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Botany JoVE (Open Access) Citable · DOI

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

DOI: 10.3791/3629-v
What you'll learn
  • ✓ Perform high-throughput and low-throughput nematode infection screening on plant roots
  • ✓ Extract and hatch root-knot nematode eggs for inoculation
  • ✓ Evaluate plant resistance to Meloidogyne spp. infection nondestructively
  • ✓ Assess nematode-induced root damage in tomato and cowpea
Protocol

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Difficulty
intermediate
Total time
~8–10 weeks (from seedling growth to infection evaluation and gall assessment)
Model organism
Tomato (Solanum lycopersicum), Cowpea (Vigna unguiculata); Meloidogyne spp. (root-knot nematodes)
Biosafety
BSL-1

Steps

1
Grow tomato seedlings in pots and trays

Establish uniform tomato seedling populations in controlled pot and tray systems to prepare plants for nematode inoculation. Maintain appropriate moisture and light conditions during seedling development.

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2
Cultivate cowpea seedlings in pouches

Grow cowpea seeds in seedling pouches using controlled watering and light conditions. Seedling pouches allow transparent observation of root development and nematode infection progression.

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3
Extract nematode eggs from infected roots

Isolate root-knot nematode eggs from naturally or artificially infested plant root material using established extraction protocols. Process roots to obtain a pure egg suspension for controlled inoculation.

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4
Induce hatching of extracted nematode eggs

Incubate extracted eggs under controlled temperature and moisture conditions to synchronize second-stage juvenile (J2) emergence. Prepare infective nematode inoculum for plant infection experiments.

▶ 05:45
5
Inoculate tomato roots and evaluate infection

Apply synchronized J2 nematodes to tomato seedling root systems in pots or trays. Monitor and quantify root gall formation and infection severity at defined time intervals using visual assessment.

▶ 06:46
6
Infect cowpea roots in pouches and assess damage

Inoculate cowpea seedling roots with J2 nematodes in transparent pouches. Evaluate gall development and root pathology nondestructively through pouch walls, allowing plants to continue growth for breeding programs.

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7
Examine representative nematode-infected root tissues

Visualize and compare characteristic root galls and nematode damage in tomato and cowpea roots at various infection stages. Document morphological indicators of susceptibility and resistance phenotypes.

▶ 10:05
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