Name: high-throughput siderophore screening from environmental samples: plant tissues, bulk soils, and rhizosphere soils
Uploaded: 2019-02-09T15:00:00
Description: Watch this Scientific Journal Video about High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils at JoVE.com

The authors wish to thank Kalyani Muhunthan for assistance in laboratory procedures, Lee Opdahl for wheat genotype harvesting, the Washington State Concord Grape Research Council, and the Washington State University Center for Sustaining Agriculture and Natural Resources for a BIOAg grant to support this work. Additional funding was provided by the USDA/NIFA through Hatch project 1014527.

NOTE: Location of Field Site: Washington State University, Plant Pathology Farm (46&#176;46&#8217;38.0&#8221;N 117&#176;04&#8217;57.4&#8221;W). Seeds were sown using a mechanical planter on October 19, 2017. Each wheat genotype was planted in headrows, approximately 1 meter apart to avoid overlapping of root system. Plant and soil samples were collected on August 9, 2018, when plants were ready for harvest. Samples were gathered from three replicates of four wheat genotypes: PI561727, PI561725, Madsen, Lewjain.
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<ol>
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The primary result of this work is the production of a new methodology that can be used to rapidly enrich for siderophore producing microbes while quantitatively measuring siderophore production/activity in the environmental sample. The methodology is quick, simple, and cost-effective, and the results show how it can be used to detect siderophore activity from complex and novel sample types (e.g., soil and plant tissue). The protocol also results in the production of glycerol stocks of the enrichment cultures, w...

Siderophores are important biomolecules involved primarily in iron-chelation for bioavailability, but with a wide array of additional purposes in terrestrial and aquatic ecosystems ranging from microbial quorum sensing, signaling to microbial plant-hosts, plant growth promotion, cooperation and competition within complex microbial communities1,2. Siderophores can be broadly classified according to their active sites and structural features, creating four basic types: carboxylate, hydroxamate, catecholate, and mixed types3,4. Many microorganisms are capable of excreting more than one type of siderophore5 and in complex communities, a vast majority of organisms biosynthesize the membrane receptors to allow the uptake of an even wider variety of siderophores1,6. Recent work indicates that siderophores are particularly important at the community level, and even in inter-kingdom communications and biogeochemical transfers7,8,9,10,11.
Chrome azurol sulphonate (CAS) has been used for over 30 years as a chelating agent to bind iron (Fe) in such a way that addition of ligands (i.e., siderophores) can result in dissociation of the CAS-Fe complex, creating an easily identifiable color change in the medium12. When the CAS is bound with Fe, the dye appears as a royal blue color, and as the CAS-Fe complex dissociates, the medium changes color according to the type of ligand used to scavenge the Fe13. The initial, liquid-based medium established by Schwyn and Neilands in 1987, has been modified in many ways to accommodate changing microbial targets14, growth habits and limitations15, as well as a variety of metals besides Fe, including aluminum, manganese, cobalt, cadmium nickel, lithium, zinc16, copper17, and even arsenic18.
Many human pathogens, as well as plant growth promoting microorganisms (PGPM) have been identified as siderophore-producing organisms3,19,20, and important rhizosphere and endophytic PGPM often test positive for siderophore-production4. The traditional Fe-based liquid method has been adapted to microtiter testing of isolates in cultivation for siderophore production21. However, these techniques fail to recognize the importance of the microbial community as a whole (the microbiome), in cooperation and potential regulation of siderophore production in soils and plant systems22. For that reason, we have developed a high-throughput community-level assessment of siderophore production from a given environment, based on the traditional CAS assay, but with replication, ease of measurement, reliability, and repeatability in a microplate assay.
In this study, a cost-effective, high-throughput CAS-Fe assay for detecting siderophore production is presented to assess the enrichment of siderophore production from complex samples (i.e., soil and plant tissue homogenates). Bulk, loosely-bound, and tightly-bound rhizosphere soil (in terms of how the soil was bound to the root) were obtained along with grain, shoot, and root tissues from four distinct wheat (Triticum aestivum L.) genotypes: Lewjain, Madsen, PI561725, and PI561727. It was hypothesized that fundamental differences in the wheat genotypes could result in differences in recruitment and selection of siderophore producing communities. Of particular interest is the difference between microbial communities associated with the PI561725 isogenic line, which is aluminum tolerant because it possesses ALMT1 (Aluminum-activated Malate Transporter 1), compared with the aluminum sensitive PI561727 isogenic line, which possesses a non-aluminum responsive form of the gene, almt123,24,25,26. The chief objective of the study was to develop a straightforward, rapid method of quantitatively assessing siderophore production in siderophore enrichment cultures of complex sample types while preserving the cultures for future work.

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Apex</td>
			<td>LF451320014</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Baking Pan</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride, granular</td>
			<td>Fiesher Scientific</td>
			<td>152315A</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave and Sterilizer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fiesher Scientific</td>
			<td>171428</td>
			<td></td>
		</tr>
		<tr>
			<td>CAS (Chrome Azurol S)</td>
			<td>Chem-Impex Int&#39;l Inc)</td>
			<td>000331-27168</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose Monohydrate (glucose), crystalline powder</td>
			<td>Fiesher Scientific</td>
			<td>1521754</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, disodium salt, dihydrate, Crystal</td>
			<td>J.T.Baker</td>
			<td>JI2476</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol, Anhydrous</td>
			<td>Baker Analyzed</td>
			<td>C22634</td>
			<td></td>
		</tr>
		<tr>
			<td>HDTMA (Cetyltrimethylammomonium Bromide</td>
			<td>Reagent World</td>
			<td>FZ0941</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloride acid</td>
			<td>ACROS Organic</td>
			<td>B0756767</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite M200 PRO plate reader</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (III) chloride hexahydrate, 99%</td>
			<td>ACROS Organic</td>
			<td>A0342179</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Fume Hood</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Incubator</td>
			<td>VWR Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fiesher Scientific</td>
			<td>27855</td>
			<td></td>
		</tr>
		<tr>
			<td>Niric Acid, (69-70)%</td>
			<td>J.T.Baker</td>
			<td>72287</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES buffer, 98.5%</td>
			<td>ACROS Organic</td>
			<td>A0338723</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibaisc,powder</td>
			<td>J.T.Baker</td>
			<td>J48594</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyoverdine</td>
			<td>SIGMA-ALDRICH</td>
			<td>078M4094V</td>
			<td></td>
		</tr>
		<tr>
			<td>Sand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SI-600R Shaker</td>
			<td>Lab Companion</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, granular</td>
			<td>Fiesher Scientific</td>
			<td>136539</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, pellets</td>
			<td>J.T.Baker</td>
			<td>G48K53</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic heptahydrate, 99%</td>
			<td>ACROS Organic</td>
			<td>A0371705</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput siderophore screening from environmental samples: plant tissues, bulk soils, and rhizosphere soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

A pyoverdine mixture biosynthesized by Pseudomonas fluorescens was used as a standard to interpret and quantify absorbance (at 420 nm) of samples in terms of pyoverdine equivalents in &#181;M. Figure 1 shows the relationship between absorbance (420 nm) and starting concentration of pyoverdine (Log10 molarity in &#181;M). EDTA did not provide an adequate standard because samples exhibited greater absorbance measurements than were attainable...

Watch this Scientific Journal Video about High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils at JoVE.com

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

Siderophores are low molecular weight, metal-chelating biomolecules involved in iron cycling in the environment. This protocol allows for rapid high throughput assessment of siderophore activity in soil and plant samples. Previous methods for siderophore detection have eliminated the crucial environment of the microbial community.Our technique allows detection within the relatively intact microbial community and the habitat in which it involved. Because iron availability is crucial to agriculture productivity, so in this protocol could be used to investigate the role of microbes in modulating the availability of iron to plants. Additionally, this method could be used to assess the impact of farm management practices on soil health and siderophore-producing communities as well as the development of such communities over time.To begin, acid wash all glassware in 100 millimolar hydrochloric 100 millimolar nitric acid for a minimum of two hours prior to utilization in the CAS assay. Prepare an aluminum baking pan filled with laboratory-grade sand and cover it with aluminum foil. Autoclave at 121 degrees Celsius for 30 minutes and set aside.Prepare HDTMA by adding 0365 grams to 20 milliliters of double deionized water, and place the solution in a water bath at 37 degrees Celsius to promote solubilization. Add 0302 grams of CAS to 25 milliliters of double deionized water while gently stirring with a sterile magnetic stir bar. Then add five milliliters of one molar iron chloride hexahydrate to the 25 milliliters of CAS solution while continuing to gently stir.Now, slowly add the 20 milliliters of HDTMA solution into the iron CAS complex solution while gently stirring. Prepare the buffer solution by dissolving 15.12 grams of PIPES into 375 milliliters of double deionized water with gently stirring. Adjust the pH to 6.8 with five molar sodium hydroxide.Then add water to bring the volume to 450 milliliters. Now add five grams of agarose to the solution. Autoclave the PIPES buffer solution and the iron CAS complex solution at 121 degrees Celsius for 30 minutes.Carefully add the entirety of the iron CAS complex solution to the entirety of the PIPES buffer in the biosafety cabinet after each of them has been autoclaved. Place the mixed solution in a water bath at 50 degrees Celsius. Now place a sterile reagent boat in the sterile sand within the biosafety cabinet and heat to 50 degrees Celsius.Transfer the iron CAS complex agar to the boat, then quickly aliquot 100 microliters to each well of a clear flat-bottomed sterile 96-well microplate. Prepare 800 micromolar pyoverdine standard in previously prepared modified M9 medium. Dilute the solution into 400, 200, 100, 50, 25, 12.5, and 6.25 micromolar solutions.Add EDTA to 500 milliliters of previously prepared modified M9 medium to prepare the 3.2 millimolar EDTA standard. Dilute this solution into 1600, 800, 400, 200, 100, 50, 25, 12.5, and 6.25 micromolar solutions. To generate a standard curve, add 100 microliters of each concentration of pyoverdine and EDTA to separate wells of a 96-well microplate containing 100 microliters of iron CAS complex agar medium.Make duplicate technical replications of each concentration. Also add blank wells with only M9.Using a microplate reader, measure absorbance after one, six, and 24 hours incubation at 22 degrees Celsius, and use absorbance measurements to generate standard curves. Wash the sampling equipment with 22 micron filter double deionized water followed by 70%ethanol and wipe with paper towels.Perform the wash before sampling and in between samples to maintain sterile technique and reduce cross-contamination. After excising plant tissues and excavating a small root ball from plants in the field, place the root ball in a separate labeled plastic bag for sample separation in the laboratory environment. Place all samples directly on ice and keep at four degrees Celsius until samples are processed for the siderophore production assay.Separate root associated soil samples into bulk, loosely bound rhizosphere soil and tightly bound rhizosphere soil. To do so, take the root balls out of the bags and gently shake off soil from the root ball. Shaken off soil, along with the soil left in the bag, comprise the bulk soil.This is the loosely bound rhizosphere soil. Generate tightly bound rhizopshere soil by taking roots with the tightly bound sample and putting them in a centrifuge tube. Add 30 milliliters of double deionized water and vortex for two to three minutes.Remove the roots to get the tightly bound rhizosphere soil slurry dilution. Homogenize each soil sample within the sample bag by mixing and turning the soil as much as possible without opening the bag. After each sample has been thoroughly mixed, aliquot and suspend two grams of each soil sample in 20 milliliters of modified M9 medium within a sterile 50 milliliter centrifuge tube.Dilute the sample and then seal the tube with a sterile foam plug to allow aeration. For tightly bound rhizosphere samples, add two milliliters of the rhizosphere soil slurry to 20 milliliters of modified M9 medium within a sterile 50 milliliter centrifuge tube. Dilute the sample and then seal the tube with a sterile foam plug to allow aeration.To prepare the tissue sample, surface sterilize the root, shoot, and grain with 70%ethanol. Macerate two grams of fresh tissue in 20 milliliters of modified M9 medium using a blender on high for 30 seconds. Then, transfer the sample to a sterile 50 milliliter centrifuge tube, dilute, and seal the tube with a sterile foam plug.To enrich siderophore production through iron limitation incubate the 50 milliliter centrifuge tubes at room temperature and shake at 160 RPM. At 24, 48, and 72 hours after initiating the enrichment culture, remove one milliliter subsamples from the enrichment tubes using sterile technique. Centrifuge the subsamples at 10, 000 times G for one minute in two milliliter centrifuge tubes to pelletize the cells.Using sterile technique, add 100 microliters of the supernatant to a 100 microliter solution of iron CAS complex agar in duplicate or triplicate within the microplate. Also add 100 microliters of sterile M9 medium as blanks. Then incubate the plate at 28 degrees Celsius.Each plate should be sealed and covered with foil prior to placement in the incubator. Suspend the remaining supernatant and pellet for each sample into its own sterile two milliliter centrifuge tube. Add 400 microliters of sterile glycerol into each sample supernatant tube and re-suspend the pellet to create glycerol stocks.Freeze the stock at minus 80 degrees Celsius for later analysis. Measure the absorbance at six, 24, 48, and 72 hours at a wavelength of 420 nanometers. A pyoverdine mixture biosynthesized by Pseudomonas fluorescens was used as a standard to interpret and quantify absorbance of samples in samples of pyoverdine equivalence in micromolar.The relationship between absorbance at 420 nanometers and the starting concentration of pyoverdine is shown here. Siderophore activity of the 72 hour enrichment was assessed at 48 hours incubation to determine the influence of genotype and sample type on siderophore isolation. Siderophore activity in bulk soil samples was relatively low and did not exhibit differences between the wheat genotype from which the bulk soil was sampled.However, enrichments of loosely bound soil isolated from the 725 genotype exhibited greater siderophore production as compared with the loosely bound soil from Madsen and 727 but not from Lewjain. Conversely, siderophore production in enrichments from tightly bound soil was not heavily influenced by genotype. Enrichment cultures of grain tissue yielded relatively low siderophore production regardless of genotype.Enrichments of Lewjain shoot tissue had significantly lower siderophore production than the other genotypes, and 725 shoot tissue cultures resulted in more variable siderophore production. The greatest difference in siderophore activity was observed in root tissue enrichment cultures where 725 had more than 200%greater siderophore activity than all other genotypes. One of the most taxing aspects of executing the protocol is maintaining aseptic conditions while completing the many steps to generate the CAS iron agar microplate and testing samples for siderophore activity.A wide array of chromatographic culture-based or DNA-based techniques can be applied either to the actual microtiter well or the glycerol preserved culture for further biochemical tests or genetic characterization and metabolic modeling. By using this protocol siderophore activity can later be linked with specific organisms responsible for that activity or subsequently targeted as a mechanism for larger ecological impacts. This protocol could easily be modified to assess siderophore production in enrichment cultures generated from samples gathered from other environmental compartments including air, water, and sediments.

Research

JoVE Journal

Environment

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