This work was financially supported by the National Natural Science Foundation of China (42307173 and 42107328) and the National Key Research and Development Program (2022YFD1500202 and 2022YFF1001800). P.W, Z.X designed the study. P.W, B.X, Z.C, J.X, and N.Z analyzed the data and created the figures. P.W wrote the first draft of the manuscript. Z.X., R.Z., and Q.S. revised the manuscript.

This protocol is generally applicable to rhizosphere soil microbiota from various plants. Here, cucumber is used as an example. Details of the reagents and equipment used for the study are listed in the Table of Materials.
1. Bacterial isolation
<ol>
	<li>Rhizosphere soil isolation
	<ol>
		<li>Cucumber culture
		<ol>
			<li>Disinfect cucumber seeds with 75% ethanol and 2% sodium hypochlorite (NaClO) solution, then rinse them in sterile water11.</li>
			<li>Germinate the seeds under sterile conditions and select those that reach the two-leaf stage12. Subsequently, transplant the consistently sprouted cucumber seedlings into pots containing 2 kg of black soil. 
			NOTE: Two types of black soil from different locations in Northeast China are used to reduce systematic errors.</li>
		</ol>
		</li>
		<li>Soil suspension preparation
		<ol>
			<li>Prepare PBS-S buffer using the formulation: 6.33 g of NaH2PO4&#183;H2O, 16.5 g of Na2HPO4&#183;7H2O, 200 &#181;L of Silwet L-77, and 1 L distilled water.</li>
			<li>When the seedlings reach the four-leaf stage12, invert the pot and isolate the roots along with 2 mm-thick rhizosphere soil using sterilized gloves.</li>
			<li>Place the roots in 30 mL of PBS-S buffer in a 50 mL centrifuge tube. After shaking for 20 min, filter the suspension through a 100 &#181;m nylon cell filter to remove roots and large sediments.</li>
			<li>Transfer the filtrate to another 50 mL centrifuge tube, centrifuge it at 3200 x g for 15 min at room temperature, and temporarily store it at 4 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extraction of rhizosphere soil bacterial cells. 
	NOTE: This methodology adopts the differential centrifugation method13, although the Nycodenz density gradient centrifugation method14 also applies for this step.
	<ol>
		<li>Dilute the soil suspension with a 0.2% Na4P2O7 solution to 500 mL and centrifuge at 1,000 x g for 10 min to initially separate organisms from the soil.</li>
		<li>Re-homogenize the precipitate, centrifuge it at the same low speed for 60 s, and repeat the process three times. Combine the supernatants, centrifuge them at 12,000 x g for 20 min, and then discard the supernatant.</li>
		<li>Resuspend the precipitate in 50 mL of PBS-S buffer, constituting the rhizosphere soil bacterial cell suspension.</li>
	</ol>
	</li>
</ol>
2. Sequencing and identification of rhizosphere biofilm microbiota
<ol>
	<li>Media preparation 
	NOTE: Tryptic soy broth (TSB) and Minimal salts glycerol glutamate (MSgg) media are commercially available. The formulations below are for reference only.
	<ol>
		<li>Prepare TSB medium using the following formulation: 15 &#181;g/mL tryptone, 5 &#181;g/mL soy peptone, 5 &#181;g/mL NaCl, pH = 7.</li>
		<li>Prepare MSgg medium using the following formulation: 5 mM KH2PO4, 100 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 2 mM MgCl2, 700 &#181;M CaCl2, 50 &#181;M MnCl2, 50 &#181;M FeCl3, 1 &#181;M ZnCl2, 2 &#181;M thiamine, 0.5% glycerol, 0.5% glutamate, 50 &#181;g/mL tryptophan, 50 &#181;g/mL phenylalanine, pH = 7.</li>
		<li>To prepare TSB-MSgg media, mix TSB media with MSgg media in a 1:1 ratio (v/v)15.</li>
	</ol>
	</li>
	<li>Pellicle biofilm coculture
	<ol>
		<li>Incubate the cell suspension in TSB medium at 30 &#176;C overnight (usually 12 h) to activate the bacteria.</li>
		<li>Place 100 &#181;m nylon cell filters on 24-well plates. Incubate the bacteria in TSB media and set up three repetitions. Cultivate the pellicle biofilm for 36 h at 30 &#176;C. 
		NOTE: If the pellicle biofilm from the last step is not well-cultivated, the TSB-MSgg medium is an effective substitute for the TSB medium.</li>
		<li>Remove the filter containing the pellicle biofilm. Add 2 mL of PBS-S buffer to a new 24-well cell plate, place the 24-well filter into it to wash the biofilm, and remove the free cells. Repeat the washing process three times.</li>
	</ol>
	</li>
	<li>Extraction of pellicle biofilm DNA
	<ol>
		<li>Extract the biofilm genome using a DNA kit following the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>High-throughput sequencing 
	NOTE: Biotech companies can help complete sequencing experiments for most labs. If microbial composition in the remaining solution is needed, sequence it at this step. The sequencing step varies depending on the instruments. Please follow the manufacturer&#39;s instructions. Step 2.4.1 is for reference only.
	<ol>
		<li>Amplify the V3-V4 regions of the 16S rRNA gene based on the database with full-length 16S rRNA gene sequences. Create species OTUs according to the minimum number of sequences per sample16. Cluster the species classification annotations and acquire the compositions of communities from each sample.</li>
		<li>Based on the sequencing data, select the top five strains in terms of their relative abundance.</li>
	</ol>
	</li>
	<li>High-throughput bacterial isolation and culture 
	NOTE: This step is designed according to the latest microbiological and bioinformatic methodology17. The dilution-coated plate method is available for bacterial isolation here, although it is more time-consuming and less targeted compared to high-throughput techniques.
	<ol>
		<li>Mix the cultivated biofilm. Use limiting dilution to ensure that no more than 30% of the wells in the 24-well plate show bacterial growth. 
		NOTE: To determine the most suitable dilution, perform preliminary experiments using a wide range of dilution rates. The optimal dilution should contain no more than two culturable bacteria per milliliter, representing bacterial incubation in less than 30% of the wells.</li>
		<li>Amplify the 16S rRNA gene of the bacteria while adding labels for the wells and plates and sequence them using the high-throughput Illumina platform17.</li>
		<li>Conduct bioinformatic analyses using commercially available software to obtain purity and species taxonomic information for each culture.</li>
		<li>Cultivate the top five abundant pure bacteria by continuous line culture and use glycerol to preserve the bacteria at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Construction of synthetic microbial communities
<ol>
	<li>Re-constructed biofilm culture 
	NOTE: For details, please refer to Ren et al.18. The 5 strains correspond to 31 synthetic combinations, including 5 single-species, 10 two-species, 10 three-species, 5 four-species, and 1 five-species consortia.
	<ol>
		<li>Incubate the 5 strains in the TSB medium until their OD600 reaches 1 to activate them.</li>
		<li>Prepare several 24-well plates with TSB medium and place 100 &#181;m nylon cell filters on them. 
		NOTE: If the biofilm from the last step is not well-cultivated, the TSB-MSgg medium is an effective substitute for the TSB medium.</li>
		<li>Incubate the 31 combinations of synthetic communities in the medium. Ensure the inoculation number of bacteria is consistent in each well. Set up three repetitions for each combination.</li>
		<li>Cultivate the biofilm at 30 &#176;C for 36 h.</li>
	</ol>
	</li>
	<li>Quantification of pellicle biofilm
	<ol>
		<li>Follow the same procedure as in step 2.2.3.</li>
		<li>Transfer the biofilm to a new 24-well plate containing 180 &#181;L of crystal violet solution and stain it for 20 min.</li>
		<li>Transfer the stained sample to another 24-well plate containing 200 &#181;L of 96% ethanol, elute it for 30 min, and measure OD590.</li>
	</ol>
	</li>
</ol>
4. Pellicle cell number quantification
NOTE: To determine the proportion of each species and the cell counts in the pellicle and remaining solution, quantitative PCR is necessary. For details, refer to Sun et al.16.
<ol>
	<li>Design strain-specific primers for the selected synthetic microbial communities.</li>
	<li>Use Roary to perform genome comparisons and find out the strain-specific single-copy genes of each isolate. Ensure that primers are targeted to these genes.</li>
	<li>Ligase the amplified fragments to PMD19T plasmids. Generate standard curves using the plasmids containing corresponding fragments as templates.</li>
	<li>Follow the same steps as mentioned in step 2.2.</li>
	<li>Prepare reaction components as follows: 7.2 &#181;L of H2O, 10 &#181;L of 2x qPCR Master mix, 0.4 &#181;L of 10 &#181;M of each primer, and 2 &#181;L template DNA.</li>
	<li>Perform the PCR with a Real-time PCR Instrument under the following conditions: 95 &#176;C for 10 min, 40 cycles of 95 &#176;C for 30 s, and 60 &#176;C for 45 s, followed by a standard melting curve segment.</li>
	<li>Perform six biological replicates for each treatment.</li>
</ol>

Developing Synergistic Multispecies Biofilms from Rhizosphere Soil

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<li>Compant, S., Clément, C., Sessitsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+growth-promoting+bacteria+in+the+rhizo-+and+endosphere+of+plants%3A+Their+role%2C+colonization%2C+mechanisms+involved+and+prospects+for+utilization%5BTitle%5D)+AND+%22Soil+Biol+Biochem%22%5BJournal%5D)">Plant growth-promoting bacteria in the rhizo- and endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization.</a> Soil Biol Biochem. 42, 669-678 (2010).</li>
<li>Bais, H. P., Fall, R. R., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biocontrol+of+Bacillus+subtilis+against+infection+of+Arabidopsis+roots+by+Pseudomonas+syringae+is+facilitated+by+biofilm+formation+and+surfactin+production%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production.</a> Plant Physiol. 134 (1), 307-319 (2004).</li>
<li>Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant-microbiome+interactions%3A+From+community+assembly+to+plant+health%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Plant-microbiome interactions: From community assembly to plant health.</a> Nat Rev Microbiol. 18, 607-621 (2020).</li>
<li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacillus+velezensis+stimulates+resident+rhizosphere+Pseudomonas+stutzeri+for+plant+health+through+metabolic+interactions%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions.</a> ISME J. 16, 774-787 (2021).</li>
<li>Pandhal, J., Noirel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+microbial+ecosystems+for+biotechnology%5BTitle%5D)+AND+%22Biotechnol+Lett%22%5BJournal%5D)">Synthetic microbial ecosystems for biotechnology.</a> Biotechnol Lett. 36, 1141-1151 (2014).</li>
<li>Grosskopf, T., Soyer, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+microbial+communities%5BTitle%5D)+AND+%22Curr+Opin+Microbiol%22%5BJournal%5D)">Synthetic microbial communities.</a> Curr Opin Microbiol. 18, 72-77 (2014).</li>
<li>Lundberg, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+core+Arabidopsis+thaliana.+root+microbiome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Defining the core Arabidopsis thaliana. root microbiome.</a> Nature. 488, 86-90 (2012).</li>
<li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+overlap+of+the+Arabidopsis+leaf+and+root+microbiota%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Functional overlap of the Arabidopsis leaf and root microbiota.</a> Nature. 528, 364-369 (2015).</li>
<li>Bakken, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+and+purification+of+bacteria+from+soil%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Separation and purification of bacteria from soil.</a> Appl Environ Microbiol. 49 (6), 1482-1487 (1985).</li>
<li>Yang, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+cell+extraction+from+fresh+and+stored+soil+samples%3A+Impact+on+microbial+viability+and+community+compositions%5BTitle%5D)+AND+%22Soil+Biol+Biochem%22%5BJournal%5D)">Direct cell extraction from fresh and stored soil samples: Impact on microbial viability and community compositions.</a> Soil Biol Biochem. 155, 108178(2021).</li>
<li>Ren, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+screening+of+multispecies+biofilm+formation+and+quantitative+PCR-based+assessment+of+individual+species+proportions%2C+useful+for+exploring+interspecific+bacterial+interactions%5BTitle%5D)+AND+%22Microb+Ecol%22%5BJournal%5D)">High-throughput screening of multispecies biofilm formation and quantitative PCR-based assessment of individual species proportions, useful for exploring interspecific bacterial interactions.</a> Microb Ecol. 68, 146-154 (2013).</li>
<li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+interactions+affect+the+biomass+of+synthetic+bacterial+biofilm+communities%5BTitle%5D)+AND+%22mSystems%22%5BJournal%5D)">Metabolic interactions affect the biomass of synthetic bacterial biofilm communities.</a> mSystems. 8, e01045-e01123 (2023).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+cultivation+and+identification+of+bacteria+from+the+plant+root+microbiota%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">High-throughput cultivation and identification of bacteria from the plant root microbiota.</a> Nat Protoc. 16, 988-1012 (2021).</li>
<li>Ren, D., Madsen, J. S., Sørensen, S. J., Burnolle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+prevalence+of+biofilm+synergy+among+bacterial+soil+isolates+in+cocultures+indicates+bacterial+interspecific+cooperation%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation.</a> ISME J. 9, 81-89 (2015).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Root+colonization+by+beneficial+rhizobacteria%5BTitle%5D)+AND+%22FEMS+Microbiol+Rev%22%5BJournal%5D)">Root colonization by beneficial rhizobacteria.</a> FEMS Microbiol Rev. 48 (1), 066(2024).</li>
<li>Palková, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicellular+microorganisms%3A+Laboratory+versus+nature%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">Multicellular microorganisms: Laboratory versus nature.</a> EMBO Rep. 5, 470-476 (2004).</li>
<li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+communication+in+plant-microbe+beneficial+interactions%3A+a+toolbox+for+precise+management+of+beneficial+microbes%5BTitle%5D)+AND+%22Curr+Opin+Microbiol%22%5BJournal%5D)">Chemical communication in plant-microbe beneficial interactions: a toolbox for precise management of beneficial microbes.</a> Curr Opin Microbiol. 72, 102269(2023).</li>
</ol>

The authors have no conflicts of interest to disclose.

Following the protocol, a series of robust synthetic multispecies biofilm communities are constructed based on the microbiota in rhizosphere soil from different plants. Using quantitative PCR techniques, the composition of each community is deciphered clearly. The biomass of the biofilm indicates the strength of their potential metabolic interactions, although various properties and mechanisms behind the cooperation could not be revealed here19. Given the size and complexity of natural microbial communities, synthetic communities (SynComs) represent a model system for overcoming existing obstacles14. This protocol highlights the systematic and fundamental methodology of constructing synthetic biofilm communities from complex rhizosphere soil microbiota.
It is evident from the last part that SQR9, XL-1, and XL-2 show strong potential for stable colonization in the actual rhizosphere. Researchers can start investigating the metabolic relationships and co-colonization mechanisms based on these strains. Subsequent research on microbial inoculants for cucumber is also accessible thereafter.
Despite the advantages mentioned, it is important not to overlook the drawbacks of the protocol. Due to differences between laboratory culture and in situ conditions, the results of each synthetic community might not accurately represent the microbiota in real rhizosphere soil20. Confirmation through further rhizosphere inoculation is necessary. Additionally, the reproducibility of these results is relatively poor. Pellicle biofilm species in the laboratory may have undergone mutations over many generations compared to wild types in the soil, leading to some uncontrollable inaccuracies.
There are several alternatives to certain steps in the protocol. For instance, in step 1.2, the Nycodenz density gradient centrifugation method is relatively more efficient for collecting soil cell samples13. Additionally, in step 2.4, the latest high-throughput cultivation and identification of bacteria significantly increase experimental efficiency, albeit at a higher cost. Therefore, the traditional dilution-coated plate method remains suitable for selecting a few species. Finally, in step 3.2, the biomass of cultivated biofilm can be assessed both by crystal violet staining and by directly weighing it on an analytical balance.
This protocol extends beyond merely creating basic experimental materials for microbial and environmental research. Identifying the most robust synthetic bacterial multispecies biofilm communities is just the first step in unraveling the open and complex rhizosphere ecosystem. By focusing on the core species of SynComs, researchers can gain insight into real in situ situations in a more targeted and rapid way. This approach may also offer a novel perspective for exploring biofilms on a larger scale, such as rhizosphere soil aggregates, and for precise management of rhizosphere microbial communities for plant growth and health21.
Most significantly, this methodology will shed light on further research into interspecies interactions that govern the assemblage and dynamics of rhizosphere soil microbiota. For instance, when dealing with a complex rhizosphere community, other analytical methods such as co-occurrence network analysis or genome-scale metabolic modeling15 are often combined with these fundamental steps to artificially construct a representative biofilm community. This includes considerations of interspecies relationships.

Biofilms represent intricate microbial communities either affixed to surfaces or linked with interfaces. There is broad acknowledgment that the majority of bacteria encountered in various environments, such as natural habitats, clinical settings, and industrial contexts, endure within biofilm communities1. In soil, the rhizosphere&#8212;encompassing the root surface and its adjacent 2 mm thick region&#8212;forms an ecological niche with heightened nutrient availability. Specific bacteria have evolved strategies to exploit this niche, fostering microbial proliferation and fostering the formation of biofilm communities in the rhizosphere2.
Rhizosphere microbes are considered the &#39;second genome&#39; of plants3. Plant growth-promoting microorganisms (PGPM) engage in mutualistic symbiosis with plants, providing significant growth promotion and biocontrol functions4. On the one hand, PGPM can supply nutrients to plant roots, enhance nutrient uptake, defend against pathogens, and degrade pollutants in the rhizosphere soil5. On the other hand, PGPM utilizes plant root exudates as a nutrient source for growth and evolution, thereby influencing the rhizosphere soil and root system through their own metabolism. It is worth noting that the prerequisite for all these functions is the effective colonization of PGPM in the plant rhizosphere, forming stable biofilms6.
Rhizosphere biofilm affects the assembly process of rhizosphere microbes, and the temporal and spatial characteristics of rhizosphere microbe assembly are closely related to the interaction of multispecies biofilm7. It serves as the hub of plant-microbiome interactions, providing a stable and controllable niche for them&#160;to interact effectively. Therefore, studying rhizosphere biofilm is also an important direction to gain insight into the interaction between plants and microbes.
However, there is currently little research on rhizosphere multispecies biofilms. The high complexity of microbiome composition makes it challenging to answer fundamental ecological questions surrounding natural microbial communities8. In the process of experiments, many conditions, such as soil type, plant type, and the large number of microbial communities, need to be considered. Various factors limit the research of rhizosphere multispecies biofilms.
To further investigate multispecies biofilm communities from rhizosphere soil, such as interspecies synergistic interactions or metabolite cross-feeding8, it is desirable to artificially construct a simplified, representative, stable, and tractable synthetic microbial community under laboratory conditions9,10. Here, a standardized protocol combines several of the latest microbiological and bioinformatic techniques.

<table><tbody><tr><td>0.2 % Na&lt;sub&gt;4&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt; solution</td><td>Sinopharm Chemical Reagent Co.,Ltd.</td><td>20041418</td><td>Used to dilute the soil suspension.</td></tr><tr><td>100 &amp;mu;m Nylon Cell Filter</td><td>Sangon Biotech (Shanghai) Co.,Ltd.</td><td>F613463-0001</td><td>Used to filter culture solution and separate pellicle biofilm.</td></tr><tr><td>2% NaClO solution</td><td>Sinopharm Chemical Reagent Co.,Ltd.</td><td>L03336903</td><td>Used to sterilize the seeds.</td></tr><tr><td>24-well Plate</td><td>Merck &amp;amp; Co., Inc.</td><td>PSRP010</td><td>Used for micobial cultivation and pellicle biofilm separation.</td></tr><tr><td>3-(N-morpholino) propane sulfonic acid (MOPS)</td><td>Bioolook Scientific Research Special</td><td>2360010</td><td>Used to prepare MSgg media.</td></tr><tr><td>50 mL Centrifuge Tube</td><td>Beijing Su-bio Biotech Co., Ltd.</td><td>62.547.254</td><td>Properly-sized centrifuge tubes for our protocol.</td></tr><tr><td>75% C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;OH solution</td><td>Sinopharm Chemical Reagent Co.,Ltd.</td><td>801769680</td><td>Used to sterilize the seeds.</td></tr><tr><td>&lt;em&gt;Acinetobacter baumannii &lt;/em&gt;XL-1</td><td>Nanjing Agricultural University</td><td>N/A</td><td>Strains isolated from rhizosphere soil.</td></tr><tr><td>&lt;em&gt;Bacillus velezensis SQR9&lt;/em&gt;</td><td>Nanjing Agricultural University</td><td>N/A</td><td>Strains isolated from rhizosphere soil.</td></tr><tr><td>Bacteria DNA Kit</td><td>Nanjing Dinsi Biotechnology Co.,Ltd.</td><td>D3350020000K04U021</td><td>Used to extract bacterial DNA.</td></tr><tr><td>Black Soil I</td><td>Nanjing Agricultural University</td><td>N/A</td><td>Black soil from Jilin Province.</td></tr><tr><td>Black Soil II</td><td>Nanjing Agricultural University</td><td>N/A</td><td>Black soil from Heilongjiang Province.</td></tr><tr><td>&lt;em&gt;Burkholderia cenocepacia&lt;/em&gt; XL-2</td><td>Nanjing Agricultural University</td><td>N/A</td><td>Strains isolated from rhizosphere soil.</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Xilong Scientific Co.,Ltd.</td><td>10035048</td><td>Used to prepare MSgg media.</td></tr><tr><td>Centrifuge</td><td>Sangon Biotech (Shanghai) Co.,Ltd.</td><td>G508009-0001</td><td>High-speed centrifuge.</td></tr><tr><td>ChamQ SYBR qPCR Master Mix</td><td>Vazyme Biotech Co.,Ltd.</td><td>Q311-02</td><td>Used for DNA amplification in qPCR.</td></tr><tr><td>Clean Bench</td><td>Suzhou Antai Airtech Co., Ltd.</td><td>NB026143</td><td>Used for aseptic operation.</td></tr><tr><td>Constant Temperature Incubator</td><td>ShangHai CIMO Medical Instrument Co.,Ltd</td><td>GNP-9080BS-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66926/66926eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td>Used for microbial cultivation.</td></tr><tr><td>Cristal Violet Dye</td><td>Sangon Biotech (Shanghai) Co.,Ltd.</td><td>A600331</td><td>Used for pellicle biofilm staining and quantifivation.</td></tr><tr><td>Cucumber Seed</td><td>Nanjing Agricultural University</td><td>N/A</td><td>&quot;Jinchun I&quot; cucumber seed.</td></tr><tr><td>Culturome v 1.0</td><td>The Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences</td><td>N/A</td><td>Used for high-throughput rhizosphere soil bacterial cultivation and identification</td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10011918</td><td>Used to prepare MSgg media.</td></tr><tr><td>Fridge</td><td>Hefei Midea Refrigerator Co.,Ltd.</td><td>BCD-556WKPM(Q)</td><td>Used for strain preservation.</td></tr><tr><td>Glutamate</td><td>Bioolook Scientific Research Special</td><td>2180020</td><td>Used to prepare MSgg media.</td></tr><tr><td>Glycerol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10010618</td><td>Used to prepare MSgg media.</td></tr><tr><td>Hydroponic Planting Basket</td><td>Yulv Furniture Store</td><td>6526262626</td><td>Used for cucumber hydroponics.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Xilong Scientific Co.,Ltd.</td><td>10017618</td><td>Used to prepare MSgg media.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Xilong Scientific Co.,Ltd.</td><td>7791186</td><td>Used to prepare MSgg media.</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Xilong Scientific Co.,Ltd.</td><td>10400101</td><td>Used to prepare MSgg media.</td></tr><tr><td>Msgg Medium</td><td>Shanghai Bioesn Biotechnology Co.,Ltd.</td><td>BES20791KB</td><td>Pellicle biofilm culture medium.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck &amp;amp; Co., Inc.</td><td>S9390-100G</td><td>Used to prepare TSB media.</td></tr><tr><td>NaCI</td><td>Chinasun Specialty Products co., Ltd.</td><td>HS0416</td><td>Used to prepare TSB media.</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck &amp;amp; Co., Inc.</td><td>S9638-25G</td><td>Used to prepare TSB media.</td></tr><tr><td>PBS-S Buffer</td><td>Sangon Biotech (Shanghai) Co.,Ltd.</td><td>E607008-0001</td><td>Basic buffer for living tissues.</td></tr><tr><td>Phenylalanine</td><td>RYON</td><td>RT3486L005</td><td>Used to prepare MSgg media.</td></tr><tr><td>Pipette</td><td>Beijing Labgic Technology Co.,Ltd.</td><td>BS-1000-T</td><td>Used for strain inoculation.</td></tr><tr><td>PMD19T Plasmid</td><td>Takara Biomedical Technology (Beijing) Co.,Ltd.</td><td>D102A</td><td>Used to generate qPCR standard curves.</td></tr><tr><td>Pot</td><td>Sinopharm Chemical Reagent Co.,Ltd.</td><td>YHHWS2401</td><td>Used for cucumber soil culture.</td></tr><tr><td>Primer for qPCR</td><td>Sangon Biotech (Shanghai) Co.,Ltd.</td><td>N/A</td><td>Used for DNA amplification in qPCR.</td></tr><tr><td>Real-Time PCR Instrument</td><td>Thermo Fisher Scientific (China) Co.,Ltd.</td><td>4484073</td><td>Used to perform qPCR on selected pellicle biofilm.</td></tr><tr><td>Roary</td><td>The Wellcome Trust SangerInstitute</td><td>N/A</td><td>Used to design primers of qPCR</td></tr><tr><td>Shaker</td><td>Shanghai Zhichu Instrument Co.,Ltd</td><td>ZQTY-50S</td><td>Used for solution mixing and microbial cultivation.</td></tr><tr><td>Silwet L-77</td><td>Cytiva Bio-technology(Hangzhou) Co., Ltd.</td><td>SL77080596</td><td>Used to prepare TSB media.</td></tr><tr><td>Soy peptone</td><td>Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd</td><td>HB8275</td><td>Used to prepare TSB media.</td></tr><tr><td>Spectrophotometer</td><td>Shanghai Yidian Analysis Instrument Co.,Ltd.</td><td>76713100010</td><td>Used for pellicle biofilm cristal violet quantifivation.</td></tr><tr><td>Sterile Water</td><td>Sinopharm Chemical Reagent Co.,Ltd.</td><td>SW150302</td><td>Used to wash the pellicle biofilm.</td></tr><tr><td>Sterilized Nitrile Gloves</td><td>Beijing Labgic Technology Co.,Ltd.</td><td>223016852LLZA</td><td>Used for basic experimental operations.</td></tr><tr><td>Template DNA</td><td>Sangon Biotech (Shanghai) Co.,Ltd.</td><td>N/A</td><td>Used for DNA amplification in qPCR.</td></tr><tr><td>Thiamine</td><td>Bioolook Scientific Research Special</td><td>2180020</td><td>Used to prepare MSgg media.</td></tr><tr><td>Tryptone</td><td>Thermo Fisher Scientific</td><td>LP0042B</td><td>Used to prepare TSB media.</td></tr><tr><td>Tryptophan</td><td>Bioolook Scientific Research Special</td><td>2190020</td><td>Used to prepare MSgg media.</td></tr><tr><td>TSB Medium</td><td>Guangdong Huankai Microbial Sci. &amp;amp; Tech. Co.,Ltd.</td><td>024048</td><td>Broad-spectrum bacterial medium.</td></tr><tr><td>TSB-Msgg Meidium</td><td>Nanjing Agricultural University</td><td>N/A</td><td>Mixed medium for pellicle biofilm culture with wider applicability.</td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Nanjing Chemical Reagent Co.,Ltd</td><td>C0310520123</td><td>Used to prepare MSgg media.</td></tr></tbody></table>

an approach to constructing multispecies biofilm communities from rhizosphere soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

Following the mentioned procedure, significant gradients in biofilm-forming capacity from cucumber rhizosphere soil microbiota are observed (Figure 1). Biofilm amplicon sequencing confirmed the species in the pellicle (Figure 2). Based on the heat mapping of sequencing data, the top five bacterial species whose abundance in the pellicle is larger than that in the remaining solution were selected (Figure 3). Here, a typical group of synthetic communities was taken as an example. Crystal violet staining and quantitative PCR directly visualize the differences in pellicle biomass and the relative abundance of each species in the communities. Increased coculture biomass indicates the success of constructing synergistic multispecies biofilm communities from rhizosphere soil (Figure 4 and Figure 5).
The results show that Bacillus velezensis SQR9, Acinetobacter baumannii XL-1, and Burkholderia cenocepacia XL-2 can form prominent synergistic consortia, with the most robust community consisting of all three species. They are predicted to exhibit strong synergism in biofilm formation. It is also noteworthy that XL-1 serves as a &#39;dedicator.&#39; It promotes the biofilm formation of XL-2 and SQR9, despite its poor growth and minimal abundance in the consortia (Figure 5). While a number of multispecies coculture biofilms represent a synergistic phenotype, some species, such as Chryseobacterium sp., are always negatively related to other strains by reducing the biomass of synthetic biofilm (Figure 2). Although this phenomenon is not related to the target of this protocol, it might be worth further investigation in the future.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66926/66926fig01.jpg" /> 
Figure 1: Biofilm formation of different synthetic communities (SynComs). Here, the samples are in standard 24-well plates. The photographs are captured without any magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66926/66926fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66926/66926fig02.jpg" /> 
Figure 2: Bacterial composition in pellicle and solution. Graphs are based on 16S rRNA gene amplicon sequencing data, mentioned in protocol step 2.3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66926/66926fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66926/66926fig03.jpg" /> 
Figure 3: Heatmap of biofilm-forming capacity in pellicle and solution. Red represents the abundance in biofilm, which is higher than the solution, and blue represents the opposite. Genus showed no difference are not shown. Species of the top 5 abundance are ticked on the left. bb: black biofilm; bs: black solution. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66926/66926fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66926/66926fig04v2.jpg" /> 
Figure 4: Biofilm biomass quantification of synthetic communities in Figure 1. Data is from crystal violet quantification with OD = 590 nm. The data presented are the mean &#177; SD, n = 5. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66926/66926fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66926/66926fig05.jpg" /> 
Figure 5: Cell numbers and species relative abundance of synthetic communities in Figure 1. Data is from quantitative PCR. The data presented are the mean &#177; SD, n = 6. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66926/66926fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Developing Synthetic Microbial Communities for Generating Second-Generation Biofertilizers

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

an-approach-to-constructing-multispecies-biofilm-communities-from

Universidad San Sebastian

Research

JoVE Journal

Biology

730 Views.  Nanjing Agricultural University. In ons onderzoek richten we ons op rhizosfeerbacteriën en biofertilizers, en we streven ernaar synthetische bacteriegemeenschappen te ontwikkelen die zullen worden gebruikt als biofertilizers van de tweede generatie. De focus van ons onderzoek naar bacteriën in de rhizosfeer is verschoven van enkelvoudige stam naar gemeenschapsniveau. Er zijn echter geen standaardmethoden voor het bouwen van synthetische bacteriegemeenschappen.Momenteel is er nog ste...

Video: Auteur in de schijnwerpers: ontwikkeling van synthetische microbiële gemeenschappen voor het genereren van biofertilizers van de tweede generatie

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genetics

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Environment

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Bioengineering

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Culturing Arbuscular Mycorrhizal Fungi Using SAP-AS for Enhanced Observation

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.

Author Spotlight: Enhancing AM Fungi Research with SAP-AS &#8212; A Novel Technique for Single-Spore Cultures

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Immunology and Infection

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

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