This work was supported by the National Natural Science Foundation of China (Nos. 31870493), the Key Research and Development Projects in Heilongjiang, China (GA21B007), and the Basic Research Fees of Universities in Heilongjiang Province, China (No. 135409103).

1. Materials and equipment
NOTE: Bacillus altitudinis LZP02 (CP075052) was isolated from the rhizosphere of rice in northeast China12,13 for this study.
<ol>
	<li>Culture B. altitudinis LZP02 in Luria-Bertani (LB) medium (peptone, 10 g L-1; NaCl, 8 g L-1 and yeast extract, 5 g L-1) for 10 h. Collect cells by centrifugation at 9,569 x g for 2 min at 4 &#176;C. and store with 15% glycerol at -80 &#176;C. 
	NOTE: For this experiment, rice seeds (Oryza sativa Longgeng 46) were provided by the Rice Research Institute of Heilongjiang Academy of Agricultural Sciences.</li>
</ol>
2. Collection of root exudates
<ol>
	<li>Randomly distribute rice seeds in a growth chamber. 
	NOTE: Rice seeds were sterilized with 30% H2O2 for 30 min and soaked in water overnight. The conditions were as follows: the controlled light (16/8 h light/dark cycle), temperature (22 &#177; 2 &#176;C) and relative humidity (&#8128;70%).</li>
	<li>Culture rice seeds for a week and add sterile water twice.</li>
	<li>Select rice seedlings of similar size and plant in 50 mL of Murashige and Skoog (MS) liquid medium. Incubate for 48 h at 22 &#176;C under aseptic conditions. 
	NOTE: Rice root exudates will be released into the MS medium14,15,16.</li>
</ol>
3. Liquid chromatography-mass spectrometry analysis of root exudates
<ol>
	<li>Collect 100 &#181;L of the sample (MS medium containing root exudates) in a 1.5 mL centrifuge tube. Add 20 &#181;L of the extraction solvent (acetonitrile-methanol-water, 2:2:1, including internal standard). Homogenize the sample at 45 Hz for 4 min, followed by ultrasonication on ice for 5 min in a water bath.</li>
	<li>Repeat the homogenization and ultrasonic cycle three times. Incubate the sample at -20 &#176;C for 1 h, followed by centrifugation at 133,778 x g and 4 &#176;C for 15 min.</li>
	<li>Transfer the resulting supernatant to LC-MS vials and store at -80 &#176;C until UHPLC-QE analysis. Prepare the quality control (QC) samples by mixing equal portions of the supernatant of all samples. 
	NOTE: Each sample volume was 600 &#181;L (six replicates per experiment) in the presented experiment.</li>
	<li>Perform LC-MS/MS analysis using UHPLC system, UPLC HSS T3 column (2.1 mm x 100 mm, 1.8 &#181;m) and Q Exactive12.
	<ol>
		<li>Use 0.1% formic acid aqueous solution and 5 mmol/L ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B. Use formic acid and ammonium acetate as positive and negative ion modes, respectively.</li>
		<li>Set the elution gradient as follows: 0 min, 1% B; 1 min, 1% B; 8 min, 99% B. 10 min, 99% B. 10.1 min, 1% B; 12 min, 1% B. Set the flow rate and injection volume to 0.5 mL/min and 2 &#181;L, respectively. 
		NOTE: The macromolecule (&#62;1,000 Daltons) cannot be detected.</li>
	</ol>
	</li>
</ol>
4. Chemotaxis assay
<ol>
	<li>Prepare the chemoattractant aqueous solution. Ensure that it is sterile. Filter the chemoattractant solution with a 0.22 &#181;m bacteria filter. 
	NOTE: The chemoattractant aqueous solution was the single substance obtained from the LC-MS studies dissolved in water. The concentration and volume can be adjusted appropriately according to different studies. Citric acid solution was used as an example. All actions must be performed by the side of a lamp.</li>
	<li>Mark the middle position of the glass slide at an interval of 1 cm. Ensure that the glass slide is sterilized several times on the flame.</li>
	<li>Add the 30 &#181;L of chemoattractant solution on the left of the glass slide. Ensure that bacteria were cultured to the logarithmic stage (2 x 108 CFU/mL) in LB medium. Add 30 &#181;L of the bacterial solution on the right of the glass slide. 
	NOTE: Prepare a negative control group with an equal volume of sterile water. The saline solution (0.9% NaCI) was used as the positive control in order to eliminate the changes caused by intermolecular force on the experiment.</li>
	<li>Sterilize an inoculating loop several times on the flame. Use the inoculating loop to connect the chemoattractant aqueous solution to the bacterial solution and keep it at room temperature for 20 min on a clean bench. 
	NOTE: The experiment must be carried out in a windless environment. Adjust the time before disconnecting the connection line for strains of different athletic abilities appropriately.</li>
	<li>After 20 min, disconnect the connecting line with filter paper.</li>
	<li>Ensure that the 1.5 mL centrifuge tube is sterile. Collect the chemoattractant aqueous solution on the left of the glass slide. Transfer the solution to the 1.5 mL sterile centrifuge tube.</li>
	<li>Add the appropriate volume of safranin to the centrifuge tube. After 2 min, collect the miscible liquids for counting bacteria and microscopic observation with a blood counting chamber.</li>
</ol>
5. Results analysis
<ol>
	<li>Determine the number of viable microorganisms attracted using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/63249/63249eq01v2.jpg" style="margin: auto;" /> 
	where NBC: Total number of bacteria cells; N: Number of bacteria in 80 grids. 
	NOTE: Statistical analysis software was used for data analysis. The error was based on three different replicated experimental values and was calculated using one-way ANOVA followed by Turkey&#39;s post-hoc analysis. P&#160;&#8804; 0.05 was considered significant.</li>
</ol>

<ol>
	<li>Belas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms,+flagella,+and+mechanosensing+of+surfaces+by+bacteria.">Biofilms, flagella, and mechanosensing of surfaces by bacteria.</a> Trends in Microbiology. 22 (9), 517-527 (2014).</li><li>Haichar, Z., Santaella, C., Heulin, T., Achouak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudates+mediated+interactions+belowground.">Root exudates mediated interactions belowground.</a> Soil Biology and Biochemistry. 77 (7), 69-80 (2014).</li><li>Zhang, N., 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=Effects+of+different+plant+root+exudates+and+their+organic+acid+components+on+chemotaxis,+biofilm+formation+and+colonization+by+beneficial+rhizosphere-associated+bacterial+strains.">Effects of different plant root exudates and their organic acid components on chemotaxis, biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains.</a> Plant and Soil. 374 (1-2), 689-700 (2014).</li><li>Zhang, N., 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=Whole+transcriptomic+analysis+of+the+plant-beneficial+rhizobacterium+Bacillus+amyloliquefaciens+SQR9+during+enhanced+biofilm+formation+regulated+by+maize+root+exudates.">Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates.</a> BMC Genomics. 16 (1), 685 (2015).</li><li>Lui, Y., 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=Induced+root-secreted+D-galactose+functions+as+a+chemoattractant+and+enhances+the+biofilm+formation+of+Bacillus+velezensis+SQR9+in+an+mcpa-dependent+manner.">Induced root-secreted D-galactose functions as a chemoattractant and enhances the biofilm formation of Bacillus velezensis SQR9 in an mcpa-dependent manner.</a> Applied Microbiology and Biotechnology. 104 (17), 785-797 (2020).</li><li>Vora, S. M., Joshi, P., Belwalkar, M., Archana, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudates+influence+chemotaxis+and+colonization+of+diverse+plant+growth-promoting+rhizobacteria+in+the+Cajanus+cajan+-+Zea+mays+intercropping+system.">Root exudates influence chemotaxis and colonization of diverse plant growth-promoting rhizobacteria in the Cajanus cajan - Zea mays intercropping system.</a> Rhizosphere. 18 (12), 100331 (2021).</li><li>Sampedro, I., 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=Effects+of+halophyte+root+exudates+and+their+components+on+chemotaxis,+biofilm+formation+and+colonization+of+the+halophilic+bacterium+halomonas+anticariensis+FP35T.">Effects of halophyte root exudates and their components on chemotaxis, biofilm formation and colonization of the halophilic bacterium halomonas anticariensis FP35T.</a> Microorganisms. 8 (4), 575 (2020).</li><li>Liu, X. L., Raza, W., Ma, J. H., Huang, Q. W., Shen, Q. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+role+amino+acid+from+sesbania+rostrata+seed+exudates+in+the+chemotaxis+response+of+Azorhizobium+caulinodans+ORS571.">A dual role amino acid from sesbania rostrata seed exudates in the chemotaxis response of Azorhizobium caulinodans ORS571.</a> Molecular Plant-Microbe Interactions. 32 (9), 1134-1147 (2019).</li><li>Ling, N., Raza, W., Ma, J. H., Huang, Q. W., Shen, Q. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+role+of+organic+acids+in+watermelon+root+exudates+for+recruiting+Paenibacillus+polymyxa+SQR-21+in+the+rhizosphere.">Identification and role of organic acids in watermelon root exudates for recruiting Paenibacillus polymyxa SQR-21 in the rhizosphere.</a> European Journal of Soil Biology. 47 (6), 374-379 (2011).</li><li>Rudrappa, T., Czymmek, K. J., Pare, P. W., Bais, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root-secreted+malic+acid+recruits+beneficial+soil+bacteria.">Root-secreted malic acid recruits beneficial soil bacteria.</a> Plant Physiology. 148 (3), 1547-1556 (2008).</li><li>Shen, C., 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=Bacterial+chemotaxis+on+Slipchip.">Bacterial chemotaxis on Slipchip.</a> Lab on a Chip. 14 (16), 3074-3080 (2014).</li><li>Liu, H., 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=Bacillus+pumilus+LZP02+promotes+rice+root+growth+by+improving+carbohydrate+metabolism+and+phenylpropanoid+biosynthesis.">Bacillus pumilus LZP02 promotes rice root growth by improving carbohydrate metabolism and phenylpropanoid biosynthesis.</a> Molecular Plant-Microbe Interactions. 33 (10), 1222-1231 (2020).</li><li>Goswami, M., Deka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+novel+rhizobacteria+having+multiple+plant+growth+promoting+traits+and+antifungal+activity+against+certain+phytopathogens.">Isolation of a novel rhizobacteria having multiple plant growth promoting traits and antifungal activity against certain phytopathogens.</a> Microbiological Research. 240, 126516 (2020).</li><li>Kaiira, M., Chemining'Wa, G., Ayuke, F., Baguma, Y., Nganga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiles+of+compounds+in+root+exudates+of+rice,+cymbopogon,+desmodium,+mucuna+and+maize.">Profiles of compounds in root exudates of rice, cymbopogon, desmodium, mucuna and maize.</a> Journal of Agricultural Sciences Belgrade. 64 (4), 399-412 (2019).</li><li>Shi, Y., 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=Effect+of+rice+root+exudates+and+strain+combination+on+biofilm+formation+of+Paenibacillus+polymyxa+and+Paenibacillus+macerans.">Effect of rice root exudates and strain combination on biofilm formation of Paenibacillus polymyxa and Paenibacillus macerans.</a> African Journal of Microbiology Research. 6 (13), 3343-3347 (2012).</li><li>Lee, H. W., Ghimire, S. R., Shin, D. H., Lee, I. J., Kim, K. U. <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+the+root+exudates+of+K21,+a+potent+allelopathic+rice.">Allelopathic effect of the root exudates of K21, a potent allelopathic rice.</a> Weed Biology and Management. 8 (2), 85-90 (2008).</li><li>Belimov, A. A., 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=Rhizobacteria+that+produce+auxins+and+contain+1-amino-cyclopropane-1-carboxylic+acid+deaminase+decrease+amino+acid+concentrations+in+the+rhizosphere+and+improve+growth+and+yield+of+well-watered+and+water-limited+potato+(Solanum+tuberosum).">Rhizobacteria that produce auxins and contain 1-amino-cyclopropane-1-carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well-watered and water-limited potato (Solanum tuberosum).</a> Annals of Applied Biology. 167 (1), 11-25 (2015).</li><li>Ankati, S., Podile, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolites+in+the+root+exudates+of+groundnut+change+during+interaction+with+plant+growth+promoting+rhizobacteria+in+a+strain-specific+manner.">Metabolites in the root exudates of groundnut change during interaction with plant growth promoting rhizobacteria in a strain-specific manner.</a> Journal of Plant Physiology. 243, 153057 (2019).</li><li>Gordillo, F., Chavez, F., Jerez, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+and+chemotaxis+of+Pseudomonas+sp.+B4+towards+polychlorobiphenyls+and+chlorobenzoates.">Motility and chemotaxis of Pseudomonas sp. B4 towards polychlorobiphenyls and chlorobenzoates.</a> FEMS Microbiology Ecology. 60 (2), 322-328 (2007).</li><li>Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S., Vivanco, J. M. <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+in+rhizosphere+interactions+with+plants+and+other+organisms.">The role of root exudates in rhizosphere interactions with plants and other organisms.</a> Annual Review of Plant Biology. 57, 233-266 (2006).</li><li>Badri, D. V., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+root+exudates.">Regulation and function of root exudates.</a> Plant Cell Environment. 32 (6), 666-681 (2009).</li><li>Badri, D. V., Weir, T. L., Lelie, D., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizosphere+chemical+dialogues:+plant-microbe+interactions.">Rhizosphere chemical dialogues: plant-microbe interactions.</a> Curr Opin Biotech. Current Opinion in Biotechnology. 20 (6), 642-650 (2009).</li><li>Kamilova, F., Kravchenko, L. V., Shaposhnikov, A. I., Makarova, N., Lugtenberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+tomato+pathogen+Fusarium+oxysporum+f.+sp.+radicis-lycopersici+and+of+the+biocontrol+bacterium+Pseudomonas+fluorescens+WCS365.">Effects of the tomato pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365.</a> Molecular Plant-Microbe Interactions. 19 (10), 1121-1126 (2006).</li><li>Kamilova, F., 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=Organic+acids,+sugars,+and+L-tryptophane+in+exudates+of+vegetables+growing+on+stonewool+and+their+effects+on+activities+of+rhizosphere+bacteria.">Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria.</a> Molecular Plant-Microbe Interactions. 19 (3), 250-256 (2006).</li><li>Badri, D. V., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+root+exudates.">Regulation and function of root exudates.</a> Plant Cell Environment. 32 (6), 666-681 (2009).</li><li>Hao, W. Y., Ren, L. X., Ran, W., Shen, Q. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathic+effects+of+root+exudates+from+watermelon+and+rice+plants+on+Fusarium+oxysporum+f.sp.+Niveum.">Allelopathic effects of root exudates from watermelon and rice plants on Fusarium oxysporum f.sp. Niveum.</a> Plant and Soil. 336 (1-2), 485-497 (2010).</li><li>Hao, Z. P., Wang, Q., Christie, P., Li, X. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelopathic+potential+of+watermelon+tissues+and+root+exudates.">Allelopathic potential of watermelon tissues and root exudates.</a> Scientia Horticulturae. 112 (3), 315-320 (2007).</li></ol>

The authors have nothing to disclose.

Increasing research indicates that plant-bacteria interactions mainly occur in the rhizosphere and are influenced by root exudates20,21,22,23,24. Plant root exudates include a diverse array of primary metabolites, including phenolic acids, organic acids and amino acids as well as more complex secondary compounds25,<sup class=...

Chemotaxis is important for colonization of plant growth-promoting rhizobacterial (PGPR) on roots and for understanding plant-microbe interactions1. A class of low molecular weight compounds (chemoattractants) in plant root exudates induce the chemotactic movement of PGPR to the rhizosphere2. Malic acid, citric acid, and other components in the root exudates stimulate chemotaxis of Bacillus strains3. For example, glucose, citric acid, and fumaric acid in maize root exudates recruit bacteria&#160;to the root surface4. D-galactose, which&#160;is derived from root exudates,induces the chemotaxis of Bacillus velezensis SQR95. Organic acids, including fumarate, malic acid, and succinate, influence chemotaxis and colonization of various PGPR in the Cajanus cajan - Zea mays intercropping system6. Oleanolic acid in rice root exudates, acts as a chemoattractant for the strain FP357. Other plant exudates (including histidine, arginine, and aspartate) can play a crucial role in the chemotactic response of bacteria8. Plant exudates function as a signal to direct the movement of bacteria, which is the first step during rhizosphere colonization. Plant colonization by PGPR is a process of enormous relevance, as PGPR are beneficial for the plant host.
Many methods have been used for analyzing bacterial chemotaxis. The swimming plate method is one of the methods described previously9. In this method, plates were made with a semisolid medium. A chemotactic buffer containing agar (1.0%, w/v) was added to the plate. The buffer is heated, and then mixed with the chemoattractant. Then, 8 &#181;L of bacteria suspension was added dropwise to the middle of the plate and the plate was placed in an incubator at 28 &#176;C. The plate was regularly observed and photographed. However, the experimental cycle of the swimming plate method was very long. In the capillary-like method10, a pipette tip serves as a chamber for holding 100 &#181;L of bacterial suspension. 1 mL syringe needle was used as a capillary. A syringe needle containing chemoattractants with different concentration gradients was inserted into the 100 &#181;L pipette tip. After incubation at room temperature for 3 h, the syringe needle was removed, the content was diluted and plated on the medium. The bacterial accumulation in the syringe was represented by colony-forming units (CFUs) in the plates. However, the experimental error within replicates for the capillary-like method was large. Another method used a microfluidic SlipChip device11. Briefly, bovine serum albumin (BSA) solution was injected into all channels and removed using a vacuum. The solutions containing different chemoattractants (1 mM concentration for qualitative detection only), bacterial cells suspended in phosphate-buffered saline and phosphate buffered saline buffer (negative control) were added to the top, middle, and bottom microwells, respectively. Incubation was then performed in a dark environment at room temperature for 30 min. The bacterial cells were then detected in the microwells. The microfluidic SlipChip device, however, was expensive. Therefore, each of the methods described above had advantages and disadvantages.
We established an improved chemotaxis assay for the rapid identification of rhizobacterial chemoattractants in root exudates using sterile glass slides without complicated steps. 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. Therefore, if used to identify a chemoattractant substance, this new method can save 2-3 days and reduce the cost of experimental materials.

<table><tbody><tr><td>2,5-dihydroxybenzoic acid</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>490-79-9</td><td></td></tr><tr><td>Acetonitrile</td><td>CNW Technologies</td><td>75-05-8</td><td></td></tr><tr><td>Ammonium acetate</td><td>CNW Technologies</td><td>631-61-8</td><td></td></tr><tr><td>Caffeic acid</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>331-39-5</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific</td><td>Heraeus Fresco17</td><td></td></tr><tr><td>Citric acid</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>77-92-9</td><td></td></tr><tr><td>Clean bench</td><td>Shanghai Boxun Industrial Co., Ltd.</td><td>BJ-CD</td><td></td></tr><tr><td>Ferulic acid</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>1135-24-6</td><td></td></tr><tr><td>Formic acid</td><td>CNW Technologies</td><td>64-18-6</td><td></td></tr><tr><td>Fructose</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>57-48-7</td><td></td></tr><tr><td>Galactose</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>59-23-4</td><td></td></tr><tr><td>Glycine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>56-40-6</td><td></td></tr><tr><td>Grinding Mill</td><td>Shanghai Jingxin Industrial Development&lt;br/&gt; Co., Ltd.</td><td>JXFSTPRP-24</td><td></td></tr><tr><td>Histidine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>71-00-1</td><td></td></tr><tr><td>Internal standard: 2-Chloro-L-phenylalanine</td><td>Shanghai Hengbai Biotech C.,Ltd.</td><td>103616-89-3</td><td></td></tr><tr><td>Leucine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>61-90-5</td><td></td></tr><tr><td>Malic acid</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>6915-15-7</td><td></td></tr><tr><td>Mannose</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>3458-28-4</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Thermo Fisher Scientific</td><td>Q Exactive Focus</td><td></td></tr><tr><td>Methanol</td><td>CNW Technologies</td><td>67-56-1</td><td></td></tr><tr><td>Optical Microscope</td><td>Olympus</td><td>BX43</td><td></td></tr><tr><td>Phenylalanine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>63-91-2</td><td></td></tr><tr><td>Proline</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>147-85-3</td><td></td></tr><tr><td>Scales</td><td>Sartorius</td><td>BSA124S-CW</td><td></td></tr><tr><td>Serine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>56-45-1</td><td></td></tr><tr><td>Threonine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>72-19-5</td><td></td></tr><tr><td>UHPLC</td><td>Agilent</td><td>1290 UHPLC</td><td></td></tr><tr><td>Ultrasound Instrument</td><td>Shenzhen Leidebang Electronics&lt;br/&gt; Co., Ltd.</td><td>PS-60AL</td><td></td></tr><tr><td>Valine</td><td>Beijing InnoChem Science &amp;amp; Technology C.,Ltd.</td><td>7004-03-7</td><td></td></tr></tbody></table>

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.

A total of 584 and 937 known metabolites were detected in the positive and negative ion indices, respectively. Previous studies have shown that chemoattractants are typically organic acids, amino acids, and carbohydrates17,18.
In this study, 16 kinds of chemoattractants from the LC-MS studies in the rice rhizosphere exudates were selected for subsequent experiments (Table 1). Using the swimming plate method, we screene...

Watch this Scientific Journal Video about An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates at JoVE.com

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

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

4.4K Views.  Qiqihar University. 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.

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

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Behavior

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Environment

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Immunology and Infection

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Source: Campbell, J. C., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55939/a-caenorhabditis-elegans-nutritional-status-based-copper-aversion">A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay.</a> J. Vis. Exp. (2017).
This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

Source: Campbell, J. C., et al. A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay. J. Vis. Exp. (2017).
This video introduces ...

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

Summary

Explore More Videos

Chapters in this video

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Measurement of Cellular Chemotaxis with ECIS/Taxis

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

C. elegans Chemotaxis Assay

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms