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).

<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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+causality:+opportunities+of+synthetic+communities+for+plant+microbiome+research.">Establishing causality: opportunities of synthetic communities for plant microbiome research.</a> Cell Host &amp; Microbe. 22 (2), 142-155 (2017).</li></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 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 ...

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

Research

JoVE Journal

Environment

5.8K Views.  Julius-Maximilians-Universität Würzburg. 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.

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

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 ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

Operation of a 25 KWth Calcium Looping Pilot-plant with High Oxygen Concentrations in the Calciner

Calcium looping (CaL) is a post-combustion CO2 capture technology that is suitable for retrofitting existing power plants. The CaL process uses limestone as a cheap and readily available CO2 sorbent. While the technology has been widely studied, there are a few available options that could be applied to make it more economically viable. One of these is to increase the oxygen concentration in the calciner to reduce or eliminate the amount of recycled gas (CO2, H2O and impurities); therefore, decreasing or removing the energy necessary to heat the recycled gas stream. Moreover, there is a resulting increase in the energy input due to the change in the combustion intensity; this energy is used to enable the endothermic calcination reaction to occur in the absence of recycled flue gases. This paper presents the operation and first results of a CaL pilot plant with 100% oxygen combustion of natural gas in the calciner. The gas coming into the carbonator was a simulated flue gas from a coal-fired power plant or cement industry. Several limestone particle size distributions are also tested to further explore the effect of this parameter on the overall performance of this operating mode. The configuration of the reactor system, the operating procedures, and the results are described in detail in this paper. The reactor showed good hydrodynamic stability and stable CO2 capture, with capture efficiencies of up to 70% with a gas mixture simulating the flue gas of a coal-fired power plant.

Operation of a 25 KWth Calcium Looping Pilot-plant with High Oxygen Concentrations in the Calciner

This manuscript describes a procedure for operating a calcium looping pilot-plant for post-combustion carbon capture with high oxygen concentrations in the calciner in order to reduce or eliminate the flue gas recycle.

Calcium looping (CaL) is a post-combustion CO2 capture technology that is suitable for retrofitting existing power plants. The CaL process uses limestone ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.