Name: inoculation strategies to infect plant roots with soil-borne microorganisms
Uploaded: 2022-03-01T13:20:00
Description: Watch this Scientific Journal Video about Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms at JoVE.com

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).

1. Media for fungal cultures and plant inoculation systems
<ol>
	<li>Liquid Potato Dextrose Broth (PDB): Prepare 21 g/L PDB in ultrapure water in a heat-stable flask.</li>
	<li>Liquid Czapek Dextrose Broth (CDB): Prepare 42 g/L CDB in ultrapure water in a heat-stable flask.</li>
	<li>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...

<ol>
	<li>Mendes, R., Garbeva, P., Raaijmakers, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rhizosphere+microbiome:+significance+of+plant+beneficial,+plant+pathogenic,+and+human+pathogenic+microorganisms.">The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms.</a> FEMS Microbiology Review. 37 (5), 634-663 (2013).</li><li>Yadeta, K. A., Thomma, B. P. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+xylem+as+battleground+for+plant+hosts+and+vascular+wilt+pathogens.">The xylem as battleground for plant hosts and vascular wilt pathogens.</a> Frontiers in Plant Science. 4, 97 (2013).</li><li>Delgado-Baquerizo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proportion+of+soil-borne+pathogens+increases+with+warming+at+the+global+scale.">The proportion of soil-borne pathogens increases with warming at the global scale.</a> Nature Climate Change. 10 (6), 550-554 (2020).</li><li>Berendsen, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-induced+assemblage+of+a+plant-beneficial+bacterial+consortium.">Disease-induced assemblage of a plant-beneficial bacterial consortium.</a> The ISME Journal. 12 (6), 1496-1507 (2018).</li><li>Yuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudates+drive+the+soil-borne+legacy+of+aboveground+pathogen+infection.">Root exudates drive the soil-borne legacy of aboveground pathogen infection.</a> Microbiome. 6 (1), 156 (2018).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+plant+recruitment+of+beneficial+microbes+to+suppress+soil-borne+pathogens.">Evidence for the plant recruitment of beneficial microbes to suppress soil-borne pathogens.</a> New Phytologist. 229 (5), 2873-2885 (2021).</li><li>Wang, H., Liu, R., You, M. P., Barbetti, M. J., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+biocontrol+using+plant+growth-promoting+bacteria+(PGPR):+role+of+bacterial+diversity.">Pathogen biocontrol using plant growth-promoting bacteria (PGPR): role of bacterial diversity.</a> Microorganisms. 9 (9), 1988 (2021).</li><li>Inderbitzin, P., Subbarao, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verticillium+systematics+and+evolution:+how+confusion+impedes+Verticillium+wilt+management+and+how+to+resolve+it.">Verticillium systematics and evolution: how confusion impedes Verticillium wilt management and how to resolve it.</a> Phytopathology. 104 (6), 564-574 (2014).</li><li>Eynck, C., Koopmann, B., Grunewaldt-Stoecker, G., Karlowsky, P., von Tiedemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+interactions+of+Verticillium+longisporum+und+V.+dahliae+with+Brassica+napus+with+molecular+and+histological+techniques.">Differential interactions of Verticillium longisporum und V. dahliae with Brassica napus with molecular and histological techniques.</a> European Journal of Plant Pathology. 118 (3), 259-274 (2007).</li><li>Floerl, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defence+reactions+in+the+apoplastic+proteome+of+oilseed+rape+(Brassica+napus+var.+napus)+attenuate+Verticillium+longisporum+growth+but+not+disease+symptoms.">Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms.</a> BMC Plant Biology. 8, 129 (2008).</li><li>Leonard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verticillium+longisporum+elicits+media-dependent+secretome+responses+with+capacity+to+distinguish+between+plant-related+environments.">Verticillium longisporum elicits media-dependent secretome responses with capacity to distinguish between plant-related environments.</a> Frontiers in Microbiology. 11, 1876 (2020).</li><li>Depotter, J. R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verticillium+longisporum,+the+invisible+threat+to+oilseed+rape+and+other+brassicaceous+plant+hosts.">Verticillium longisporum, the invisible threat to oilseed rape and other brassicaceous plant hosts.</a> Molecular Plant Pathology. 17 (7), 1004-1016 (2016).</li><li>Fr&#246;schel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gain-of-function+screen+reveals+redundant+ERF+transcription+factors+providing+opportunities+for+resistance+breeding+toward+the+vascular+fungal+pathogen+Verticillium+longisporum.">A gain-of-function screen reveals redundant ERF transcription factors providing opportunities for resistance breeding toward the vascular fungal pathogen Verticillium longisporum.</a> Molecular Plant-Microbe Interactions. 32 (9), 1095-1109 (2019).</li><li>Zhou, L., Hu, Q., Johansson, A., Dixelius, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verticillium+longisporum+and+V.+dahliae:+infection+and+disease+in+Brassica+napus.">Verticillium longisporum and V. dahliae: infection and disease in Brassica napus.</a> Plant Pathology. 55 (1), 137-144 (2006).</li><li>Ralhan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+pathogen+Verticillium+longisporum+requires+a+jasmonic+acid-independent+COI1+function+in+roots+to+elicit+disease+symptoms+in+Arabidopsis+shoots.">The vascular pathogen Verticillium longisporum requires a jasmonic acid-independent COI1 function in roots to elicit disease symptoms in Arabidopsis shoots.</a> Plant Physiology. 159 (3), 1192-1203 (2012).</li><li>Reusche, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+cytokinin+levels+enhances+Arabidopsis+resistance+against+Verticillium+longisporum.">Stabilization of cytokinin levels enhances Arabidopsis resistance against Verticillium longisporum.</a> Molecular Plant-Microbe Interactions. 26 (8), 850-860 (2013).</li><li>Iven, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+activation+and+production+of+tryptophan-derived+secondary+metabolites+in+Arabidopsis+roots+contributes+to+the+defense+against+the+fungal+vascular+pathogen+Verticillium+longisporum.">Transcriptional activation and production of tryptophan-derived secondary metabolites in Arabidopsis roots contributes to the defense against the fungal vascular pathogen Verticillium longisporum.</a> Molecular Plant. 5 (6), 1389-1402 (2012).</li><li>Reusche, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+with+the+vascular+pathogens+Verticillium+longisporum+and+Verticillium+dahliae+induce+distinct+disease+symptoms+and+differentially+affect+drought+stress+tolerance+of+Arabidopsis+thaliana.">Infections with the vascular pathogens Verticillium longisporum and Verticillium dahliae induce distinct disease symptoms and differentially affect drought stress tolerance of Arabidopsis thaliana.</a> Environmental and Experimental Botany. 108, 23-37 (2014).</li><li>Fr&#246;schel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+evaluation+of+root+infection+systems+using+the+vascular+fungus+Verticillium+longisporum+as+soil-borne+model+pathogen.">In-depth evaluation of root infection systems using the vascular fungus Verticillium longisporum as soil-borne model pathogen.</a> Plant Methods. 17 (1), 57 (2021).</li><li>Karapapa, V. K., Bainbridge, B. W., Heale, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+molecular+characterization+of+Verticillium+longisporum+comb,+nov.,+pathogenic+to+oilseed+rape.">Morphological and molecular characterization of Verticillium longisporum comb, nov., pathogenic to oilseed rape.</a> Mycological Research. 101 (11), 1281-1294 (1997).</li><li>Poncini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+roots+of+Arabidopsis+thaliana,+the+damage-associated+molecular+pattern+AtPep1+is+a+stronger+elicitor+of+immune+signalling+than+flg22+or+the+chitin+heptamer.">In roots of Arabidopsis thaliana, the damage-associated molecular pattern AtPep1 is a stronger elicitor of immune signalling than flg22 or the chitin heptamer.</a> PLoS One. 12 (10), 1-21 (2017).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Fradin, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+Verticillium+wilt+resistance+mediated+by+tomato+Ve1.">Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.</a> Plant Physiology. 150 (1), 320-332 (2009).</li><li>Singh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+host+Brassica+napus+induces+in+the+pathogen+Verticillium+longisporum+the+expression+of+functional+catalase+peroxidase+which+is+required+for+the+late+phase+of+disease.">The plant host Brassica napus induces in the pathogen Verticillium longisporum the expression of functional catalase peroxidase which is required for the late phase of disease.</a> Molecular Plant-Microbe Interactions. 25 (4), 569-581 (2012).</li><li>Zeise, K., von Tiedemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+RAPD-PCR+for+virulence+type+analysis+within+Verticillium+dahliae+and+Verticillium+longisporum.">Application of RAPD-PCR for virulence type analysis within Verticillium dahliae and Verticillium longisporum.</a> Journal of Phytopathology. 150 (10), 557-563 (2002).</li><li>Fr&#246;schel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+roots+employ+cell-layer-specific+programs+to+respond+to+pathogenic+and+beneficial+microbes.">Plant roots employ cell-layer-specific programs to respond to pathogenic and beneficial microbes.</a> Cell Host &amp; Microbe. 29 (2), 299-310 (2021).</li><li>Gigolashvili, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+HIG1/MYB51+regulates+indolic+glucosinolate+biosynthesis+in+Arabidopsis+thaliana.">The transcription factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis thaliana.</a> The Plant Journal. 50 (5), 886-901 (2007).</li><li>Back, M. A., Haydock, P. P. J., Jenkinson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+complexes+involving+plant+parasitic+nematodes+and+soilborne+pathogens.">Disease complexes involving plant parasitic nematodes and soilborne pathogens.</a> Plant Pathology. 51 (6), 683-697 (2002).</li><li>Behrens, F. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+abscisic+acid+biosynthesis+at+the+early+infection+stage+of+Verticillium+longisporum+in+oilseed+rape+(Brassica+napus).">Suppression of abscisic acid biosynthesis at the early infection stage of Verticillium longisporum in oilseed rape (Brassica napus).</a> Molecular Plant Pathology. 20 (12), 1645-1661 (2019).</li><li>Vorholt, J. A., Vogel, C., Carlstr&#246;m, C. I., M&#252;ller, D. 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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 differen...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar (Gelrite)</td>
			<td>Carl Roth</td>
			<td>Nr. 0039</td>
			<td>all systems described require Gelrite</td>
		</tr>
		<tr>
			<td>Arabidopsis thaliana 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>BlattFlaeche</td>
			<td>software to determine leaf areas</td>
		</tr>
		<tr>
			<td>Brassica napus wild-type</td>
			<td>see Floerl et al., 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>KNO3</td>
			<td>Carl Roth</td>
			<td>P021.1</td>
			<td>&#8805; 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 &#8805; 99 %</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Carl Roth</td>
			<td>T888.1</td>
			<td>Magnesiumsulfate-Heptahydrate</td>
		</tr>
		<tr>
			<td>Murashige &#38; 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 &#216;xH: 92 &#215; 16 mm</td>
		</tr>
		<tr>
			<td>Plastic cups (500 mL, transparent)</td>
			<td>Pro-pac, salad boxx</td>
			<td>5070</td>
			<td>size: 108 &#215; 81 &#215; 102 mm</td>
		</tr>
		<tr>
			<td>Pleated cellulose filter</td>
			<td>Hartenstein</td>
			<td>FF12</td>
			<td>particle retention level 8&#8211;12 &#956;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&#246;ppelmann GmbH</td>
			<td>TO 7 D or TO 9,5 D</td>
			<td>&#216; 7 cm resp. &#216; 9.5 cm</td>
		</tr>
		<tr>
			<td>PromMYB51::YFP</td>
			<td>see Poncini et al., 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&#252;hler</td>
			<td>SM 30 C control</td>
			<td></td>
		</tr>
		<tr>
			<td>Sand (bird sand)</td>
			<td>Pet Bistro, M&#252;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>Solanum lycopersicum wild-type</td>
			<td>see Chavarro-Carrero et al., 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 mm2</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>Verticillium dahliae</td>
			<td>see Reusche et al., 2014</td>
			<td>isolate JR2</td>
			<td></td>
		</tr>
		<tr>
			<td>Verticillium longisporum</td>
			<td>Zeise and von Tiedemann, 2002</td>
			<td>strain Vl43</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 evalu...

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 ...

Although countless microbes colonize plant roots, these interactions are difficult to analyze as they happen underground. To facilitate this, this protocol collection provides an overview of strategies to inoculate plant roots with soil-borne microbes. Root inoculation is the basis for studying root microbe interactions, and by using the following techniques, interactions can be studied at a molecular, cytological or histological level.We explain three inoculation systems using verticillium as root invading microbe, and the model plant, arabidopsis. However, transfer of the methods to other plants such as tomato or oilseed rape, and to other root microbes, for example beneficial serendipita, is possible. Prepare the verticillium inoculum in advance by culturing the mycelia in PDB and inducing sporulation and Czapek dextrose broth.Then begin by pouring the autoclaved medium into Petri dishes. After hardening of the medium, repack the Petri dishes in a sterile plastic bag and store them upside down overnight in the refrigerator at four to 10 degrees Celsius. Cut and remove an infection channel in the upper third of the solidified medium with a scalpel.Avoid getting liquid or air under the agar medium while cutting. Next, use a sterile pipette tip to distribute 50 to 100 surface sterilized arabidopsis seeds on the cut upper surface. Put the seeds in the angle where the cut agar surface contacts the Petri dish wall so that the roots can grow in between them.Close the Petri dishes and seal them with a breathable adhesive tape to allow gas exchange. Keep the Petri dishes in the dark at four degrees Celsius for two days for stratification to ensure synchronized and efficient germination. Then place the plates vertically in a rack and grow the plants in a growth chamber under long day conditions.After nine to 11 days when the roots reach the infection channel, lay the plates horizontally and open them. Then add 500 microliters of freshly harvested Verticillium conidia directly into the infection channel. Prepare control plates by adding 500 microliters of a mock solution instead of spores.Incubate both control and spore inoculated plates horizontally until the liquid has soaked in. Then close the lid and seal the plates with breathable adhesive tape. Cover the root parts with black paper boxes to darken roots and soil-borne fungus.Incubate the plates vertically in the growth chamber. Perform the analyses at the preferred time points after inoculation. First, sterilize transparent 500 milliliter plastic cups into 70 to 75%ethanol bath for 20 minutes.Dry the cups in the lamina flow hood. Pour the autoclaved medium into the plastic cups. Prepare a plastic layer with five pre-fabricated holes one large in the center and four smaller ones in each corner.Place the plastic layer on the medium before it solidifies. Cut the agar with a scalpel through the pre-fabricated center hole to a depth of about 1.5 centimeters. Remove the cut agar to create an infection channel to add the fungal spores later.Scratch the agar medium slightly with a pipette tip in the four smaller holes to interrupt the solidified skin. Place the seeds using a pipette tip into the smaller holes. Close the plastic cup with a second inverted plastic cup and seal with breathable adhesive tape.Keep the plants at four degrees Celsius for three days in the dark for stratification. Then incubate the cup systems in the growth chamber. To inoculate plantlets with verticillium, add one milliliter of conidia solution into the infection channel.For controls, add one milliliter of germ free one fourth MS medium as a mock solution. Perform the analyses at the preferred time points after inoculation. First, thoroughly mix soil and sand in a three to one volumetric ratio which facilitates washing the substrate from the roots later.Pour the mixture into an autoclave bag and steam at 80 degrees Celsius for 20 minutes in an autoclave. Fill pots with the soil-sand mixture and transfer them into trays. Add water into the trays about one third the height of a pot for uniform soaking of the soil-sand mixture.Water spray the mixture to ensure wet starting conditions. Sow three to four seeds in each pot at enough distance from each other. Keep them for stratification at four degrees Celsius for three days in the dark.Allow the seedlings to grow under long day conditions with regular watering, then pick plants of similar size and age to perform the root dip inoculation. Take the soil out of the pots and excavate the roots. Gently wash the roots in a water container and keep the rosettes out of the water.Incubate the roots for 60 minutes in a Petri dish containing the verticillium spore solution or mock solution. Prepare new pots with moist steam sterilized soil without sand. Use a pipette tip to make a hole in the center of the soil in each pot.Place the roots directly into the hole and refill the holes gently with soil to avoid additional stress to the plants. Cultivate the plants under long day conditions with regular watering. Perform the analyses at the preferred time points after inoculation.In the Petri dish-based system, successful infection of verticillium was visualized in the roots of arabidopsis. Two days post inoculation, the marker gene MYB51 was strongly induced in infected roots compared to control, which was confirmed by QRTPCR analysis. In the cup-based system, a high amount of fungal DNA was found in the leaves of infected plants as the graph shows in comparison to mock control after 12 days of inoculation.QRTPCR analysis showed enriched transcript abundance of the marker gene MYB51 in the arabidopsis roots. Using this system, verticillium was inoculated to other model plant species. In oilseed rape, the fungal DNA was detectable in stem segments cut at the base of the seedlings 12 days after inoculation at the roots.Fungal DNA was also detected in tomato stems indicating propagation of the pathogen within the plant. 21 days post inoculation using the soil-based system, the rosettes of mock treated arabidopsis plants looked healthy and green while infected plants showed yellowish or necrotic leaves and had lesser green leaf area relative to mock indicating successful infection. Pay attention when cutting the infection channel in the Petri dish system to prevent the agar from sliding.Applying the soil-based system, don't compress the soil around the roots while repotting to avoid further stress to the plants. After root inoculation, for example, pathogenic tests, analysis of gene expression, functional genomics or agrochemical tests can be performed providing new insights into root microbe interactions and tools to improve agriculture.

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