Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

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.

Although countless microbes colonize plant roots, these interactions are difficult to analyze as they happen underground. To facilitate this, this protocol collection provides an overview of strategies to inoculate plant roots with soil-borne microbes. Root inoculation is the basis for studying root microbe interactions, and by using the following techniques, interactions can be studied at a molecular, cytological or histological level.

We explain three inoculation systems using verticillium as root invading microbe, and the model plant, arabidopsis. However, transfer of the methods to other plants such as tomato or oilseed rape, and to other root microbes, for example beneficial serendipita, is possible. Prepare the verticillium inoculum in advance by culturing the mycelia in PDB and inducing sporulation and Czapek dextrose broth.

Then begin by pouring the autoclaved medium into Petri dishes. After hardening of the medium, repack the Petri dishes in a sterile plastic bag and store them upside down overnight in the refrigerator at four to 10 degrees Celsius. Cut and remove an infection channel in the upper third of the solidified medium with a scalpel.

Avoid getting liquid or air under the agar medium while cutting. Next, use a sterile pipette tip to distribute 50 to 100 surface sterilized arabidopsis seeds on the cut upper surface. Put the seeds in the angle where the cut agar surface contacts the Petri dish wall so that the roots can grow in between them.

Close the Petri dishes and seal them with a breathable adhesive tape to allow gas exchange. Keep the Petri dishes in the dark at four degrees Celsius for two days for stratification to ensure synchronized and efficient germination. Then place the plates vertically in a rack and grow the plants in a growth chamber under long day conditions.

After nine to 11 days when the roots reach the infection channel, lay the plates horizontally and open them. Then add 500 microliters of freshly harvested Verticillium conidia directly into the infection channel. Prepare control plates by adding 500 microliters of a mock solution instead of spores.

Incubate both control and spore inoculated plates horizontally until the liquid has soaked in. Then close the lid and seal the plates with breathable adhesive tape. Cover the root parts with black paper boxes to darken roots and soil-borne fungus.

Incubate the plates vertically in the growth chamber. Perform the analyses at the preferred time points after inoculation. First, sterilize transparent 500 milliliter plastic cups into 70 to 75%ethanol bath for 20 minutes.

Dry the cups in the lamina flow hood. Pour the autoclaved medium into the plastic cups. Prepare a plastic layer with five pre-fabricated holes one large in the center and four smaller ones in each corner.

Place the plastic layer on the medium before it solidifies. Cut the agar with a scalpel through the pre-fabricated center hole to a depth of about 1.5 centimeters. Remove the cut agar to create an infection channel to add the fungal spores later.

Scratch the agar medium slightly with a pipette tip in the four smaller holes to interrupt the solidified skin. Place the seeds using a pipette tip into the smaller holes. Close the plastic cup with a second inverted plastic cup and seal with breathable adhesive tape.

Keep the plants at four degrees Celsius for three days in the dark for stratification. Then incubate the cup systems in the growth chamber. To inoculate plantlets with verticillium, add one milliliter of conidia solution into the infection channel.

For controls, add one milliliter of germ free one fourth MS medium as a mock solution. Perform the analyses at the preferred time points after inoculation. First, thoroughly mix soil and sand in a three to one volumetric ratio which facilitates washing the substrate from the roots later.

Pour the mixture into an autoclave bag and steam at 80 degrees Celsius for 20 minutes in an autoclave. Fill pots with the soil-sand mixture and transfer them into trays. Add water into the trays about one third the height of a pot for uniform soaking of the soil-sand mixture.

Water spray the mixture to ensure wet starting conditions. Sow three to four seeds in each pot at enough distance from each other. Keep them for stratification at four degrees Celsius for three days in the dark.

Allow the seedlings to grow under long day conditions with regular watering, then pick plants of similar size and age to perform the root dip inoculation. Take the soil out of the pots and excavate the roots. Gently wash the roots in a water container and keep the rosettes out of the water.

Incubate the roots for 60 minutes in a Petri dish containing the verticillium spore solution or mock solution. Prepare new pots with moist steam sterilized soil without sand. Use a pipette tip to make a hole in the center of the soil in each pot.

Place the roots directly into the hole and refill the holes gently with soil to avoid additional stress to the plants. Cultivate the plants under long day conditions with regular watering. Perform the analyses at the preferred time points after inoculation.

In the Petri dish-based system, successful infection of verticillium was visualized in the roots of arabidopsis. Two days post inoculation, the marker gene MYB51 was strongly induced in infected roots compared to control, which was confirmed by QRTPCR analysis. In the cup-based system, a high amount of fungal DNA was found in the leaves of infected plants as the graph shows in comparison to mock control after 12 days of inoculation.

QRTPCR analysis showed enriched transcript abundance of the marker gene MYB51 in the arabidopsis roots. Using this system, verticillium was inoculated to other model plant species. In oilseed rape, the fungal DNA was detectable in stem segments cut at the base of the seedlings 12 days after inoculation at the roots.

Fungal DNA was also detected in tomato stems indicating propagation of the pathogen within the plant. 21 days post inoculation using the soil-based system, the rosettes of mock treated arabidopsis plants looked healthy and green while infected plants showed yellowish or necrotic leaves and had lesser green leaf area relative to mock indicating successful infection. Pay attention when cutting the infection channel in the Petri dish system to prevent the agar from sliding.

Applying the soil-based system, don't compress the soil around the roots while repotting to avoid further stress to the plants. After root inoculation, for example, pathogenic tests, analysis of gene expression, functional genomics or agrochemical tests can be performed providing new insights into root microbe interactions and tools to improve agriculture.