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

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.