The overall goal of this procedure is to screen plants infested with root knot nematodes. First, grow healthy seedlings in sandy soil or growth pouches. Next, produce an inoculum of highly infected second stage juveniles of root, not nematodes.
Then properly inoculate and infect the seedlings. Finally evaluate the development of nematode egg masses on the host root system. We will be demonstrating a high and low throughput screens of plants with root, not nematodes.
And the main advantages of the high throughput system is the need for limited space for plant growth and ease of evaluation of the infection. So these approaches will result in reproducible and uniform infection, nematode infection of plant roots, and therefore a better evaluation of the host status of the plant. So generally, individuals new to evaluating plants with root, not nematodes are likely to struggle because of not using the proper developmental stage to infect the roots with nematodes.
And also by using several day old infective stage juveniles, which typically have low infectivity Demonstrating the procedure will be hug up atian and graduate student. In my laboratory, I will be demonstrating the procedure for screening in the growth pouches For pot assays. Plant tomato seeds in a single pot filled with organic rich soil, such as sunshine mix, maintaining a greenhouse at 22 to 28 degrees Celsius after germination.
Ize once a week with miracle grow about two weeks after germination at the two true leaf stage transplant individual seedlings into pots filled with sterile sandy soil, 90%sand and 10%organic matter. Add osmo coat slow release fertilizer and maintain in a greenhouse at 22 to 28 degrees Celsius for two weeks. Continue to fertilize once a week with miracle grow.
For high throughput screens, plant seeds directly in trays in sandy soil. Cover with plastic wrap until germination and maintain as previously described. After germination, add Osmo coats slow release fertilizer and fertilize once a week with miracle grow germinates seeds in a Petri dish lined with several layers of Kim White paper maintained in an incubator and 25 to 28 degrees Celsius, and then transfer the seedlings to individual pouches.
Alternatively, place seeds in the paper groove to germinate directly in individual pouches. Place the pouches in a plastic hanging file folder to per folder and arrange the folders on a rack. In a vertical position.
Place the rack in a controlled environment chamber maintained at a temperature of 25 to 28 degrees Celsius and 16 hours light. Eight hours dark cycle water the pouches once or twice per day with reversals mosis water about 10 to 14 days after seeding. When an adequate root system with tertiary root tips has developed, the seedlings are ready for inoculation Before starting the egg extraction, wash the work area and all tools thoroughly with hot water to avoid contamination.
Place a bucket in a sink to collect, run through solution and position a wire mesh support over the top. Stack three sieves from top to bottom in order of decreasing aperture size, such that the eggs collect in the 25 micron sieve. Cut the tops of the infected plants and discard Carefully.
Remove the plant from the pot and wash the roots by dipping in a plastic bucket full of water. Rinse the roots further under running water until the soil particles are washed away. Then extract root knot nematode eggs from infected tomato roots.
First, cut the roots into small pieces using scissors disposing of the tap root. Put the chopped roots from a single plant in a large plastic jar with a lid and add just enough 10%bleach solution to cover the roots. Close the lid and shake the jar for two minutes.
Open the jar and pour the roots onto the top sieve and wash with a hose attached to a misting nozzle wash. Well until all the bleach smell is gone. And use a mallet to tap the sides of the sieves to avoid clogging the sieve.
Pause, remove the top sieve and rinse the debris. And the second sieve. Remove the second sieve and collect the fine debris, which includes the eggs from the last sieve with a gentle stream from a water bottle.
Move the debris to one side of the sieve. Collect the debris and eggs into a clean beaker with a minimum amount of water. Run the water collected in the bucket through the 25 micron sieve to collect any eggs that may have escaped.
Rinse well with water and collect in the same beaker. Line a clean metal basket with a few layers of Kim. Wipe paper and fit on a glass petri dish, leaving a one centimeter distance between the bottom of the and the dish.
Pour the extracted nematode eggs onto the paper. In the wire basket. Add enough liquid so that the bottom of the wire basket touches the water surface but is not immersed in water.
Cover the top with a plastic lid every day. Check for water evaporation and add some water in the Petri dish so that the bottom of the basket touches the water. In order to prevent the eggs from drying out every other day over six to eight days, collect the water that contains the second stage juveniles or J twos from the Petri dish into a beaker.
If not used, immediately aerate the collected inoculum at room temperature using a lab, air supply or air generated by an aquarium pump. Begin the next step by stirring the inoculum on a magnetic stirring plate at low speed. Use three aliquots of the collected nematodes.
To count the number of J twos in accounting. Slide or dish, calculate the average and determine the desired volume for inoculation. These are J twos as viewed under a microscope.
Typically, 3000 J twos are used per plant in pots and 500 J twos for plants grown in seedling trays. Before inoculating the plants, make sure that the soil is moist but not too wet. For pot inoculation, make three holes of about half pot depth in the sand around each tomato root system.
Using a pencil, inoculate each plant by delivering the J twos into the three holes using a pipette. Afterwards, cover the holes for the high throughput screens in trays. Use a modified sealed tip needle with holes on the sides glued to a five milliliter pipette.
Tip along a pipeter to deliver the J twos into the soil. Maintain the plants in a greenhouse at 24 to 27 degrees Celsius for six to eight weeks. Continue to fertilize twice per month with miracle grow.
For evaluation of infection. Cut the tops and carefully remove the plants from the pots and wash the roots. Then stain the egg masses blue by submerging the roots in one milligram per liter.
Aerio glossing for 15 minutes. Rinse the roots in water and evaluate by counting the stained egg masses on individual root systems. An illuminated death magnifier can be used to help visualize the egg masses, count the nematode, inoculum, and stir on a magnetic stirring plate as previously described.
Remove the pouches from the hanging folders and place on a horizontal surface. Inoculate each pouch with 1, 500 J twos in five milliliters. Lift the plastic cover of the pouch and distribute the nematodes evenly over the surface of the roots.
Keep the pouches in a horizontal position for 24 hours after inoculation covered with dark paper to exclude light. Afterwards, return the pouches to the hanging folders in the growth chamber water. The plants is needed once or twice daily for about three days with half strength hoagland solution until a response to the fertilizer is observed.
Typically enhanced foliage greening and more vigorous shoot growth. After three days, keep the pouches moist with water. Approximately 30 days after inoculation, infuse each pouch with about 10 to 20 milliliters of 75 milligrams liter.
Aero glossing. Keep the pouches flooded with the dye in a horizontal position overnight. The next day, drain the pouches and evaluate the root systems by counting the egg masses under an illuminated desk.
Magnifier shown here are the roots of a well infected tomato plant with the widespread egg masses stained blue. The use of trays enables screening of hundreds of plants in a small growth space. Growth pouches also allow fast and deficient non-destructive evaluation of root, not nematode infections with no need for washing roots.
This method also enables high throughput screening of thousands of plants in a small growth space. Here the root system of a cow P plant grown in a pouch shows blue stained egg masses following inoculation. Timing of the infection is very critical for plant assays with nematodes.
So delaying infection even by a few days, can result in very poor infection levels. So after watching this video, you should now have a good understanding of how to infect and evaluate plants with root knot nematodes. Don't forget that nematode infected roots need to be handled properly.
Autoclaving the roots before disposing them will limit the spread of the nematode. Also, be careful using aerial glysine as it high doses. It is carcinogenic.
Therefore, you need to use gloves, handling roots infected with this chemical as well as in preparation of this chemical.
