Periodic Reporting for period 1 - ALIENinSoil (Microbial community response to the invasion of a non-endemic fungal bio-inoculant in soil)
Okres sprawozdawczy: 2021-01-04 do 2023-01-03
Fungi and bacteria are utilised to produce microbial-based fertilisers and pesticides worldwide. These bioinoculants have a vast potential in agriculture because they can help increase crop yields and quality and reduce the application of chemicals. While the effectiveness of bio-inoculants as biofertilisers or biopesticides is widely tested for crop yield and pathogens control, little is known about the effect of bioinoculants on microbial assemblages in the non-rhizospheric soil of agroecosystems. ALIENinSoil project evaluated the impact of a fungal inoculum, the globally used biofertiliser Trichoderma afroharzianum T22, on microbial assemblages of a model soil system. The fertiliser industry has selected this fungus to be very competitive and exhibit specific functions. Like all bioinoculants, it can influence soil properties, crops and pathogens by producing hormone-like compounds, antibiotics, secondary metabolites, siderophores, and organic acids. The fungus has proven effective in supporting many crops; however, it is not known how these interactions and compounds might affect the native soil microbial biology in agroecosystems.
Furthermore, the size of such a change can depend on many variables, like the initial alpha and beta diversity of the community or the bioavailability of nutrients. The project applied a rapid metagenomic approach based on long-read Oxford Nanopore Technology to assess the effects of the fungal inoculum on soil microbial communities and functions in a laboratory-based microcosms experiment. Metagenomics is based on the analysis of all DNA present in environmental samples and, thus, the genetic material belonging to all organisms present in that environment, which in the case of this project is soil. Innovative and cutting-edge techniques were used to understand 1) to what extent the native microbial community richness and relative abundance are influenced by a competitive fungal strain introduced to soil; 2) whether the keystone microbial taxa are resilient to the disturbance by the introduced fungus 3) how far the bioinoculant impacts the functions of soil microorganisms. A better knowledge of the interaction mechanisms between a massively introduced fungal species and the resident communities can stimulate innovation in soil bioinoculants and agriculture through competitive technology transfer. We showed that Oxford Nanopore sequencing has great potential for real-time diagnostics in agricultural surveys and the development of indicators for monitoring soil biodiversity and functionality.
Furthermore, the size of such a change can depend on many variables, like the initial alpha and beta diversity of the community or the bioavailability of nutrients. The project applied a rapid metagenomic approach based on long-read Oxford Nanopore Technology to assess the effects of the fungal inoculum on soil microbial communities and functions in a laboratory-based microcosms experiment. Metagenomics is based on the analysis of all DNA present in environmental samples and, thus, the genetic material belonging to all organisms present in that environment, which in the case of this project is soil. Innovative and cutting-edge techniques were used to understand 1) to what extent the native microbial community richness and relative abundance are influenced by a competitive fungal strain introduced to soil; 2) whether the keystone microbial taxa are resilient to the disturbance by the introduced fungus 3) how far the bioinoculant impacts the functions of soil microorganisms. A better knowledge of the interaction mechanisms between a massively introduced fungal species and the resident communities can stimulate innovation in soil bioinoculants and agriculture through competitive technology transfer. We showed that Oxford Nanopore sequencing has great potential for real-time diagnostics in agricultural surveys and the development of indicators for monitoring soil biodiversity and functionality.
The experiment consisted of adding Trichoderma T22 spores to soil: the fungus was added to two treatments, one with native soil and one with sterilised soil mixed with the native soil at a ratio of 1:9, representing a disturbance event that decreases the overall abundance of species. Soil bioreactors were incubated for seven weeks, and genomic DNA was extracted from a set of samples every week (one sacrificed bioreactor per sample). DNA extraction has been optimised to obtain high quantities of pure, long DNA filaments. The quantity and quality of DNA extracted each week were compared, and extracts from weeks zero (pre-incubation), three, five and seven were used for Illumina barcoding of fungi and bacteria (three replicates each). Soil DNA from the seventh week of incubation was then used in a metagenomic sequencing experiment on the GridION® platform (ONT, Oxford, UK) based on the Rapid Sequencing Kit (ONT, Oxford, UK) without PCR step. Six flow cells were used, and in each one, a library consisting of 4 multiplexed samples (one for each condition of the experiment) was sequenced. The DNA raw reads were basecalled via the Oxford Nanopore Technology pipeline "Guppy" supplied with the GridION®, using a GPU accelerated server available at NHM. Early downstream analysis components such as barcoding/demultiplexing, adapter trimming and alignment were contained within Guppy. The microbial community analysis was performed with Kraken2 software, used with a default database, and OmicsBox (Blast2Go) software was used to assemble the reads and align them to genes. MEGAHIT was applied for the assembly, and Prodigal was run for (procaryotic) gene prediction. Pfam-scan and Eggnog mapper were also used to obtain some annotations.
The introduction of the fungus had a negative impact on the abundance of taxa of bacteria, but it stimulated the presence of others, metabolically or physically linked to the fungus. The results suggest that more than an impact on bacteria's overall biodiversity, the fungus has favoured some groups at the expense of others, even creating new food webs and trophic niches. It was also found that the fungus led to more significant differences in the community composition compared to the control when incubated for seven weeks in the soil with an initial lower biodiversity. Therefore the level of resilience of the soil microbial community to T22 introduction appeared to be linked to the initial soil biodiversity. It also emerged that certain taxa of bacteria behaved differently in the presence of the fungus, depending on whether there were competitors or not. Thus, the success and resilience of certain groups of bacteria appeared to depend on the competition for space and nutrients. The positive effects of the fungus on specific taxa, in the sense of a population increase, were also only appreciated in samples with a lower abundance of microorganisms. At the functional level, the most striking differences were between intact soil and soil artificially depleted of its initial biodiversity. A differential abundance of a few dozen gene families was observed in the bioreactors where the fungus had been added compared to the control.
The introduction of the fungus had a negative impact on the abundance of taxa of bacteria, but it stimulated the presence of others, metabolically or physically linked to the fungus. The results suggest that more than an impact on bacteria's overall biodiversity, the fungus has favoured some groups at the expense of others, even creating new food webs and trophic niches. It was also found that the fungus led to more significant differences in the community composition compared to the control when incubated for seven weeks in the soil with an initial lower biodiversity. Therefore the level of resilience of the soil microbial community to T22 introduction appeared to be linked to the initial soil biodiversity. It also emerged that certain taxa of bacteria behaved differently in the presence of the fungus, depending on whether there were competitors or not. Thus, the success and resilience of certain groups of bacteria appeared to depend on the competition for space and nutrients. The positive effects of the fungus on specific taxa, in the sense of a population increase, were also only appreciated in samples with a lower abundance of microorganisms. At the functional level, the most striking differences were between intact soil and soil artificially depleted of its initial biodiversity. A differential abundance of a few dozen gene families was observed in the bioreactors where the fungus had been added compared to the control.
In addition to the results obtained on the project's initial questions concerning the dynamics of soil microbial communities following the introduction of the fungus, essential insights were also obtained from the analytical apparatus used in the project. The results obtained in optimising the protocol for extracting DNA from soil to prepare Nanopore sequencing libraries will help transfer the technology to other contexts. In addition, the in-depth investigations required for the use of metagenomics with Nanopore technology in the study of the effects of bioinoculants on agricultural soils have highlighted some critical aspects, such as the need to build customised reference databases for taxonomy that includes the genomes of fungal and bacterial species that are used as bioinoculants.
The project's outcome contributes new fundamental knowledge on biofertilisers impact on soil microbial biodiversity and function and shows how this knowledge is essential for applications and bio-based products in an early development stage. It is foreseen that soil metagenomics and Nanopore technology will support the development of regulations for biofertilisers in EU. The recent EU regulation on biofertiliser marketing seeks methods to define standards linked to environmental impact and quality. Published articles and data from AlienInSoil will directly contribute knowledge to implement these actions, making the project's output particularly timely and relevant to current European research trends and policies, with a real contribution to the knowledge-based economy and society.
The project's outcome contributes new fundamental knowledge on biofertilisers impact on soil microbial biodiversity and function and shows how this knowledge is essential for applications and bio-based products in an early development stage. It is foreseen that soil metagenomics and Nanopore technology will support the development of regulations for biofertilisers in EU. The recent EU regulation on biofertiliser marketing seeks methods to define standards linked to environmental impact and quality. Published articles and data from AlienInSoil will directly contribute knowledge to implement these actions, making the project's output particularly timely and relevant to current European research trends and policies, with a real contribution to the knowledge-based economy and society.