Name: repeatable stair-step assay to access the allelopathic potential of weedy rice (oryza sativa ssp.)
Uploaded: 2020-01-28T15:00:00
Description: Watch this Scientific Journal Video about Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp. at JoVE.com

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...

<ol>
	<li>Weston, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+and+Current+Trends+in+the+Use+of+Allelopathy+for+Weed+Management.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathy+in+annual+grasses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+root+exudates+and+allelochemicals+in+the+rhizosphere.">The role of root exudates and allelochemicals in the rhizosphere.</a> Plant and Soil. 256 (1), 67-83 (2003).</li><li>Stevenson, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+Use+and+World+Food+Production:+Risks+and+Benefits.+Environmental+Fate+and+Effects+of+Pesticides.">Pesticide Use and World Food Production: Risks and Benefits. Environmental Fate and Effects of Pesticides.</a> American Chemical Society. , 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathic+Effect+of+Echinochloa+colona+L.+and+Cyperus+iria+L.+Weed+Extracts+on+the+Seed+Germination+and+Seedling+Growth+of+Rice+and+Soybean.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathy+for+weed+control+in+agricultural+systems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Cereal+Crops+for+Enhanced+Weed+Suppression:+Optimizing+Allelopathy+and+Competitive+Ability.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corrigendum+to+"Clearfield+(R)+rice:+Its+development,+success,+and+key+challenges+on+a+global+perspective.".">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+global+rice+production,+supply,+trade,+and+consumption.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathy+Comparison+of+Allelopathic+Potential+of+Rice+Leaves,+Straw,+and+Hull+Extracts+on+Barnyardgrass.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+crop+competitive+ability+using+allelopathy+&#208;+an+example+from+rice.">Improving crop competitive ability using allelopathy &#208; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weed-suppressing+rice+cultivars-does+allelopathy+play+a+role.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locating+Genes+Controlling+Allelopathic+Effects+against+Barnyardgrass+in+Upland+Rice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+phenotyping:+from+component+trait+in+the+lab+to+breeding.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+allelopathy+in+cool+season+turfgrass+species.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+active+secondary+metabolites+of+barley:+Developing+techniques+and+assessing+allelopathy+in+barley.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evaluation of Herbicide Tolerance and Interference Potential among Weedy rice germplasm. , (2018).</li><li>Kim, K. U., Shin, D. H., Olofsdotter, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rice+allelopathy+research+in+Korea.+Allelopathy+in+Rice.">Rice allelopathy research in Korea. Allelopathy in Rice.</a> IRRI. , (1998).</li><li>Quasem, J. R., Hill, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+difficulties+with+allelopathy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mulching,+intercropping+with+Sesbania+and+herbicide+use+for+weed+management+in+dry-seeded+rice+(Oryza+sativa+L.).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelochemicals+released+by+rice+roots+and+residues+in+soil.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelochemical+autotoxicity+in+the+emergent+wetland+macrophyte+Juncus+effusus+(Juncaceae).">Allelochemical autotoxicity in the emergent wetland macrophyte Juncus effusus (Juncaceae).</a> American Journal of Botany. 87 (6), 853-860 (2000).</li></ol>

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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			<td>1.25 in by 6 in by 8 ft standard severe weather wood board</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>489248</td>
			<td>N/A</td>
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			<td>2 in by 4 in by 8 ft white wood stud</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>6005</td>
			<td>Cut into appropriate sizes</td>
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		<tr>
			<td>63 mm (2.5 in) corner braces</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>809449</td>
			<td>N/A</td>
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			<td>Asporto 16 oz Round Black Plastic To Go Box - with Clear Lid, Microwavable &#8211; 6.25 in by 6.25 in by 1.75 in - 100 count box</td>
			<td>Restaurantware.com, Chicago, IL</td>
			<td>RWP0191B</td>
			<td>black</td>
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			<td>ATP vinyl-flex PVC food grade plastic tubing, clear, .125 in id by .25 in od, 100 ft</td>
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			<td>B00E6BCV0G</td>
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			<td>Ccm-300 chlorophyll content meter</td>
			<td>Opti-Sciences, Inc. Hudson, NH</td>
			<td>ccm/300</td>
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			<td>Common 1 in by 2 in by 8 ft pine board</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>1408</td>
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			<td>Contractors choice contractor 24-pack 42-gallon black outdoor plastic construction trash bag</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
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			<td>Cut to cover collection tanks</td>
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			<td>EURO POTS</td>
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			<td>CN-EU</td>
			<td>15 cm short black 6 in diameter 4.25 in height 1.37qt volume</td>
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			<td>Fisher brand petri dish with clear lid</td>
			<td>Fisher Scientific, Waltham, MA</td>
			<td>FB0857513</td>
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			<td>Aexit Ac 220 V-240 V electrical equipment US plug 21 W 1000 L/hr multipurpose submersible pump</td>
			<td>Amazon, Seattle WA</td>
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			<td>Walmart, Bentonville, AR</td>
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			<td>20 settings</td>
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			<td>GE silicone 2+ 10.1 oz almond silicone caulk</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>48394</td>
			<td>Sealant for edges of any attached tubing</td>
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			<td>Great Value Distilled Water</td>
			<td>Walmart, Bentonville, AR</td>
			<td>565209428</td>
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			<td>Great Value White Basket coffee filters 200 count</td>
			<td>Walmart, Bentonville, AR</td>
			<td>562723371</td>
			<td>Size may vary</td>
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			<td>Grip-rite primgaurd plus #9-3 in pollimerdex screws</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>323974</td>
			<td>N/A</td>
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			<td>Hoagland's No. 2 basal salt mixture</td>
			<td>Caisson Laboratories, INC. Smithfield, UT</td>
			<td>HOP01/50LT</td>
			<td>&#189; strength rate</td>
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			<td>JMP (14)</td>
			<td>SAS Institute Inc. North Carolina State University, NC</td>
			<td></td>
			<td>N/A</td>
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			<td>Project source flat black spray paint</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>282254</td>
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			<td>Project source utility 1.88 in by 165 ft gray duct tape</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>488070</td>
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			<td>Rubbermaid 2 qt square food storage canister clear</td>
			<td>Walmart, Bentonville, AR</td>
			<td>555115144</td>
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			<td>Sealproof unreinforced PVC clear vinyl tubing, food-grade .5 in id by .625 in od, 100 ft</td>
			<td>Amazon, Seattle WA</td>
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			<td>Connects to pump</td>
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			<td>Short Mountain Silica 50 lb Play sand</td>
			<td>Lowe&#39;s, Mooresville, NC</td>
			<td>10392</td>
			<td>Sand should be purified</td>
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			<td>Steve Spangler&#39;s 1 Liter Soda Bottles - 6 Pack - For Science Experiment Use</td>
			<td>Amazon, Seattle WA</td>
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			<td>Top step tank discard lid</td>
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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 ...

The stair-step system allows us to identify if a plant is allelopathic or not, eliminating the competition among and between plants. Additionally, it can also identify the allelochemicals that's involved or associated with allelopathy. This method is efficient in time, space, and resources.Additionally, you can customize this assay or method to fit other studies that require a different donor or acceptor species. One challenge in allelopathic research is isolating allelopathy. If plants do not compete for resources and weeds are still being suppressed, then it must be due to chemical production.Analysis of the solution allows for identification of the actual compound or compounds that are being produced by the plants. These compounds can be used as potential bioherbicides. In the greenhouse, fight of outgrowth and mold is a challenge.By using distilled water, opaque materials, and autoclaving the sand, outgrowths in the tanks and tubes can be suppressed. Cut wood into appropriate sizes and amounts as listed in the text protocol. For the tallest level, stand one 2.44 meter board across two 0.91 meter pieces on each end of the edge and drill two screws vertically into each of the 0.91 pieces.Screw one more 0.91 meter piece 1.22 meters from each end for support. Then place a 2.44 meter board across the back of the 0.91 meter stands and screw into place for support. Repeat these steps for the next bench level with the 0.76 meter pieces and then again for the next bench with the 0.61 meter pieces down to the sixth bench at 0.15 meters.No supporting 2.44 meter board is needed for benches three through six. Line benches in descending height order with the overhanging lip facing the backside touching the bench above it allowing for a gap between levels. Line a 0.91 centimeter board on each of the bottom edges of the benches along the ground and screw the benches in place.Screw three corner braces onto the front facing ends and center of the tallest bench. Screw one 2.54 centimeter by 5.08 centimeter by 20.32 centimeter wooden piece across the braces 2.54 centimeters from the base of the bench. The final stand has six benches with three vertical supports each, one on each end and one in the middle.At the bottom of each soda bottle, drill a small hole just large enough to embed a 0.35 centimeter inner diameter, 0.64 centimeter outer diameter, 5.08 centimeter long plastic PVC tube. After insertion of the tube, smear a layer of silicone waterproof sealant around the edge of the hole to prevent any leaks. Let it dry completely.Prepare the plastic dishes used to hold the pots with tubes in the same manner. Four dishes will be needed for one column. Drill a small hole in the upper backside of the canister.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. Just below the soda bottle on the next bench, place one plastic dish with its tube hanging over the rim of the bench. Repeat this step for the next two benches.Place the canister on the bottom bench with the hole facing the back. Connect the canister with the dish above it by stringing the tube from the dish through the hole in the back of the canister. Smear waterproof sealant around the edge of the canister where the tube runs through to prevent leaks.Place a 21 watt 1, 000 liter per hour submersible electric pump inside the bottom canister. Connect a 1.07 meter long, 1.27 centimeter ID, 1.59 centimeter OD PVC tube to the nozzle of the electrical pump. String the tube through the gap between the benches and over the back of the soda bottle at the top of the system.Plug the pump into a digital timer and set the timer setting as needed. Perform the planting as described in the text protocol. Place four pots of one accession of donor plants in the four dishes of column one, a single pot per row.Column one consists of donor plants only. Place two pots of the same accession of donor plants in the dishes of column two on the first and third row of the column. Place two pots of receiver plants in the dishes of column two on the second and fourth row in the column.Two columns, the first consisting of donor plants only and the second alternating donors and receivers, make one treatment. Repeat these steps for each treatment of donor plant accession. Each replication requires one column of receiver plant samples to serve as a control for one replication.Treatments were replicated three times in a randomized complete block design. On the day after treatment one, fill the collection tank at the bottom of each column with half-strength Hoagland solution in distilled water approximately 1, 500 milliliters. Set the timers to run as desired in the auto off setting.Cover the collection tanks with black plastic to limit light exposure and evaporation. Fill the tanks every two days with 500 milliliters of Hoagland solution to keep the system flowing constantly. Maintain the greenhouse temperatures at 28 degrees Celsius during the day and 24 degrees Celsius at night respectively with a 16 to eight-hour split and humidity at 53%Measure and record the heights of each plant in the stair-step system on day after treatment one by placing a ruler at the base of each plant and observing the tallest leaf stand.Continue with data collection and analysis as described in the text protocol. Two preliminary screenings using this method were performed on nine weedy rice accessions and five cultivated rice lines. Height measurements recorded at day after treatment 14 were used to calculate the barnyard grass height reduction percent.Five weedy rice accessions displayed more significant barnyard grass height reduction than rondo, the allelopathic rice standard. Weedy rice accessions B8, S97, B14, S33 reduced barnyard grass height by 25 to 30%Weedy rice accession B81 exhibited the most considerable barnyard grass height reduction by 74%which was nearly three times as much as the standard allelopathic rice rondo. Biomass reduction percent from data collected at day after treatment 21 displayed a range in barnyard grass biomass reduction percent from 0 to 86%Among the weedy rice accessions that reduced barnyard grass height the most, S33 reduced barnyard grass biomass the most at approximately 84%compared to rondo at 60%It is important that there are no leaks in the whole system as this will disrupt the flow and cause variation in the results.Growing the plants in a hydroponic system could be an alternative and effective method to identify compounds produced by the plant and screened for allelopathic activity. This technique paved the way in understanding what genes are controlling allelopathic production in these well-performing weedy rice biotypes. It is crucial to use gloves, safety glasses, and appropriate footwear while using power tools.Additionally, make sure the area is well-ventilated when spray painting tanks.

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

Research

JoVE Journal

Biology

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to observe plant tissues in three dimensions (3D) while preserving their internal structures. In rice (Oryza sativa L.), a monocot model plant and a globally important crop, clearing technology has been reported in organs that are relatively easy to observe, such as the roots and leaves. Applications of clearing technology in shoot apical meristem (SAM) and stems have also been reported, but only to a limited degree because of the poor penetration of the clearing solution (CS) in these tissues. The limited efficiency of the clearing solutions in these tissues has been attributed to auto-fluorescence, thickening, and hardening of the tissues in the stem as the vascular bundles and epidermis develop and layering of the SAM with water-repellent leaves. The present protocol reports the optimization of a clearing approach for continuous and 3D observation of gene expression from the SAM/young panicle to the base of the shoots during development. Fixed tissue samples expressing a fluorescent protein reporter were trimmed into sections using a vibrating micro-slicer. When an appropriate thickness was achieved, the CS was applied. By specifically targeting the central tissue, the penetration rate and uniformity of the CS increased, and the time required to make the tissue transparent decreased. Additionally, clearing of the trimmed sections enabled the observation of the internal structure of the whole shoot from a macro perspective. This method has potential applications in deep imaging of tissues of other plant species that are difficult to clear.

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The present protocol describes a clearing technique for rice shoots, which are difficult to prepare for internal structural observations owing to the hard, thick, or layered nature of the tissues. This method facilitates continuous and deep fluorescence observations, even in adult rice plants.

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to ...

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 ...