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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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).

Abstract

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.

Introduction

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 progres....

Protocol

1. Determining race by root-dip method (RDM)

  1. Preparation of the experimental environment
    1. 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).
      1. Set the temperature to 26-28 °C, relative humidity to 50-75%, and set a 16 h photoperiod to ensure adequate plant growth and health.
        N.......

Representative Results

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 "resistant" 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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
100% Fuller’s EarthSigma-AldrichF200-5KG
1 L glass Erlenmeyer FlaskPYREX4980-1L
15 mL falcon tubesFisher Scientific14-959-49B
50 mL graduated cylinderLab Safety Supply41121805
50 mL Eppendorf Conical TubesFisher Scientific05-413-921
Aluminum foil wrapReynolds Wrap720
BleachWalmart587192290
Bunsen burnerFisher Scientific03-391-301
CaCO3sigma-Aldrich239216
cell spreadersFisher Scientific08-100-11
CheeseclothLions Services, Inc8305-01-125-0725
Clear plastic dishesVisions Wave999RP6CLSS~15 cm diameter
Clear vinyl tubing for mushroom bag clampsShroom Supply6" for small bag, 8" for medium bag, 10" for large bag
Cotton BallsFisherbrand22-456-885Sterile
EthanolFisher ChemicalA4094100%, then combine with water to make 70% for use
Flourescent Tube LightsMaxLumeModel T52800 K Color Temperature, 24'' or 48'' long
granulated agarVWR International90000-786
Hand-held Spray BottleAbility One24122002~0.95 L
hemacytometerFisher Scientific02-671-55ATwo chamber hemacytometer
Lab traysFisher Scientific15-236-2A
Large, sealable plastic bagsZiploc43080538 cm x 38 cm
Mister / watering canBar5FB10H22
Mushroom Bag ClampShroom Supply6" for small bag, 8" for medium bag, 10" for large bag
Nitrile GlovesFisher Scientific19-130-1597D
Organic Rye BerriesShroom Supply0.5 gallon or 25 lb bags
P1000 pipette and tipsFisher Scientific14-388-100
Petri dishesFisherbrandFB0875713Round, 100 mm diameter, 15 mm height
Planting mediaJolly GardenerPro-Line C/B
Plastic PitcherBrandTechUX06008501 L or larger
Plastic planting potsNeo/SCI01-1177~15 cm diameter and ~10 cm height
Plastic, autoclave-safe binThermo ScientificUX06010223 L
Quarter-strength potato dextrose agar mediaCole-ParmerUX1420028Use powder in combination with recipe for QPDA
Scientific Balance Scale, measuring in gOhaus30208458Any precise scale that can hold and measure 200g will work
Size #4 cork boreCole-ParmerNC9585352
Small Mushroom grow bagShroom Supply0.5 micron filter, also comes in medium and large sizes
Soil trowelWalmart563876946
Styrofoam flats (6 x 12 cells)SpeedlingModel TR72A
Styrofoam flats (8 x 16 cells)SpeedlingModel TR128A
Syringe (5 or 10 mL)fisher Scientific14-829-19C
TimerWalmartTM-01
V8 Original 100% Vegetable JuiceWalmart564638212
vortexFisher Scientific02-215-418
Watermelon Seed - Black DiamondWillhite Seed Inc17
Watermelon Seed - Calhoun GrayHolmes Seed Company4440
Watermelon Seed - Charleston GrayBonnie Plants7.15339E+11
Watermelon Seed - PI 296341-FRContact authorsContact authors
Wheat Kernels (Maxie var.) (optional)Alachua County Feed & Seed

References

  1. Edel-Hermann, V., Lecomte, C. Current status of Fusarium oxysporum formae speciales and races. Phytopathology. 109 (4), 512-530 (2019).
  2. Everts, K. L., Himmelstein, J. C. Fusarium wilt of watermelon:....

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