The authors acknowledge Tim Iven and Jaqueline Komorek for previous work on these methods, the group of Wolfgang Dr&#246;ge-Laser (Department of Pharmaceutical Biology, University of W&#252;rzburg, Germany) for providing the equipment and the resources needed for this work, and Wolfgang Dr&#246;ge-Laser as well as Philipp Kreisz (both University of W&#252;rzburg) for critical proofreading of the manuscript. This study was supported by the &#34;Deutsche Forschungsgemeinschaft&#34; (DFG, DR273/15-1,2).

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</ol>

The authors have nothing to disclose.

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 (&#62;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 &#34;-omics&#34; 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 &#34;semi-sterile&#34; 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.

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.

<table><tbody><tr><td>Agar (Gelrite)</td><td>Carl Roth</td><td>Nr. 0039</td><td>all systems described require Gelrite</td></tr><tr><td>&lt;em&gt;Arabidopsis thaliana&lt;/em&gt; wild-type</td><td>NASC stock</td><td>Col-0 (N1092)</td><td></td></tr><tr><td>Autoclave</td><td>Systec</td><td>VE-100</td><td></td></tr><tr><td>BlattFlaeche</td><td>Datinf GmbH</td><td>&lt;em&gt;BlattFlaeche&lt;/em&gt;</td><td>software to determine leaf areas</td></tr><tr><td>&lt;em&gt;Brassica napus&lt;/em&gt; wild-type</td><td>see Floerl &lt;em&gt;et al.&lt;/em&gt;, 2008</td><td>rapid-cycling rape</td><td>genome ACaacc</td></tr><tr><td>Cefotaxime sodium</td><td>Duchefa</td><td>C0111</td><td></td></tr><tr><td>Chicanery flask 500 mL</td><td>Duran Group / neoLab</td><td>E-1090</td><td>Erlenmeyer flask with four baffles</td></tr><tr><td>Collection tubes 50 mL</td><td>Sarstedt</td><td>62.547.254</td><td>114 x 28 mm</td></tr><tr><td>Czapek Dextrose Broth medium</td><td>Duchefa</td><td>C1714</td><td></td></tr><tr><td>Digital camera</td><td>Nikon</td><td>D3100 18-55 VR</td><td></td></tr><tr><td>Exsiccator (Desiccator )</td><td>Duran Group</td><td>200 DN, 5.8 L</td><td>Seal with lid to hold chlorine gas</td></tr><tr><td>Fluorescence Microscope</td><td>Leica</td><td>Leica TCS SP5 II</td><td></td></tr><tr><td>HCl</td><td>Carl Roth</td><td>P074.3</td><td></td></tr><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Carl Roth</td><td>P021.1</td><td>&amp;ge; 99 %</td></tr><tr><td>KOH</td><td>Carl Roth</td><td>6751</td><td></td></tr><tr><td>Leukopor</td><td>BSN medical GmbH</td><td>2454-00 AP</td><td>non-woven tape 2.5 cm x 9.2 m</td></tr><tr><td>MES (2-(N-morpholino)ethanesulfonic acid)</td><td>Carl Roth</td><td>4256.2</td><td>Pufferan &amp;ge; 99 %</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>T888.1</td><td>Magnesiumsulfate-Heptahydrate</td></tr><tr><td>Murashige &amp;amp; Skoog medium (MS)</td><td>Duchefa</td><td>M0222</td><td>MS including vitamins</td></tr><tr><td>NaClO</td><td>Carl Roth</td><td>9062.1</td><td></td></tr><tr><td>Percival growth chambers</td><td>CLF Plant Climatics GmbH</td><td>AR-66L2</td><td></td></tr><tr><td>Petri-dishes</td><td>Sarstedt</td><td>82.1473.001</td><td>size &amp;Oslash;xH: 92 &amp;times; 16 mm</td></tr><tr><td>Plastic cups (500 mL, transparent)</td><td>Pro-pac, salad boxx</td><td>5070</td><td>size: 108 &amp;times; 81 &amp;times; 102 mm</td></tr><tr><td>Pleated cellulose filter</td><td>Hartenstein</td><td>FF12</td><td>particle retention level 8&amp;ndash;12 &amp;mu;m</td></tr><tr><td>poly klima growth chamber</td><td>poly klima GmbH</td><td>PK 520 WLED</td><td></td></tr><tr><td>Potato Dextrose Broth medium</td><td>SIGMA Aldrich</td><td>P6685</td><td>for microbiology</td></tr><tr><td>Pots</td><td>P&amp;ouml;ppelmann GmbH</td><td>TO 7 D or TO 9,5 D</td><td>&amp;Oslash; 7 cm resp. &amp;Oslash; 9.5 cm</td></tr><tr><td>&lt;em&gt;Prom&lt;sub&gt;MYB51&lt;/sub&gt;::YFP&lt;/em&gt;</td><td>see Poncini &lt;em&gt;et al.&lt;/em&gt;, 2017</td><td>MYB51 reporter line</td><td>YFP (i.e. 3xmVenus with NLS)</td></tr><tr><td>Reaction tubes 2 mL</td><td>Sarstedt</td><td>72.695.400</td><td>PCR Performance tested</td></tr><tr><td>Rotary (orbital) shaker</td><td>Edmund B&amp;uuml;hler</td><td>SM 30 C control</td><td></td></tr><tr><td>Sand (bird sand)</td><td>Pet Bistro, M&amp;uuml;ller Holding</td><td>786157</td><td></td></tr><tr><td>Soil</td><td>Einheitserde spezial</td><td>SP Pikier (SP ED 63 P)</td><td></td></tr><tr><td>&lt;em&gt;Solanum lycopersicum&lt;/em&gt; wild-type</td><td>see Chavarro-Carrero &lt;em&gt;et al.&lt;/em&gt;, 2021</td><td>Type: Moneymaker</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>Marienfeld</td><td>642710</td><td>depth 0.020 mm; 0.0025 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Ultrapure water (Milli-Q purified water)</td><td>MERK</td><td>IQ 7003/7005</td><td>water obtained after purification</td></tr><tr><td>&lt;em&gt;Verticillium dahliae&lt;/em&gt;</td><td>see Reusche &lt;em&gt;et al.&lt;/em&gt;, 2014</td><td>isolate JR2</td><td></td></tr><tr><td>&lt;em&gt;Verticillium longisporum&lt;/em&gt;</td><td>Zeise and von Tiedemann, 2002</td><td>strain &lt;em&gt;Vl&lt;/em&gt;43</td><td></td></tr></tbody></table>

inoculation strategies to infect plant roots with soil-borne microorganisms

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.

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.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63446/63446fig02.jpg" /> 
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 &#181;m). 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; &#177; 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; &#177; 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; &#177; 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; &#177; 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; &#177; 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; &#177; SD). (B-F,H) For primer pairs see19; statistics: student &#39;s t-test relative to mock, * p&#8804; 0.05, ** p&#160;&#8804; 0.01, *** p&#160;&#8804; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63446/63446fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms at JoVE.com

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.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

inoculation-strategies-to-infect-plant-roots-with-soil-borne

Nanyang Technological University

Research

JoVE Journal

Environment

6.1K Views.  Julius-Maximilians-Universität Würzburg. Hoewel talloze microben plantenwortels koloniseren, zijn deze interacties moeilijk te analyseren omdat ze ondergronds plaatsvinden. Om dit te vergemakkelijken, biedt deze protocolcollectie een overzicht van strategieën om plantenwortels te enten met bodemmicroben. Wortelinenting is de basis voor het bestuderen van interacties tussen wortelmicroben en door de volgende technieken te gebruiken, kunnen interacties worden bestudeerd op moleculair, cytologisch of histologisch niveau.We verk...

Video: Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Bioengineering

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

Genetics

Rhizobacterial Colonization Detection on Arabidopsis thaliana

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

Culturing Arbuscular Mycorrhizal Fungi Using SAP-AS for Enhanced Observation

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.

Author Spotlight: Enhancing AM Fungi Research with SAP-AS &#8212; A Novel Technique for Single-Spore Cultures

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Biology

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

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