Absolute Quantitative 16s/18s/ITS Amplicon Sequencing

Partial results are shown below:

The taxonomy distribution of all sample in Phylum classification level.

Species abundance Heatmap.

Rarefaction curve of the sequenced reads for samples (The above figure) & The depth of the sequencing samples (The below figure).

Boxplot analysis based on bray Curtis (A), binary jaccard (B), unweighted unifrac (C), and weighted unifrac (D).

PCoA analysis based on bray Curtis (A), binary jaccard (B), unweighted unifrac (C), and weighted unifrac (D).

UPGMA clustering tree.

Mean proportion of treated and control group.

Cladogram.

LDA SCORE.

1. How does Absolute Quantitative Amplicon Sequencing differ from traditional amplicon sequencing?

Traditional amplicon sequencing provides relative abundance data, which can be misleading as it does not account for variations in total microbial load between samples. Absolute Quantitative Amplicon Sequencing uses internal or external standards to provide absolute counts of microbial taxa, giving a more accurate picture of microbial community structure.

2. Which kinds of samples are amenable to analysis utilizing Absolute Quantitative Amplicon Sequencing?

This methodology is versatile and can be effectively employed across a myriad of sample types encompassing soil, water, air, human and animal tissues, plant specimens, as well as an array of environmental and industrial matrices.

3. What is the unit of measurement for absolute quantification in 16S amplicon sequencing?

The unit of measurement for absolute quantification can be either 16S copies/g of sample or 16S copies/ng of DNA. However, using 16S copies/g of sample is generally more accurate and preferable. This approach accounts for variations in the amount of starting material, leading to more reliable and interpretable results in microbial community analysis.

4. What are OTUs and ASVs?

Operational Taxonomic Units (OTUs) serve as consolidated clusters of akin sequences, delineated based on a predefined threshold of similarity, frequently set at the 97% mark. Conversely, Amplicon Sequence Variants (ASVs) represent sequences of utmost precision at the single-nucleotide level; they are derived via denoising algorithms, therefore embodying distinct sequence variants.

5. What are the advantages of integrating absolute quantification analysis with metabolomics?

Absolute quantification analysis provides the absolute abundance of species, while current metabolomics techniques, such as targeted and untargeted metabolomics, yield the absolute concentrations of metabolites. Integrating microbiome and metabolome data typically involves correlating species' relative abundances with metabolites' absolute concentrations, which can lead to erroneous conclusions. Therefore, it is strongly recommended to base subsequent correlation analyses on species' absolute abundances and metabolites' absolute concentrations. This approach yields more accurate and reliable results.

Synthetic community derived from grafted watermelon rhizosphere provides protection for ungrafted watermelon against Fusarium oxysporum via microbial synergistic effects

Journal: Microbiome
Impact factor: 16.837
Published: 05 June 2024

Background

Research has highlighted the significant role of rhizosphere microbial communities in plant health and growth. Strategies such as resistant crop cultivation and Rhizosphere Microbiota Transplantation (RMT) are recognized as effective tools for managing plant diseases. Recent studies have emphasized the concept of the "core microbiome" in plant rhizospheres, comprising microbial species closely linked to the host plant across diverse environments. The emergence of Synthetic Communities (SynComs) offers a novel approach to enhancing plant health in non-sterile environments. This study aimed to investigate the impact of grafted watermelon rhizosphere microbial communities on disease resistance and evaluate the effectiveness of SynComs in promoting plant health. Additionally, it developed an efficient methodology for constructing and simplifying functional SynComs based on synergistic interactions among community members.

Methods

Results

The protective effects of the SynCom were investigated by comparing rhizosphere microbial community compositions and metagenomic profiles across different treatment groups. SynCom inoculation normalized microbial diversity in response to pathogen infestation, with notable increases in the relative abundances of beneficial genera like Pseudomonas and Streptomyces. Additionally, SynCom inoculation led to more complex microbiome networks with improved stability and enhanced community cohesion, potentially facilitating disease resistance.

Fig 1. Changes in the rhizosphere microbial community composition and function profile in plants inoculated with SynCom.

Metagenomic analysis revealed distinct functional properties triggered by the SynCom in the rhizosphere community. Significant differences in KEGG pathway abundance were observed between SynCom-treated and control groups, with enriched pathways related to the two-component system and biofilm formation. Particularly, Pseudomonas showed a remarkable increase in abundance post-SynCom inoculation, demonstrating effective colonization and a negative correlation with rhizosphere pathogens. Further analysis highlighted the enrichment of genes associated with Pseudomonas biofilm formation pathway, primarily originating from SynCom members, suggesting a pivotal role of Pseudomonas in disease suppression and growth promotion in watermelon plants.

The synergistic interactions between SynCom members and Pseudomonas contribute to Pseudomonas growth and colonization in the rhizosphere, enhancing plant growth. Analysis of the competitive potential and interactions among core strains revealed low competition and positive correlations. In vitro co-culture experiments demonstrated that certain SynCom strains promoted the growth of others, with metabolomic analyses identifying potential cross-feeding metabolites. Overall, these findings highlight the importance of metabolic facilitation in driving synergistic cooperation among SynCom members and Pseudomonas.

Fig 2. In vitro interaction matrix and substrate depletion profiles between individual SynCom strains.

Conclusion

This study reveals that core rhizosphere bacteria from grafted watermelon plants form a SynCom, effectively controlling disease and promoting growth in ungrafted plants. Increased abundance of Pseudomonas and enhanced biofilm formation suggest its pivotal role in plant health. In vitro assays demonstrate metabolic facilitation between SynCom members and Pseudomonas, leading to a simplified SynCom with retained growth-promoting effects. This highlights the potential of SynComs in protecting plants from biotic stresses, emphasizing the importance of understanding and manipulating microbial interactions in the rhizosphere for ecological plant health management.

Reference

1. Qiao Y, Wang Z, Sun H, et al. Synthetic community derived from grafted watermelon rhizosphere provides protection for ungrafted watermelon against Fusarium oxysporum via microbial synergistic effects. Microbiome, 2024, 12(1): 101.