Funding for this project was provided by the Special Research Initiative Grant sponsored by the Mississippi Agricultural and Forestry Experiment Station and is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under accession number 230060.

1. Stand Construction
NOTE: Measurements for the wood are listed as thickness (cm) x width (cm) x length (m).
<ol>
	<li>Cut wood into appropriate sizes and amounts as follows: five 10.16 cm x 5.08 cm x 0.91 m wooden pieces, three 10.16 cm x 5.08 cm x 0.76 m wooden pieces, three 10.16 cm x 5.08 cm x 0.61 m wooden pieces, five 10.16 cm x 5.08 cm x 0.46 m wooden pieces, three 10.16 cm x 5.08 cm x 0.3 m wooden pieces, and three 10.16 cm x 5.08 cm x 0.15 m wooden pieces.</li>
	<li>For the tallest level, stand one 2.44 m board across two 0.91 m pieces on each end at the edge and drill two screws vertically into each of the 0.91 pieces. Screw one more 0.91 m piece 1.22 m from each end for support, and place a 2.44 m board across the back of the 0.91 m stands and screw into place for support. 
	NOTE: The eight 3.175 cm x 15.24 cm x 2.44 m are kept as is and uncut to serve as the benchtop for each bench level.</li>
	<li>Repeat step 1.2 for the next bench level with the 0.76 m pieces.</li>
	<li>Repeat step 1.2 for the next bench with the 0.61 m pieces down to the sixth bench at 0.15 m. 
	NOTE: No supporting 2.44 m board is needed for benches 3&#8211;6. The final stand has six benches with three vertical supports each, one on each end and one in the middle.</li>
	<li>Line benches in descending height order with the overhanging lip facing the backside touching the bench above it, allowing for a gap between levels.</li>
	<li>Line a 0.91 cm board on each of the bottom edges of the benches along the ground and screw the benches in place.</li>
	<li>Screw a 0.46 m board horizontally for support on the tallest three benches on each side of the structure 0.61 m from the ground.</li>
	<li>Screw three corner braces onto the front-facing ends and center of the tallest bench.</li>
	<li>Screw one 2.54 cm x 5.08 cm x 20.32 cm wooden piece across the braces 2.54 cm from the base of the bench. 
	NOTE: Make one 0.91 m by 0.91 m by 2.44 m structure. Refer to Figure 1 for the final base product. Dimensions are subject to change with the experimental needs. The structure described was designed to fit 15.24 cm pots. The heights between benches were designed to fit the pots and plant material used in this experiment in order to maintain a steady flow of allelochemicals and solution from one pot to another down the benches by gravity.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60764/60764fig1a.jpg" /> 
Figure 1: Front view of the wooden base stand. A wooden base serves as the stand for the plant samples. Materials for the system are to be assembled and added depending on the number of samples needed for the experiment. In this study, two stands served as a base for 31 samples. <a href="https://www.jove.com/files/ftp_upload/60764/60764fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. System Assembly
<ol>
	<li>Remove the cap from a 1 L soda bottle and spray paint with black paint. 
	NOTE: The soda bottles will serve as a reservoir at the top of the system for one column. The paint provides a block for the light, decreasing or preventing algae growth.</li>
	<li>At the bottom of each soda bottle, drill a small hole, just large enough to embed a 0.35 cm inner diameter (ID), 0.64 cm outer diameter (OD), 5.08 cm long plastic PVC tube.</li>
	<li>Smear a layer of silicone waterproof sealant around the edge of the hole after insertion to prevent any leaks. Let it dry completely.</li>
	<li>Repeat steps 2.2 and 2.3 on each of the plastic dishes used to hold the pots. 
	NOTE: Four dishes will be needed for one column.</li>
	<li>Remove the lid and spray paint the outside of 2.27 L plastic canisters with black paint. These canisters will serve as the collection tanks at the base of each column.</li>
	<li>Drill a small hole in the upper backside of the canister. 
	NOTE: The supplies listed in steps 2.1&#8211;2.6 make one column. The number of columns is subject to the number of samples needed for the experiment desired. Two columns are needed for one sample. All dimensions are subject to change depending on the experimental needs.</li>
	<li>After the supplies have been prepared and dried, place the soda bottle on the highest bench so that the PVC tube is hanging over the rim facing the stairs.</li>
	<li>Just below the soda bottle on the next bench, place one plastic dish with its tube hanging over the rim of the bench.</li>
	<li>Repeat step 2.8 for the next two benches.</li>
	<li>Place the canister on the bottom bench with the hole facing the back.</li>
	<li>Connect the canister with the dish above it by stringing the tube from the dish through the hole in the back of the canister.</li>
	<li>Smear waterproof sealant around the edge of the canister where the tube runs through to prevent leaks.</li>
	<li>Place a 21 W 1,000 L/hr submersible electric pump inside the bottom canister.</li>
	<li>Connect a 1.07 m long, 1.27 cm ID, 1.59 cm OD PVC tube to the nozzle of the electric pump.</li>
	<li>String the tube through the gap between the benches and over the back of the soda bottle at the top of the system.</li>
	<li>Plug the pump into a digital timer and set the timer setting as needed. 
	NOTE: The timer was set to run for 1 min every 3 h throughout the entire experiment. The selected timing allowed for the maximum amount of liquid in the collection tank to be cycled and allowed for approximately 10 min of flow each time the pump was turned on while avoiding flooding and spillovers.</li>
</ol>
3. Planting
<ol>
	<li>Sterilize all the rice seeds needed by rinsing in 70% ethanol for 30 s, soaking in 5% bleach for 20 min, and rinsing 6x with distilled water.</li>
	<li>Pregerminate the sterilized rice seeds in Petri dishes lined with filter paper filled with 5 mL of distilled water in a growth chamber set at 25 &#176;C.</li>
	<li>After the seeds germinate, line the bottom of each pot with two large coffee filters by placing them inside the pots in their natural cupped form.</li>
	<li>Fill each pot to the top of the filter (approximately 75% of the pot) with autoclaved, washed, and screened specially graded quartz sand. Dampen the sand with distilled water by pouring water over the top of the sand or by placing pots in planting trays filled just slightly with distilled water to allow the pots to soak up the water and remain damp. Transplant six pregerminated donor plant seedlings into sand, evenly spaced.</li>
	<li>Cover the seedlings with sand.</li>
	<li>Let the seedlings establish for 3 weeks. 
	NOTE: The sand dries very quickly. Therefore, placing pots in trays is an efficient watering technique. Changing water out constantly will help prevent mold.</li>
	<li>Pregerminate the receiver plant seedlings (E. crus-galli) in Petri dishes 3 weeks after planting donor plants by lining the bottom of the dish with filter paper and along with 5 mL of distilled water. Place the dishes in a growth chamber at 25 &#176;C for 3&#8211;5 days.</li>
	<li>Prepare the pots as described in steps 3.1&#8211;3.2.</li>
	<li>After the seedlings germinate, transplant three seedlings into the prepared pots and cover with sand. 
	NOTE: The experiment begins one day after treatment (DAT), or the day that the receiver plant seedlings emerge and are transplanted and placed in the system.</li>
</ol>
4. Sample Placement
<ol>
	<li>Place four pots of one accession of donor plants in the four dishes of column 1, a single pot per row. Column 1 consists of donor plants only.</li>
	<li>Place two pots of the same accession of donor plants in the dishes of column 2 on the first and third row of the column.</li>
	<li>Place two pots of receiver plants in the dishes of column 2 on the second and fourth row in the column.</li>
	<li>For each replication, ensure that only one row of receiver plants is added. Two columns, the first consisting of donor plants only and the second alternating donors and receivers, make one treatment (Figure 2).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60764/60764fig2a.jpg" /> 
Figure 2: Placement map. Diagram depicting placements of donor (WR/R) and receiver plants (BYG) in respective positions in the stair-step system. Two columns of the stair-step system with plants in place comprise one treatment. A single column of receiver plants served as a control for one replication (far right), a single column of donor plants as a control for each accession (center), and the treatment column consisted of alternating donor and receiver plants (far left). <a href="https://www.jove.com/files/ftp_upload/60764/60764fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Repeat steps 4.1&#8211;4.4 for each treatment or donor plant accession (Figure 3). 
	NOTE: Each replication requires one column of receiver plant samples to serve as a control for one replication. Treatments were replicated 3x in a randomized complete block design.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60764/60764fig3a.jpg" /> 
Figure 3: Final stair-step structure. The stair-step system assembled with the plants in place. The system contained four rows of plant samples and a collection tank at the bottom for the solution to cycle to the top bottle and downward by gravity through each respective pot. <a href="https://www.jove.com/files/ftp_upload/60764/60764fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Operation
<ol>
	<li>On DAT 1, fill the collection tank at the bottom of each column with half-strength Hoagland solution17 in distilled water, approximately 1,500 mL.</li>
	<li>Set the timers to run as desired in the auto off setting.</li>
	<li>Cover the collection tanks with black plastic to limit light exposure and evaporation.</li>
	<li>Fill the tanks every 2 days with 500 mL of Hoagland&#39;s solution to keep the system flowing constantly.</li>
	<li>Maintain the greenhouse temperatures at 28 &#176;C during the day and 24 &#176;C at night respectively with a 16/8 h split and humidity at 53%.</li>
</ol>
6. Data Collection
<ol>
	<li>Measure and record the heights of each plant in the stair-step system on DAT 1 and once every week up to DAT 21 by placing a ruler at the base of each plant and observing the tallest leaf stand.</li>
	<li>Measure and record the chlorophyll levels of each plant on DAT 7 and 14 using the chlorophyll content meter.</li>
	<li>On the last day of the experiment (i.e., DAT 21) label one paper bag for each pot.</li>
	<li>Cut plant samples at the base and place in separate bags.</li>
	<li>Place all samples in an oven dryer set at 60 &#176;C for 48 h16.</li>
	<li>Remove dried samples and empty contents individually onto a scale and record the weight in grams.</li>
</ol>
7. Data Analysis
<ol>
	<li>Calculate the allelopathic potential of the donor plants based on the percent inhibition of the receiver plant using this equation: 
	height reduction (%) = [height of control (cm) &#8211; height of treated (cm)] &#215; 100</li>
	<li>Calculate the donor plant height reduction as a check for any reverse effect the receiver plant may have on the target plants.</li>
	<li>Analyze accessions as the fixed effect while replications and runs are the random effects18.</li>
	<li>Analyze the data using a general linear model with mean values separated using Fisher&#39;s protected least significant difference at or below a 0.05 probability level in a statistical software (e.g., JMP 14).</li>
	<li>Visualize the correlation among the original variables using principle component analysis of by uploading data.
	<ol>
		<li>Select the Analyze tab in the toolbar, select Fit Y by X. Under columns, Highlight the response (i.e., percent height reduction) then click Y, response to specifythe factor being observed for Y, (i.e., percent height reduction). For the X factor, Hightlight accession and click X, factor, then select OK.</li>
		<li>Select the red down arrow on the Oneway Analysis bar, select Means/ANOVA. Again select the down arrow on the Oneway Analysis bar and highlight compare means then select each pair, student&#39;s T.</li>
	</ol>
	</li>
</ol>

<ol><li>Weston, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((History+and+Current+Trends+in+the+Use+of+Allelopathy+for+Weed+Management%5BTitle%5D)+AND+%22HortTechnology%22%5BJournal%5D)">History and Current Trends in the Use of Allelopathy for Weed Management.</a> HortTechnology. 15 (3), 529-534 (2005).</li>
<li>Pratley, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allelopathy+in+annual+grasses%5BTitle%5D)+AND+%22Plant+Protection+Quarterly%22%5BJournal%5D)">Allelopathy in annual grasses.</a> Plant Protection Quarterly. 11, 213-214 (1996).</li>
<li>Bertin, C., Yang, X., Weston, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+root+exudates+and+allelochemicals+in+the+rhizosphere%5BTitle%5D)+AND+%22Plant+and+Soil%22%5BJournal%5D)">The role of root exudates and allelochemicals in the rhizosphere.</a> Plant and Soil. 256 (1), 67-83 (2003).</li>
<li>Stevenson, G. R. Pesticide Use and World Food Production: Risks and Benefits. Environmental Fate and Effects of Pesticides. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aStevenson%2C+GR+%22American+Chemical+Society%22">American Chemical Society</a>. , Chapter 15 261-270 (2003).</li>
<li>Chopra, N., Tewari, G., Tewari, L. M., Upreti, B., Pandey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allelopathic+Effect+of+Echinochloa+colona+L.+and+Cyperus+iria+L.+Weed+Extracts+on+the+Seed+Germination+and+Seedling+Growth+of+Rice+and+Soybean%5BTitle%5D)+AND+%22Advances+in+Agriculture%22%5BJournal%5D)">Allelopathic Effect of Echinochloa colona L. and Cyperus iria L. Weed Extracts on the Seed Germination and Seedling Growth of Rice and Soybean.</a> Advances in Agriculture. 2017, 1-5 (2017).</li>
<li>Jabran, K., Mahajan, G., Sardana, V., Chauhan, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allelopathy+for+weed+control+in+agricultural+systems%5BTitle%5D)+AND+%22Crop+Protection%22%5BJournal%5D)">Allelopathy for weed control in agricultural systems.</a> Crop Protection. 72, 57-65 (2015).</li>
<li>Worthington, M., Reberg-Horton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breeding+Cereal+Crops+for+Enhanced+Weed+Suppression%3A+Optimizing+Allelopathy+and+Competitive+Ability%5BTitle%5D)+AND+%22Journal+of+Chemical+Ecology%22%5BJournal%5D)">Breeding Cereal Crops for Enhanced Weed Suppression: Optimizing Allelopathy and Competitive Ability.</a> Journal of Chemical Ecology. 39, 213-231 (2013).</li>
<li>Sudianto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corrigendum+to+%22Clearfield+%28R%29+rice%3A+Its+development%2C+success%2C+and+key+challenges+on+a+global+perspective.%22%5BTitle%5D)+AND+%22Crop+Protection%22%5BJournal%5D)">Corrigendum to "Clearfield (R) rice: Its development, success, and key challenges on a global perspective.".</a> Crop Protection. 55, 142-144 (2014).</li>
<li>Gressel, J., Valverde, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+strategy+to+provide+long-term+control+of+weedy+rice+while+mitigating+herbicide+resistance+transgene+flow%2C+and+its+potential+use+for+other+crops+with+related+weeds%5BTitle%5D)+AND+%22Pest+Management+Science%22%5BJournal%5D)">A strategy to provide long-term control of weedy rice while mitigating herbicide resistance transgene flow, and its potential use for other crops with related weeds.</a> Pest Management Science. 65, 723-731 (2009).</li>
<li>Muthayya, S., Sugimoto, J. D., Montgomery, S., Maberly, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+global+rice+production%2C+supply%2C+trade%2C+and+consumption%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">An overview of global rice production, supply, trade, and consumption.</a> Annals of the New York Academy of Sciences. 1324, 7-14 (2014).</li>
<li>Chung, I. M., Kim, K. H., Ahn, J. K., Lee, S. B., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allelopathy+Comparison+of+Allelopathic+Potential+of+Rice+Leaves%2C+Straw%2C+and+Hull+Extracts+on+Barnyardgrass%5BTitle%5D)+AND+%22Agronomy+Journal%22%5BJournal%5D)">Allelopathy Comparison of Allelopathic Potential of Rice Leaves, Straw, and Hull Extracts on Barnyardgrass.</a> Agronomy Journal. 95 (4), 1063-1070 (2003).</li>
<li>Olofsdotter, M., Jensen, L. B., Courtois, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+crop+competitive+ability+using+allelopathy+%C3%90+an+example+from+rice%5BTitle%5D)+AND+%22Journal+of+Plant+Breeding%22%5BJournal%5D)">Improving crop competitive ability using allelopathy Ð an example from rice.</a> Journal of Plant Breeding. 121, 1-9 (2002).</li>
<li>Olofsdotter, M., Navarez, D., Rebulanan, M., Streibig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Weed-suppressing+rice+cultivars-does+allelopathy+play+a+role%5BTitle%5D)+AND+%22Weed+Research%22%5BJournal%5D)">Weed-suppressing rice cultivars-does allelopathy play a role.</a> Weed Research. 39 (6), 441-454 (1999).</li>
<li>Jensen, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Locating+Genes+Controlling+Allelopathic+Effects+against+Barnyardgrass+in+Upland+Rice%5BTitle%5D)+AND+%22Agronomy+Journal%22%5BJournal%5D)">Locating Genes Controlling Allelopathic Effects against Barnyardgrass in Upland Rice.</a> Agronomy Journal. 93 (1), 21-26 (2001).</li>
<li>Kuijken, R. C., Eeuwijk, F. A. V., Marcelis, L. F., Bouwmeester, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Root+phenotyping%3A+from+component+trait+in+the+lab+to+breeding%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Root phenotyping: from component trait in the lab to breeding.</a> Journal of Experimental Botany. 66 (18), 5389(2015).</li>
<li>Lickfeldt, D. W., Voigt, T. B., Branham, B. E., Fermanian, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+allelopathy+in+cool+season+turfgrass+species%5BTitle%5D)+AND+%22International+Turfgrass+Society%22%5BJournal%5D)">Evaluation of allelopathy in cool season turfgrass species.</a> International Turfgrass Society. 9, 1013-1018 (2001).</li>
<li>Liu, D. L., Lovett, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biologically+active+secondary+metabolites+of+barley%3A+Developing+techniques+and+assessing+allelopathy+in+barley%5BTitle%5D)+AND+%22Journal+of+Chemical+Ecology%22%5BJournal%5D)">Biologically active secondary metabolites of barley: Developing techniques and assessing allelopathy in barley.</a> Journal of Chemical Ecology. 19, 2217-2230 (1993).</li>
<li>Shrestha, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Evaluation+of+Herbicide+Tolerance+and+Interference+Potential+among+Weedy+rice+germplasm%22">Evaluation of Herbicide Tolerance and Interference Potential among Weedy rice germplasm</a>. , (2018).</li>
<li>Kim, K. U., Shin, D. H. Rice allelopathy research in Korea. Allelopathy in Rice. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKim%2C+KU+%22IRRI%22">IRRI</a>. Olofsdotter, , Chapter 4 (1998).</li>
<li>Quasem, J. R., Hill, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+difficulties+with+allelopathy%5BTitle%5D)+AND+%22Weed+Research%22%5BJournal%5D)">On difficulties with allelopathy.</a> Weed Research. 29, 345-347 (1989).</li>
<li>Singh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+mulching%2C+intercropping+with+Sesbania+and+herbicide+use+for+weed+management+in+dry-seeded+rice+%28Oryza+sativa+L.%29%5BTitle%5D)+AND+%22Crop+Protection%22%5BJournal%5D)">Evaluation of mulching, intercropping with Sesbania and herbicide use for weed management in dry-seeded rice (Oryza sativa L.).</a> Crop Protection. 26, 518-524 (2007).</li>
<li>Kong, C. H., Li, H. B., Hu, F., Xu, X. H., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allelochemicals+released+by+rice+roots+and+residues+in+soil%5BTitle%5D)+AND+%22Plant+and+Soil%22%5BJournal%5D)">Allelochemicals released by rice roots and residues in soil.</a> Plant and Soil. 288 (1-2), 47-56 (2006).</li>
<li>Ervin, G. N., Wetzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allelochemical+autotoxicity+in+the+emergent+wetland+macrophyte+Juncus+effusus+%28Juncaceae%29%5BTitle%5D)+AND+%22American+Journal+of+Botany%22%5BJournal%5D)">Allelochemical autotoxicity in the emergent wetland macrophyte Juncus effusus (Juncaceae).</a> American Journal of Botany. 87 (6), 853-860 (2000).</li>
</ol>

The authors have nothing to disclose.

Exploiting allelopathy may potentially serve as a biological control for weeds that are difficult to manage1,7,13. Allelopathy has shown great potential as a possible solution to the weed crisis in rice and serves as an alternative or supplement to chemicals and manual weed control practices5,13,19. Identifying allelopathic varieties or...

Allelopathy is a natural and complex phenomenon that has been the focus of many plant scientists in the past few decades. The mechanisms relating to allelopathy for use in crops have been the subject of much research since the 1930s, when Molisch observed that a plant has a direct or indirect effect on a neighboring plant through the production and secretion of chemical compounds into the environment1. Allelopathy is the production of secondary metabolites that have inhibitory effects on the growth and germination of some plant species. Released allopathic chemical compounds help provide the donor plants with a competitive advantage by adding phytotoxins to the environment around them2. Many factors contribute to the allelopathic activity. It is selective in its effectiveness and varies between varieties, environmental conditions, growth stage, stress, environment, and nutrient availability3.
In recent years, allelopathy has been highlighted in research as a possible supplement to the constant and growing weed control crisis. With the growing global population, the demand for sustainable food and fiber production has increased4. Weed control is one of the biggest threats to production faced by agronomists5,6. Traditional weed control methods focus on mechanical, chemical, and cultural practices. The continuous usage of herbicides, while effective, useful, and efficient, has promoted the evolution of resistant weed populations at an alarmingly fast pace7. Genetic engineering and breeding practices have been used effectively to give crops competitive advantages over weeds by designing them to withstand chemical applications that their neighbors cannot survive7,8. Although effective, these technologies are not always sustainable and sometimes pose outcrossing concerns9. Supplemental weed management practices need to be introduced if the goal of increasing food production is to be met10. Allelopathy shows excellent promise as a new defense tool for crops to improve their quality and outlive their competitors1,7.
Allelochemicals are often secondary products, and because their production is highly influenced by environmental factors, the specific compounds associated with plant suppression can be difficult to identify3. Production factors include genetics and the joint action of secondary metabolites that may act synergistically11,12. It is challenging to separate allelopathic activity from the competition that naturally exists within crop-weed interactions, and due to this, when screening for allelopathy there must be a standard set of outcomes that qualify the assay as valid and repeatable. Below is a set of criteria that qualifies findings of allelopathy as outlined by Olofsdotter et al.12 1) One plant must demonstrate suppression of another plant in a pattern; 2) The chemicals that are released into the environment in bioactive amounts must be produced by the donor plant; 3) The chemicals produced must be transportable to the receiver plant; 4) Some mechanism of uptake must be present in the receiver plant; 6) The pattern of inhibition observed must have no other exclusive explanation (e.g, competition for resources)12.
In an effort to overcome the barrier between the lack of knowledge of the mechanisms supporting allelopathy and variety development, phenotypic traits associated with allelopathic varieties can be identified and selected for further research and use. Some plants known to have allelopathic qualities are rye, sorghum, rice, sunflower, rapeseed, and wheat13. During the early observations of allelopathy in crops, due to distinguished borders of weed growth in field experiments, it was proposed that chemicals were involved rather than competition for resources14. However, most studies were field experiments that made it impossible to eliminate competition as a factor14. Competition elimination efforts gave way to lab and greenhouse experiments in attempts to prove and quantify allelopathic activity in rice and other crops. Field and greenhouse methods to screen plants for allelopathy demonstrate that allelopathic tendencies are present in both growing conditions11,15. Some critics believe that laboratory screenings may only hold limited value due to the lack of natural conditions, which may affect the results15.
The proposed method for screening allelopathic potential in plants provides adequate resources and space and eliminates resource competition with the use of a stair-step structure11,17. The method was adapted and modified from previous experiments exploring allelopathy in turfgrass and barley17,18. These studies found that a similar system was able to produce accurate results on the allelopathic potential of a target plant while removing any doubts that the observations could be attributed to natural competition. The stair-step method creates a circulatory system where a nutrient solution from a reservoir can cycle through each plant to an incubation tray through a few steps. An electric pump then recycles the solution along with any allelochemicals produced18. A method such as this is efficient in both time, space, and resources. It also provides similar field conditions for the plants and eliminates any resource competition. The methods and tools used for screening are easily manipulated to fit the desired study goals, conditions, and specific species. The objective of this study is to confirm weedy rice allelopathy through height suppression measurements on barnyardgrass with the use of the stair-step method.

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repeatable stair-step assay to access the allelopathic potential of weedy rice (oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously applied herbicides has presented the need for additional management methods. Allelopathy is a physiological process that some plant species possess that provide the plant with an advantage over its neighbors. Allelopathic crop varieties would be equipped with the ability to suppress the growth of surrounding competitors, thus reducing potential yield loss due to weed interference. This paper focuses on the construction and operation of a stair-step assay used for the screening of the allelopathic potential of a donor species (Oryza sativa) against a receiver weed species (Echinochloa crus-galli) in a greenhouse setting. The structure described in this paper serves as a stand for the plant samples and incorporates a timed watering system for the accumulation and distribution of allelochemicals. Allelochemicals produced by the plant roots are allowed to flow downward through a series of four pots separately into a collection tank and recycled back to the top plant through electric pumps. This method of screening provides an avenue for the allelochemicals from the donor plant to reach receiver plants without any resource competition, thus allowing quantitative measurement of the allelopathic potential of the selected donor plant. The allelopathic potential is measurable through the height reduction of the receiver plants. Preliminary screening data for the effectiveness of this method demonstrated height reduction in the receiver species, barnyardgrass (E. crus-galli), and thus the presence of allelopathic residues from the donor plant, weedy rice (Oryza sativa).

Two preliminary screenings using this method were performed on nine weedy rice accessions (B2, S33, B83, S97, S94, B81, B8, B34, B14) and five cultivated rice lines (PI338046, Rex, Rondo, PI312777, CL163). Weedy rice accessions and rice lines were selected based on their performance in previous allelopathic screenings conducted by Shrestha (2018)18. The weedy rice seeds were collected from across the state of Arkansas. The rice lines selected are commonly grown lines in the US, some known to expre...

Watch this Scientific Journal Video about Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp. at JoVE.com

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Allelopathy has shown promise as a useful supplemental weed control strategy in cropping systems. To determine the allelopathic potential of a desired plant specimen, a stair-step screening method is provided.

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

repeatable-stair-step-assay-to-access-allelopathic-potential-weedy

Research

JoVE Journal

Biology

7.1K Views.  Mississippi State University. De trap-stap systeem stelt ons in staat om te identificeren of een plant is allelopathisch of niet, waardoor de concurrentie tussen en tussen planten. Bovendien kan het ook de allelochemicals identificeren die betrokken zijn of geassocieerd zijn met allelopathie. Deze methode is efficiënt in tijd, ruimte en resources.Bovendien u deze test of methode aanpassen aan andere studies waarvoor een andere donor of acceptor soort nodig is. Een uitdaging in allelopathisch onderzoek is het isole...

Video: Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Environment

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

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 ...

Preference Assessment of White-backed Planthopper Feeding on Rice

Exploiting insect-resistant rice germplasm resources and related genes is the primary need for breeding insect-resistant varieties, but the accuracy of the identification of insect-resistant phenotypes of rice is a major difficulty. It is urgent to develop a new method or improve existing methods to screen rice for insect resistance. This article describes a simple and feasible method to assess nonpreference-type resistance of rice to the white-backed planthopper (WBPH), Sogatella furcifera, in the laboratory. The preference of adult WBPHs feeding or inhabiting on maturing rice plants is continuously analyzed by pairwise comparison. The dynamic changes of WBPHs on rice plants are recorded and compared as an index of resistance identification. The current method is simply operable and easily observable and has a short cycle. The use of this method could be extended to investigate the feeding and oviposition preference of similar hemipterans, such as the brown planthopper (BPH), Nilaparvata lugens(St&#229;l).

This study presents a simple and feasible method to assess the nonpreference resistance to white-backed planthoppers who are feeding on rice under laboratory conditions. Improvement of the strategies and makeup of the current method of identification of the resistance to white-backed and brown planthoppers are discussed.

Exploiting insect-resistant rice germplasm resources and related genes is the primary need for breeding insect-resistant varieties, but the accuracy of ...

CRISPR-Based Efficient Genome Editing for Resistant Starch in Rice Breeding

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

Author Spotlight: Streamlining Rice Breeding with CRISPR/Cas for Obtaining Optimal Phenotypic and Agronomic Traits 

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Bioengineering

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions

Rice (Oryza sativa L.) is a vital food crop for more than half of the global population. However, its growth is severely impacted by saline soils, which present a significant challenge to crop production worldwide. Arbuscular mycorrhizal (AM) fungi, which form mutualistic symbiotic relationships with over 90% of agricultural plants and 80% of terrestrial plant species, have been shown to enhance the salt tolerance of rice plants. AM fungi are obligate symbionts that cannot complete their life cycle without a host root. Therefore, effectively utilizing plants to produce AM fungal inoculum is crucial for advancing research in this field. In this study, we present a series of robust methods that begin with generating sand inoculum containing spores of Rhizophagus irregularis using Allium tuberosum L. These methods include inoculating rice seedlings with the sand inoculum, analyzing the growth phenotype of mycorrhizal rice, and quantifying fungal colonization levels using trypan blue staining under salt stress. These approaches can efficiently generate AM fungal inoculum for further investigation into how AM symbiosis enhances the salinity tolerance of rice.

This article describes a protocol for generating arbuscular mycorrhizal (AM) fungi inoculum to investigate AM-enhanced salt stress tolerance in rice.

Rice (Oryza sativa L.) is a vital food crop for more than half of the global population. However, its growth is severely impacted by saline soils, which ...

Establishment of Rice Plant Leaf Infection through Fungal Inoculation via Wounding

Source: Zhang, M. et al., <a href="https://app.jove.com/v/57675">The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen&#160;Magnaporthe Grisea.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the establishment of rice plant leaf infection through wounding inoculation with Magnaporthe grisea mycelium. The fungal spore-derived germ tube penetrates the epidermal layer, resulting in tissue infection and lesions on the leaves. This method aids pathotype screening and improves plant resistance understanding for molecular breeding.

1. Fungal culture for&#160;M.grisea

	Prepare the oatmeal tomato agar (OTA) culture medium for fungal strains.
	Weigh 30 - 50 g of oatmeal, add this to ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

An Assay for Detecting Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation

Source: Wang, Y., et al. <a href="https://app.jove.com/v/64776">Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay.</a> J. Vis. Exp. (2022).
This video demonstrates an assay for real-time detection of reactive oxygen species (ROS) production in rice tissues upon pathogen-associated molecular pattern (PAMP) elicitation. Cut sheath segments and leaf discs are exposed to a bacterial flagella-derived peptide functioning as a PAMP. Upon recognition of the peptide via pattern recognition receptors (PRRs) on plant cells, a rapid and transient production of ROS occurs, which is detected via a chemiluminescence assay.

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

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