The paper reported identification of chemoattractant of rhizobacteria in root exudates using an improved chemotaxis assay. Chemotaxis implant root actuates is significant for root colonization for plant growth, promoting rhizobia bacteria and for providing the helpful factions of plant-microbe interactions. Many methods were used for analyzing bacterial chemotaxis.
For instance, swimming plate method, Cappilary like method, microfluidic slipchip device. The experiment cycle of swimming plate method was long. The experimental error of Cappilary like method was launched, microfluidic slipchip device is expensive to experiment.
We established a straightforward method to quickly identify the chemoattractant that could induce the chemotactic movement of rhizosphere growth-promoting bacteria using sterile glass slides without complicated steps. The method reduced the error of the plate counting and shortened the experiment cycle. In regard to identifying a chemoattractant substance, the new method could save 2-3 days and also reduce the cost of experimental materials.
Randomly distribute rice seeds in the grows chamber culture arises for a week and add sterile water twice. Select the rice seedlings of similar size and plant in a 50 milliliter of Ms liquid medium. Incubate for 48 hours at 22 degrees Celsius and a septic conditions.
Rice root exudates will be released into the Ms medium. Liquid Chromatography Mass Spectrometry Analysis was performed using UHPLC system. Prepare chemoattractant aqueous solution ensure it is sterile.
Filter the chemoattractant solution with a 0.22 micrometer bacteria filter. The chemoattractant aqueous solution was the single substance obtained from the LCMS studies dissolved in water. Citrix acid solution was used as example.
All actions must be performed by the side of the lamp. Mark the middle position of glass slide at an interval of 1 centimeter. Ensure that the glass slide is sterilized several times on the flame.
At the 30 microliter chemoattractant solution on the left of the glass slide ensure that bacteria was cultured to the logarithmic stage. Add the 30 microliter bacteria solution on the right of the glass slide. Sterilize inoculating loop several times on the flame using the inoculating loop to connect the chemoattractant aqueous solution to the bacteria solution and keep it at a room temperature for 20 minutes on a clean bench.
After 20 minutes disconnect the connecting line with filter paper. Ensure that the 1.5 milliliter centrifuged tube is sterile. Collect the chemoattractant aqueous solution on the left of the glass slide.
Transfer the solution to the 1.5 milliliters sterile centrifuged tube. Add the appropriate volume of the cell front dye to the centrifuged tube. After 2 minutes, collect the missible liquids for bacteria counting and microscopic observation with a blood counting chamber.
Determine the number of the viable microorganisms attracted using a following equation. A higher RCI indicates a strong reaction to the chemoattractant. The results indicated that citric acid and Caffeic acid have the highest chemotaxis indices.
The new chemotaxis assay method results wrote that bacterial can concentration of Citric acid, Caffeic acid and Galactose experiments were significantly higher than that of the control group and positive control. Citric acid wrote the strongest chemotaxis. The experimental results were in agreement with results of traditional methods.
This video protocol can be presented as a valuable resource for clarification of plant-microbe interactions.
