Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

Summary

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.

Abstract

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.

Introduction

Natural soils are inhabited by an astonishing number of microbes that can be neutral, harmful, or beneficial to plants¹. Many plant pathogens are soil-borne, surround the roots, and attack the subterranean organ. These microorganisms belong to a wide variety of clades: fungi, oomycetes, bacteria, nematodes, insects, and some viruses^1,2. Once environmental conditions favor infection, susceptible plants will become diseased and crop yields decline. The effects of climate change, such as global warming and weather extremes, will increase the proportion of soil-borne plant pathogens³. Therefore, it will become more and more important to study these destructive microbes and their impact on food and feed production, but also on natural ecosystems. Additionally, there are microbial mutualists in the soil that tightly interact with roots and promote plant growth, development, and immunity. When confronted with pathogens, plants can actively recruit specific opponents in the rhizosphere that can support host survival by suppressing pathogens^4,5,6,7. However, mechanistic details and pathways involved in beneficial root-microbe interactions are often still unknown⁶.

It is, therefore, essential to expand the general understanding of root-microbe interactions. Reliable methods for inoculating roots with soil-borne microorganisms are necessary to perform model studies and transfer the findings to agricultural applications. Beneficial interactions in the soil are studied, for example, with Serendipita indica (formerly known as Piriformospora indica), nitrogen-fixing Rhizobium spp., or mycorrhizal fungi, while known soil-borne plant pathogens include Ralstonia solanacearum, Phytophthora spp., Fusarium spp., and Verticillium spp.¹. The latter two are fungal genera that are globally distributed and cause vascular diseases². Verticillium spp. (Ascomycota) can infect hundreds of plant species - largely dicotyledons, including herbaceous annuals, woody perennials, and many crop plants^2,8. Hyphae of Verticillium enter the root and grow both intercellularly and intracellularly toward the central cylinder to colonize the xylem vessels^2,9. In these vessels, the fungus remains for most of its life cycle. As the xylem sap is nutrient-poor and carries plant defense compounds, the fungus must adapt to this unique environment. This is accomplished by the secretion of colonization-related proteins that enable the pathogen to survive in its host^10,11. After reaching the root vasculature, the fungus can spread within the xylem vessels acropetally to the foliage, which leads to systemic colonization of the host^9,12. At this point, the plant is negatively affected in growth^9,10,13. For instance, stunting and yellow leaves occur as well as premature senescence^13,14,15,16.

One member of this genus is Verticillium longisporum, which is highly adapted to brassicaceous hosts, such as the agronomically important oilseed rape, cauliflower, and the model plant Arabidopsis thaliana¹². Several studies combined V. longisporum and A. thaliana to gain extensive insights into soil-borne vascular diseases and the resulting root defense responses^13,15,16,17. Straightforward susceptibility testing can be realized by using the V. longisporum / A. thaliana model system and well-established genetic resources are available for both organisms. Closely related to V. longisporum is the pathogen Verticillium dahliae. Although both fungal species perform a similar vascular life-style and invasion process, their propagation efficiency from roots to leaves and the elicited disease symptoms in A. thaliana are different: while V. longisporum usually induces early senescence, V. dahliae infection results in wilting¹⁸. Recently, a methodological summary presented different root inoculation strategies for infecting A. thaliana with V. longisporum or V. dahliae, assisting in planning experimental setups¹⁹. In the field, V. longisporum occasionally causes significant damage in oilseed rape production¹², whereas V. dahliae has a very broad host range comprising several cultivated species, such as grapevine, potato, and tomato⁸. This makes both pathogens economically interesting models to study.

Thus, the following protocols use both V. longisporum and V. dahliae as model root pathogens to exemplify possible approaches for root inoculations. Arabidopsis (Arabidopsis thaliana), oilseed rape (Brassica napus), and tomato (Solanum lycopersicum) were chosen as model hosts. Detailed descriptions of the methodologies can be found in the text below and the accompanying video. Advantages and disadvantages for each inoculation system are discussed. Taken together, this protocol collection can help to identify a suitable method for specific research questions in the context of root-microbe interactions.

Protocol

1. Media for fungal cultures and plant inoculation systems

1. Liquid Potato Dextrose Broth (PDB): Prepare 21 g/L PDB in ultrapure water in a heat-stable flask.
2. Liquid Czapek Dextrose Broth (CDB): Prepare 42 g/L CDB in ultrapure water in a heat-stable flask.
3. Medium for the Petri dish inoculation system: Prepare a heat-stable flask with 1.5 g/L Murashige and Skoog medium (MS) and 8 g/L agar in ultrapure water.
   NOTE: Avoid sugar in this medium as it will lead to excessive fungal growth after inoculation.
4. Medium for the plastic-cup-based inoculation system: Prepare a heat-stable flask with 4.4 g/L MS, 0.2 g/L MgSO₄, 1 g/L KNO₃, 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES), and 6.0 g/L agar in ultrapure water and adjust pH to 5.7 with 5 M KOH.
   NOTE: Avoid sugar in this medium as it will lead to excessive fungal growth after inoculation.
5. ¼ MS medium: Prepare 1.2 g/L MS in ultrapure water.
6. Use the autoclave to sterilize all the above solutions. Put the glass flasks into the basket, close the lid and sterilize for 15 min at 121 °C and 98.9 kPa.

2. Sterilizing the surface of plant seeds

NOTE: Use the below protocol always to sterilize the surface of seeds from Arabidopsis, oilseed rape, and tomato prior to sowing.

1. Transfer the seeds into a 2 mL reaction tube. Place the tube in an exsiccator with an internal capacity of 5.8 L.
2. Generate chlorine gas in the exsiccator by adding 6 mL of 33% hydrochloric acid (HCl) into 100 mL of 12% aqueous sodium hypochlorite (NaClO).
3. Immediately close the lid of the exsiccator and incubate the seeds for 3 h in the gas.

3. Preparing the inoculum with Verticillium spores (asexual derived conidia)

NOTE: Cultivate V. dahliae (strain JR2) in the same way as V. longisporum (strain Vl43)^17,18,19. Ensure that all the equipment and media are germ-free and that all steps are performed in a laminar flow hood to keep the inoculum axenic.

1. Fill 150 mL of liquid PDB (step 1.1) in a 500 mL chicanery flask and supplement the medium with 500 mg/L cefotaxime.
2. Add Verticillium conida from glycerol-stock storage to the PDB medium. Close the flask with a sterile foam stopper.
3. Incubate the culture for 7-10 days in a dark box at room temperature (RT) under continuous, horizontal shaking (rotary shaker; 60 rpm). This results in small, white mycelia spheres.
4. Remove and discard the PDB supernatant carefully. Most of the mycelia should remain in the flask.
5. Add 100 mL of liquid CDB (step 1.2) on the mycelia in the chicanery flask and supplement the medium with 500 mg/L cefotaxime.
6. Incubate another 4-5 days in a dark box at RT under continuous, horizontal shaking (rotary shaker, 60 rpm) to induce sporulation. The supernatant will turn yellowish-greyish as conidia are released.
7. Filter a portion (5-10 mL) of the conidia-containing liquid through a filter paper (particle retention level of 8-12 µm) into a sterile 50 mL collection tube. This separates spores from mycelia.
8. Determine the spore concentration by using a cell counting chamber and a microscope. Dilute with germ-free ¼ MS medium in ultrapure water until the spore concentrations given below are obtained.
   NOTE: Under the microscope, the conidia from V. longisporum are mostly long-drawn and 7.1-8.8 µm in size, while V. dahliae conidia are shorter (3.5-5.5 µm) and rather spherical²⁰.
9. Use these freshly harvested conidia as inoculum. Ensure to conduct the experiments always with freshly harvested conidia and not with frozen stocks, as freezing significantly reduces the number of viable spores¹⁹.
10. For long-term storage, freeze the spores as high concentrated spore solution (approximately 1 x 10⁸ spores/mL) in 25% glycerol at -80 °C (storable up to 1 year). For the next experiments, use these glycerol stocks to inoculate the PDB medium in step 3.2.

4. A sterile in vitro inoculation system based on Petri dishes

NOTE: For the Petri dish system¹⁷, ensure that all equipment and media are germ-free and that all steps are performed in a laminar flow hood.

1. After autoclaving, pour the medium (see step 1.3) into Petri dishes.
2. After the hardening of the medium, repack the Petri dishes in a sterile plastic bag and store them upside-down overnight in the refrigerator (4-10 °C). A chilled medium helps to prevent sliding of the medium in the next steps.
3. Cut and remove an infection channel and the upper third of the solidified medium with a scalpel (Figure 1A). Avoid getting liquid or air under the agar medium while cutting; otherwise, the medium will slip and close the infection channel.
4. Distribute 50-100 surface-sterilized Arabidopsis seeds with a sterile pipette tip on the cut upper surface. Put the seeds in the angle where the cut agar surface contacts the wall of the Petri dish so that roots can grow between the medium and the Petri dish wall. This will facilitate the inoculation later.
5. Close the Petri dishes and seal them with air-permeable adhesive tape to allow gas exchange.
6. After stratification for 2 days in the darkness at 4 °C, place the plates vertically in a suitable rack and grow the plants at 22 °C ± 1 °C under long-day conditions (16 h light / 8 h darkness) in a growth chamber.
7. When the majority of the roots reach the infection channel (about 9-11-day old seedlings), lay the plates horizontally, open them and add 500 µL of freshly harvested Verticillium conidia with a concentration of 4 x 10⁵ spores/mL directly into the infection channel, making sure that the liquid is evenly distributed in the channel.
8. Similarly, prepare control plates by adding 500 µL of a mock solution instead of spores (germ-free ¼ MS medium).
9. Incubate the plates horizontally for a couple of minutes until the liquid has soaked in and cannot leak out when the plates are set up vertically again. Then, close the lid and seal the plates with air-permeable adhesive tape.
10. Incubate the plates vertically in the growth chamber. Optionally, cover the root parts with black paper-boxes to darken roots and soil-borne fungus (see¹⁹).
11. Perform the analyses at the preferred time points after inoculation depending on the research question (refer to the figure legends for the exact time points used here). Following are some suggestions.
    1. Cut the leaves from the roots and harvest both separately. Take the agar strips out of the Petri dishes to easily access the roots and carefully pull them out of the agar using forceps. Freeze all plant material immediately in liquid nitrogen.
       1. Grind the samples in liquid nitrogen. Extract total DNA from 100 mg of leaf material to determine via a quantitative PCR (qPCR) the amount of fungal DNA relative to plant DNA (see¹⁹).
       2. Grind the samples in liquid nitrogen. Take 100 mg of plant material and extract total RNA. Conduct quantitative reverse transcription PCR (qRT-PCR) to determine the expression of plant genes (or fungal genes) during infestation (see¹⁹).
    2. Carefully remove the roots from the agar avoiding injury and examine them under the fluorescent microscope.
       1. Determine induction of marker genes in plant reporter lines (e.g., luciferase, β-glucuronidase, or fluorescent reporters^17,19,21).
       2. Visualize fungal propagation at the root by using fungal reporter lines (e.g., V. longisporum constitutively expressing enhanced Green Fluorescent Protein, Vl-sGFP⁹) or by staining techniques (e.g., through 5-bromo-4-chloro-3-indoxyl-N-acetyl-beta-d-glucosaminide (X-beta-D-Glc-Nac)¹⁸).

5. A sterile in vitro inoculation system organized with plastic cups

NOTE: As noted in the first description of this technique¹⁹, ensure that all equipment and media are germ-free and that all steps are performed in a laminar flow hood.

1. Utilize transparent plastic cups with a total volume of 500 mL and sterilize them in a 70%-75% ethanol bath for at least 20 min. Dry the cups in the laminar flow hood.
2. Pour the autoclaved medium (see step 1.4) into the plastic cups. Optionally, add cefotaxime (final concentration of 50 mg/L) to the autoclaved medium to prevent bacterial contaminations. Use 150 mL of medium per cup for experiments with Arabidopsis or more medium (250-300 mL per cup) for experiments with larger plant species (oilseed rape, tomato).
3. Place a plastic layer (sterilized before by incubating in 70%-75% ethanol for 20 min) on the medium before it solidifies (Figure 1B).
   NOTE: This plastic layer contains four prefabricated holes at the corners for placing surface-sterilized seeds. This allows the seeds to access the medium. Later, this separating layer prevents the leaves from touching the fungus-containing medium, so that the microbes cannot directly attack the leaves and must take the root pathway. Another hole is in the center, enabling cutting the infection channel.
4. When the medium has solidified, cut the agar with a scalpel through the prefabricated center hole to a depth of about 1.5 cm. Remove the cut agar to create an infection channel into which the fungal spores can be added later.
5. Slightly scratch the agar medium with a pipette tip in the four smaller holes to interrupt the solidified skin (this allows the seeds to soak up water from the watery agar medium). Place the seeds using a pipette tip into the smaller holes.
6. Close the plastic cup with a second, inverted plastic cup and seal with air-permeable adhesive tape. The tape must allow gas exchange.
7. After stratification for 3 days in darkness at 4 °C, incubate the cup systems under 12 h light / 12 h darkness (Arabidopsis, oilseed rape) or 16 h light / 8 h darkness conditions (tomato) in growth chambers at a constant temperature of 22 °C and 60% humidity.
8. Follow the recommended age of plants for inoculation: 21 days for Arabidopsis; 5-7 days for oilseed rape; 12 days for tomato.
9. Inoculate plantlets with Verticillium by adding 1 mL of conidia solution (recommended concentration: 4 x 10⁵ spores/mL) into the infection channel. To prepare control samples, add 1 mL of mock solution without spores (germ-free ¼ MS medium) into the channel.
10. Perform the analyses at the preferred time points after inoculation depending on the research question (refer to the figure legends for the exact time points used here). Following are some suggestions.
    1. Take photographs of the plants with a digital camera from above keeping the distance the same for each photo. Quantify the leaf area (e.g., with ImageJ²² or BlattFlaeche^17,19; use the length of the cups to set the scale) and compare infected and control groups. Categorize the development of disease symptoms (e.g., smaller, more yellowish, or necrotic leaves).
       NOTE: If there are any stems on Arabidopsis, remove them to get better photos of the rosettes.
    2. Remove the roots and define the biomass (fresh weight) of shoots from infected and control samples by weighing. Determine the relative fresh weight¹⁹.
    3. Collect the samples for molecular analyses as follows.
    4. Arabidopsis: Remove stems if there are any. Cut the rosettes at the base from the roots. Make sure to exclude all root material from the sample and harvest whole rosettes. Combine 4-5 rosettes from different plants into one sample and freeze the leaf material in liquid nitrogen.
    5. Pull the roots carefully out of the medium with forceps, press and dab them with a paper towel to remove agar remains, and combine 4-5 roots from different plants into one sample. Freeze immediately in liquid nitrogen.
    6. Oilseed rape/tomato: Cut stem segments from the hypocotyl (e.g., 1 cm in length; always take the same stem region). Combine material from 4-5 plants into each sample and freeze in liquid nitrogen.
    7. Grind the samples in liquid nitrogen. Extract total DNA from 100 mg of the leaf or stem material to determine via qPCR the amount of fungal DNA relative to plant DNA (see¹⁹).
    8. Grind the samples in liquid nitrogen, take 100 mg of plant material and extract total RNA. Conduct qRT-PCR to determine the expression of plant genes (or fungal genes) upon infestation (see¹⁹).

6. A soil-based inoculation system in pots

1. Thoroughly mix soil and sand in a 3:1 (soil:sand) volumetric ratio to facilitate washing the substrate off the roots. Pour the mixture into an autoclave bag. If the mixture is too dry, add an appropriate amount of water and mix it into the substrate. Steam at 80 °C for 20 min in an autoclave to minimize microbial contaminations.
   NOTE: Avoid heating to over 80 °C, as this may affect organic soil nutrients.
2. Fill pots with the soil-sand mixture and transfer them into trays. Add water into the trays about 1/3 the height of a pot, so that the soil-sand mixture gets thoroughly soaked with water. Additionally, water-spray the substrate with a spray bottle to ensure wet starting conditions.
3. Sow 3-4 seeds in each pot (Figure 1C) ensuring that the seeds have enough distance from each other. Keep them for 3 days in darkness at 4 °C for stratification to synchronize germination.
   NOTE: Pre-cultivate an excess of plants, which enables a selection of plants of similar size for the inoculation experiments and reduces deviations due to individual differences.
4. Let the seedlings grow under long-day conditions (16 h light / 8 h darkness; constant temperature of 22 °C; 60% humidity) with regular watering.
5. Follow the recommended age of plants for inoculation: 21 days for Arabidopsis, 7 days for oilseed rape, and 10 days for tomato. Pick plants of similar size to perform the "root dip inoculation"^15,17,23,24. Take the soil out of the pots and carefully excavate the roots.
6. Gently wash only the roots in a water container and keep the rosettes out of the water. Incubate the washed roots for 60 min in a Petri dish containing the Verticillium spore solution (recommended concentration: 2 x 10⁶ spores/mL). For the non-infected control group, incubate the roots for 60 min in the mock solution without spores (germ-free ¼ MS medium).
7. Prepare new pots with moist, steam-sterilized soil (80 °C for 20 min) without sand. Use a pipette tip to make one hole in the center of the soil in each pot.
8. Directly place the roots into the hole (transfer only one plant per pot). After inserting the roots, make sure to refill the holes carefully with soil. Avoid pressing the soil, otherwise repotting can cause stress symptoms such as purple leaves.
9. Cultivate infected and control groups under long-day conditions (16 h light / 8 h darkness; a constant temperature of 22 °C; 60% humidity) with regular watering.
10. Perform the analyses at the preferred time points after inoculation depending on the research question (refer to the figure legends for the exact time points used here). Following are some suggestions.
    1. Take photographs of the plants with a digital camera from above, keeping the distance the same for each photo. Quantify the leaf area (e.g., with ImageJ²² or BlattFlaeche^17,19; use the diameter of the pots to set the scale) and compare infected and control groups. Categorize the development of disease symptoms (e.g., smaller, more yellowish, or necrotic leaves)¹³.
       NOTE: Removing stems of Arabidopsis facilitates taking photos of the rosettes.
    2. Remove the roots and define the biomass (fresh weight) of shoots from infected and control samples by weighing. Determine the relative fresh weight¹⁹.
    3. Alternatively, measure the plant height or categorize fungal outgrowth from stem segments to evaluate disease severity¹³.
    4. Collect samples for molecular analyses as follows.
    5. Arabidopsis: Remove the stems. Cut the rosettes at the root crown. Combine 4-5 rosettes from different plants into one sample. Freeze the leaf material in liquid nitrogen.
       NOTE: In the case of roots, it is difficult to clean them sufficiently from the soil without reprogramming gene expression through washing.
    6. Oilseed rape/tomato: Cut stem segments from the hypocotyl (e.g., 1 cm in length; always take the same stem region). Combine material from 4-5 plants into one sample and freeze it in liquid nitrogen.
    7. Grind the samples in liquid nitrogen. Extract total DNA from 100 mg leaf or stem material to determine via qPCR the amount of fungal DNA relative to plant DNA (see¹⁹).
    8. Grind the samples in liquid nitrogen, take 100 mg of plant material and extract total RNA. Conduct qRT-PCR to determine the expression of plant genes (or fungal genes) upon infestation (see¹⁹).

7. Analyzing the data

1. Calculate the average and standard deviation (± SD) based on the biological replicates.
2. Calculate the relative values by dividing all the results from the infected group by the result of the control. Display the average as, for example, "relative to mock" or "relative to wild-type".
3. Determine statistical significance between groups.

Figure 1: Compilation of the three inoculation systems and individual steps in the protocols. These figures illustrate the systems with the model plant Arabidopsis thaliana. For other plant species, the timing must be adjusted. Orange boxes highlight, for which subsequent analyses are most recommended with the respective system. (A) For the inoculation system in Petri dishes¹⁷, pour the medium and let it solidify. Keep the plates in the fridge overnight. Then, cut and remove the upper third as well as the infection channel (IC) with a scalpel (white areas in the illustration were removed from the agar, while bluish areas represent the agar). Place the seeds on the cut surface and close the Petri dishes. After stratification, place the plates vertically and let the plants grow. Once most of the roots have reached the infection channel, add the spore solution with a pipette directly into the channel. Make sure that the solution is evenly distributed. Close the Petri dishes and incubate them vertically in a growth chamber. Approaches that may follow are expressional analysis with quantitative reverse transcription PCR (qRT-PCR), microscopy with reporter lines, and quantification of microbial DNA. (B) For the inoculation system in plastic cups¹⁹, pour the medium and transfer the separating plastic layer with the prefabricated holes (four small holes in the corners for placing the seeds and one large hole in the center for the infection channel). Let the medium solidify. Cut and remove the agar medium in the center hole with a scalpel to obtain the infection channel (IC). Scratch the medium in the smaller holes and transfer the seeds. Close the cup with an inverted cup and seal with air-permeable tape (symbolized in yellow). Let the plants grow. For inoculation, add the spore solution with a pipette directly into the infection channel. Close the system and continue cultivation in the growth chamber. Approaches that may follow are expressional analysis with qRT-PCR, quantification of microbial DNA, and determination of fresh weight, leaf area, or other disease characteristics. (C) "Root dip inoculation"^15,17,23,24: for the soil-based inoculation system, fill pots with a soil:sand mixture. Transfer the seeds and let the seedlings grow. Excavate plants of similar size and wash the roots in water. Place the washed roots in a Petri dish holding the solution with the spores. After incubation, insert single plants in pots with soil. Approaches that may follow are expressional analysis in leaves with qRT-PCR, quantification of microbial DNA, and determination of fresh weight, leaf area, or other disease characteristics. Please click here to view a larger version of this figure.

Representative Results

Following the protocol, the plants were cultivated and inoculated with V. longisporum (strain Vl43²⁵) or V. dahliae (isolate JR2¹⁸). Various scenarios were designed to prove the effectiveness and to highlight some capabilities of the given protocols. Representative outcomes are shown.

Expressional induction of genes involved in the antimicrobial indol-glucosinolate (IG) biosynthesis is a reliable indicator for the evaluation of a Verticillium infection^17,19,26. In Arabidopsis, MYB51 (MYB domain protein 51; AT1G18570) encodes a transcription factor involved in the activation of genes necessary for IG biosynthesis²⁷. MYB51 can serve as a marker gene that indicates successful infestation as it is consistently induced in roots by V. longisporum²⁶ or other soil-borne fungi such as Phytophthora parasitica²⁶ and Fusarium oxysporum²¹. Two days post-inoculation (2 dpi) in the Petri dish based system, induction of MYB51 was visualized in roots of Arabidopsis. The reporter plant line Prom_MYB51::YFP²¹ disclosed a promoter activation and a qRT-PCR analysis confirmed a significant transcriptional induction of this gene (Figure 2A-B). Such experiments aim to determine expressional changes in roots during infection.

Because the Verticillium species used in this study perform a vascular lifestyle and spread from the root to the shoot via the xylem vessels, the amount of fungal DNA can be determined in leaves as a parameter for the degree of fungal propagation. Arabidopsis was root inoculated in the in vitro system in plastic cups and the rosettes were harvested 12 days later. Compared to the background value detected in the mock control, substantial amounts of fungal DNA have been found in leaves from infected plants (Figure 2C). This demonstrates that the infection has progressed successfully. For further examinations, the amount of fungal DNA can be quantified in different plant genotypes to gain insight into root defense responses. In addition, roots were collected at this time point to test the induction of the marker gene via qRT-PCR. MYB51 transcript abundance was significantly enriched (Figure 2D). This illustrates that susceptibility tests and expressional analyses can easily be performed in parallel with the cup system, which underlines the great advantage of this procedure.

To include evidence that other model plant species can also be introduced, oilseed rape was infected with V. longisporum and tomato with V. dahliae in the system in plastic cups. On day 12 after inoculation at the roots, the amount of fungal DNA was quantified in stem segments cut at the base of the seedlings (Figure 2E-F). Fungal DNA was detectable in both plant species indicating propagation of the pathogens within the plant. Again, different plant genotypes could be tested to gain knowledge on defense mechanisms.

If the root colonizing microbe of interest does not spread from roots to leaves, it is not possible to quantify the amount of microbial DNA in leaves as a parameter for disease severity. Another option to measure disease severity is the assessment and extrapolation of symptoms at the host. To exemplify this, Arabidopsis was root-dip inoculated with V. dahliae in the soil-based system and the green leaf area was evaluated (Figure 2G-H). While the rosettes of mock-treated plants looked healthy and green, pathogen-infected plants had a reduced leaf size and yellowish or even necrotic leaves. In this manner, the susceptibility of different plant genotypes can be analyzed and confirmed, for example, by quantifying the fresh weight.

Figure 2: Representative results obtained by following the protocols. (A,B) Arabidopsis roots were inoculated with V. longisporum in the Petri dish system. The reporter line Prom_MYB51::YFP²¹ revealed a strong activation of the MYB51 promoter in infected roots compared with the mock control (microscopy at 2 dpi; scale bar: 50 µm). Transcriptional induction of MYB51 in wildtype (WT) roots was further confirmed with qRT-PCR analysis (2 dpi). Values of infected samples are given relative to mock samples (set to 1) (n = 5; ± SD). (C) Systemic colonization through V. longisporum: After inoculating Arabidopsis roots in the cup system, the relative amount of fungal DNA was determined in leaves by quantitative PCR (qPCR; 12 dpi). Values of infected samples are given relative to background level in mock samples (set to 1) (n = 3; ± SD). (D) V. longisporum infected and mock-treated roots were harvested from the cup system and qRT-PCR analysis was performed. Values of infected samples are given relative to mock samples (set to 1) (n = 3; ± SD). MYB51 was found to be transcriptionally induced (12 dpi). (E) Oilseed rape was inoculated in the cup system with V. longisporum and the relative amount of fungal DNA was quantified through qPCR in stem segments (12 dpi). Values of infected samples are given relative to background level in mock samples (set to 1) (n = 3; ± SD). (F) Tomato was inoculated in the cup system with V. dahliae and the relative amount of fungal DNA was quantified through qPCR in stem segments (12 dpi). Values of infected samples are given relative to background level in mock samples (set to 1) (n = 5; ± SD). (G,H) Arabidopsis was root dip inoculated with V. dahliae in the soil-based system and representative pictures of infected and mock-treated plants are shown (21 dpi). The green leaf area was examined. Relative to mock (set to 1), infected plants had less green leaf area (n = 5; ± SD). (B-F,H) For primer pairs see¹⁹; statistics: student 's t-test relative to mock, * p≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Please click here to view a larger version of this figure.

Discussion

Due to the tremendous yield losses caused by soil-borne phytopathogens¹, an improvement of farming strategies or crop varieties is required. The limited insight into the pathogenesis of soil-borne diseases hinders the development of more resistant plants. Underlying pathomechanisms need to be explored, for which a robust methodological platform is required. Reported inoculation procedures have shown that multifactorial events in root-microbe interactions can be well dissected by combining different systems¹⁹. The protocols described above are intended to provide a routine workflow for both experts and researchers new to this field. The handling is straightforward, allows independent replication, and the required technical equipment exists in standard plant science laboratories.

Early Verticillium infection events (hours post inoculation to 6 dpi) can be examined in the Petri dish system, late events (>21 dpi) in the soil-based system, and in the cup system temporally in between (4 to 21 dpi). After adding the spores to the infection channel of the Petri dish system, they are in direct contact with the roots. This enables the examination of early defense responses. As exemplified with the MYB51 induction, expressional changes can be easily studied with gene reporter constructs, qRT-PCR, or even genome-wide "-omics" approaches^17,26. Compared with the other two infection systems, defense responses are more vigorous in the Petri dishes. Due to their small size in the Petri dishes, the plantlets are quickly overgrown by the fungus and the intensity of the defense responses may be rather unnatural in such assays¹⁹. Expressional changes can also be studied in roots of the plastic cup system leading to results comparable to the Petri dish system, although with lower induction values for marker genes. In the experimental setup in cups, the inoculum is not in immediate contact with the roots, the spores must germinate, and the pathogen must grow through the medium toward the roots. Progression of infection is slower compared to the Petri dish system and, therefore, closer to natural conditions. Regarding roots, the soil-based system rather has a disadvantage, because they must be sufficiently cleared from the soil for analysis without reprogramming gene expression by washing. Thus, studying expressional changes in roots is more complicated in soil. However, it is possible to test in leaves whether the root infestation triggers responses there.

In both in vitro inoculation systems (Petri dishes and plastic cups), external contaminations can be prevented as long as each step is executed under the laminar flow hood with sterile equipment. Thus, bilateral interactions can be examined undisturbed. Conversely, the soil-based system is just "semi-sterile" as the plants are not hermetically isolated in the growth chamber. Nevertheless, it can be considered as the one closest to natural conditions as plants grow in soil. However, roots are damaged in the soil-based system due to up-rooting, which offers microbial access to the tissues. Albeit this might be a bit artificial, this could mimic natural conditions that injure roots, such as nematode feeding²⁸.

The Petri dish system is well suited to visualize the dynamics of pathogen spread using fluorescently labeled strains (e.g., V. longisporum strain Vl-sGFP⁹). Root phenotypes resulting from colonization are well observable. On the other hand, quantification of disease symptoms at leaves/shoots in Petri dishes is hardly feasible as the system is quite small. Moreover, there might be not enough space for plant species larger than Arabidopsis. Alternatively, Behrens et al.²⁹ established an infection system on plates suitable for oilseed rape, where a brush is dipped in a spore suspension and used to distribute the inoculum along roots growing on agar. For assessing symptoms, the cup- and soil-based systems are certainly preferable. Here, the development of symptoms (e.g., reduced leaf area, loss in fresh weight, decreased plant height, the extent of necrotic tissue) can be evaluated at leaves/shoots. Investigation of larger plant species, such as oilseed rape and tomato, is not a problem in the cup- and soil-based systems as demonstrated in the representative results. If the soil-borne microbe under investigation spreads from root to shoot, pathogen-specific DNA can be PCR-amplified relative to plant DNA in shoot/leaf tissue. This can serve as a marker for conducting pathogenicity tests with different plant genotypes^13,19 and is an advantage when using vascular pathogens such as Verticillium as a model. Vice versa, different genotypes of pathogens can be applied to identify virulence genes necessary for successful colonization.

In all cases, the best timing for analyses depends on genotype, plant species, and microbe. The most critical step is to define the best time point for each research question through preliminary testing. Furthermore, when employing other microbes than Verticillium, adequate concentrations for inoculation should initially be figured out.

Besides the possibilities already mentioned, the cup system offers the potential to expand it for screenings of agrochemicals that might be applied to restrict colonization with specific parasitic microbes. The impact of biocidal chemicals on plant microbial colonization could be tested by adding the putative antimicrobial compound directly into the agar medium before (or during) inoculation and monitoring symptom development at the host. This may facilitate the implementation of screenings to accelerate the discovery of novel treatments against soil-borne diseases.

Although several members of the soil microbiome are pathogenic, the vast majority are neutral or even beneficial for plant growth¹. There is the opportunity to use the protocols to inoculate plants with beneficial microorganisms. Previously, S. indica spores were added in the Petri dish system to examine the subsequent responses in roots²⁶. This broadens the spectrum of the explained infection systems to study not only pathogenic but also beneficial interactions.

Since it is known that microbes in the rhizosphere influence each other⁶, this can be simulated by a parallel inoculation with different microbial species (or treatment of the plants first with one and later with another). This enables co-, triple-, or even multi-culture models. A more advanced extension to adequate synthetic communities (SynComs, collections of microorganisms) is conceivable as this helps to understand the influences of complex microbial compositions³⁰.

In summary, the inoculation systems allow several combinations for subsequent analyses and support a range of applications. This collection of methods is broadly applicable to various root-colonizing microbes (both beneficial and pathogenic) and offers a robust platform for analyzing root-microbe interactions.

Materials

Name	Company	Catalog Number	Comments
Agar (Gelrite)	Carl Roth	Nr. 0039	all systems described require Gelrite
Arabidopsis thaliana wild-type	NASC stock	Col-0 (N1092)
Autoclave	Systec	VE-100
BlattFlaeche	Datinf GmbH	BlattFlaeche	software to determine leaf areas
Brassica napus wild-type	see Floerl et al., 2008	rapid-cycling rape	genome ACaacc
Cefotaxime sodium	Duchefa	C0111
Chicanery flask 500 mL	Duran Group / neoLab	E-1090	Erlenmeyer flask with four baffles
Collection tubes 50 mL	Sarstedt	62.547.254	114 x 28 mm
Czapek Dextrose Broth medium	Duchefa	C1714
Digital camera	Nikon	D3100 18-55 VR
Exsiccator (Desiccator )	Duran Group	200 DN, 5.8 L	Seal with lid to hold chlorine gas
Fluorescence Microscope	Leica	Leica TCS SP5 II
HCl	Carl Roth	P074.3
KNO3	Carl Roth	P021.1	≥ 99 %
KOH	Carl Roth	6751
Leukopor	BSN medical GmbH	2454-00 AP	non-woven tape 2.5 cm x 9.2 m
MES (2-(N-morpholino)ethanesulfonic acid)	Carl Roth	4256.2	Pufferan ≥ 99 %
MgSO4	Carl Roth	T888.1	Magnesiumsulfate-Heptahydrate
Murashige & Skoog medium (MS)	Duchefa	M0222	MS including vitamins
NaClO	Carl Roth	9062.1
Percival growth chambers	CLF Plant Climatics GmbH	AR-66L2
Petri-dishes	Sarstedt	82.1473.001	size ØxH: 92 × 16 mm
Plastic cups (500 mL, transparent)	Pro-pac, salad boxx	5070	size: 108 × 81 × 102 mm
Pleated cellulose filter	Hartenstein	FF12	particle retention level 8–12 μm
poly klima growth chamber	poly klima GmbH	PK 520 WLED
Potato Dextrose Broth medium	SIGMA Aldrich	P6685	for microbiology
Pots	Pöppelmann GmbH	TO 7 D or TO 9,5 D	Ø 7 cm resp. Ø 9.5 cm
PromMYB51::YFP	see Poncini et al., 2017	MYB51 reporter line	YFP (i.e. 3xmVenus with NLS)
Reaction tubes 2 mL	Sarstedt	72.695.400	PCR Performance tested
Rotary (orbital) shaker	Edmund Bühler	SM 30 C control
Sand (bird sand)	Pet Bistro, Müller Holding	786157
Soil	Einheitserde spezial	SP Pikier (SP ED 63 P)
Solanum lycopersicum wild-type	see Chavarro-Carrero et al., 2021	Type: Moneymaker
Thoma cell counting chamber	Marienfeld	642710	depth 0.020 mm; 0.0025 mm2
Ultrapure water (Milli-Q purified water)	MERK	IQ 7003/7005	water obtained after purification
Verticillium dahliae	see Reusche et al., 2014	isolate JR2
Verticillium longisporum	Zeise and von Tiedemann, 2002	strain Vl43