Name: Forfatter Spotlight: Forbedring af rhizobakteriers kolonisering på planterødder for forbedret mikrobiel gødningseffektivitet
Uploaded: 2024-03-01T14:10:00
Description: Watch this Scientific Journal Video about Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency at JoVE.com

Dette arbejde blev finansieret af National Natural Science Foundation of China (32370135), innovationsprogrammet fra det kinesiske akademi for landbrugsvidenskab (CAAS-CSAL-202302), videnskabs- og teknologiprojektet fra Jiangsu Vocational College of Agriculture and Forestry (2021kj29).

1. Steril hydroponisk dyrkning af A. thaliana
<ol><li>Forbered A. thaliana frøplanter.<ol><li>Forbered kultur A. thaliana frøplanter medium, som består af 1/2 MS medium (Murashige og Skoog) med 2% (vægt / vol) saccharose og 0,9% (vægt / vol) agar.</li>
		<li>Hæld det tilberedte steriliseringsmedium i sterile firkantede petriskåle (13 cm x 13 cm) inden størkning. Undgå lufttørring for at opretholde fugtigheden.</li>
		<li>Nedsænk A. thaliana</em...

Rhizobakteriel koloniseringsdetektion på Arabidopsis thaliana

<ol>
	<li>Wang, B., Wan, C., Zeng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+on+cotton+plants+with+a+GFP+labeled+strain+of+Bacillus+axarquiensis.">Colonization on cotton plants with a GFP labeled strain of Bacillus axarquiensis.</a> Curr Microbiol. 77 (10), 3085-3094 (2020).</li><li>Zhai, Z., 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=A+genetic+tool+for+production+of+GFP-expressing+Rhodopseudomonaspalustris+for+visualization+of+bacterial+colonization.">A genetic tool for production of GFP-expressing Rhodopseudomonaspalustris for visualization of bacterial colonization.</a> AMB Express. 9 (1), 141 (2019).</li><li>Synek, L., Rawat, A., L'Haridon, F., Weisskopf, L., Saad, M. M., Hirt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+strategies+of+plant+colonization+by+beneficial+endophytic+Enterobacter+sp.+SA187.">Multiple strategies of plant colonization by beneficial endophytic Enterobacter sp. SA187.</a> Environ Microbiol. 23 (10), 6223-6240 (2021).</li><li>Zhang, 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+velezensis+tolerance+to+the+induced+oxidative+stress+in+root+colonization+contributed+by+the+two-component+regulatory+system+sensor+ResE.">Bacillus velezensis tolerance to the induced oxidative stress in root colonization contributed by the two-component regulatory system sensor ResE.</a> Plant Cell Environ. 44 (9), 3094-3102 (2021).</li><li>Liu, 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=Plant+commensal+type+VII+secretion+system+causes+iron+leakage+from+roots+to+promote+colonization.">Plant commensal type VII secretion system causes iron leakage from roots to promote colonization.</a> Nat Microbiol. 8 (8), 1434-1449 (2023).</li><li>Feng, 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=Identification+of+chemotaxis+compounds+in+root+exudates+and+their+sensing+chemoreceptors+in+plant-growth-promoting+Rhizobacteria+Bacillus+amyloliquefaciens+SQR9.">Identification of chemotaxis compounds in root exudates and their sensing chemoreceptors in plant-growth-promoting Rhizobacteria Bacillus amyloliquefaciens SQR9.</a> Mol Plant Microbe Interact. 31, 995-1005 (2018).</li><li>Woo, S. L., Hermosa, R., Lorito, M., Monte, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichoderma:+a+multipurpose,+plant-beneficial+microorganism+for+eco-sustainable+agriculture.">Trichoderma: a multipurpose, plant-beneficial microorganism for eco-sustainable agriculture.</a> Nat Rev Microbiol. 21 (5), 312-326 (2023).</li><li>Nongkhlaw, F. M., Joshi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+study+on+colonization+and+antimicrobial+property+of+endophytic+bacteria+associated+with+ethnomedicinal+plants+of+Meghalaya.">Microscopic study on colonization and antimicrobial property of endophytic bacteria associated with ethnomedicinal plants of Meghalaya.</a> J Microsc Ultrastruct. 5 (3), 132-139 (2017).</li><li>Ravelo-Ortega, G., Raya-Gonz&#225;lez, J., L&#243;pez-Bucio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+rhizosphere+microbes+that+promote+plant+growth.">Compounds from rhizosphere microbes that promote plant growth.</a> Curr Opin Plant Biol. 73, 1369-5266 (2023).</li><li>Schulz-Bohm, K., Gerards, S., Hundscheid, M., Melenhorst, J., de Boer, W., Garbeva, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calling+from+distance:+attraction+of+soil+bacteria+by+plant+root+volatiles.">Calling from distance: attraction of soil bacteria by plant root volatiles.</a> ISME J. 12 (5), 1252-1262 (2018).</li><li>Sharifi, R., Lee, S. M., Ryu, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-induced+plant+volatiles.">Microbe-induced plant volatiles.</a> New Phytol. 220 (3), 684-691 (2018).</li><li>Eckshtain-Levi, N., Harris, S. L., Roscios, R. Q., Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+community+members+increase+Bacillus+subtilis+maintenance+on+the+roots+of+Arabidopsis+thaliana.">Bacterial community members increase Bacillus subtilis maintenance on the roots of Arabidopsis thaliana.</a> Phytobiomes J. 4, 303-313 (2020).</li><li>Liu, 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=Root+colonization+by+beneficial+rhizobacteria.">Root colonization by beneficial rhizobacteria.</a> FEMS Microbiol Rev. 48, (2024).</li><li>Yahya, M., 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=Differential+root+exudation+and+architecture+for+improved+growth+of+wheat+mediated+by+phosphate+solubilizing+bacteria.">Differential root exudation and architecture for improved growth of wheat mediated by phosphate solubilizing bacteria.</a> Front Microbiol. 12, 744094 (2021).</li><li>Husna, K. B. -. E., Won, M. -. H., Jeong, M. -. I., Oh, K. -. K., Park, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+genomic+insight+of+surfactin-producing+Bacillus+velezensis+and+its+biocontrol+potential+against+pathogenic+contamination+in+lettuce+hydroponics.">Characterization and genomic insight of surfactin-producing Bacillus velezensis and its biocontrol potential against pathogenic contamination in lettuce hydroponics.</a> Environ Sci Pollut Res Int. 30 (58), 121487-121500 (2023).</li></ol>

For at opnå god reproducerbarhed er der fire kritiske trin til koloniseringsdetekteringsprocessen for denne protokol. For det første er det nødvendigt at sikre, at antallet af podede bakterieceller er nøjagtigt det samme i hvert forsøg. For det andet er det også nødvendigt at kontrollere den ukoloniserede bakterierensningsintensitet med sterilt vand. For det tredje skal hver prøvefortyndingsproces hvirvelbehandles, før den udføres, for at lade prøven være i en fuldstændig blandingstilstand for at undgå abso...

Der er kvantitative og kvalitative metoder til påvisning af rhizosfærekolonisering af en enkelt bakteriestamme. Til den kvalitative metode bør der anvendes en stamme, der konstitutivt udtrykker fluorescens, og fluorescensfordelingen og -intensiteten bør undersøges ved fluorescensmikroskopi eller laserkonfokale instrumenter 1,2. Disse strategier kan godt afspejle bakteriel kolonisering in situ3, men de er ikke så nøjagtige som traditionelle pladetællingsmetoder til kvantificering. På grund af begrænsningen ved kun at vise delvise rodzoner under mikroskopet kan det undertiden påvirkes af subjektiv bias.
Her beskriver vi en kvantitativ metode, som omfatter indsamling af de koloniserede bakterieceller og tælling af de bakterielle CFU'er på en plade. Denne metode er baseret på fortynding og plettering, hvorved de koloniserede stammer, der blev fjernet fra planterødder, kan tælles, og det samlede koloniserede bakterieantal på roden kan beregnes 4,5.
For det første blev A. thaliana dyrket under hydroponiske forhold, og derefter blev bakterieceller inokuleret i rhizosfæren i en endelig koncentration på 0, 01 OD600. De inficerede rodvæv blev høstet 2 dage efter podning og vasket i sterilt vand for at fjerne de ukoloniserede bakterieceller. Endvidere blev bakterieceller koloniseret på roden opsamlet, fortyndet i fosfatbufret saltvand (PBS) buffer og belagt på et Luria-Bertani (LB) agarmedium. Efter inkubation ved 37 °C i 10 timer blev enkelte kolonier på LB-plader talt og normaliseret for at bestemme bakteriecellerne koloniseret på rødder.
Denne metode er meget anvendelig, har god repeterbarhed og er mere egnet til nøjagtig bestemmelse af rhizosfærens bakterielle kolonisering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cell stainer</td>
			<td>Solarbio</td>
			<td>F8200-40&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader&#160;</td>
			<td>Tecan</td>
			<td>Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog medium</td>
			<td>Hopebio</td>
			<td>HB8469-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaClO</td>
			<td>Alfa</td>
			<td>L14709</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>Square petri dish</td>
			<td>Ruiai Zhengte</td>
			<td>PYM-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie2</td>
			<td>Scientific Industries</td>
			<td>G560E</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring bacterial colonization on arabidopsis thaliana roots in hydroponic condition

Måling af bakteriekolonisering på Arabidopsis thaliana rod er et af de hyppigste forsøg i plante-mikrobe interaktionsundersøgelser. En standardiseret metode til måling af bakteriekolonisering i rhizosfæren er nødvendig for at forbedre reproducerbarheden. Vi dyrkede først steril A. thaliana under hydroponiske forhold og podede derefter bakteriecellerne i rhizosfæren ved en endelig koncentration på OD600 på 0,01. 2 dage efter podning blev rodvævet høstet og vasket tre gange i sterilt vand for at fjerne de ukoloniserede bakterieceller. Rødderne blev derefter vejet, og bakteriecellerne koloniseret på roden blev opsamlet af hvirvel. Cellesuspensionen blev fortyndet i en gradient med en fosfatbufret saltvandsbuffer (PBS) efterfulgt af plettering på et Luria-Bertani (LB) agarmedium. Pladerne blev inkuberet ved 37 °C i 10 timer, og derefter blev de enkelte kolonier på LB-plader talt og normaliseret for at indikere bakteriecellerne koloniseret på rødder. Denne metode anvendes til at detektere bakteriekolonisering i rhizosfæren under monointeraktionsbetingelser med god reproducerbarhed.

There are quantitative and qualitative methods for detecting rhizosphere colonization by a single bacterial strain. For the qualitative method, a strain that constitutively expresses fluorescence should be used, and the fluorescence distribution and intensity should be examined under fluorescence microscopy or laser confocal instruments1,2. Those strategies can well reflect bacterial colonization in situ3, but they are not as accurate as traditional plate counting methods in quantification. Besides, due to the limitation of only displaying partial root zones under the microscope, sometimes it can be influenced by subjective bias. 
Here, we describe a quantitative method, which includes collecting the colonized bacterial cells and counting the bacterial CFUs on a plate. This method is based on dilution and plating by which the colonized strains that were stripped from plant roots can be counted, and the total colonized bacteria number on the root can be calculated4,5. 
First, A. thaliana was cultured in hydroponic conditions, and then bacterial cells were inoculated in the rhizosphere at a final concentration of 0.01 OD600. The infected root tissues were harvested 2 days post-inoculation and washed in sterile water to remove the uncolonized bacterial cells. Further, bacterial cells colonized on the root were collected, diluted in phosphate-buffered saline (PBS) buffer, and plated onto a Luria-Bertani (LB) agar medium. After incubation at 37 &#176;C for 10 h, single colonies on LB plates were counted and normalized to determine the bacterial cells colonized on roots.
This method is highly applicable, has good repeatability, and is more suitable for accurate rhizosphere bacterial colonization determination.

Forfatter Spotlight: Forbedring af rhizobakteriers kolonisering på planterødder for forbedret mikrobiel gødningseffektivitet

Måling af bakteriel kolonisering på Arabidopsis thaliana rødder i hydroponisk tilstand

Our research focuses on the interaction of beneficial rhizobacteria with plant roots. We are looking for the key mechanisms to enhance the colonization of beneficial bacteria, thus improving the efficiency and the stability of microbial fertilizers in the field. The methods for bacterial colonization of plant roots have been extensively studied, but there are still problems.For example, it's hard to measure the colonization on different sites distinctively, In the future we think applying microscope and high throughput counting will help to solve the problems. Our protocol is highly applicable with good repeatability, and it is more suitable for accurate rhizosphere bacteria colonization determination.

For at teste nøjagtigheden af bakteriernes koloniseringsevne påvist ved denne metode i A. thaliana rhizosfæren, podede vi Bacillus velezensis SQR9 WT og en afledt mutant Δ8mcp i A. thaliana rhizosfæren separat. Δ8mcp er en mutant, der mangler alle kemoreceptorkodende gener, og den har en signifikant nedsat kolonisering6. Vi målte deres kolonisering 2 dage efter podning ved hjælp af den nuværende rodkoloniseringsanalyse. Resultaterne viste en si...

Watch this Scientific Journal Video about Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency at JoVE.com

Kolonisering af plantevækstfremmende rhizobakterier (PGPR) i rhizosfæren er afgørende for dens vækstfremmende virkning. Det er nødvendigt at standardisere metoden til påvisning af bakteriel rhizosfærekolonisering. Her beskriver vi en reproducerbar metode til kvantificering af bakteriekolonisering på rodoverfladen.

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

Vores forskning fokuserer på samspillet mellem gavnlige rhizobakterier og planterødder. Vi leder efter nøglemekanismerne til at forbedre koloniseringen af gavnlige bakterier og dermed forbedre effektiviteten og stabiliteten af mikrobiel gødning i marken. Metoderne til bakteriel kolonisering af planterødder er blevet grundigt undersøgt, men der er stadig problemer.For eksempel er det svært at måle koloniseringen på forskellige steder markant, I fremtiden tror vi, at anvendelse af mikroskop og høj gennemstrømningstælling vil hjælpe med at løse problemerne. Vores protokol er meget anvendelig med god repeterbarhed, og den er mere velegnet til nøjagtig bestemmelse af rhizosfærebakteriekolonisering.

measuring-bacterial-colonization-on-arabidopsis-thaliana-roots

Research

JoVE Journal

Environment

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.