Module 8 – Genomics approach to develop soil biomarkers

1. INTRODUCTION

Soil is the universal source of vital for all Earth ecosystem services due to its significance:

The largest deposit of nutrients and water for plants;
The biggest regulator of gas emissions;
The major participant in cycling and recycling biogenic elements and molecules.

The negative consequences of climate change, e.g., the intense flooding or long drought periods and the extensive anthropogenic activities related to industry and agriculture, all impose accelerated soil fragmentation. Urgent measures are needed to minimize these negative impacts on soil structure and to protect soil health.

One of the strategies that comprise a potentially impactful set of protective measures is the employment of bioindicators that characterize fluctuations in soil health and provide valuable information that complements that accumulated by the physical/chemical characteristics.

The environmental studies explore a variety of bioindicators. They encompass different kingdoms of life and range from microorganisms to invertebrates. Typical examples are the

Earthworms (Moreiera et al., 2012);
Nematodes (Martin et al., 2022);
Beetles (Menta and Remelli, 2020);
Microorganisms (Schloter et al., 2018).

Among all, the microorganisms possess the potential to be useful bioindicators due to the regulated methods established by the International Standardization Organization (ISO) for their use in analyses of soil quality (ISO 14240-1:1997, ISO 16072:2002, ISO/TS 10832:2009, ISO/TS 29843-1:2010, ISO 17601:2016, ISO 18400-206:2018, ISO 11063:2020). However, just one of these methods (ISO 11063:2020) is related to genomics techniques, applicable for direct DNA extraction from soil.

Thus, there is a need for knowledge and practical skills for the use of metagenomic techniques in soil quality and health assessment through the identification of microbial soil communities in distinct samples.

2. FINDINGS

2.1. Microorganisms – biomarkers of soil health

The soil bio-transformations (e.g., biogeochemical cycles, accumulation, and/or degradation of organic matter) are attributed to the biodiversity hosted by the soil. Among the representatives of various kingdoms, those of Bacteria and Fungi are the most abundant and with the best contribution: over 80% of the above-mentioned processes are directly related to the microbial component of the global ecosystem.

The soil microbiome – the soil microbial communities and their genetic material, is a sensitive system that reflects the fluctuations in the environment and rapidly responds to any environmental changes in a way that those individuals who are better adapted to the said changes can survive and persist in the soil.

The microbial biodiversity in soil depends on the physical-chemical characteristics of the soil itself, its state of pollution, and the land management techniques used. Thus,

pH: Fierer and Jackson, 2006;
Heavy-metal contaminations: Feng et al., 2018; and
Land use management: Bhowmik et al., 2019;

determine changes in the microbial populations, and these changes can be used as indicators for soil health. Since soil health is defined by USDA as the … continued capacity of soil to function as a vital living ecosystem to sustain plants, animals, primary productivity, and ecological biodiversity …., the microorganisms in soil can serve as a sharp and reliable bioindicator.

Historically, the knowledge about soil microorganisms dates back to the XIX century. However, there are several important research milestones that highlighted their potential as soil health bioindicators:

The first one is the establishment of thesoil microorganisms’ role in nutrient cycling and their use in the quantification of nutrient dynamics. The experimental part demanded cultivation and identification of microbial species, and consequently – over 97% of the bacterial and archaeal species remained unidentifiable because were nonculturable. Thus, the soil microorganisms’ abundance remained hardly estimated and studied.
The next is the use of the soil mobilome. This is the so-called volatile gene pool represented by plasmids, bacteriophages, and extracellular DNA acquired through gene transfer (transformation). Among all, the plasmids are those gaining attention since they carry genes that confer valuable traits to their hosts (and the soil microbiome in general), operable under stress conditions (e.g., the presence of antibiotics or other chemicals). Researchers have proved that the plasmids’ frequency and diversity increase as a response to changed environmental conditions and stressor activation. This dependence allows for establishing soil mobilome biomarkers as reporters of stress occurrence in these soils and a marker for soil status per se. Furthermore, mobile genetic elements like Ti and Sym plasmids are responsible for the microbe-plant interactions, and as such they account for the horizontal gene transfer that takes place at high speed in the plant rhizosphere. The same is valid for the bacteria-fungi interaction, for instance, Burkholderia terraeand soil fungi, in which certain bacterial gene clusters may be used as reporters on bacteria interacting with fungi in the soil.
The most recent is the molecular revolution that allows the use of metagenomics technique for the extraction of nucleic acids directly from soil samplesand omitting the cultivation step to characterize microorganisms.

The metagenomics methodology comprises:

Extracting DNA/RNA directly from a soil sample;
Making genomic libraries for all microbial species found in a defined soil study site;
Sequencing and bioinformatic processing.

Currently, the metagenomics technique is implicated as a bioindicator tool for soil health analysis.

Learn: How to effectively extract soil DNA (video)

Learn: How to make genomic libraries (video)

Learn: What is a genomic sequencing (video)

2.2. Genomics/Metagenomic studies of soil microbial communities

The application of metagenomic techniques to study soil health revealed the great diversity of non-culturable microorganisms and decoded new genomes. It is now possible to relate particular members of microbial communities to certain soil transformations. For instance:

Impact of anthropogenic activity – variations in the taxonomic groups of microorganisms;
Conversion of natural lands to agricultural ones (e.g., forest soils to grassland) – alterations in soil microbiome;
Introduction of bacterial taxa through agricultural activities – alterations in soil microbiome;
Use of pesticides and fertilizers – reduced abundance of some microbial phyla.

The adaptation and interaction among soil microbial communities and the effect of environmental changes on these phenomena can be also studied through metagenomic approaches. Two types of metagenomics can be specified for these purposes.

Guided metagenomics (or metabarcoding) searches for taxonomic identification at a large scale through DNA analysis of a single or a small number of genes. This approach is applied for studying the relative abundance of a certain gene, e.,a specific molecular marker in a soil sample, and allows the identification of distinct taxonomic groups. The molecular markers are different depending on the organisms searched. For microorganisms, these are rRNA genes for 16S for bacteria and the ITS elements for fungi.
Shotgun metagenomics determines the total genomic content of a sample via sequencing libraries. This technique is applied for the identification of microbial communities’ functional potential. Besides, it can help in the assignment of taxonomic identities since it sequences all the genetic material present in a soil sample. In this way, shotgun metagenomics can achieve reconstruction of complete genes and genomes contributing to their annotation and interpretation of metabolic routes.

In a comparative context, both techniques differ in the product they achieve and the bioinformatic methods they employ. For metabarcoding and shotgun bioinformatic pipelines, follow Duque-Zapata et al., 2023.

Learn: How to perform metaboarcoding (video)

Learn: Shotgun metagenomic sequencing principle (video)

3. ALTERNATIVES (DISCUSSION)

3.1. Soil metagenomics limitations and challenges

The input of metagenomics in our understanding of soil microbiome is indisputable. However, there are certain limitations and challenges that have to be considered.

Soil physical/chemical characteristics. The physical and chemical properties of the soil influence the soil microbiome composition and functioning. The classical metagenomic analyses count for the soil pH, temperature, moisture, and organic C. The influence of the elements K, P, and Fe is neglected or underestimated.
Soil structure, depth of sampling, and seasonal fluctuations. The above-mentioned properties vary depending on the soil depth and the seasonal changes in the environment, a fact that makes the conclusions launched on a specific soil sample hardly acceptable for identifying the microorganisms at an ecosystem o global scale.
Technical difficulties. Soil metagenomics generates a vast amount of raw sequencing data. This volume of data is making the replication of a research study a real challenge. Moreover, a relatively large number of sequencings or taxonomic units lack currently available information. The metabarcoding is limited to the PCR use and to the bias that can occur during bioinformatic processing. Shotgun metagenomics is dependent on the sequencing depth, which should be high enough to cover the entire genomic contents of every microbe present in a soil sample to allow integral analysis of its functional potential.

4. SOLUTIONS

Metagenomics analyses impact the discovery of microorganisms in different areas, resulting in the development of ambitious projects that contribute to the invention and implementation of microbial biomarkers for the assessment of soil health. Some of these areas are as follows.

4.1. One-Health approach towards soil health

OneHealth is a summative term referencing the interactions among microorganisms, plants, animals, and humans, how they influence the ecosystem’s homeostasis, the effects they exert on human health, and the detection, prevention, and control of risks that affect human health. Among the OneHealth actors, microorganisms play a vital role within ecosystems. Their effects encompass harmful actions to their hosts, executing the role of virulence factors, or reservoirs of illness/drug resistance genes. Since the great majority of microorganisms are non-culturable, the metagenomics approach for their study reveals prospects for the enrichment of our knowledge of ‘OneHea;th’ due to its capability to explore how microbes interact with their surroundings. Useful applications in this sense can be found in other areas related to global ecosystem health:

Human Microbiome project– epidemiological surveillance of human beings and their microbiome interactions: comparative characterization of the intestinal microbiome of healthy and various illnesses suffering individuals.
ListeriaInfection project – recognition of more than 33 novel small RNAs and 53 novel antisense RNAs with putative involvement in the inhibition of the expression of various regulatory operons in Listeria monocytogenes.
2009 Pandemic Influenza survey– application of metagenomics-based approaches (pan-viral microarray and deep sequencing) for studying variations of H1N1 flu virus and discovery of the ability to assembly its genome almost (90%) de novo. These techniques are proposed as a preventive strategy and diagnostic tool to investigate outbreaks of new pathogens.
Antimicrobial resistance global monitoring– application of metagenomics approach to perform continuous global surveillance and prediction of antimicrobial resistance genes using sewage – an ethically acceptable and economically feasible decision.

The One Health approach has been deepened and widened by the Planetary Health concept that considers the planetary ecosystems and their disturbance related to climate change, C and N biogeochemical cycle disorders, loss of biodiversity at a global scale, and air pollution. Although the research on this concept is focused on soil damage, the connection between soil health and Planetary Health is well recognized. The antimicrobial resistance and encouragement of plant-based diets to diminish greenhouse gas emissions are among those that are worth attention. Pesticide use reduction and crop biodiversity promotion stimulate the biodiversity of farm fields and soil microbiome. Thus, the global biodiversity can be enriched and protected. On the other hand, better soil health management and raising public awareness of the need for soil microbiome management through good agricultural practices could help reduce soil-associated pathogens and slow down the antimicrobial resistance spreading.

The relationships among One Health and Planetary Health approaches and priorities, good agricultural practices, and their reflection on soil health are outlined in the flow diagram in Fig. 8.1.

4.2. Ecogenomics

Ecogenomics (from ecological genomics) is a genomics branch that seeks to reveal the effect of genes/genomes functioning on organisms’ interaction in their natural environments. Using metagenomics as an integrative approach, the researchers pointed out the importance of the relationships between representatives of different kingdoms:

Plants-pollinators– application of metabarcoding to evaluate and study the interactions between plants and different insects;
Microorganisms-Black Soldier Fly– application of metagenomics to analyze the Black Soldier Fly gut microbiota and identify causal pathogens that can compromise the harvest of these flies for feeding purposes due to the risk of zoonotic diseases.
Plants-Phyllospheric microorganisms– the metagenomic study of rice phyllosphere with emphasis on the phytopathology potential of the microorganisms colonizing the plants’ exterior.
Medicinal and Aromatic Plants-rhizobacteria– metagenomics approach to identify bacteria contributing to the MAP properties of pharmaceutical importance.
Technical crops-rhizobacteria– metagenomic analysis of promoter genes regulating the transcription of genes involved in the vegetative growth and C-cycle in corn fields of different farming and management histories.
Animals-microorganisms-plants– metabarcoding of wild animals’ faces and determination of the fungal and plant species to characterize their diet and design conservation strategies based on assurance of proper food resources.

Figure 8.1. Good Agricultural Practices approaches to support soil heal and their reflection of One Health and Planetary Health approaches and priorities. (Source: Montgomery et all., 2024)

4.3. A global inventory of soil microbiome

Applying metagenomics to study the soil microbiome is not only an ambitious goal. It is crucially important for establishing strategies for the well-being, production, and conservation of plants, animals, and ecosystems. The biodiversity and metabolic performance of soil microbiome is an object of targeted projects focusing on the application of metagenomic studies for these purposes. These projects aim to reveal the abundance and functions of soil microorganisms through genomics techniques.

TerraGenome project

TerraGenome project is an international research consortium unifying 23 countries and operating to establish a full genetic fingerprint of all the bacterial species present in reference soil samples from one and the same location – Rothamstead, UK. This site is used because its use and management have been well-regulated for more than 1.5 centuries. The project has two aims:

To make a catalog of all genes in the soil and
To reveal their functions and their interactions with one another.

The idea behind the characterization of a single soil metagenome in detail is to use such soil as a reference for future metagenomic analyses.

The research playground of the TerraGenome project started in 2008 with DNA isolation and a year later continued with analysis of millions of DNA sequences from the soil that lasted for more than 10 years.

The scale of the TerraGenome project is enormous; similar are the technical challenges since the thousands of individual species genomes have to be sequenced, annotated, and compared. TerraGenome project not only persuades its objectives related to soil microbiome sequencing but also serves as a standard platform for other soil metagenomic sequencing efforts.

Earth Microbiome Project (EMP)

The EMP started in 2010 in the USA as a joint initiative to study the microorganisms’ components in distinct ecosystems and their surroundings all over the world under standard experimental conditions. This open, collaborative project objective was to compare multiple microbial communities via amplicon sequencing and metagenomics.

The EMP project mission was to construct a microbial map for our planet employing the environmental parameter space in different biomes and study them through samples gathered worldwide.

The first results of the EMP project were published in 2017, based on nearly 28,000 samples gathered around the globe (7 continents, 43 countries), processing of over two billion sequences, and identifying 308 species. Currently, the second phase of the EPM project is active – the Earth Microbiome Project 500 (EMP500) focused on metagenomic sequencing and metabolic profiling of microbial communities from various sites on Earth. The EMP500 upgraded EMP with new protocols for metagenomic sequencing and assembly. It enlarges the experimental sites’ nature, including a range of habitats and a total of 500 samples that meet certain criteria that guarantee comparability and reproducibility of results.

5. RECOMMENDATIONS (CONCLUSION)

To assess soil health and quality, an integrated approach that unifies the exploitation of a set of variables into a common framework. The parameters that describe the soil microbiome could be grouped in such a network and used to discriminate the soil status under normal and stressful conditions.

This network should unify both traditional and innovative indicators. The former includes an assessment of visible soil qualities and measurable physical and chemical characteristics. The latter comprises advanced innovative tools, an outcome of research knowledge, and technology development that resulted in our deeper understanding of microbial processes in the soil. Metagenomics is one of the most impactful approaches and techniques in this connection since it allows the measurement of soil microorganisms’ abundance and diversity. Thus, it enables the soil microbiome to serve as a reliable indicator for the state of soil health evaluation.

Metagenomics allows for the study of the structure, biodiversity, and functions of microbial communities from distinct environments – in norma and under stress, and contributes not only to the evaluation of soil health but also to shaping bioremediation strategies, agricultural practices, and human health.

6.&nbspREFERENCES

Duque-Zapata J.D., Lopez-Alvarez D., Muñoz J. E. Metagenomics approaches to understanding soil health in environmental research – a review. Soil Science Annual · April 2023 DOI: 10.37501/soilsa/163080

Ma B., Lu C., Wang Y. et al. (2023) A genomic catalogue of soil microbiomes boosts mining of biodiversity and genetic resources. Nat Commun 14, 7318. https://doi.org/10.1038/s41467-023-43000-z.

Montgomery R. R., Rabinowitz P., Sipos Y., Wheat E. E. (2024) Soil health: A common focus for one health and planetary health interventions. One Health, 18, 100673 https://doi.org/10.1016/j.onehlt.2023.100673

Xu Z, Hansen MA, Hansen LH, Jacquiod S, Sørensen SJ (2014) Bioinformatic Approaches Reveal Metagenomic Characterization of Soil Microbial Community. PLoS ONE 9(4): e93445. doi:10.1371/journal.pone.0093445

Vogel, T., Simonet, P., Jansson, J. et al. TerraGenome: a consortium for the sequencing of a soil metagenome. Nat Rev Microbiol 7, 252 (2009). https://doi.org/10.1038/nrmicro2119