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.

The authors declare that they have no competing financial interests.

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 not face the same degree of infection or exposure, and equally high levels of disease may be needed to test the resistance of the cultivars of interest. This is the case because inoculum produced in this manner is not well quantified, and the proportion of viable propagules, or the number of infectious propagules that reach the root zone, is not well regulated31. Additionally, this method is limited by inconsistencies in the proximity of the planted kernels to the root zone. If too distant, the spores may not germinate, or hyphae may not develop enough to reach the roots.
The root-dip method32,33 is more laborious and time-consuming; however, because the quantity of viable propagules interacting with the plant is more accurately measured, host resistance can be more accurately described, facilitating resistance screening. Moreover, differences in virulence within the same race can be more easily detected. This method has the added benefit that generally, plants become symptomatic earlier and more expressively than in the kernel method. One variant of the root-dip method using chlamydospores in the inoculum suspension instead of conidia may lack this benefit6. Similarly, the modified tray-dip method22 is labor-intensive but allows for high-throughput phenotyping when many isolates and seedlings need to be screened.
Shared factors for the three methods include cultivar selection, growing conditions, and requirements for hygiene. Depending on what is commercially available, certain cultivars can be substituted21,34. Sugar Baby and Black Diamond can both be used to determine race 0 isolates, while Charleston Gray, Allsweet, and Dixielee have been described as resistant to race 0 but susceptible to race 1. Calhoun Gray and Sunsugar are resistant to races 0 and 1 and susceptible to races 2 and 3. Fon disease development is highly dependent on temperature. Care should be taken to ensure that the experimental conditions control for this variable. When choosing a planting medium, general commercial mixes that include peat moss and/or gypsum and allow for good aeration should be satisfactory. Precautions should be taken to prevent cross-contamination of the planting media in both methods, especially from the source bag.
After using one of the described methods, disease must be accurately and consistently assessed. Previous researchers have typically decided on a threshold at which plants are categorized as either susceptible or resistant35,36. For example, if less than 33% of plants of a specific cultivar were symptomatic, then that cultivar would be categorized as resistant with the isolate defined in relation to the susceptible profile of the cultivar. The threshold set is defined by the researcher and the question they wish to address. Variability between raters and by the same rater between plants has been widely reported37,38. Factors such as quality of the seed used, soil quality, inoculum density, storage age of isolates, and rater bias39,40 all contribute to this variability8. Due to this variability in inoculation and PI cultivar responses, multiple experimental replications are needed; ideally, at least three but five replications are recommended, with 6 plants per replication per variety.
While molecular assays have been developed to detect Fon isolates41,42,43, results have not been consistent due to the polyphyletic nature of F. oxysporum and the geographical and genomic variability of the species complex44,45,46. Furthermore, although prior research has established the importance of the Secreted in Xylem (SIX) effectors in full virulence, the exact complement of effectors that define the racial structure of Fon isolates has yet to be determined13. Molecular diagnostics for race are still being developed, for which these phenotypic techniques are critical to assessing their accuracy and utility in Fon race typing19,47.

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><tbody><tr><td>100% Fuller&amp;rsquo;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>CaCO&lt;sub&gt;3&lt;/sub&gt;</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&quot; for small bag, 8&quot; for medium bag, 10&quot; 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&#039;&#039; or 48&#039;&#039; 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&quot; for small bag, 8&quot; for medium bag, 10&quot; 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 &amp;amp; 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 Black Diamond and Charleston Grey cultivars died or showed serious symptoms, while the Calhoun Grey and PI cultivars showed resistance (Table 2 and Figure 8A).
All methods showed that when the seedlings were infected with a Race 3 isolate, nearly all the plants from all cultivars died or showed serious symptoms (Figure 8B). These results demonstrate how bioassays using both inoculation methods successfully differentiate between races of Fon. The appearance of diseased plants should be the same for all methods. The only difference is in how the cultivars are grouped spatially. For the root-dip and modified dray-dip methods, the cultivars will be organized by columns of the tray, whereas in the kernel method, the cultivars will be grouped in their own pots.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63181/63181fig01.jpg" /> 
Figure 1: Experimental area for RDM. Due to symptom variability, which is highly dependent on environmental conditions such as relative humidity, temperature, photoperiod, and light intensity, maintaining a regulated experimental area is important. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63181/63181fig02.jpg" /> 
Figure 2: Preparing the starting flats for RDM. Fill 8 x 16-cell (25 cm width x 50 cm length) starting flats with planting medium and tap down to slightly compress the soil. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63181/63181fig03.jpg" /> 
Figure 3: Preparation of conidial suspension for RDM. (A) Isolation and culturing. Either from a stored or newly collected sample, isolate and culture a F. oxysporum f. sp. niveum strain of interest on a plate of qPDA to the point that its growth covers half the plate. This demonstrates that it is active and viable, which is necessary for substantial infestation of the grain in later steps. (B)&#160;Dislodging conidia. Dislodge conidia by scraping a sterile cell spreader across the medium surface. (C) Suspension deposition. Pool the liquid conidia suspension and transfer it to a sterile 50 mL culture tube. Abbreviation: qPDA = one-quarter strength potato dextrose agar medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63181/63181fig04.jpg" /> 
Figure 4: Organization and vortexing of seedlings for RDM. (A) Separation of cultivars. Temporarily store rinsed plants in clean containers with tap water until use, keeping cultivars separate. (B)&#160;Vortexing of seedlings. Vortex the tubes with plantlet roots submerged for 30 s for planting a single plantlet per cell in the 6 x 12 styrofoam flats. Plants of the same cultivar are placed in the same column in the tray. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63181/63181fig05.jpg" /> 
Figure 5: Preparation and infestation of kernels for IKM. (A) Imbibition of rye berries. On a scale, measure out 200 g of rye (Secale spp.) berries (or Maxie var. wheat (Triticum spp.) kernels) in any sufficiently large container and pour them into one or more 1 L glass Erlenmeyer flasks. Add sterile tap water into the flasks to completely cover the grains up to at least 5 cm. (B)&#160;Draining the flasks. Drain the water from the flasks, plug the opening with a piece of cotton roll wrapped in cheesecloth, and cover the opening with aluminum foil wrap. (C)&#160;Autoclave setup. Place the bag in a plastic, autoclave-safe bin. Do not use a metal bin when autoclaving the grains in the bags, as that may cause the bags to melt. Cover the bin with aluminum foil wrap. (D) Storage of bags.&#160;Store the bag upright. Ensure the filter is pulled away from the opposing side of the bag to enable maximum gas exchange. (E) Measure 14 grains of infested kernels into a large plastic bag. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63181/63181fig06.jpg" /> 
Figure 6: Sowing and germination of watermelon seeds. (A) Sowing cultivar seeds in pots. Sow six seeds in each pot. Ensure that each pot only contains seeds from one cultivar. Position the seeds with the apex end of the seed facing up to allow proper growth during emergence. (B) Seed germination. Using a spray bottle, wet the upper 0.3-0.6 cm of soil with water. Place a clear plastic dish (15 cm diameter) under and over each pot to create a humid environment for seed germination. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63181/63181fig07.jpg" /> 
Figure 7: Inoculum preparation and seedling inoculation for MTDM. (A) Preparation of inoculum. Determine the microconidial concentration in the flasks using a hemocytometer as previously described. Prepare a 7 L inoculum suspension in a plastic tub (40.6 cm width &#215; 67.3 cm length &#215; 16.8 cm depth) by transferring the correct volume of spore suspension into sterile water for a final spore concentration of 1 &#215; 106 mL&#8722;1. (B)&#160;Inoculating the seedlings. Fourteen days after sowing&#160;(at least first true leaf stage), transfer the cell inserts with the seedlings into webbed trays (26.9 cm width &#215; 53.7 cm length &#215; 6.28 cm depth). Gently place the webbed trays with the seedlings into a plastic tub containing the 7 L inoculum suspension. Inoculate each tray one at a time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63181/63181fig08.jpg" /> 
Figure 8: Phenotypic results of race identification methods. (A) Race 1 results. The results of the bioassays using (A) all methods show that when the seedlings were infected with a Race 1 isolate, the Black Diamond and Charleston Grey cultivars died or showed serious symptoms, while the Calhoun Grey and PI cultivars showed resistance. (B)&#160;Race 3 results. All methods showed that when the seedlings were infected with a Race 3 isolate, nearly all the plants from all cultivars died or showed serious symptoms. (The appearance of diseased plants should be the same for all methods. Order of planting (left to right) shown by arrows: Black Diamond (blue arrow), Charleston Grey (purple arrow), Calhoun Grey (brown arrow), Plant Introduction 296341-FR (green arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63181/63181fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cultivar</td>
			<td>Race 0</td>
			<td>Race 1</td>
			<td>Race 2</td>
			<td>Race 3</td>
		</tr>
		<tr>
			<td>Sugar Baby, Black Diamond</td>
			<td>S</td>
			<td>S</td>
			<td>S</td>
			<td>S</td>
		</tr>
		<tr>
			<td>Charleston Gray, Allsweet, Dixielee</td>
			<td>R</td>
			<td>S</td>
			<td>S</td>
			<td>S</td>
		</tr>
		<tr>
			<td>Calhoun Gray, Sunsugar</td>
			<td>R</td>
			<td>R</td>
			<td>S</td>
			<td>S</td>
		</tr>
		<tr>
			<td>PI-296341-FR</td>
			<td>R</td>
			<td>R</td>
			<td>R</td>
			<td>S</td>
		</tr>
	</tbody>
</table>
Table 1: Race of Fusarium oxysporum f. sp. Niveum. The race of Fusarium oxysporum f. sp. niveum is determined by susceptible or resistant reactions to a set of watermelon differentials. The cultivars listed in each row are the most used to represent each level of resistance during the evaluation of an isolate&#39;s race. This table has been modified from 4. Abbreviations: S = susceptible; R = resistant.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Isolate</td>
			<td>Method</td>
			<td colspan="2">BD</td>
			<td colspan="2">CH. G</td>
			<td colspan="2">Cal G.</td>
			<td colspan="2">PI</td>
			<td>Race</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>S</td>
			<td>AS</td>
			<td>S</td>
			<td>AS</td>
			<td>S</td>
			<td>AS</td>
			<td>S</td>
			<td>AS</td>
			<td></td>
		</tr>
		<tr>
			<td>X</td>
			<td>Dip</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>1</td>
		</tr>
		<tr>
			<td>X</td>
			<td>Kernel</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>1</td>
		</tr>
		<tr>
			<td>X</td>
			<td>MTD</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Y</td>
			<td>Dip</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Y</td>
			<td>Kernel</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Y</td>
			<td>MTD</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>6</td>
			<td>0</td>
			<td>3</td>
		</tr>
		<tr>
			<td colspan="11">S = Symptomatic; AS = Asymptomatic</td>
		</tr>
	</tbody>
</table>
Table 2: Identification of races. Values used in this table reflect incidence or the number of symptomatic plants, compared to the healthy control, and the number of dead plants as a proportion of the total number of plants in that cultivar. The numbers in each cell reflect the incidence reported at the end of the observation period. A cultivar is deemed susceptible when at least 1/3rd or 33% of the plants of that cultivar are symptomatic or dead. The race of the pathogen is then determined based on which cultivars have been deemed susceptible. In other words, how the pathogen performs against cultivars with increasing resistance determines the isolate&#39;s race. These results are not from an actual trial and are rather shown to convey how races are identified from the results of these methods. Abbreviations: MTD = modified tray-drip method; BD = Black Diamond; CH. G = Charleston Grey; Cal G. = Calhoun Grey; PI = Plant Introduction 296341-FR; S = symptomatic; AS = asymptomatic.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Biology

3.2K Views.  University of Florida. Dit protocol kan degenen die betrokken zijn bij de watermeloengemeenschap helpen bij het evalueren van de fusariumverwelkingspathogeen, en dit zal helpen bij het bepalen van de pathogene populaties die aanwezig zijn en helpen deze effectief te beheren. Race-typing fusarium verwelking is complex en tijdrovend om te voltooien, en het hebben van drie levensvatbare race-typerende bioassay-opties stelt gebruikers in staat om een methode te kiezen die het meest geschikt is voor hun experimentele om...

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

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Environment

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

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

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