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.
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.
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 viruses1,2. 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 pathogens4,5,6,7. 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 plants2,8. Hyphae of Verticillium enter the root and grow both intercellularly and intracellularly toward the central cylinder to colonize the xylem vessels2,9. 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 host10,11. After reaching the root vasculature, the fungus can spread within the xylem vessels acropetally to the foliage, which leads to systemic colonization of the host9,12. At this point, the plant is negatively affected in growth9,10,13. For instance, stunting and yellow leaves occur as well as premature senescence13,14,15,16.
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 responses13,15,16,17. 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.
1. Media for fungal cultures and plant inoculation systems
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.
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.
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.
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.
6. A soil-based inoculation system in pots
7. Analyzing the data
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.
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 infection17,19,26. 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 PromMYB51::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 PromMYB51::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.
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" approaches17,26. 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 genotypes13,19 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.
The authors have nothing to disclose.
The authors acknowledge Tim Iven and Jaqueline Komorek for previous work on these methods, the group of Wolfgang Dröge-Laser (Department of Pharmaceutical Biology, University of Würzburg, Germany) for providing the equipment and the resources needed for this work, and Wolfgang Dröge-Laser as well as Philipp Kreisz (both University of Würzburg) for critical proofreading of the manuscript. This study was supported by the "Deutsche Forschungsgemeinschaft" (DFG, DR273/15-1,2).
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 |