Name: an easy and flexible inoculation method for accurately assessing powdery mildew-infection phenotypes of arabidopsis and other plants
Uploaded: 2021-03-09T14:00:00
Description: Watch this Scientific Journal Video about An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants at JoVE.com

The work was supported by the National Science Foundation (IOS-1901566) to S. Xiao. The authors would like to thank F. Coker and C. Hooks for the maintenance of the plant growth facility, and Jorge Zamora for technical help associated with fabrication of the inoculation box and chamber.

1. Making a standard inoculation box with a removable top lid mounted with a mesh
<ol>
	<li>Purchase a roll of 50 &#181;m nylon membrane mesh or 48 &#181;m stainless steel mesh (recommended) from stores. Make sure to order enough for cutting into multiple pieces of 14 in x 26 in for replacement of worn mesh.</li>
	<li>Purchase one 1/4 in x 2 ft x 4 ft medium density fiberboard or plywood, and cut two 24-1/2 in x 10 in pieces and two 12 in x 10 in pieces for making an inoculation box.Use 8 corner clamps for supporting t...

<ol>
	<li>Huckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bi+ology+of+the+plant-powdery+mildew+interaction.">Cell bi ology of the plant-powdery mildew interaction.</a> Current Opinion in Plant Biology. 14 (6), 738-746 (2011).</li><li>Dean, R., 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+top+10+fungal+pathogens+in+molecular+plant+pathology.">The top 10 fungal pathogens in molecular plant pathology.</a> Molecular Plant Pathology. 13 (4), 414-430 (2012).</li><li>Sakurai, H., Hirata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+the+relation+between+the+penetration+hypha+and+haustorium+of+barley+powdery+mildew+and+host+cell.+V.+Influence+of+water+spray+on+the+pathogen+and+host+tissue.">Some observations on the relation between the penetration hypha and haustorium of barley powdery mildew and host cell. V. Influence of water spray on the pathogen and host tissue.</a> Annual Phytopathology Society of Japan. 24, 239-245 (1959).</li><li>Shomari, S. H., Kennedy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+Oidium+anacardii+on+cashew+(Anacardium+occidentale)+in+southern+Tanzania.">Survival of Oidium anacardii on cashew (Anacardium occidentale) in southern Tanzania.</a> Plant Pathology. 48 (4), 505-513 (1999).</li><li>Sitterly, W. R., Spencer, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Powdery Mildews. , 369 (1978).</li><li>Reuveni, M., Agapov, V., Reuveni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+systemic+resistance+to+powdery+mildew+and+growth+increase+in+cucumber+by+phosphates.">Induction of systemic resistance to powdery mildew and growth increase in cucumber by phosphates.</a> Biological Agriculture &amp; Horticulture. 9 (4), 305-315 (1993).</li><li>Reifschneider, F. J. B., Boiteux, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vacuum-operated+settling+tower+for+inoculation+of+powdery+mildew+fungi.">A vacuum-operated settling tower for inoculation of powdery mildew fungi.</a> Phytopathology. 78 (11), 1463-1465 (1988).</li><li>Chowdhury, A., Bremer, G. B., Salt, D. W., Miller, P., Ford, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+delivering+Blumeria+graminis+f.+sp+hordei+spores+for+laboratory+experiments.">A novel method of delivering Blumeria graminis f. sp hordei spores for laboratory experiments.</a> Crop Protection. 22 (7), 917-922 (2003).</li><li>Xiao, 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=Broad-spectrum+mildew+resistance+in+Arabidopsis+thaliana+mediated+by+RPW8.">Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8.</a> Science. 291 (5501), 118-120 (2001).</li><li>Xiao, S., Ellwood, S., Findlay, K., Oliver, R. P., Turner, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+three+loci+controlling+resistance+of+Arabidopsis+thaliana+accession+Ms-0+to+two+powdery+mildew+diseases.">Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms-0 to two powdery mildew diseases.</a> The Plant Journal. 12 (4), 757-768 (1997).</li><li>Reuber, T. 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=Correlation+of+defense+gene+induction+defects+with+powdery+mildew+susceptibility+in+Arabidopsis+enhanced+disease+susceptibility+mutants.">Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants.</a> The Plant Journal. 16 (4), 473-485 (1998).</li><li>Xiao, 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+atypical+resistance+gene,+RPW8,+recruits+components+of+basal+defence+for+powdery+mildew+resistance+in+Arabidopsis.">The atypical resistance gene, RPW8, recruits components of basal defence for powdery mildew resistance in Arabidopsis.</a> The Plant Journal. 42 (1), 95-110 (2005).</li></ol>

Our meshed-box-based inoculation method has several advantages over other inoculation methods. First, it can achieve even distribution of spores if operated properly, as demonstrated in Figure 5. Second, the use of ~50 &#181;m mesh, plus spore-dislodging by gentle shaking of infected leaves can reduce plant infection by thrips or other plant-infecting insects that are present in source plants. Third, the use of different-sized inoculation boxes for inoculating plants or detached leaves insid...

Powdery mildew is one of the most common and important diseases of numerous food crops and ornamental plants1. Studies of powdery mildew diseases have been very popular, as evidenced by over 10,500 publications as the search result with "powdery mildew" as key word at the Web of Science (as of November 2020). Indeed, powdery mildew (represented by Blumeria graminis) is considered to be one of the top 10 fungal pathogens by the journal of Molecular Plant Pathology2. Quantification of disease susceptibility is a necessary step in characterization of plant genes contributing to disease resistance or susceptibility, or functional identification of candidate effector genes in powdery mildew. However, reliable disease phenotyping is far more challenging with powdery mildew compared to that with most other fungal pathogens, partly because, unlike spores of the latter, spores of powdery mildew species (such as Golovinomyces cichoracearum UCSC1 based on our lab experience) show reduced viability after going through a water-suspension process3,4. Inadequate and/or uneven inoculation of test plants with a particular powdery mildew pathogen may lead to inaccurate phenotyping results.
A number of inoculation methods were reported for powdery mildew studies. These include (i) brushing spores directly from infected leaves to test plants5, (ii) spraying a spore suspension to test plants6, (iii) blowing spores using a vacuum-operated settling tower to plants at the bottom of the tower7, and (iv) spore delivery by the combinatorial use of a nylon mesh membrane and sound-based vibration8. The spore-brushing (or dusting) method is easy to perform but uneven in nature, thus it may not be accurate for quantitative assessment. Spore-spraying is convenient and even, but as stated above may result in poor spore germination4. The latter two (i.e., iii-iv) are much-improved methods capable of achieving even inoculation; however, both are not flexible in adjusting their inoculation capacity in terms of the number of plants to be inoculated in a single event, making either apparatus is not trivial, and their operation is restricted to lab areas where there is a vacuum and/or electricity source.
Our lab has been working with plant-powdery mildew interaction for over 20 years9,10. Over the past decade, we tested a number of inoculation methods and recently developed a simple and yet effective powdery mildew inoculation method. This mesh-based spore-brushing method can ensure even inoculation, and is simple and scalable, thus should be easily adopted by any laboratory working with powdery mildew.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >&#160;48 &#181;m stainless steel grid mesh screen; Size: 24&#34; X 48&#34;&#160;</td>
			<td >Amazon</td>
			<td >NA</td>
			<td >For making the lid of an inoculation box</td>
		</tr>
		<tr >
			<td >#6-32 x &#190;&#34; machine screws, flat washers and nuts&#160;</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >#6-32 zinc plated nylon lock nut (4-Pack)</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >#6-32x3/8&#8221; Phillips flat head machine screws, flat washers and nuts&#160;</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For securing&#160; magnet door catch plates</td>
		</tr>
		<tr >
			<td >#8-32x1/2&#34; machine screws, flat washers and nuts</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For securing corner braces and door hinge</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet;&#160; Size: 17 &#189;&#34; X 20&#34;</td>
			<td >Professional Plastics</td>
			<td >SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet; Size: 18&#34; X 20&#34;&#160;&#160;</td>
			<td >Professional Plastics</td>
			<td >&#160;SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet; Size: 18&#34; X 30&#34;&#160;</td>
			<td >Professional Plastics</td>
			<td >SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet; Size: 20&#34; X 29 &#189; &#34;</td>
			<td >Professional Plastics</td>
			<td >SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >1-5/8&#34; cabinet door magnetic catch white</td>
			<td >Home Depot</td>
			<td >Model #P110-W</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >2&#34; steel zinc-plated corner brace (8-Pack)&#160;</td>
			<td >Home Depot</td>
			<td >&#160;Model #13611&#160;</td>
			<td>For making an inoculation box &#38; chamber</td>
		</tr>
		<tr >
			<td >3&#34; Corner Clamp</td>
			<td>Harbor Freight Tools</td>
			<td>SKU 63653, 1852, 60589</td>
			<td>For making inoculation chamber</td>
		</tr>
		<tr >
			<td >3/4&#34;&#160; steel zinc plated corner brace (4-Pack)</td>
			<td >Home Depot</td>
			<td >Model #13542</td>
			<td>For making an inoculation box &#38; chamber</td>
		</tr>
		<tr >
			<td >4-7/8&#34; zinc-plated light duty door pull handles</td>
			<td >Home Depot</td>
			<td >Model #15184</td>
			<td>For making an inoculation box</td>
		</tr>
		<tr >
			<td >Fine fan-blender brushes</td>
			<td>Michaels Store</td>
			<td>M10472846&#160;</td>
			<td>For inoculation</td>
		</tr>
		<tr >
			<td >Kelleher 3/4&#34; x 3/4&#34; x 36&#34; wood square dowel&#160;</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For making the lid of an inoculation box</td>
		</tr>
		<tr >
			<td >Medium density fiberboard (1/4&#34; x 2&#39; x 4&#39;);&#160;</td>
			<td >Home Depot</td>
			<td >Model# 1508104</td>
			<td>For making an inoculation box</td>
		</tr>
		<tr >
			<td >Round glass coverslips with a 500 &#181;m grid</td>
			<td >ibidi USA Inc.</td>
			<td >10816</td>
			<td>For determining&#160; spore density</td>
		</tr>
	</tbody>
</table>

an easy and flexible inoculation method for accurately assessing powdery mildew-infection phenotypes of arabidopsis and other plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

Here, we present a new powdery mildew spore inoculation method that is easy to prepare, operate and adjust. Figure 1 shows the assembly of the standard inoculation box with emphasis on the make of the removable lid mounted with a 50 &#181;m membrane mesh. Figure 2 shows the assembly of the inoculation chamber. Figure 3 illustrates the key steps of the inoculation process using this system. Figure 4 show...

Watch this Scientific Journal Video about An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants at JoVE.com

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Powdery mildew is an important plant disease. Accurate determination of the disease phenotype requires even inoculation of the pathogen. Since existing methods are inefficient, we have developed this protocol.Our spore inoculation method can easily achieve even inoculation, and it is simple and scalable. Hence, any lab working with powdery mildew can easily use this method. The researchers can also mount mesh over a cardboard box and use it for spore inoculation.How to dislodge spores and brush them through mesh is a critical step. So demonstrating this on video could be very helpful. For this protocol, get a roll of 50 micrometer nylon membrane mesh or 48 micrometer stainless steel mesh.Cut large sized, medium-density plywood into pieces of 24.5X10 inches and 12X10 inches for making an inoculation box. To make a removable top lid with a mesh, cut the long wooden square dowel into two sets of 26 inches and 12.75 inches. Glue and nail the four pieces to fit around the box.Take acrylic sheets and assemble all the acrylic sheets into a box, using eight corner clamps. Cut a 7X2 inch opening window on the front side of the acrylic sheet of the box. To prepare fresh powdery mildew spores as inocula, grow clean Arabidopsis immune-compromised pad4-1 mutant plants in a growth chamber at 22 degrees Celsius and 65%relative humidity for six to eight weeks.Grow the plants to determine infection phenotypes in standard flats. Inoculate one or half of pad4-1 plants to build sufficient fresh powdery mildew spores. When fresh spores from infected plants are available, move a flat of plants into the inoculation box inside the inoculation chamber for an infection test.Cut 15 to 20 heavily infected Arabidopsis leaves, and let them dry if there is condensed water. Hold one to two leaves upside down in one hand with a pair of long forceps. Then put both hands through the opening of the chamber and gently hit the arm of the forceps with a pair of scissors, while slowly moving the leaves above the mesh mounted on the inoculation box.Brush the spores with fine fan blender brushes through the entire mesh surface in different directions to maximize the even distribution of spores onto the plants. Gently pat the mesh a few times with the brushes to shake off the spores stuck in the mesh. Once the spores settle, move out the flat containing the inoculated plants from the inoculation box.Cover the flat with a plastic dome and place it in a growth chamber at 22 degrees Celsius and 65%relative humidity. Disease phenotyping can be performed at 5 or 8 to 12 days post inoculation. Convert cardboard boxes to an inoculation box by removing the top and bottom sides of the box, taping the corner to firm it, and mounting a 50 micrometer mesh on the top with glue.Move the flat containing the plants inside the inoculation chamber, and place the smaller inoculation box on top of the flat to cover the plants. Then dislodge and brush the spores. Make a mini inoculation box slightly bigger than a Petri dish.Store the plates at four degrees Celsius in a fridge until use. Cut one or two fully expanded leaves from the base of the petiole for each plant to be tested. Insert the petiole of the detached leaves into the agar medium at an angle of 30 degrees.Inoculate the leaves inside the inoculation chamber, and move the plate with the lid to a growth chamber. For assessing infection phenotypes, transfer the leaves to a fresh aseptic MS agar plate at four or five days post inoculation. Cut the base of the petiole before inserting the leaves into the fresh agar medium.Assess the disease phenotypes at eight or nine days post inoculation. The microscopic analysis displayed even distribution of spores on the microscopic slides on the bottom of the flat. The calculated density of spores, distributed in six different locations after light inoculations, showed no significant differences.Similar results were achieved following heavy inoculations with spores. The inoculation evenness was examined among different plant genotypes at 12 days after heavy inoculation. The pad4-1 plants showed enhanced disease susceptibility compared to Columbia 0, the wild type plants.Using adequate fresh spores, produced around 10 days after inoculation, on susceptible plants, are important, because fresh spores are easier to dislodge and more likely to establish colonization. Powdery mildew only colonizes plant epidermal cells. Isolation of powdery mildew infected cells by peeling the epidermis, or laser micro dissection, would enable a more targeted transcriptomic and proteomic study of host responses.This method will help researchers more efficiently determine powder mildew infection phenotypes, and therefore should facilitate more effective functional studies on plant powdery mildew interactions.

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