Name: a plate competition assay as a quick preliminary assessment of disease suppression
Uploaded: 2018-10-28T15:00:00
Description: Watch this Scientific Journal Video about A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression at JoVE.com

The Vermont Agricultural Experiment Station Competitive Hatch Program VT-HO1609 funded the research. Lynn Fang used the method as part of her M.S. thesis at University of Vermont6.

1. Prepare in Advance
<ol>
	<li>Master fungal culture (test organism)
	<ol>
		<li>Order from the American Type Culture Collection, Microbiology Collection9, by its teleomorph Thanatephorus cucumeris (Frank) (ATCC 10154) or MYA 4031.</li>
		<li>Alternatively: Collect local isolates as done by the authors. Seedlings of red radish (Raphanus sativus) work well as a bait plant; collect soil from a location known to have a history of damping-off or root rot caused by R. solani</e...

<ol>
	<li>Gonzalez Garcia, G., Onco, M. A. P., Susan, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review.+Biology+and+systematics+of+the+form+genus+Rhizoctonia.">Review. Biology and systematics of the form genus Rhizoctonia.</a> Spanish Journal of Agricultural Research. 4 (1), 55-79 (2006).</li><li>Bonanomi, G., Antignani, V., Pane, C., Scala, E. <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+soilborne+fungal+diseases+with+organic+amendments.">Suppression of soilborne fungal diseases with organic amendments.</a> Journal of Plant Pathology. 89, 311-324 (2007).</li><li>Noble, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+and+benefits+of+soil+amendment+with+composts+in+relation+to+plant+pathogens.">Risks and benefits of soil amendment with composts in relation to plant pathogens.</a> Australasian Plant Pathology. 40, 157-167 (2011).</li><li>Wichuk, K. M., McCartney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compost+stability+and+maturity+evaluation+-+a+literature+review.">Compost stability and maturity evaluation - a literature review.</a> Canadian Journal of Civil Engineering. 37, 1505-1523 (2010).</li><li>Alfano, G., Lustrato, G., Lima, G., Vitullo, D., Ranalli, 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+composted+olive+mill+wastes+to+predict+potential+plant+disease+suppressiveness.">Characterization of composted olive mill wastes to predict potential plant disease suppressiveness.</a> Biological Control. 3, 199-207 (2011).</li><li>Fang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological indicators of compost-mediated disease suppression against the soilborne plant pathogen Rhizoctonia solani. , (2015).</li><li>Bonanomi, G., Antignani, V., Capodilupo, M., Scala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+characteristics+of+organic+soil+amendments+that+suppress+soilborne+plant+diseases.">Identifying the characteristics of organic soil amendments that suppress soilborne plant diseases.</a> Soil Biology and Biochemistry. 42, 136-144 (2010).</li><li>Neher, D. A., Fang, L., Weicht, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecoenzymes+as+indicators+of+compost+to+suppress+Rhizoctonia+solani.">Ecoenzymes as indicators of compost to suppress Rhizoctonia solani.</a> Compost Science and Utilization. 25 (4), 251-261 (2017).</li><li>. ATCC&#174; Mycology Culture Guide Available from: <a target="_blank" href="https://www.atcc.org/~/media/PDFs/Culture%20Guides/Mycology_Guide.ashx">https://www.atcc.org/~/media/PDFs/Culture%20Guides/Mycology_Guide.ashx</a> (2018)</li><li>Berns, A. E., Phillip, H., Narres, H. -. D., Burauel, P., Vereecken, H., Tappe, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+gamma-sterilization+and+autoclaving+on+soil+organic+matter+structure+as+studied+by+solid+state+NMR,+UV+and+fluorescence+spectroscopy.">Effect of gamma-sterilization and autoclaving on soil organic matter structure as studied by solid state NMR, UV and fluorescence spectroscopy.</a> European Journal of Soil Science. 59, (2008).</li></ol>

We know from previous research, that certain composts are effective at suppressing R. solani and that the suppressive effects are due to the microbes living in the compost, not the abiotic properties of compost6,8. The use of autoclaving as a means to &#39;kill&#39; microbiota has been criticized because it affects the carbon chemistry of the media10. We compared the use of autoclaving to vacuum filtration through Whatman No. 1 pa...

Rhizoctonia represents a broad complex of fungi, of which Thanatephorus cucumeris (Frank) Donk (anamorph = Rhizoctonia solani K&#252;hn) is the pathogen causing root rot and damping-off1. Rhizoctonia solani is an aggressive pathogen and a saprophyte that can survive as sclerotia under adverse environmental conditions1. As a result, it has a global distribution and can cause disease on wide range of plant hosts including Solanaceae, Fabaceae, Asteraceae, and Brassicaceae resulting in serious economic losses.
Compost has the capacity to harbor biocontrol agents for certain plant pathogens2. However, not all composts are alike nor do they affect all pathogens similarly3. Wood-based carbon has higher lignin to cellulose ratios than hay or straw-carbon based composts. R. solani prefers readily available carbon found in straw. In contrast, biological control fungi, such as Trichoderma spp., are more effective when carbon is less readily available. Beneficial fungi and bacteria into compost can suppress plant disease through competition, antagonism or regulating plant growth3. The proposed assay primarily detects antagonism created by production of antibiotics, ecoenzymes or chelators that are detrimental to the pathogen.
Plant bioassays are a gold standard to determine whether composts favor or deter plant growth4. However, plant bioassays are time-consuming (weeks to months) to complete which may be longer than desired and requires more labor to extract plants with roots to quantify severity of root systems. Comparably robust, but quicker (days) assays would be ideal for quality control programs. The goal of this paper is to demonstrate a relatively quick and accurate test to predict suppressive potential of compost. The method was patterned after Alfano et al.5 with two exceptions, compost extracts were diluted and water agar was used in place of potato dextrose agar (PDA). R. solani grows rapidly on simple water agar media whereas PDA promoted growth of bacteria and other fungi that contaminated the culture6.
This plate assay serves as an indicator of suppression that applies to a range of plant pathogens that survive in soil as saprophytes including R. solani7, Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot. The plate competition assay is useful to screen a range of compost products for containing communities of microbes that serve as biological control agents of soil pathogens. The assay was one of the most consistent indicators of disease suppression in commercial compost products6,8. Products were chosen for their variation in recipe, maturity, and production process.

<table>
	<tbody>
		<tr>
			<td>autoclavable narrow-neck glass conical flask</td>
			<td>Fisher Scientific</td>
			<td>10-040D</td>
			<td>125-mL</td>
		</tr>
		<tr>
			<td>dehydrated granulated agar</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td>500 g quantity</td>
		</tr>
		<tr>
			<td>heat resistant gloves</td>
			<td>Fisher Scientific</td>
			<td>MEMGG1314WL</td>
			<td>several brands available</td>
		</tr>
		<tr>
			<td>Parafilm (strips of 2-3 cm wide)</td>
			<td>Fisher Scientific</td>
			<td>PM992 13-374-16</td>
			<td>5 or 10 cm widths work</td>
		</tr>
		<tr>
			<td>disposable polystyrene petri dishes</td>
			<td>Fisher Scientific</td>
			<td>R80116</td>
			<td>comes in sleeves of 20/ea or cases of 500</td>
		</tr>
		<tr>
			<td>dehydrated potato dextrose agar</td>
			<td>Fisher Scientific</td>
			<td>DF0013-15-8</td>
			<td>comes in quantities of 100, 500 and 2000 grams</td>
		</tr>
		<tr>
			<td>benchtop reciprocal shaker</td>
			<td>Thomas Scientific</td>
			<td>1227Y31</td>
			<td>other models will work</td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>ThermoScientific</td>
			<td>S37363</td>
			<td>5L general purpose</td>
		</tr>
		<tr>
			<td>clear ruler, flat, at least 10 cm</td>
			<td>Any</td>
			<td></td>
			<td>use metric rule</td>
		</tr>
		<tr>
			<td>ATCC culture</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 10154</td>
			<td>teleomorph Thanatephorus cucumeris (Frank) (ATCC 10154) or MYA 4031;</td>
		</tr>
		<tr>
			<td>lab tape</td>
			<td>Fisher Scientific</td>
			<td>15935</td>
			<td>autoclavable and removable, 1&#34; wide preferred</td>
		</tr>
		<tr>
			<td>water resistant marker</td>
			<td>office or scientific supply</td>
			<td>Sharpie fine tip</td>
			<td>write sample number on tape</td>
		</tr>
	</tbody>
</table>

a plate competition assay as a quick preliminary assessment of disease suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Finished compost should be stable and mature, two terms that are often used interchangeably, so it can be safely packaged and transported, and not cause adverse effects during its end use4. Stability is a resistance to decomposition and is usually determined using indices of microbial activity. General measures of microbial respiration may measure compost stability but not necessarily disease suppression of an aggressive pathogen and saprophyte such as...

Watch this Scientific Journal Video about A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression at JoVE.com

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

This method can help answer key questions related to the biological control of soilborne plant pathogens. The gold standard for testing materials like compost is to have a plant bioassay to test whether it's toxic to the plants. However, that takes three weeks.This method can be done in about two to three days. The main advantage of this technique is that it is simple, affordable, and effective. This assay is a rapid screening of specific compost to suppress the growth of Rhizoctonia solani.However, this assay can also be used against other soilborne plant pathogens, such as Fusariums, Pythiums, and Phytophthoras. Visual demonstration of this method is critical because it takes care to isolate and maintain a pure culture of a fungus while avoiding contamination. Demonstrating the isolation procedure will be Taylor Readyhough, a graduate student from my laboratory.Emma Wright, an undergraduate student from my laboratory, will demonstrate the competition assay. To begin, sow red radish seeds in soil known to have a history of disease caused by R.solani. After three to four weeks, remove the seedlings from the soil, and rinse them with tap water.Using a razor blade, cut one-centimeter segments of the hypocotyl and root that have a brown color. Then, use flame-sterilized forceps to dip the segments into 10%bleach solution for one minute. After this, rinse the segments in sterile water.Next, use flame-sterilized forceps to transfer the segments to a paper towel. Pat the segments dry, and transfer them to a Petri dish with water agar. Place the Petri dish inside of a container with a lid, and incubate the segments at room temperature.To establish a daughter culture, transfer one of the segments to the center of a fresh plate of potato dextrose agar. After this, autoclave 25-milliliter test tubes containing 10 milliliters of water. Add two 0.5-gram samples of each test sample to a pair of autoclaved test tubes with 10 milliliters of sterile water, and shake the test tubes overnight.After 24 hours, add 1.5 grams of plain agar and 90 milliliters of distilled water to a pair of conical flasks. After autoclaving the flasks, place them into a 45-degree Celsius water bath for 30 minutes. Next, pour the autoclaved reference sample and the living sample into the molten agar.Pour the mixtures from each flask into five Petri dishes. To protect the indigenous microbes, it is important not to add the compost while the agar is too hot. Therefore, allow the agar to cool to 45 degrees Celsius before adding the compost.Using aseptic technique, transfer pieces of R.solani to each pair of sample plates. Then, incubate the plates at room temperature for one to two days. Finally, use a clear, flat ruler and a stereo microscope to measure the radius of the mycelium in each plate to the nearest millimeter.In this protocol, extracts from living composts suppressed R.solani growth significantly more than autoclaved samples, demonstrating that the effect was microbial and not nutrient-based. Suppressiveness is measured as a reduction in growth compared to the autoclaved control. Growth was not affected by the duration of maturation or curing of the compost.Growth was significantly affected by compost method and recipe ingredients, specifically with vermicompost, windrow processes, and recipes with hardwood bark. After its development, this technique may be a candidate for commercial certification of compost for disease-suppressive properties.

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