>sigma-Aldrich</td>
			<td>239216</td>
			<td></td>
		</tr>
		<tr>
			<td>cell spreaders</td>
			<td>Fisher Scientific</td>
			<td>08-100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>Lions Services, Inc</td>
			<td>8305-01-125-0725</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear plastic dishes</td>
			<td>Visions Wave</td>
			<td>999RP6CLSS</td>
			<td>~15 cm diameter</td>
		</tr>
		<tr>
			<td>Clear vinyl tubing for mushroom bag clamps</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>6&#34; for small bag, 8&#34; for medium bag, 10&#34; for large bag</td>
		</tr>
		<tr>
			<td>Cotton Balls</td>
			<td>Fisherbrand</td>
			<td>22-456-885</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Chemical</td>
			<td>A4094</td>
			<td>100%, then combine with water to make 70% for use</td>
		</tr>
		<tr>
			<td>Flourescent Tube Lights</td>
			<td>MaxLume</td>
			<td>Model T5</td>
			<td>2800 K Color Temperature, 24&#39;&#39; or 48&#39;&#39; long</td>
		</tr>
		<tr>
			<td>granulated agar</td>
			<td>VWR International</td>
			<td>90000-786</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-held Spray Bottle</td>
			<td>Ability One</td>
			<td>24122002</td>
			<td>~0.95 L</td>
		</tr>
		<tr>
			<td>hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>02-671-55A</td>
			<td>Two chamber hemacytometer</td>
		</tr>
		<tr>
			<td>Lab trays</td>
			<td>Fisher Scientific</td>
			<td>15-236-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Large, sealable plastic bags</td>
			<td>Ziploc</td>
			<td>430805</td>
			<td>38 cm x 38 cm</td>
		</tr>
		<tr>
			<td>Mister / watering can</td>
			<td>Bar5F</td>
			<td>B10H22</td>
			<td></td>
		</tr>
		<tr>
			<td>Mushroom Bag Clamp</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>6&#34; for small bag, 8&#34; for medium bag, 10&#34; for large bag</td>
		</tr>
		<tr>
			<td>Nitrile Gloves</td>
			<td>Fisher Scientific</td>
			<td>19-130-1597D</td>
			<td></td>
		</tr>
		<tr>
			<td>Organic Rye Berries</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>0.5 gallon or 25 lb bags</td>
		</tr>
		<tr>
			<td>P1000 pipette and tips</td>
			<td>Fisher Scientific</td>
			<td>14-388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisherbrand</td>
			<td>FB0875713</td>
			<td>Round, 100 mm diameter, 15 mm height</td>
		</tr>
		<tr>
			<td>Planting media</td>
			<td>Jolly Gardener</td>
			<td>Pro-Line C/B</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pitcher</td>
			<td>BrandTech</td>
			<td>UX0600850</td>
			<td>1 L or larger</td>
		</tr>
		<tr>
			<td>Plastic planting pots</td>
			<td>Neo/SCI</td>
			<td>01-1177</td>
			<td>~15 cm diameter and ~10 cm height</td>
		</tr>
		<tr>
			<td>Plastic, autoclave-safe bin</td>
			<td>Thermo Scientific</td>
			<td>UX0601022</td>
			<td>3 L</td>
		</tr>
		<tr>
			<td>Quarter-strength potato dextrose agar media</td>
			<td>Cole-Parmer</td>
			<td>UX1420028</td>
			<td>Use powder in combination with recipe for QPDA</td>
		</tr>
		<tr>
			<td>Scientific Balance Scale, measuring in g</td>
			<td>Ohaus</td>
			<td>30208458</td>
			<td>Any precise scale that can hold and measure 200g will work</td>
		</tr>
		<tr>
			<td>Size #4 cork bore</td>
			<td>Cole-Parmer</td>
			<td>NC9585352</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Mushroom grow bag</td>
			<td>Shroom Supply</td>
			<td></td>
			<td>0.5 micron filter, also comes in medium and large sizes</td>
		</tr>
		<tr>
			<td>Soil trowel</td>
			<td>Walmart</td>
			<td>563876946</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam flats (6 x 12 cells)</td>
			<td>Speedling</td>
			<td>Model TR72A</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam flats (8 x 16 cells)</td>
			<td>Speedling</td>
			<td>Model TR128A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (5 or 10 mL)</td>
			<td>fisher Scientific</td>
			<td>14-829-19C</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Walmart</td>
			<td>TM-01</td>
			<td></td>
		</tr>
		<tr>
			<td>V8 Original 100% Vegetable Juice</td>
			<td>Walmart</td>
			<td>564638212</td>
			<td></td>
		</tr>
		<tr>
			<td>vortex</td>
			<td>Fisher Scientific</td>
			<td>02-215-418</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - Black Diamond</td>
			<td>Willhite Seed Inc</td>
			<td>17</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - Calhoun Gray</td>
			<td>Holmes Seed Company</td>
			<td>4440</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - Charleston Gray</td>
			<td>Bonnie Plants</td>
			<td>7.15339E+11</td>
			<td></td>
		</tr>
		<tr>
			<td>Watermelon Seed - PI 296341-FR</td>
			<td>Contact authors</td>
			<td>Contact authors</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Kernels (Maxie var.) (optional)</td>
			<td>Alachua County Feed &#38; Seed</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a contrast of three inoculation techniques used to determine the race of unknown fusarium oxysporum f.sp. niveum isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the southeastern USA, especially in Florida. Deployment of integrated pest management strategies, such as race-specific resistant cultivars, requires information on the diversity and population density of the pathogen in growers' fields. Despite some progress in developing molecular diagnostic tools to identify pathogen isolates, race determination often requires bioassay approaches.
Race typing was conducted by root-dip inoculation, infested kernel seeding method, and the modified tray-dip method with each of the four watermelon differentials (Black Diamond, Charleston Grey, Calhoun Grey, Plant Introduction 296341-FR). Isolates are assigned a race designation by calculation of disease incidence five weeks after inoculation. If less than 33% of the plants for a particular cultivar were symptomatic, they were categorized as resistant. Those cultivars with incidence greater than 33% were regarded as susceptible. This paper describes three different methods of inoculation to ascertain race, root-dip, infested kernel, and modified tray-dip inoculation, whose applications vary according to the experimental design.

These experiments help define the relative resistance of commonly grown cultivars (Table 1). This information can then be used to guide management recommendations based on local Fon populations. In other words, if race 0 or 1 is known to be present in a commercial field, then the farmer may be inclined to grow a &#34;resistant&#34; variety such as Calhoun Gray, Sunsugar, or equivalent. The results of the bioassays using all methods show that when the seedlings were infected with a Race 1 isolate, the Bla...

Watch this Scientific Journal Video about A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates at JoVE.com

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

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

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

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the ...

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.

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

Abstract