sequencing and identification of multispecies biofilm microbiota from rhizosphere samples

Developing Synergistic Multispecies Biofilms from Rhizosphere Soil

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.

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

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

In our research, we focus on rhizosphere bacteria and biofertilizers, and we aim to develop a synthetic bacterial communities that will be used as second-generation biofertilizers. The focus of our research on rhizosphere bacteria has shift from single-strain to community-level. However, there is no standard methods for building synthetic bacterial communities.Currently, there is still uncertainty regarding the compatibility and colonization ability of synthetic bacterial communities predicted by microbiome analysis. Thus, it is also a major bottleneck in the development of second-generation biofertilizers. Compared to large sequencing data of conventional methods, our culture first, identify later approach greatly reduces the amount of work involved in 60S rRNA gene sequencing.It is cost-effective to create synthetic bacterial communities that colonize roots quickly and efficiently. Our future research will focus on biological function of bacterial communities based on multispecies biofilms, and we will pay particular attention to promoting plant growth, improve plant stress resistance, and protecting plant health.

Sequencing and Identification of Multispecies Biofilm Microbiota from Rhizosphere Samples

To begin, establish a multi-species rhizosphere biofilm, and obtain the microbiota cell suspension. Mix triptych soy broth media with minimal salts glycerol glutamate media in a one-to-one ratio. Incubate the cell suspension in the medium at 30 degrees Celsius overnight to activate the bacteria.Place 100 micrometer nylon cell filters on a 24 well plate. Inoculate the bacterial suspension in three replicates and cultivate the pellicle biofilm for 36 hours at 30 degrees Celsius. Remove the filter containing the pellicle biofilm and add two milliliters of PBSS buffer to a new 24 well cell plate.Place the filter into the plate to wash the biofilm and remove the free cells. After identification and cultivation of the main five species in the biofilm, incubate the 31 combinations of synthetic communities in the medium. Following the mentioned procedure, significant gradients in biofilm-forming capacity from cucumber rhizosphere soil microbiota were observed.Biofilm amplicon sequencing confirmed the presence of various species in the pellicle.

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Research

JoVE Journal

Biology

46 Views. Per iniziare, stabilisci un biofilm della rizosfera multispecie e ottieni la sospensione cellulare del microbiota. Mescolare il brodo di soia a trittico con sali minimi, glicerolo glutammato in un rapporto uno a uno. Incubare la sospensione cellulare nel terreno a 30 gradi Celsius durante la notte per attivare i batteri.Posizionare i filtri cellulari in nylon da 100 micrometri su una piastra a 24 pozzetti. Inoculare la sospensione batterica in tre repl...

Video: Sequenziamento e identificazione di microbiota di biofilm multispecie da campioni di rizosfera

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.

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.

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

Constructing Multispecies Biofilms from Rhizosphere Samples for Developing Advanced Biofertilizers

To begin, obtain cucumber seeds and disinfect them with 75%ethanol, followed by 2%sodium hypochlorite solution. After rinsing the seeds in sterile water, germinate them under sterile conditions. Transplant the consistently sprouted, two-leaf stage seedlings into pots containing two kilograms of black soil.When the seedlings reach the four-leaf stage, invert the pot and isolate the roots along with two millimeter thick rhizosphere soil using sterilized gloves. Place the roots in 30 milliliters of PBSS buffer in a 50 milliliter centrifuge tube. Shake the tube for 20 minutes and filter the suspension through a 100 micrometer nylon cell filter to remove roots or large sediments.Transfer the filtrate to another 50 milliliter centrifuge tube and centrifuge it at 3, 200 G for 15 minutes at room temperature. Temporarily store the tube at four degrees Celsius before extraction of rhizosphere soil bacterial cells.