Method for identifying bacteria capable of colonizing plant roots

Abstract

The present invention relates to a method of producing a microbial consortium of bacteria capable of colonizing plant roots.

Description

[0001] P7198PC00

[0002] Method for identifying bacteria capable of colonizing plant roots

[0003] Technical field

[0004] The present invention relates to a method of producing a microbial consortium of bacteria capable of colonizing plant roots.

[0005] Background

[0006] Plants interact with the surrounding microbiome. Many of these interactions are beneficial for the plant. Plant-associated soil microbes may comprise plant growthpromoting traits.

[0007] In monocultures, which is the norm in agriculture today, the soil microbiome may be depleted. Moreover, a buildup of specific soil-borne pathogens may occur, since the continuous cultivation of the same crop species creates favourable conditions for certain pathogens. Fertilizers and biocides used to maintain productivity can disrupt the natural microbial consortia by harming beneficial microbes and altering microbial processes, such as nutrient cycling and organic matter breakdown.

[0008] Inoculants prepared as microbial consortia, derive from singular inoculants or from so- called synthetic communities or SynComs. SynComs are constructed by co-culturing multiple taxa isolated from plants grown in the soil, under well-defined conditions to mimic the structure and function of a microbiome. The underlying principle is to reduce the complexity of the original soil microbial community, while still preserving some of the essential interactions between the microbes and their hosts. These SynComs are used to identify principles of microbiome assembly and to predict functions that enable individual bacteria to colonise plants tissues. These can be applied as individual isolates in an inoculum or as an artificial consortium containing more than one isolate. Single isolate-based inoculants or consortia can be added by farmers to crops to increase crop growth and promote crop health.

[0009] SynComs comprise a collection of living microbial cells of multiple strains, capable of increasing plant capacity to mobilise nutrients from the soil, to acquire inaccessible nutrients or to protect them against pathogens, or to survive in stressful abiotic conditions like drought, salinity, etc. A variety of microorganisms, including beneficial bacteria but also fungi, can be used. P7198PC00

[0010] Summary

[0011] Related disclosures, such as Wippel et al. (2021) have described the construction of synthetic microbial communities based on taxonomic selection strategies, such as choosing one bacterial isolate per family according to their abundance in natural root microbiomes. While these approaches have provided insights into host-specific colonisation patterns, they do not identify the genetic features required for successful colonisation, nor do they enable systematic selection of bacterial isolates based on function.

[0012] There remains a need for methods that enable the identification and assembly of microbial consortia with enhanced root colonisation capacity, based on conserved genetic traits that are predictive of function across diverse plant hosts. In particular, methods that allow selection of bacterial strains using genomic criteria associated with colonisation, competitiveness, and beneficial impact on plant health would offer improved precision, reproducibility and agronomic utility.

[0013] Accordingly, the present disclosure relates to methods for obtaining a microbial consortium to be used as bioinoculant for plants, wherein said microbial consortium is characterized by having the capacity of colonizing the roots of a plant species and consequently affecting the growth of the colonized plant species, for example by enhancing its growth, enhancing its metabolic activity, enhancing its nutrient uptake, changing its chemical composition, changing its physical properties, enhancing its resistance to environmental stress and / or enhancing its resistance to behaviour changes.

[0014] In contrast to taxonomic selection approaches, the present invention utilises genomebased screening to select microbial entities. Due to high inter-isolate variation in gene content, even between members of the same species or genus, taxonomic identity alone is insufficient to predict root colonisation capacity. Instead, isolates are selected based on the presence of specific KO-classified functions identified as enriched in rootcolonising strains across multiple plant hosts.

[0015] The method disclosed herein are based on the finding that bacteria that successfully colonize the roots of taxonomically different plant species have largely overlapping functions that can be encoded by taxonomically diverse bacterial isolates. P7198PC00

[0016] The inventors of the present disclosure have developed a method for producing a microbial consortium, the method comprising identifying one or more bacterial isolates that have those functions, defined as homologous protein sequences, or the nucleotide sequences encoding such homologous protein sequences, which the present inventors have identified as being necessary to successfully colonize the roots of a plant.

[0017] In one aspect, the present disclosure relates to methods of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - 1, and ii. the same biological function as the corresponding sequence in Table A-l, in the obtained genomic sequence, d. Selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0018] In another aspect, the present disclosure relates to a methods of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table A- I and having the same biological function as the corresponding sequence in Table A-l; P7198PC00 wherein the presence of protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— I , as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

[0019] In another aspect, the present disclosure relates to methods of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i) at least 30% sequence identity with the sequences as defined in Table A - II, and ii) the same biological function as the corresponding sequence in Table A— 11, in the obtained genomic sequence, d. selecting a combination of said bacterial entities such that the combination collectively comprises nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0020] In another aspect, the present disclosure relates to methods of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i. at least 30% sequence identity with the sequences as defined in Table A

[0021] - II, and ii. the same biological function as the corresponding sequence in Table A— 11, in the obtained genomic sequence; wherein the presence of nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species. P7198PC00

[0022] In another aspect, the present disclosure relates to methods of improving growth, productivity and / or resilience towards biotic and abiotic stresses of a plant, the method comprising inoculating root(s) of the plant with the microbial communities produced by the methods of the present disclosure.

[0023] Description of Drawings

[0024] Figure 1 : Total number of KEGG Orthology functions (KOs) identified in four microbial culture collections. AtSC, HvSC or LjSC represent culture collections from Arabidopsis thaliana, Hordeum vulgare and Lotus japonicus, respectively. SSC denotes all three beforementioned collections together. The overlap of KOs across the three culture collections (AtSC, HvSC or LjSC) is shown in the Venn diagram.

[0025] Figure 2: Gene sequence variation within KEGG Orthology functions (KOs). The distribution of number of genes per KO in each of the four inocula is shown on the y-axis. The x-axis displays the gene diversity, calculated by summing all the branch lengths of a gene dendrogram of each KO. A higher gene diversity indicates a larger variation in nucleotide sequence between genes in a specific KO

[0026] Figure 3: The experimental protocol for reconstitution experiments. The culture collections have been subjected to whole-genome sequencing and the number of unique genomes is displayed between brackets. Culture collections are assembled into hostspecific inocula (AtSC, HvSC, LjSC) and the SuperSynCom (all collections together) to be inoculated with Arabidopsis, Barley, and Lotus. The DNA extracted from root and rhizoplane compartments was subjected to metagenome sequencing, from which the microbiome composition was inferred. Integration of bacterial genomic data and microbial composition provided the composition of bacterial functions through the KEGG Orthology database (KOs). (https: / / www.kegg.jp / )

[0027] Figure 4: Within community alpha diversity: the number of observed isolates (A) and KOs (B) in the dataset. Input indicates the number of bacterial isolates that were present in the inoculum at the start. Statistical differences were computed using ANO As and post-hoc test (p<0.05) at the inoculum level. P7198PC00

[0028] Figure 5: Heatmaps displaying the correlation between the functional diversity of bacterial isolates and their relative abundance in the root microbiome subsetted per bacterial family. (A) The family relative abundance in the root microbiome is represented on the right of the plot and extended at the genus level for the large Burkholderiaceae family in (B). Significant correlations (p<0.05 with multiple test correction) indicating high abundance and functional diversity of isolates belonging to a certain family in each of the four communities (AtSC, HvSC, LjSC, and SSC) are displayed with a (*) symbol. Values between brackets indicate the number of isolates in the family, while (-) represents non-computable correlations because of a too low number of isolates in a given inoculum.

[0029] Figure 6: Piedonut plots dissecting host R2of the original dataset (A) or without the dominating strains such as Mesorhizobium sp. nodule symbionts from Lotus and Rhizobacter P2_G4 (B). The inner circle represents the combined plant species’ impact on host R2between inocula. The outer circle represents the contribution of every plant species to host R2in every inoculum.

[0030] Figure 7: (A) Sankey diagram depicting the relation between filtering steps and respective KO enrichments. Starting from 5,981 root-enriched bacterial KOs, to 852 common host-enriched KOs, to 266 strictly general KOs of which 203 carrying pathway-level annotations. (B) Function enrichment index method calculated as a fold change ratio based on the change in cumulative relative abundance of bacterial populations with or without the respective pathway function between root communities and inocula.

[0031] Figure 8: Density distribution of common host-enriched KOs (n=266) across the three hosts based on the functional enrichment index.

[0032] Figure 9: Density plots showing the distribution of the 266 strictly general KOs in the bacterial isolates across the three culture collections used in this study.

[0033] Figure 10: Density plots showing the distribution of the 266 strictly general KOs in the bacterial genome database categorized as soil, non-plant associated or plant- associated bacteria (Levy et al., 2018). The Venn diagram displays the overlap P7198PC00 between the identified plant-associated KO functions in this study and Levy et al. (2018).

[0034] Figure 11 : Sankey diagram to investigate host-specific KOs. KOs were considered host-specific if only for that host, the function enrichment index was >3, while being <3 for the other two hosts. The function enrichment index value for each host was calculated by taking the median value for each host across the four inocula.

[0035] Figure 12: The function enrichment indices of host-specific KOs in the ternary plane confirm the host-specificity. For Lotus, an additional analysis was performed with the dataset in which the genes of the six Lotus-nodulating Mesorhizobium sp. are removed. Lotus-specific KOs in the dataset with the nodulators are indicated by closed coordinates while the open coordinates indicate KOs that are Lotus-specific when nodulators are excluded. The number of host-specific KOs is added in the corners of the host-specific ternary plot. The number of Lotus-specific KOs in the dataset without the nodulators is 61 as compared to 355 when they are included.

[0036] Figure 13. Host-specific ABC transporters with observed species-specific enrichment, categorized by the putative substrate they transport across the cell membrane. Multiple gene cassettes that together make up an ABC transporter were combined for this analysis. Dot size represents the cumulative relative abundance of isolates carrying these ABC transporters in their genomes (average of gene cassettes) and the color gradient is an indication of the DESeq2 p-value between root microbiome and initial inoculum (averaged by Stouffer's method across gene cassettes).

[0037] Figure 14. Using the core categorization script from Shade & Stopnisek (2019), core strains were selected in the root microbiome of every host (Arabidopsis, Barley, and Lotus) inoculated with one of the four SynComs (AtSC, HvSC, LjSC, or SSC). The Venn diagram shows the overlap in selected core isolates across the three hosts and four SynComs. Twenty-nine bacterial strains are consistently enriched in the root microbiome irrespective of host, while there are 21 , 30, and 22 Arabidopsis-, Barley-, and Lotus-specific strains.

[0038] Figure 15: Differential abundance analysis of the SynCom members across all hosts in distinct contexts. The x-axis indicates the Iog2foldchange (by ANCOM-BC) of the strain P7198PC00 in the SSC as compared to SSC inoculum at the start of the experiment and the y-axis indicates the Iog2foldchange of the strain in its respective host-specific SynCom (AtSC, HvSC or LjSC) as compared to the respective inoculum at the start of the experiment. The Iog2foldchanges were averaged across the three plants hosts: Arabidopsis, Barley, and Lotus. The shape indicates the core status of the strain, be it core, only in the SSC, or also in the host-specific SynCom (HSC).

[0039] Figure 16: Enriched gene clusters for each core strain with regard to root competence. Enriched gene clusters are shown for 20 / 29 core strains. Enrichment of gene clusters is defined by the presence of five adjacent genes being significantly associated with high root competence which in turn is defined as a large increase in abundance from inoculum to root microbiome (Figure 15). The size of the dot indicates how many gene clusters with the same predicted function are found to be enriched. Nine out of twenty- nine core strains do not have any root competence-associated gene clusters by the above defined criteria and are therefore excluded from this figure.

[0040] Figure 17: Unique genes in the SSC-core strains LjRoot3 (Devos / a), LjRoot221 (Agrobacterium'), and LjRoot149 (Flavobacterium). Percentage of genes in the core strains that is significantly associated with competitiveness and is unique compared to closely related strains in the SSC is shown for LjRoot3 (A), LjRoot221 (C), and LjRoot149 (E). Manhattan plots show p-values for association with competitiveness for significant (p-value < 0.05) unique genes (y-axis) in line with their respective location on the genome (x-axis) for LjRoot3 (B), LjRoot221 (D), and LjRoot149 (F). Assembled contigs and their encoded genes are ordered from large to small and marked grey and black one after the other. Relevant gene or gene cluster annotations are indicated by the respective labels.

[0041] Figure 18: Unique genes in the SSC-core strains LjRoot206 (Pseudoxanthomonas), P2_G4 (Rhizobacter), LjRoot135 and LjRoot38 (Acidovorax). Percentage of genes in the core strains that is significantly associated with competitiveness and is unique compared to closely related strains in the SSC is shown for LjRoot206 (A), P2_G4 (C), and LjRoot135 and LjRoot38 (E). Manhattan plots show p-values for association with competitiveness for significant (p-value < 0.05) unique genes (y-axis) in line with their respective location on the genome (x-axis) for LjRoot206 (B), P2_G4 (D), LjRoot135 (F) and LjRoot38 (G). Assembled contigs and their encoded genes are ordered from P7198PC00 large to small and marked grey and black one after the other. Relevant gene or gene cluster annotations are indicated by the respective labels.

[0042] Figure 19: Cumulative relative abundance of strains carrying gene cluster-associated competitiveness genes. A) and C) Heatmap depicting the cumulative relative abundance of the strains with notable genes and gene clusters depicted in Figures 16B, D, F, and 17B, D, F, and G. Columns indicate different plant-SynCom combinations, and significant deviations from the expected abundance based on the distribution of the respective gene is indicated by the asterisks (binomial test; * - p- value < 0.05, ** - p-value < 0.01, *** - p-value < 0.001). The final column indicates in which strain the gene was competitive (see Manhattan plots in Figures 16B, D, F, and 17B, D, F, and G). B) and D) Proportion of strains in each SynCom with the gene in the SSC data and in data published by Levy et al. (2018) in which non-plant-associated (NPA), plant-associated (PA), root-associated (RA), and soil bacteria are compared. Notable plant / root-associated genes are indicated by black boxes.

[0043] Figure 20: Community and plant-context dependent competitiveness of Pseudomonas strain KB_12. A) Percentage of genes in Pseudomonas strain KB_12 that is significantly associated with competitiveness in the Arabidopsis root microbiome and is unique compared to closely related strains in the AtSC and the SSC is shown. Manhattan plots for strain KB_12 (B) and the most closely related SSC strain LjRoot6 (C) show p-values for significance of association with competitiveness for significant (p- value < 0.05) unique genes (y-axis) in line with their respective location on the genome (x-axis). For KB_12 uniqueness is defined by the absence of related sequences in closely related strains in either the AtSC (circles) or the SSC (triangles), while for LjRoot6 uniqueness is defined by the absence of related sequences in closely related SSC strains (circles). Assembled contigs and their encoded genes are ordered from large to small and marked grey and black one after the other. Relevant gene or gene cluster annotations are indicated by the respective labels.

[0044] Figure 21 : Cumulative relative abundance of strains carrying competitiveness-related genes identified in Pseudomonas strains KB_12 and LjRoot6. Left) Heatmap depicting the cumulative relative abundance of the strains with notable genes from clusters depicted in Figure 20B and C. Columns indicate different plant-SynCom combinations, and significant deviations from the expected abundance based on the distribution of the P7198PC00 respective gene is indicated by the asterisks (binomial test; * - p-value < 0.05, ** - p- value < 0.01 , *** - p-value < 0.001). The final column indicates in which strain the gene was competitive (see Manhattan plots in Figure 20B and C). Right) Proportion of strains in AtSC and SSC with the gene. Notable differences in relevant genes between AtSC and SSC are indicated by black boxes.

[0045] Figure 22: KO diversity in "one isolate per family" 1000 simulated SynComs. The distribution of total KOs recovered across simulations (full line) and confidence intervals (dashed lines) is shown. Note that app. 6000 KOs are achieved, compared to the >8000 KOs present in the SSC collection. The inset illustrates KO representativeness across simulations.

[0046] Figure 23: 266 general plant-selected KOs across bacterial families, (a) Intrafamily distribution of the 266 KOs indicating what proportion of the 266 KOs are shared by the majority of bacterial strains within a family or by a smaller subset of bacteria. Bacteria were ranked by relative abundance in the data and only the bacteria that cumulatively amount to 90% of the total relative abundance - top colonizers - were taken for this analysis (top). KOs among this set that were statistically overrepresented among the top colonizers compared to the other bacteria in the bacterial family are shown in (b), with the level of significance indicated by the Barnard Z-statistic.

[0047] Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied to facilitate the understanding of the present invention.

[0049] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. In addition, as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of”.

[0050] As used in the specification and claims, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B". P7198PC00

[0051] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Similarly, terms such as “one or more” or “at least one” include both the singular and plural form of the respective feature.

[0052] “Synthetic Community” or “SynCom” refers to an artificially assembled group of microorganisms, including but not limited to bacteria, fungi, archaea, or other microbes, that are deliberately selected, for example from bacteria and / or other microorganisms isolated in bulk from a certain environment, for example from plant roots, and combined to function together in a controlled environment. These microorganisms are chosen based on specific characteristics or functions, for example with the help of metagenomic techniques, to achieve a desired outcome, such as promoting plant growth, degrading pollutants, or studying microbial interactions. In the present disclosure, a desired function was studying root colonization.

[0053] “Inoculum” or “bioinoculant” is one microorganism or a collection of selected microorganisms, for example bacteria, but also fungi and / or other beneficial microbes, that is introduced into a plant or soil environment, for example on seeds or plant roots, or other related environment, with the purpose of enhancing plant growth, soil fertility, resistance to pests and diseases, or other desired properties of the plant or related environment.

[0054] “Microbial consortium” or “bacterial consortium” are groups of diverse microorganisms that can act together as communities. Microbial communities are ubiquitous in their natural environment and key players in global carbon and nitrogen cycles. A microbial consortium or bacterial consortium can be derived from isolates from a SynCom or be composed of independently isolated bacterial isolates brought together in a community. Microbial consortia can be applied directly to agricultural settings.

[0055] “Plant microbiome” refers to the community of microorganisms, including bacteria, fungi, archaea, viruses, and other microorganisms, that are associated with a plant. This term encompasses microorganisms that inhabit various parts of the plant, such as the rhizosphere (e.g., the root-associated soil), phyllosphere (above-ground parts like leaves and stems), endosphere (internal tissues), and any other plant-associated environments, such as nearby soil, water, and air. The plant microbiome includes both P7198PC00 symbiotic and non-symbiotic microorganisms that interact with the plant, influencing its growth, health, and overall biology.

[0056] “Root colonization” is defined as the proliferation of microbes in, on, or around roots. It includes dispersal of microorganisms from a source of inoculum to the actively growing root, and multiplication or growth in the rhizosphere.

[0057] “Root competence” is defined as the ability of microbes to colonize the roots in the presence of complex microbial environment and persist in the root microbiome.

[0058] “Bacterial entity” refers to any form of bacteria, encompassing both individual bacterial strains, isolates and bacterial communities. This term includes, but is not limited to: i) bacterial isolates, such as individual bacterial isolates, referring to single strains or species of bacteria that have been isolated and cultured independently; and ii) bacterial communities, also referred to herein as microbial consortia or a microbial consortium, such as naturally occurring or synthetic bacterial or microbial consortia, which are assemblies of multiple bacterial strains or species that coexist in a shared environment, interacting with each other. In the context of the present disclosure, a bacterial entity may be characterised by genomic sequencing either at the individual strain level or via metagenomic analysis of a mixed community.

[0059] “Compost” refers to biologically decomposed organic material resulting from the controlled decomposition of organic matter, such as plant residues, food waste, manure, and other biodegradable materials. This process is facilitated by microorganisms, including bacteria and fungi, which break down the organic matter into a stable, humus-like substance rich in nutrients. Compost is commonly used as a soil amendment to enhance soil fertility, structure, and microbial activity, and it may also be utilized for other agricultural, horticultural, or environmental purposes.

[0060] The “rhizosphere” is a narrow zone of soil surrounding and influenced by the root of vascular plants. This zone is characterized by intense biological activity owing to the release of root exudates, which stimulate or inhibit the microorganisms present. The rhizosphere is analogous to laimosphere and spermosphere.

[0061] The “rhizoplane” represents the outer surface of the root. P7198PC00

[0062] The “endosphere” represents intracellular regions within plant tissues colonized by microbial endophytes without causing disease symptoms to host plants.

[0063] The “phyllosphere” is the total above-ground surface of a plant when viewed as a habitat for microorganisms. The phyllosphere can be further subdivided into the caulosphere (stems), phylloplane (leaves), anthosphere (flowers), and carposphere (fruits)

[0064] “Kegg orthologs” or “KO” are functional gene annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. KEGG is a widely used database that offers organism-independent annotation for genes from shotgun microbiome studies and / or whole-genome shotgun sequences of bacteria. Due to the inherent diversity within KO groups, a wide range of sequence variants may exist that perform the same function. The present invention encompasses any homologous sequence that is assigned to the relevant KO group based on accepted annotation tools (e.g. KEGG Mapper, BlastKOALA, GhostKOALA) and / or contributes to the cumulative KO coverage within a microbial consortium as defined herein.

[0065] “KEGG Orthology identifiers” (KO identifiers) refer to the unique alphanumeric codes assigned by the KEGG database to group functionally equivalent genes or proteins from diverse organisms into orthologous groups. Each KO identifier (e.g., K00001, K12345) represents a specific biological function rather than a specific sequence, and is used to categorise genes and proteins according to conserved function across species.

[0066] “KEGG Orthology functions” refer to the biological functions assigned to protein and / or nucleotide sequences based on their classification under a given KEGG Orthology identifier. These functions are defined by the KEGG database and reflect the predicted role of the encoded protein in cellular or metabolic pathways. Multiple different protein and / or nucleotide sequences may be classified under the same KEGG Orthology function, provided they perform the same annotated role.

[0067] The “orthogroups” or clusters of “orthologous sequences” or “OGs” were generated by protein-protein alignment of the protein sequences of all bacterial strains. Each orthogroup contains at least two proteins or groups of paralogs. Orthogroups represent P7198PC00 sets of genes from different species that are descended from a single gene in the last common ancestor of those species, and which are assumed to perform equivalent biological functions across taxa. Orthogroups with a certain percentage of similarity by BLASTp comparison are database-independent and derive from a frequently used protein-clustering method to perform comparative genomics analyses between phylogenetically distant organisms. In the context of the present disclosure, the orthogroups defined in Table B-l were identified using comparative genomics tools such as OrthoFinder, based on sequence similarity across multiple bacterial isolates. These orthogroups represent protein-coding functions associated with enhanced root colonisation competitiveness. Because these functions may not be fully mapped to existing KEGG Orthology identifiers, the orthogroups serve as a complementary functional classification system. Their presence can be determined using sequencebased clustering methods or in silico comparative analyses.

[0068] "Homologous protein sequences" or “homologous nucleotide sequences” as used herein refers to amino acid sequences or nucleotide sequences, respectively, that are identified as homologues and / or orthologues through various databases or classification systems, including but not limited to KEGG Orthology (KO) and orthologous groups (OGs). In the context of the present disclosure, a homologous protein sequence refers to a sequence that shares homology with a reference sequence, defined as having at least 30% full-length amino acid sequence identity to the reference sequence, such as those listed in Table A-l.

[0069] The skilled person will be able to determine whether a bacterial genome encodes homologous protein sequences as defined in Table A-l or A-l I by performing wholegenome sequencing of the isolate and annotating protein-coding sequences using tools such as KEGG Mapper, BlastKOALA, GhostKOALA or Prokka in combination with KEGG. Functional annotation assigns predicted proteins to KO identifiers, allowing determination of the presence and abundance of the 266 KOs defined herein. For large-scale screening of isolates and / or a microbial consortium, the presence of at least a defined proportion (e.g. 50%) of the 266 KOs can be evaluated computationally. Genomes may be annotated in silico, and KO coverage calculated using a script or pipeline (e.g., KEGGDecoder or in-house tools) that compares predicted KOs to the 266-member set disclosed. P7198PC00

[0070] The terms homology, identity and similarity, with respect to a polynucleotide (or polypeptide), as defined herein are used interchangeably and refer to the percentage of nucleic acids (or amino acids) in the candidate sequence that are, homolog, identical or similar, respectively, to the residues of a corresponding native nucleic acids (or amino acids), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity I similarity, and considering any conservative substitutions according to the NCIIIB rules (http: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-llIB, Eur J Biochem (1985)) as part of the sequence identity. In particular, the percentage of similarity refers to the percentage of residues conserved with similar physiochemical properties. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity or similarity. Methods and computer programs for the alignments are well known in the art. Generally, a given similarity between two sequences implies that the identity between these sequences is at least equal to the similarity; for example, if two sequences are 70% similar to one another, they cannot be less than 70% identical to one another - but could be sharing 80% identity.

[0071] When using BLASTp to identify homologous, or even orthologous, protein sequences, typically a threshold as low as 30% can be sufficient for positive identification. An approach that may be used, and is commonly used by a person of skill in the art, to find orthogroups (OGs), is Orthofinder. Orthofinder is a computational tool / software used for identifying orthologous gene and proteins across multiple species. It is widely used in comparative genomics to identify orthologous proteins, which are critical for understanding functional conservation, evolutionary processes, and cross-species biological similarities. Orthofinder considers both the protein length, the percentage (%) similarity and the phylogenetic distance. All three elements are collectively important. To find the relevant homologous sequence in a taxonomically distant organism, as in the methods disclosed herein, requires a lower threshold then when looking to find it in a sister species. Hence, a suitable percentage homology is 30%.

[0072] "Genomic sequence" refers to the complete DNA sequence of an organism's genome, including all of its genes and non-coding regions. In the context of bacterial communities, a genomic sequence may refer to the collective genetic material present within the community, often obtained through metagenomic sequencing. This encompasses the genomes of all bacterial species within the community. In the context P7198PC00 of individual bacterial isolates, a genomic sequence specifically pertains to the DNA sequence of a single bacterial strain, representing its unique genetic blueprint. The term includes complete or partial sequences, and any naturally occurring or artificial variants.

[0073] Detailed description

[0074] Method of making a microbial consortium

[0075] In one aspect, the present disclosure relates to methods of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table A -

[0076] I, and ii. the same biological function as the corresponding sequence in Table A-l, in the obtained genomic sequence, d. Selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0077] In another aspect, the present disclosure relates to a methods of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table A- I and having the same biological function as the corresponding sequence in Table A-l; P7198PC00 wherein the presence of protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— I , as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

[0078] In another aspect, the present disclosure relates to methods of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i) at least 30% sequence identity with the sequences as defined in Table A

[0079] - II, and ii) the same biological function as the corresponding sequence in Table A- II, in the obtained genomic sequence, d. selecting a combination of said bacterial entities such that the combination collectively comprises nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0080] In another aspect, the present disclosure relates to methods of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i. at least 30% sequence identity with the sequences as defined in Table A

[0081] - II, and ii. the same biological function as the corresponding sequence in Table A- II, in the obtained genomic sequence; P7198PC00 wherein the presence of nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

[0082] The term “Biological function”, as used herein, refers to the specific biochemical activity, molecular role, or physiological effect performed by a protein, as defined by functional annotation systems such as KEGG Orthology (KO). In some embodiments, the biological function is determined by the assignment of the protein to a specific KEGG Orthology group, wherein all members of the group are considered to perform the same conserved function across different organisms. Functional annotation may be based on the KO identifier assigned to the protein sequence using accepted annotation tools such as KEGG Mapper, BlastKOALA, GhostKOALA, or equivalent, which classify protein sequences into orthologous groups sharing a defined function.

[0083] The determination of biological function may further be supported by enzyme commission (EC) numbers, pathway involvement, ligand-binding specificity, or other experimentally validated or predicted roles. Two proteins may have the same biological function even if they share less than 30 % sequence identity, provided they are assigned to the same KO group or otherwise perform the same function as defined by such annotations.

[0084] Functional variant, as used herein, refers to a protein sequence that (i) meets the definition of a homologous protein sequence above ( / .e., has at least 30% full-length amino acid sequence identity to the reference sequence) and (ii) retains the same biological function as that reference protein sequence, as determined for example by KEGG Orthology annotation. A functional variant may contain one or more amino acid substitutions, deletions, insertions, or chemical modifications, provided these changes do not abolish the annotated biological function.

[0085] In the context of homologous proteins, a functional variant may differ from the reference sequence by a number of changes that do not significantly alter the protein's overall structure or functional properties. This term encompasses natural variants, engineered mutants, or proteins resulting from evolutionary divergence, provided they perform the same or substantially similar biological function as the original protein. P7198PC00

[0086] A functional variant of any of the homologous protein sequences as defined in any one of Table A - I and Table B - I as determined for example through BLASTp, and the same predicted function, which is described in the “Description / Biological function” column in Table A - I and Table B - I. In some embodiments, a functional variant is assigned to the same KEGG Orthology (KO) identifier as the homologous protein sequence. In other embodiments, a functional variant retains one or more conserved protein domains necessary for biological activity, as identified using tools such as Pfam or InterPro. In some embodiments, a functional variant may also refer to a synonymous nucleotide variant that encodes the same amino acid sequence as the homologous protein. Since these homologous protein sequences can be found in taxonomically distant bacteria, their amino acid sequences, and consequently the nucleotide sequences encoding the protein, may be very different, which the function and possible also the three-dimensional structure are conserved.

[0087] In one embodiment of the present disclosure, the steps of: a. Determining presence of the homologous protein sequences as defined in Table A - 1 in the obtained genomic sequence, b. Determining presence of the nucleotide sequences as defined in Table A - II in the obtained genomic sequence, c. Determining presence of the homologous protein sequences as defined in Table B - 1 in the obtained genomic sequence, d. Determining presence of the nucleotide sequences as defined in Table B - II in the obtained genomic sequence, correspond to: a. Determining presence of the homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - I, and ii. the same biological function as the corresponding sequence in Table A-l, in the obtained genomic sequence, b. Determining presence of the homologous sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - II, and ii. the same biological function as the corresponding sequence in Table A-l I, P7198PC00 in the obtained genomic sequence, c. Determining presence of the homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table B - I, and ii. the same biological function as the corresponding sequence in Table B-l, in the obtained genomic sequence, d. Determining presence of the homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table B - II, and ii. the same biological function as the corresponding sequence in Table B-l I, in the obtained genomic sequence, wherein the biological functions are as defined in Table A - I and Table B - I in the column “Description / biological function”.

[0088] In some embodiments, the method comprises: a. determining, for each bacterial entity, the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table B-l, and having the same biological function as the corresponding sequence in Table B-l, in the obtained genomic sequence; and b. selecting a combination of said bacterial entities such that the combination collectively comprises; i) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, and ii) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B-l, as identified in step a).

[0089] In some embodiments, the method comprises: a. Determining, for each bacterial entity, the presence of the nucleotide sequences as defined in Table B - II, and having the same biological function as the corresponding sequence in Table B-l I, in the obtained genomic sequence, P7198PC00 b. Selecting a combination of said bacterial entities such that the combination collectively comprises; i. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B - II, and ii. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II.

[0090] In some embodiments, the method comprises determining, for each bacterial entity, the presence of homologous protein sequences, as defined herein, having a sequence identity to the sequences listed in one or more of Table A— I , Table A— 11, Table B-l or Table B-l I and performing the same biological function as the corresponding sequence in the respective table. The sequence identity may be at least 35 %, such as at least 40 %, such as at least 45 %, such as at least 50 %, such as at least 55 %, such as at least 60 %, such as at least 65 %, such as at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 %, or such as at least 99 %. In some embodiments, the method further comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions meeting the sequence identity requirement for a defined proportion of entries in any one or more of Table A— I, Table A— II, Table B-l or Table B— 11, wherein the proportion may be at least 50 %, such as at least 55 %, such as at least 60 %, such as at least 65 %, such as at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, such as at least 90 %, such as at least 95 %, or such as at least 99 % of the entries in the respective table(s).

[0091] In some embodiments, the homologous protein sequences having the same biological function as the corresponding sequence in Table A-l are sequences which are assigned to the same KEGG Orthology identifier as defined in Table A-l.

[0092] In some embodiments, the homologous nucleotide sequences having the same biological function as the corresponding sequence in Table A-l I are sequences which are assigned to the same KEGG Orthology identifier as defined in Table A-l I.

[0093] In one embodiment of the present disclosure, a microbial consortium that successfully colonises the roots of a plant is a microbial consortium that has a stable and detectable P7198PC00 population on or within the plant microbiome for a period of at least 14 days postinoculation of that plant with said microbial consortium.

[0094] In one embodiment of the present disclosure, successful colonisation of the roots of the plant species results in one or more changes to the plant selected from growth, metabolic activity, nutrient uptake, chemical composition, physical properties, resistance to environmental stress and / or behaviour changes.

[0095] In addition to identifying the key functions for bacterial survival in complex communities associated with plant roots, the present inventors have also identified competitiveness- associated functions, which can be used to select a bacterial entity that will not only colonize the roots of a plant, but also thrive on them. These competitiveness- associated functions are listed as homologous protein sequences in Table B - I. In addition to homologous protein sequences structurally defining the competitiveness- associated functions, the nucleotide sequences encoding the listed competitiveness- associated homologous protein sequences are found in Table B - II.

[0096] Thus, in one embodiment of the present disclosure, the method further comprises: a. determining, for each bacterial entity, the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table B-l, and having the same biological function as the corresponding sequence in Table B-l, in the obtained genomic sequence, b. selecting a combination of said bacterial entities such that the combination collectively comprises; i) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— I , and ii) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B-l, as identified in step a).

[0097] Similarly, in another embodiment of the present disclosure the method further comprises: a. Determining, for each bacterial entity, the presence of the nucleotide sequences as defined in Table B - II, and having the same biological function as the corresponding sequence in Table B-l I, in the obtained genomic sequence, and P7198PC00 b. Selecting a combination of said bacterial entities such that the combination collectively comprises; i. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B - II, and ii. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— 11.

[0098] In some embodiments, the homologous sequences having the same biological function as the corresponding sequence in Table B-l are sequences which are assigned to the same Orthogroup identifier as defined in Table B-l.

[0099] In some embodiments, the homologous sequences having the same biological function as the corresponding sequence in Table B-l I are sequences which are assigned to the same Orthogroup identifier as defined in Table B-l I.

[0100] In one embodiment the microbial consortium produced according to said method has the capacity to successfully adapt to a plant root environment and / or ensure competitive colonisation of the roots of a plant species and / or successfully colonise roots of a plant species.

[0101] As used herein, “successfully colonise roots” refers to the ability of one or more bacterial entities, when introduced into the vicinity of a plant root system, to establish a detectable, stable, and functionally relevant presence on or within the roots of at least one plant species. In one embodiment, successful colonisation is evidenced by the sustained detection of the bacterial entity, or a genetically distinguishable derivative thereof, on or within root tissues or the rhizoplane for a period of at least 14 days postinoculation, using standard microbiological, molecular, or genomic detection techniques, such as qPCR, metagenomic sequencing, or culturing. In another embodiment, successful colonisation is determined by the recovery of the bacterial entity from the root or rhizoplane at a relative abundance of at least 0.5% within the root-associated microbiome, based on metagenomic profiling.

[0102] In another embodiment, successful colonisation is demonstrated by a statistically significant increase (e.g. p < 0.05) in the abundance of the bacterial entity in the root compartment as compared to its abundance in the inoculum or growth substrate. P7198PC00

[0103] In a further embodiment, successful colonisation is confirmed by the observation of a functional impact on the plant, such as enhanced growth, increased nutrient uptake, or improved resistance to biotic or abiotic stress, provided that such impact correlates with the detectable presence of the bacterial entity or consortium on the root surface or within root tissues. In yet another embodiment, successful colonisation is determined by the presence of a minimum number or proportion (e.g. at least 50%) of homologous protein sequences associated with root colonisation, as defined in Table A— I , within the genome of the colonising bacterial entity or consortium, in combination with measurable root association under controlled conditions. This is demonstrated, for example, in Examples 5, 6, 9 and 10, which show how community composition and host specificity can promote and drive root community assembly and competitive root colonization. In particular, Example 5 demonstrates how the identified functions in Table A - I, and consequently in Table A - II, are associated with general root community assembly. Example 6 demonstrates how functions are identified that are associated to host-specific root community assembly. Moreover, Example 9 demonstrates that the identified functions in Table B - I, and consequently in Table B - II, are associated with root colonization and competitiveness of individual bacterial isolates irrespective of host. Similarly, Example 10 demonstrates how functions associated with host-specific competitiveness may be identified. Thus, these Examples also provide examples of which methodology a skilled person can use to assess whether a microbial consortium or a SynCom has the capacity to successfully adapt to a plant root environment and / or has the capacity to ensure competitive colonisation of the roots of a plant species.

[0104] Sources of bacteria

[0105] It is known that plants can benefit from the presence of microorganisms in their surrounding environment. Through these interactions, a plant can obtain nutrients, enhance its metabolism, and get protection from pathogens. Soil microbes can decompose organic material and provide a plant with nutrients like phosphorous and nitrogen. The plant surface, such as leaves, hosts a variety of microbes that can help protect plants by outcompeting pathogens, producing antibiotics, or inducing systemic resistance in the plant. Microbial communities in surface water can form biofilms on plant roots in aquatic or semi-aquatic environments, aiding in nutrient absorption and protecting roots from harmful pathogens. P7198PC00

[0106] Hence, the plant microbiome, from which the bacterial entity is isolated, may originate from a plurality of locations and habitats.

[0107] In one embodiment, the plant microbiome comprises soil, including humus, air or water surrounding the plant.

[0108] In another embodiment, the plant microbiome comprises the endosphere, phyllosphere and / or rhizosphere of a plant. For example, in one embodiment the plant microbiome originates from the endosphere, phyllosphere and / or rhizosphere of a plant.

[0109] Certain bacteria act as decomposers and break down organic matter, releasing nutrients such as carbon, nitrogen, and phosphorus back into the soil for plant use. Dead plant material, including food or agricultural waste, manure and any decomposing plant material is also a possible source of bacterial entity.

[0110] Thus, in one embodiment, the plant microbiome comprises compost. For example, the plant microbiome may originate from compost.

[0111] The plant species

[0112] The methods of the present disclosure are suitable for any plant species, for example for any plant species that has a root system.

[0113] Angiosperms generally have more complex and varied root system that secrete a diverse array of root exudates, including sugars, amino acids, and organic acids. These exudates attract a broad range of microorganisms, including bacteria, fungi, and archaea, leading to a more diverse and dynamic root microbiome. The diversity of root exudates in angiosperms supports a wide range of microbial functions, from nutrient cycling to plant growth promotion.

[0114] Gymnosperms typically have a simple root system. Their root exudates tend to be less diverse compared to those of angiosperms, often dominated by compounds like phenolics and terpenoids, which can have antimicrobial properties. The bacterial diversity in gymnosperms is often lower compared to angiosperms. The bacterial communities in gymnosperms are well-adapted to the acidic and nutrient-poor soils where many gymnosperms grow. P7198PC00

[0115] Despite the above differences, studies have found that taxonomically different plants have overlapping requirements in terms of root microbiota. For example, Duran et al. 2022 found the existence of shared ecological principles driving the assembly of the A thaliana root and C. reinhardtii phycosphere microbiota, despite the vast evolutionary distance between these two photosynthetic organisms. Study of Takakia by Satjarak et al. 2022 considered the Earth’s most archaic modern land plant, identified to have in their microbiome taxa normally associated with higher plants including intracellular symbionts.

[0116] In one embodiment, the at least one plant species belongs to a Viridiplantae, an angiosperm or a gymnosperm. For example, the angiosperm may be a monocot or a eudicot.

[0117] In one embodiment, the at least one plant species belongs to Brassicaceae, Fabaceae or Poaceae.

[0118] In one embodiment, the at least one plant species belongs to Arabidopsis, Lotus, Hordeum, Zea, Triticum, Oryza, Solanum, Glycine, Saccharum, Manihot or Musa.

[0119] In one embodiment, the at least one plant species is Arabidopsis thaliana, Hordeum vulgare or Lotus japonicus.

[0120] It is also relevant to apply the microbial consortium obtained through the methods disclosed herein to already successfully established commercial crops.

[0121] Thus, in one embodiment, the at least one plant species is a maize species, a wheat species, a rice species, a potato species, a tomato species, a soybean species, a sugarcane species, a cassava species, or a banana species.

[0122] Bacterial entity

[0123] In one embodiment the bacterial entity isolated from certain plant species and used for production of microbial consortia is an individual bacterial strain or single bacterial strains. P7198PC00

[0124] In one embodiment the bacterial entity is one individual bacterial strain (or isolate, or species, all used herein as synonyms).

[0125] In one embodiment the bacterial entity is a plurality of bacterial strains, such as a plurality of individual bacterial strains.

[0126] In one embodiment the bacterial entity is a naturally occurring bacterial consortium.

[0127] In one embodiment the bacterial entity is a synthetic bacterial consortium.

[0128] Hence, in one embodiment, obtaining a genomic sequence of the bacterial entity corresponds to obtaining a genomic sequence of one individual bacterial isolate. In one embodiment, wherein the bacterial entity comprises a plurality of bacterial isolates, obtaining a genomic sequence of the bacterial entity corresponds to obtaining the collective genetic material present within the community, for example through metagenomic sequencing.

[0129] Homologous protein sequences

[0130] Some of the aspects of the present disclosure relate to microbial consortia that successfully colonize roots. These microbial consortia comprise at least 50% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, or of their corresponding nucleotide sequences defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II. These microbial consortia may further comprise at least 50% of the homologous protein sequences as defined in Table B - I, or functional variants of the homologous protein sequences as defined in Table B - I, or of their corresponding nucleotide sequences defined in Table B - II, or functional variants of the nucleotide sequences as defined in Table B - II.

[0131] In one embodiment, step d. of the method for producing a microbial consortium comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, such as at least 60 % of the entries listed in Table A-l, such as at least 65 % of the entries listed in Table A-l, such as at least 70 % of the entries listed in Table A-l, such as at least 75 % of the entries listed in Table P7198PC00

[0132] A— I , such as at least 80 % of the entries listed in Table A— I , such as at least 85 % of the entries listed in Table A— I, such as at least 90 % of the entries listed in Table A— I, such as at least 95 % of the entries listed in Table A— I , such as at least 99 % of the entries listed in Table A— I , or such as about 100 % of the entries listed in Table A— I , thereby producing a microbial consortium that successfully colonises roots of a plant species.

[0133] Thus, in one embodiment, the microbial consortium comprises and / or expresses at least 50% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, such as at least 55%; such as at least 60%; such as at least 65%; such as at least 70%; such as at least 80%; such as at least 85%; such as at least 86%; such as at least 88%; such as at least 90%; such as at least 92%; such as at least 94%; such as at least 98%; such as at least 99%; such as about 100% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I.

[0134] Moreover, in one embodiment, the obtained microbial consortium comprises at least 85% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, such as at least 86%; such as at least 88%; such as at least 90%; such as at least 92%; such as at least 94%; such as at least 98%; such as at least 99%; such as about 100% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I.

[0135] Similarly, in one embodiment, the obtained microbial consortium comprises and / or expresses at least 50% of the homologous protein sequences as defined in Table B - 1, or functional variants of the homologous protein sequences as defined in Table B - I, such as at least 55%; such as at least 60%; such as at least 65%; such as at least 70%; such as at least 80%; such as at least 85%; such as at least 86%; such as at least 88%; such as at least 90%; such as at least 92%; such as at least 94%; such as at least 98%; such as at least 99%; such as about 100% of the homologous protein sequences as defined in Table B - I, or functional variants of the homologous protein sequences as defined in Table B - I. P7198PC00

[0136] In yet a further embodiment, the obtained microbial consortium comprises and / or expresses at least 85% of the homologous protein sequences as defined in Table B - 1, or functional variants of the homologous protein sequences as defined in Table B - I, such as at least 86%; such as at least 88%; such as at least 90%; such as at least 92%; such as at least 94%; such as at least 98%; such as at least 99%; such as about 100%.

[0137] When the microbial consortium comprises a plurality of bacterial strains, such as a plurality of individual bacterial strains, each strain should comprise at least 50% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, or their respective nucleotide sequences as defined in Table A - II, or functional variants thereof, and the whole microbial consortium should preferably comprise at least 80% of the homologous protein sequences as defined in Table A - 1, or their respective nucleotide sequences as defined in Table A - II, or functional variants thereof, more preferably about 100% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, or their respective nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II.

[0138] In one embodiment of the present disclosure, the microbial consortium is a plurality of bacterial strains, a naturally occurring bacterial consortium or a synthetic bacterial consortium, wherein: a. each bacterial strain comprised in the microbial consortium comprises at least 50 % of the homologous protein sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l, or at least 50 % of the homologous nucleotide sequences as defined in Table A-l I and having the same biological function as the corresponding sequence in Table A-l I, and b. the microbial consortium comprises at least 80 % of the homologous protein sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l, or at least 80 % of the homologous nucleotide sequences as defined in Table A-l I and having the same biological function as the corresponding sequence in Table A-l I, preferably about 100 % of the homologous protein sequences as defined in Table A-l or the P7198PC00 homologous nucleotide sequences as defined in Table A— 11, each having the same biological function as the corresponding sequence in the respective table.

[0139] In another embodiment of the present disclosure, the microbial consortium is a plurality of bacterial strains, a naturally occurring bacterial consortium, or a synthetic bacterial consortium, wherein: a. each bacterial strain comprised in the microbial consortium comprises at least 50 % of the homologous protein sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l, or at least 50 % of the homologous nucleotide sequences as defined in Table A-ll and having the same biological function as the corresponding sequence in Table A-ll, and at least 50 % of the homologous protein sequences as defined in Table B-l and having the same biological function as the corresponding sequence in Table B-l, or at least 50 % of the homologous nucleotide sequences as defined in Table B-l I and having the same biological function as the corresponding sequence in Table B— II, and b. the microbial consortium comprises at least 80 % of the homologous protein sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l, or at least 80 % of the homologous nucleotide sequences as defined in Table A-ll and having the same biological function as the corresponding sequence in Table A-ll, preferably about 100 % of the homologous protein sequences as defined in Table A-l or the homologous nucleotide sequences as defined in Table A-ll, each having the same biological function as the corresponding sequence in the respective table.

[0140] In yet another embodiment of the present disclosure, the microbial consortium is a plurality of bacterial strains, a naturally occurring bacterial consortium, or a synthetic bacterial consortium, wherein: a. each bacterial strain in the microbial consortium comprises at least 50 % of the homologous protein sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l, or at least 50 % of the homologous nucleotide sequences as defined in Table A-ll and having the same biological function as the corresponding sequence in Table A-ll, and b. the microbial consortium comprises at least 80 % of the homologous protein sequences as defined in Table A-l and having the same biological function as P7198PC00 the corresponding sequence in Table A— I, or at least 80 % of the homologous nucleotide sequences as defined in Table A-ll and having the same biological function as the corresponding sequence in Table A-ll, and at least 80 % of the homologous protein sequences as defined in Table B-l and having the same biological function as the corresponding sequence in Table B-l, or at least 80 % of the homologous nucleotide sequences as defined in Table B-l I and having the same biological function as the corresponding sequence in Table B— 11, preferably about 100 % of the homologous protein sequences as defined in Table A-l or the homologous nucleotide sequences as defined in Table A-ll, and about 100 % of the homologous protein sequences as defined in Table B-l or the homologous nucleotide sequences as defined in Table B— II, each having the same biological function as the corresponding sequence in the respective table.

[0141] Measurement of homologous protein sequences / identification techniques Step c. of the method of the present disclosure, that is determining presence of the homologous protein sequences as defined in Table A - l, in the obtained genomic sequence, may be conducted using known methodologies. Example 1 provides an example of which methodologies can be used; however, a skilled person would know how to substitute one or more specific methodologies with other known in the field.

[0142] In one embodiment of the present disclosure, determining presence of the homologous protein sequences, as defined in Table A - l, and / or Table B - I, or their corresponding nucleotide sequences as defined in Table A - ll and / or Table B - II, in the obtained genomic sequence comprises performing proteome analysis.

[0143] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, , b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, f. Obtaining annotated proteins, P7198PC00 thereby determining the presence of the homologous protein sequences as defined in Table A-l and / or having the same biological function as the corresponding sequence in Table A-l in the bacterial entity.

[0144] In one embodiment of the present disclosure, determining presence of the homologous protein sequences, as defined in Table A - I, in an individual bacterial isolate comprises performing proteome analysis. For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, f. Obtaining annotated proteins, and g. conducting a protein-protein alignment (for example using BLASTp) thereby determining the presence of the homologous protein sequences as defined in Table A-l and / or having the same biological function as the corresponding sequence in Table A-l in the individual bacterial isolate.

[0145] In one embodiment of the present disclosure, determining presence of the homologous protein sequences, as defined in Table B - I, in the bacterial entity comprises performing proteome analysis. For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identification of protein-coding genes, c. Functional annotation of said protein-coding genes, d. Comparison of said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, and f. Obtaining annotated proteins, thereby determining the presence of the homologous protein sequences as defined in Table B - I and / or having the same biological function as the corresponding sequence in Table B - I in the bacterial entity. P7198PC00

[0146] In one embodiment of the present disclosure, determining presence of the homologous protein sequences, as defined in Table B - I, in an individual bacterial isolate comprises performing proteome analysis. For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, f. Obtaining annotated proteins, and g. conducting a protein-protein alignment (for example using BLASTp) thereby determining the presence of the homologous protein sequences as defined in Table B - I and / or having the same biological function as the corresponding sequence in Table B - I in the individual bacterial isolate.

[0147] In one embodiment of the present disclosure, determining presence of the nucleotide sequences, as defined in Table A - II, in the bacterial entity comprises performing proteome analysis. For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identification of protein-coding genes, c. Functional annotation of said protein-coding genes, d. Comparison of said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, and f. Obtaining annotated proteins and the respective nucleotide sequences, thereby determining the presence of the nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II, in the bacterial entity. thereby determining the presence of the nucleotide sequences as defined in Table A - II and / or having the same biological function as the corresponding sequence in Table A - II in the bacterial entity. P7198PC00

[0148] In one embodiment of the present disclosure, determining presence of the nucleotide sequences, as defined in Table A - II, in an individual bacterial isolate comprises performing proteome analysis. For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, f. Obtaining annotated proteins and the respective nucleotide sequences, and g. conducting a nucleotide sequence alignment (for example using nucleotide BLAST) thereby determining the presence of the nucleotide sequences as defined in Table A - II and / or having the same biological function as the corresponding sequence in Table A

[0149] - II in the individual bacterial isolate.

[0150] In one embodiment of the present disclosure, determining presence of the nucleotide sequences, as defined in Table B - II, in the bacterial entity comprises performing proteome analysis. For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identification of protein-coding genes, c. Functional annotation of said protein-coding genes, d. Comparison of said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, and f. Obtaining annotated proteins and the respective nucleotide sequences, thereby determining the presence of the nucleotide sequences as defined in Table B - II and / or having the same biological function as the corresponding sequence in Table B

[0151] - II in the bacterial entity.

[0152] In one embodiment of the present disclosure, determining presence of the nucleotide sequences, as defined in Table B - II, in an individual bacterial isolate comprises performing proteome analysis. For example, said proteome analysis comprises: P7198PC00 a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, f. Obtaining annotated proteins and the respective nucleotide sequences, and g. conducting a nucleotide sequence alignment (for example using nucleotide BLAST) thereby determining the presence of the nucleotide sequences as defined in Table B - II and / or having the same biological function as the corresponding sequence in Table B

[0153] - II in the individual bacterial isolate.

[0154] The methods disclosed herein may further comprise a step of cultivating each bacterial strain comprised in the bacterial entity prior to obtaining their genomic sequences.

[0155] In one embodiment of the present disclosure, determining presence of the homologous protein sequences as defined in Table A - I, in the bacterial entity comprises performing proteome analysis.

[0156] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences P7198PC00 as defined in Table A - I and / or having the same biological function as the corresponding sequence in Table A - I in the bacterial entity.

[0157] In one embodiment of the present disclosure, determining presence of the homologous protein sequences as defined in Table A - I, in an individual bacterial isolate comprises performing proteome analysis.

[0158] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, and g. conducting a protein sequence alignment (for example using BLASTp), thereby determining the presence of the homologous protein sequences as defined in Table A - I, and / or having the same biological function as the corresponding sequence as defined in Table A - I, in the individual bacterial isolate.

[0159] In one embodiment of the present disclosure, determining presence of the homologous protein sequences as defined in Table B - I, in the bacterial entity comprises performing proteome analysis.

[0160] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, P7198PC00 d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, and f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences as defined in Table B - I, and / or having the same biological function as the corresponding sequence as defined in Table B - I, in the bacterial entity.

[0161] In one embodiment of the present disclosure, determining presence of the homologous protein sequences as defined in Table B - I, in an individual bacterial isolate comprises performing proteome analysis.

[0162] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, and g. conducting a protein sequence alignment (for example using Blastp), thereby determining the presence of the homologous protein sequences as defined in Table B - I, and / or having the same biological function as the corresponding sequence as defined in Table B - I, in the individual bacterial isolate. P7198PC00

[0163] In one embodiment of the present disclosure, determining presence of the nucleotide sequences as defined in Table A - II, in the bacterial entity comprises performing proteome analysis.

[0164] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, and f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences as defined in Table A - II, and / or having the same biological function as the corresponding sequence in Table A - II, in the bacterial entity.

[0165] In one embodiment of the present disclosure, determining presence of the nucleotide sequences as defined in Table A - II, in an individual bacterial isolate comprises performing proteome analysis.

[0166] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER P7198PC00 search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, and g. conducting a protein sequence alignment (for example using Blastp), thereby determining the presence of the homologous protein sequences as defined in Table A - II, and / or having the same biological function as the corresponding sequence in Table A - II, in the individual bacterial isolate.

[0167] In one embodiment of the present disclosure, determining presence of the nucleotide sequences as defined in Table B - II, in the bacterial entity comprises performing proteome analysis.

[0168] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, and f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences as defined in Table B - II, and / or having the same biological function as the corresponding sequence in Table B - II, in the bacterial entity.

[0169] In one embodiment of the present disclosure, determining presence of the nucleotide sequences as defined in Table B - II, in an individual bacterial isolate comprises performing proteome analysis.

[0170] For example, said proteome analysis comprises: a. Obtaining a genomic sequence of the individual bacterial isolate, P7198PC00 b. Identifying protein-coding genes, such as open reading frames (ORFs), for example via PROKKA, c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, and g. conducting a protein sequence alignment (for example using Blastp), thereby determining the presence of the homologous protein sequences as defined in Table B - II, and / or having the same biological function as the corresponding sequence in Table B - II, in the individual bacterial isolate.

[0171] In another embodiment of the present disclosure, determining presence of the homologous protein sequences as defined in Table A - I and / or Table B - 1, and / or having the same biological function as the corresponding sequence, in the bacterial entity comprises: a. Performing metagenomic analysis comprising whole-genome shotgun sequencing in the absence of culturing, b. Metagenomic assembly to generate metagenome-assembled genomes (MAGs) and, c. Determining the presence of homologous protein sequences as disclosed herein.

[0172] In another embodiment of the present disclosure, determining presence of the nucleotide sequences as defined in Table A - II and / or Table B - II, and / or having the same biological function as the corresponding sequence, in the bacterial entity comprises: a. Performing metagenomic analysis comprising whole-genome shotgun sequencing in the absence of culturing, b. Metagenomic assembly to generate metagenome-assembled genomes (MAGs) and, P7198PC00 c. Determining the presence of nucleotide sequences as disclosed herein.

[0173] In a further embodiment of the present disclosure, determining presence of the homologous protein sequences as defined in Table A - I and / or Table B - 1, and / or those having the same biological function as the corresponding sequence, in the bacterial entity and / or in an individual bacterial isolate comprises functional annotation of the identified genes, such as determination of KEGG-orthologs (KOs) and / or cluster of Orthologous Groups (OG). Both database-dependent and database-independent methods may be used for the functional annotation.

[0174] A method of improving plant growth

[0175] Typically, the roots of a plant, or even the seeds, are inoculated with a microbial consortium or SynCom with the purpose of improving and / or enhancing plant growth, or production of valuable plant material, for example increasing crop yield. However, this can only work if the microbial consortium or SynCom colonizes the roots of that plant and is competitive in relation to the microbial community already present in the growth environment or on the roots and / or in the rhizosphere.

[0176] The present disclosure provides a solution to the problem of ensuring that a microbial consortium that is meant to be inoculated on the roots of a plant has the functions required to successfully colonize the roots of said plant and thereby positively affecting the growth of said plant.

[0177] Thus, in one aspect, the present disclosure relates to a method of improving growth, productivity and / or resilience towards biotic and abiotic stresses of a plant, the method comprising inoculating root(s) of said plant with a microbial consortium that successfully colonizes roots of at least one plant species obtained according to the method disclosed herein, that by a method comprising: a. Isolating a bacterial entity from a plant microbiome, b. Determining the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l, in the bacterial entity, c. Selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding P7198PC00 to at least 50% of the entries listed in Table A— I, thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0178] In one embodiment, the method further comprises a. Determining the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table B - I, and having the same biological function as the corresponding sequence in as defined in Table B - I, in the obtained genomic sequence, b. selecting a combination of said bacterial entities such that the combination collectively comprises; i. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— I , and ii. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B-l, as identified in step a), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0179] Similarly, in another embodiment of the present disclosure the method further comprises: a. Determining presence of the nucleotide sequences as defined in Table B - II, or the same biological function as the corresponding sequence in Table B - II, in the obtained genomic sequence, b. Selecting the bacterial entity corresponding to the obtained genomic sequence comprising at least 50% of the nucleotide sequences as defined in Table B - II, the same biological function as the corresponding sequence as defined in Table B - II, and at least 50% of the nucleotide sequences as defined in Table A - II, or the same biological function as the corresponding sequence as defined in Table A - II, thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0180] In one embodiment, said biotic stress are plant pathogens.

[0181] Nutrients in the soil are dissolved in water allowing them to be taken up by roots. When soil moisture is low, fewer nutrients are dissolved, reducing the availability of essential nutrients like nitrogen, phosphorus, and potassium for uptake by roots. Roots rely on water to move nutrients from the soil into the plant. A low soil moisture slows these P7198PC00 processed down leading to nutrient deficiencies even if the soil contains adequate nutrients. When plants are under water stress, their overall health declines, making them more susceptible to pathogens.

[0182] In one embodiment, said abiotic stress is drought, salinity, heat stress, cold stress, oxidative stress, heavy metal toxicity, low soil pH, high soil pH and / or nutrient limitation.

[0183] Salinity stress could affect a plant in many ways. Excess soluble salts, such as Na+, in the soil reduce the water potential of the soil around the root surface, thereby limiting water availability for the plant and reducing plant water uptake. Thus, salt stress leads to water deficit or osmotic stress for plants, which are significant problems for plants grown in saline soils. Water deficit reduces cell turgor, disturbs water relations, and affects water-use efficiency in plants. It also induces stomatai closure and disrupts photosynthetic pigments.

[0184] Heat stress triggers dehydration in plants, which stunts their development. Reduced photosynthetic production is a direct result of heat stress effect on the leaf relative water content and the plant's water potential. When crops are under severe heat stress, they often lose their leaves. Buds, flowers and growing fruit may fall off. This can result in large financial losses for a farmer.

[0185] Cold stress or chilling stress affects membrane rigidification in plant cells, which is considered to be the primary event that triggers downstream cold-stress responses in plants. It disturbs the stability of proteins or protein complexes and reduces the activities of enzymes such as ROS scavenging enzymes. These processes result in photo-inhibition and impaired photosynthesis, as well as considerable membrane damage. It also affects gene expression and protein synthesis. Eventually ice crystals form leading to irreversible damage. The temperature where a plant experiences damaging heat or cold stress is depends on the species.

[0186] Oxidative stress is a complex chemical and physiological phenomenon that accompanies virtually all biotic and abiotic stresses in higher plants and develops as a result of overproduction and accumulation of reactive oxygen species. P7198PC00

[0187] Oxidative stress could for example be characterized by the accumulation of reactive oxygen species in plant cells, measured as increased levels of reactive oxygen species, such as hydrogen peroxide or superoxide anion.

[0188] Heavy metal toxicity in plants depends on the bioavailability of these elements in soil solution, which is a function of pH, organic matter and cation exchange capacity of the soil. Bioaccumulation of heavy metals in excessive concentrations may replace essential metals in pigments or enzymes disrupting their function and causing oxidative stress. Heavy metal toxicity hinders the growth process of the underground and aboveground plant parts and the activity of the photosynthetic apparatus, which is often correlated with progress in senescence.

[0189] Soils can be naturally acid or alkaline. Having the correct pH is important for healthy plant growth. Some soil management practices can alter the pH over time. Soil pH affects the solubility of nutrients and therefore its availability to plants. Some nutrients are more available under acid conditions while others are more available under alkaline conditions. Since soil pH affects nutrient availability to roots, the primary symptoms of adverse soil pH are similar to those of nutrient deficiencies and toxicity.

[0190] Nutrient depletion in soils occurs when essential minerals and nutrients, such as nitrogen, phosphorus, potassium, and other trace elements, are used up faster than they can be replenished. This is often a result of intensive farming practices, overuse of chemical fertilizers, soil erosion, and lack of crop rotation. Whether a plant suffers from nutrient limitation stress depends on many factors such as the availability of the available nutrients to the plants and he general nutrient requirements of different species. Microbial consortia can be utilised to improve the cycling of essential nutrients like nitrogen, phosphorus, and potassium. For example, certain bacterial entities can fix atmospheric nitrogen into a form that plants can use, while others might solubilize phosphorus from the soil, making it more available to crops.

[0191] In one embodiment, inoculating a plant with a microbial consortium produced according to the methods disclosed herein results in improved plant growth compared to a plant not inoculated with a microbial consortium produced by the methods disclosed herein. P7198PC00

[0192] Examples

[0193] Example 1 - Assessment of taxonomic and functional diversity across culture collections of rhizobacteria isolated from Arabidopsis thaliana, Hordeum vulgare, and Lotus japonicus.

[0194] Materials and Methods

[0195] The following abbreviations are used below:

[0196] AtSC: Arabidopsis thaliana SynCom HvSC: Hordeum vulgare SynCom LjSC: Lotus japonicum SynCom SSC: SuperSynCom representing all three Syncoms together

[0197] A culture collection of bacteria associated with roots of healthy Arabidopsis thaliana grown in natural Reijerscamp soil from Utrecht, The Netherlands (AtSC) was obtained and additionally, a combined culture collection from both Barley and maize, both grown in soil from Askov, Denmark (HvSC) was obtained.

[0198] The Arabidopsis rhizobacterial culture collection was established by isolating the bacteria associated with roots of Arabidopsis seedlings at three- and seven-days posttransplantation from MS agar plate to Reijerscamp soil. Roots of soil-grown plants were washed in 10 mM MgSCU, diluted 10A6 times, and plated on 1 / 1 Oth tryptic soy agar, Reasoner’s 2A Agar, yeast extract medium, minimal medium with 2 mM coumarin, potato dextrose agar and King’s B medium. Single colonies were picked after three, five, seven, ten, fourteen and twenty-one days after plating and individually cultivated in liquid tryptic soy broth. Bacterial isolates were subjected to high throughput two-step barcoded amplicon sequencing on the V3-V4 region of the 16S rRNA gene. The isolates were narrowed down to uniqueness based on sequence identity of the V3-V4 region of the 16S rRNA gene.

[0199] Bacteria associated with roots of Barley and maize were isolated at seven weeks post germination and growth in Askov soil. Roots were washed in sterile water and diluted 2x10A6 times in Tryptic Soy Broth or Yeast extract medium and plated in 96-well plates. Pure bacterial cultures were isolated at seven- and fourteen-days post-harvest. Bacterial isolates were subjected to high throughput two-step barcoded amplicon sequencing on the V5-V7 region of the 16S rRNA gene. The isolates were narrowed down to uniqueness P7198PC00 based on sequence identity of the V5-V7 region of the 16S rRNA gene. The HvSC was established by collating isolated rhizobacteria from Barley and maize roots.

[0200] To assess the extent to which different hosts impose a selection on their root microbiota, the isolates from these collections were first subjected to whole-genome sequencing (Illumina short read sequencing). The bacterial genomes were assembled using A5 (A5- miseq version 20160825) and compared to those previously isolated from the model legume Lotus japonicus (LjSC) grown in soil from Cologne, Germany (LjSC). The chosen hosts are taxonomically diverse and represent three major plant classes: cereals, brassica, and nitrogen-fixing legumes. Subsequently, the bacterial genomes were tested for contamination using CheckM (version 1.1.3) and cleaned for any contaminating sequences with MaxBin (version 2.2.7) and MMSeqs2 (version 13.45111). Taxonomy was inferred to the cleaned genomes using GTDB (version v2.3.0) after which the genomes were subjected to PROKKA (version 1.14.6) to retrieve the gene and protein sequences. These were annotated to KO level (KEGG ontology) with EggNOG (version 2.1.4-2). Genetic variability in SynComs was assessed by number of unique KOs present and the KO variability. The KO variability was calculated by building a dendrogram of all gene sequences with the same KO annotation within a SynCom and calculating the total and average branch length of the dendrogram (gene tree).

[0201] Results

[0202] Each culture collection contained hundreds of isolates distributed across the most common root-associated phyla and families, though, also a taxonomic separation is maintained according to their origin (data not shown). Interestingly, despite the phylogenetic distance between the three hosts, a large overlap in represented bacterial families in the three rhizobacterial culture collections exists.

[0203] With respect to the functional diversity, 55% of the encoded genes were annotated with a KO. An increase in the count of isolates in a collection was mirrored by an increase in KO diversity represented by the number of KOs (Figure 1), gene-copy number and KO intravariability (Figure 2). One deviation from this overall pattern was identified; the HvSC exceeded the AtSC in the number of KOs, even if the latter contained more isolates. The analysis showed that the three collections overlapped for approximately 80% of their KOs (6,255 of 8,013 total KOs), even if originating from different plants grown in soils with different properties and located up to 500 km apart. P7198PC00

[0204] Conclusion

[0205] These results support the well-established finding that plants associate with taxonomically diverse bacterial isolates that originate from the soils in which they are grown. Importantly, the extensive functional overlap between plants and the functional complementarity of bacteria per family is shown here. This demonstrates that hosts select functionally analogous but taxonomically distinct isolates, implying that with complex culture collections, pinpointing bacterial functions which are advantageous for host association at the isolate level is achievable.

[0206] Example 2 - Assessment of isolate and KO diversity in the root microbiome of Arabidopsis thaliana, Hordeum vulgare, and Lotus japonicus.

[0207] Materials and Methods

[0208] Reconstitution experiments were conducted by exposing Arabidopsis, Barley, and Lotus to the three collections separately, as well as to an assemblage of all isolates together (SuperSynCom or SSC) (Figure 3). Bacterial isolates were cultivated independently from each other in either Tryptic soy broth or Yeast extract media (depending on the media they were isolated from) and, after five days of growth, washed with Long Ashton media, and proportionally pooled together according to the concentration (OD600 measurement). To pinpoint host-dependent community assemblies and reduce the influence of substrate-dependent bacterial enrichment, a minimal growth system was implemented, cultivating plants for three weeks with one of the host-derived inocula or the SuperSynCom in a sterile inert substrate supplemented with low nutrients. The entire experiment was performed twice and included a replicate with nutrient-rich growth media. The low nutrient condition indicated that the plants were supplemented with Long Ashton media. The high nutrient condition consisted of a modified Long Ashton media, in which the micronutrient concentration was similar to the low nutrient condition, but the macronutrient concentration (phosphate, nitrate, and potassium) was taken from the high nutrient Hoagland solution.

[0209] After three weeks, the roots and rhizospheres were harvested from these plants by washing the roots with sterile water. The first wash comprises the rhizosphere, while the root fraction was taken after five sterile water washes (shaking the roots vigorously after adding sterile water to the roots). From these fractions, the DNA was extracted and P7198PC00 subjected to shotgun metagenome sequencing. For Lotus, nodules were removed from the roots and kept as a separate fraction.

[0210] The metagenome sequencing reads were cleaned for host-derived reads (Bowtie2 version 2.2.5) and subsequently mapped to the whole genome sequences of the bacterial isolates using Salmon (version 1.9.0). Isolate numbers were subsequently extrapolated to KO numbers using the genome-encoded KOs (isolate counts multiplied with bacterial genome KO counts). To do this, the KO counts per genome table and the isolate counts per microbiome sample table were inserted into the PICRUSt2 algorithm (version 2.5.1) without using the 16S rRNA copy number normalization.

[0211] The selection at the functional level both in numbers and in diversity was assessed by performing diversity analyses for the isolates and the genome-encoded KOs. The alpha diversity analysis — a measure for within community diversity — shows the number of isolates (Figure 4A) or KOs (Figure 4B) that are present in the root microbiomes of the distinct hosts inoculated with distinct inocula. The more features (isolates or KOs) are present in the root microbiome, the more diverse it is regarded. The beta diversity analysis (data not shown) compares the microbiome composition across hosts and isolates and calculates how the host and inocula affect these compositions. The output of this analysis is often visualized in Principal component or coordinate analyses, in which clustered samples indicate a similar microbiome composition and samples that are distant from each other are more different in microbiome composition. Diversity analyses are commonly used in ecological studies, investigating the diversity of species in an ecosystem or a microbiome.

[0212] Results

[0213] The diversity analyses revealed that all hosts performed a clear selection of the isolates and KOs across all inocula when shaping their root communities. Irrespective of initial bacterial richness, between 76 to 146 isolates were identified (on average 119; >0.5% relative abundance) associated with the roots, a number that varied significantly with the plant and the inoculum (Figure 4A). With an average of 97 isolates across inocula, Lotus was the most restrictive host; Barley, on the other hand, was the most permissive, averaging 134 isolates, and Arabidopsis occupying the middle ground at 123 isolates. In addition, root communities of all hosts demonstrated a reduced number of KOs compared to inoculum (Figure 4B). A large reduction was observed, especially, in the presence of HvSC or SSC, which could reflect larger effects of microbe-microbe interactions. P7198PC00

[0214] The principal coordinate analysis (beta diversity) revealed a clear separation of the different communities based on the inoculum (R2inOcuium 0.415), and that the host had a much-reduced impact (R2piant 0.203), followed by a much-reduced effect of the combined inoculum and host (R2inocuium:Piant 0.09) (data not shown). The nutrient regimes or experimental replication had a significant but negligible effect on the overall variation. When performing the same analysis of Bray-Curtis differences at the KO level, a similar separation was identified, however the individual communities were much more clustered indicating a reduced variation at the functional compared to taxonomic level. This was further corroborated by permutational multivariate analysis of variance (PERMANOVA) revealing reduced impact of inoculum on KO composition compared to genus or isolate level (R2KO 0.266 vs R2Genus 0.415 and R2iSoiate 0.436). PERMANOVA analysis across all inocula revealed that individual hosts had a larger impact on the overall functional composition in root communities compared to that on the taxonomic composition at genus or isolate level (R2KO 0.288 vs R2Genus 0.203 and R2iSoiate 0.129).

[0215] Conclusion

[0216] These reconstitution experiments revealed a similar pattern to that observed when comparing the three independent collections isolated from the natural soil, namely a large functional overlap across hosts and inocula.

[0217] Plant roots have a highly selective capacity when accommodating taxa and functions which importantly remains largely stable across inoculum’s diversity and complexity.

[0218] Together, these results provide evidence that Arabidopsis thaliana, Hordeum vulgare, and Lotus japonicus select the isolates from the initial inoculum primarily based on the encoded functions, and this selection is not discernible when communities are analysed at a low taxonomic level, e.g. isolate or genus like currently and widely depicted by 16S rRNA amplicons.

[0219] Example 3 - Assessment of the functional diversity of bacterial strains with relation to root community assembly

[0220] Materials and Methods

[0221] The number of unique functions of the bacterial strains of a certain family was correlated to the abundance of the bacterial strains of that family on the roots across the three hosts P7198PC00 to investigate whether functionally diverse bacterial strains have a better root colonization capacity and play a larger role in root community assembly.

[0222] Results

[0223] Bacterial isolates that encode larger proportions of the respective familial functions display a higher abundance in root communities when compared to the remaining isolates within the same family (Figure 5A). This trend is visible in nine of the 17 families as well as in six genera of the largest family (Burkholderiaceae) (Figure 5B).

[0224] Conclusion

[0225] A functionally diverse bacterial strain is more likely to efficiently colonize the roots irrespective of host compared to a functionally less diverse isolate from the same family. In other words, strains that cover a larger breath of the functions encoded by the family they belong to, have an increased capacity to colonise the root when compared to other isolates from the same family encoding for fewer familial functions.

[0226] Example 4 - Assessment of host specificity of inocula by investigating the host's effect on the KO composition in the root microbiome.

[0227] Materials and Methods

[0228] To assess the host-specificity with regard to the functional (KO) composition in the root microbiome, PERMANOVA analyses were implemented to calculate what proportion of compositional differences are because of the distinct hosts. By computationally removing one host at a time and reimplementing the PERMANOVA on the dataset with the remaining two hosts, the compositional differences caused by only the two remaining hosts can be compared to the compositional differences caused by all three hosts. The size of this difference gives an indication to what degree each host affects the functional (KO) composition in the roots.

[0229] Results

[0230] Root communities associated with Lotus had the largest contribution to the overall variation in the dataset and this was largest in the presence of inocula containing nitrogen-fixing symbionts (HvSC, LjSC, or SSC) (Figure 6A). P7198PC00

[0231] By contrast, removing Barley or Arabidopsis from the analysis had minimal consequences on the observed community variation, with one exception, Arabidopsis in the presence of AtSC.

[0232] Parallel to the observed host-controlled interaction leading to the recruitment and enrichment of nitrogen-fixing symbionts by Lotus, the dataset also contains an inoculumdependent enrichment of a specific member of the community. Rhizobacter sp. isolate P2_G4 was highly enriched in root communities of all plant hosts when inoculated with HvSC. This enrichment, however, was significantly reduced when P2_G4 was a member of the SSC, indicating that HvSC provides a microbial context favouring the enrichment of this isolate.

[0233] Importantly, in the absence of the symbionts and P2_G4, each host was found to have the greatest contribution to functional variation in the presence of their own inocula (Figure 6B).

[0234] Conclusion

[0235] This example shows that different plant hosts can have a different impact on the observed variation in root communities. In this example, the presence of a host with an active nitrogen-fixing symbiosis has the largest impact on the functional variation identified in the recruited root communities identified.

[0236] Lotus and Arabidopsis have a larger impact on the observed bacterial functions compared to Barley, and this is most evident in the presence of communities that have host-compatible members. For Lotus, these would be the communities with a compatible symbiont, while for Arabidopsis this is a community previously enriched by Arabidopsis itself.

[0237] Together, the analyses provide evidence that plant hosts select commensals from the microbial environment based on their functions and that this selection can be largely affected by the presence of specific members which are host-enriched (symbionts) or inoculum-dependent (Rhizobacter sp. P2_G4). This selection has a significant consequence for the community composition and the overall enriched bacterial functions. P7198PC00

[0238] Example 5 - Assessment of general KOs associated with root colonization in complex communities irrespective of host.

[0239] Materials and Methods

[0240] Analyses of root communities provide a unique opportunity to pinpoint general functions selected by plant roots as well as those dependent on these major determinants. For this, a differential abundance analysis of bacterial KOs was conducted to discover enriched KO in the roots of the three hosts versus start inocula. DESeq2 (version 1 .40.0) was used to conduct this differential abundance analysis. Significance was tested by a Wald test and p-values were adjusted for multiple testing using the Benjamini and Hochberg method (significance p = 0.05). DESeq2 was run to find the enriched KOs in the root microbiome as compared to the initial inoculum and compare this between hosts and inocula. This analysis was run on the dataset in which the dominating strains were excluded (Lotus symbionts and HvSC-context dominant strain Rhizobacter P2_G4) to better disentangle the effect of the inoculum. When including those strains, enriched functions will mainly derive from these individual isolates and not from the community.

[0241] Results

[0242] First, it became clear that, overall, a significant share of initial KOs (45.7% to 59.3%; i.e. , 3500-4000 KOs / 7500 total) was enriched in root communities, an outcome which is tied to the root-isolated origin of the strains (data not shown). The KOs that were higher abundant in the root microbiome as compared to the start inoculum were subjected to multiple filtering steps to narrow down the number of KOs to the most interesting set (Figure 7A). This analysis showed 266 KOs (Table A - 1 and Table A - II) to be enriched in all twelve combinations of host and inoculum, which do not show any signature of hostspecificity (Figure 8). The host-specificity was assessed by calculating the proportion of two-fold changes, the fold change of bacterial strains with the KO relative to the fold change of bacterial strains without the KO. The fold change, in this regard, is the abundance increase from the start inoculum to the root microbiome (Figure 7B).

[0243] The isolates from the collections were inspected for the presence of these common 266 KOs and found that none contained them all, few encode for approximately half of them, but majority have less than a quarter (Figure 9). Bacteria belonging to different taxa contain different proportion of these common 266 KOs (Figure 23) which have different P7198PC00 functions according to KEGG database (Figure 23), illustrating that their enrichment is a communal function.

[0244] Next, these findings were compared with those revealed by a previous comprehensive and unbiased meta-analysis performed by Levy et al. (2018), on 3,837 deposited bacterial genomes. 6,581 KOs of the 8,013 KOs were included in this study and 3,566 of these were plant-associated, from which 177 (from the 266 KOs) overlap (Figure 10). The isolates analysed by Levy et al. (2018), were also inspected for the presence of the common KOs identified in the study and a similar distribution pattern was found as observed for the collections and that, unlike soil and non-plant associated bacteria, those recruited by plants can contain a larger proportion of these KOs (Figure 10). Figure 10 illustrates that the 266 functions of Table A - I can be used to pinpoint if an isolate is likely a plant associated one rather than a soil-residing one.

[0245] Table A - I: KEGG orthologues (KO) that have been found to be key biological functions for bacterial survival in complex communities associated with plant roots. These biological functions are defined by KEGG Orthology identifiers (KO) and represent protein-coding sequences that are functionally annotated under those identifiers. The SEQ ID NOs of the protein sequences as provided in the sequence listing enclosed are example sequences corresponding to each KO, listed in order as they appear in the table: SEQ ID NO: 1 to SEQ ID NO: 266. P7198PC00 P7198PC00 P7198PC00 P7198PC00 P7198PC00 P7198PC00 P7198PC00 P7198PC00

[0246] Table A - II: Nucleotide sequences encoding the protein sequences functionally annotated under the KEGG Orthology identifiers listed in Table A-l. The SEQ ID NOs of the nucleotide sequences as provided in the sequence listing enclosed are, in order as they appear in the table: SEQ ID NO: 500 to SEQ ID NO: 765. P7198PC00 P7198PC00 P7198PC00

[0247] Conclusion

[0248] This informs us that the functions recruited by plant roots were not isolated actions of single microbes but rather the collective outcome of interactions within the microbial community.

[0249] The 266 general functions found in this study could be considered key functions for bacterial survival in complex communities associated with plant roots.

[0250] Example 6 - Assessment of host-specificity of bacterial functions regarding root microbiome assembly.

[0251] Materials and Methods

[0252] To investigate KOs that were specifically enriched by a particular host and identify possible host-preferred bacterial functions within their root communities, a different filtering step was used as compared to the general enriched KOs in Table A. A threshold was implemented that categorizes a host-specific KO when it is highly enriched in one P7198PC00 host. That means a fold change of isolate with KO >3 while <3 for the other two hosts (Figure 11). The fold changes are calculated similarly as in Figure 7B.

[0253] Results

[0254] A reduced number of KOs was found to be highly enriched (FC>3 in at least three out of four inocula) in Arabidopsis (n=40), Barley (n=48) or Lotus with (n=355) or without the symbionts (n=61) (Figure 12). In Arabidopsis, genes were found involved in biosynthesis of bacteriochlorophyllides by photosynthetic bacteria to be enriched corroborating with the enrichment of isolates belonging to Rhodobacter in this host (data not shown). KOs corresponding to enzymes that add pyruvate {exoV) or acetyl groups (exoZ) to the succinoglycan polysaccharide chain were found Barley-enriched, while those for catabolism of erythritol, biosynthesis of Nod factors (nod), and for nitrogen fixation (n / 7) were Lotus specific (data not shown).

[0255] When grouping the host-specific KOs according to pathway annotation, the most frequently host-specific enriched KOs are the ABC transporter KOs (data not shown). In a similar analysis conducted in Figure 5, the correlation between ABC transporter KO diversity and abundance on the root was assessed for each host and inoculum (data not shown). This demonstrates how bacterial strains encoded with a diverse palette of ABC transporters are more abundant in the root microbiome when inoculated with the AtSC or HvSC, especially for Lotus. Evidently, having a large diversity of ABC transporter KOs are not found among the abundant bacterial strains in the root microbiome in the LjSC and SSC. Interestingly, even though this correlation is absent for the LjSC, the LjSC isolates, on average, possess a more diverse palette of ABC transporter KOs compared to AtSC and HvSC isolates.

[0256] The compounds that these enriched ABC transporter KOs transport across the bacterial cell membrane are diverse, especially for Lotus (Figure 13), though also Arabidopsis- specific compounds (e.g. Arginine) or Arabidopsis and Barley-specific compounds can be found (e.g. myo-inositol 1 -phosphate and mannopine) (Figure 13).

[0257] Conclusion

[0258] Together, a moderate but distinguishable effect of the specific hosts for recruitment of bacteria based on specific functions was found. P7198PC00

[0259] Arabidopsis and Barley are more versatile and can select necessary functions from a defined microbial environment. Alternatively, the two hosts might have a lower influence on the selected KOs, in which case they will emerge in the root communities due to microbe-microbe interactions within the individual communities.

[0260] Lotus communities contained isolates with a larger panel of functions, and since this pattern was also observed for inoculum without symbionts (AtSC) or when these were computationally excluded from the analysis, indicates that this is not a symbiosis-related pattern. This larger diversity of functions combined with the knowledge that Lotus was also more restrictive for the number of isolates enriched in its root communities indicate that Lotus selectively recruits isolates from most of the four inocula that encode a larger and more diverse panel of functions compared to Arabidopsis and Barley.

[0261] An enhanced capacity for taking up diverse metabolites from the environment could provide LjSC isolates with better survival and growth capacity in a plant-root derived environment.

[0262] Example 7 - Selection of core strains from dataset and assessment of their individual competitiveness across SynComs.

[0263] Materials and Methods

[0264] To investigate the root competence and competitiveness in individual bacterial strains (not in communities but in a complex community context), a core set of bacterial strains was selected according to the abundance-occupancy distributions computed in Shade & Stopnisek (2019), which selects both abundant and consistently present bacterial strains in the root microbiome.

[0265] To differentiate the highly competitive SynCom members in the core sets, differential abundance analysis was performed for each SynCom member abundance comparing the root microbiome and the SynCom inoculum using ANCOM-BC (version 2.0.3) with default parameters (Lin & Peddada, 2020). ANCOM-BC uses the relative abundance of SynCom members in the different samples as input, and tests for significant differences between groups (inoculum versus microbiome here) using a generalized linear model or GLM. See Lin & Peddada, 2020 for a complete description of the method. P7198PC00

[0266] Results

[0267] A total of 47, 37, 54 and 62 strains were found to be a core strain for one or multiple hosts in the AtSC, HvSC, LjSC and SSC respectively (Figure 14 and Table X - I, II, III, IV). The overlap in these SynComs across the three plants was 11 , 7, 10, and 7 strains in the AtSC, HvSC, LjSC and SSC respectively, see Table X - 1. Additionally, 6 / 7 of the general core strains in the SSC were found to also be a general core strain in their respective host-specific SynComs. Aside from strains that consistently colonize all three hosts, a large degree of overlap was also found in core sets between Arabidopsis and Barley.

[0268] Rhizobiaceae and Burkholderiaceae strains were consistently found among the best root colonizers in every combination of plant and SynCom (data not shown). Bacterial families that display host-specificity are the Beijerinckiaceae, Pseudomonadaceae and Xanthomonadaceae (Arabidopsis and Barley-specific), while other families, like the Caulobacteraceae and Xanthomonadaceae, display SynCom-specificity by colonizing all three hosts but only originating from one or two hosts.

[0269] Evidently, the SSC general core strains (LjRoot3, LjRoot221 , LjRoot149, LjRoot135, LjRoot38, LjRoot206, and P2_G4) display a high competitiveness, illustrated by their large increase in abundance in both the host-specific SynComs and the SSC as compared to the inoculum (Figure 15).

[0270] Aside from consistently dominant strains, strains that only display competitiveness in host-specific SynComs or for one host were also found. Examples of these include an Arabidopsis-derived Pseudomonas (KB_12) that is only competitive in the AtSC context (Figure 15) and an Arabidopsis-derived Rhizobium (2TAF27) that is only competitive in the Barley root microbiome (data not shown). Not surprisingly, Lotus’ top root colonizers almost all represent nitrogen-fixing Mesorhizobium strains (P2_A12, P2_D6, P1_H10 and LjNodule214) (data not shown). Such strains are capable of inducing nodule formation in Lotus which likely drive their high abundance on Lotus roots, even in the absence of nodules. P7198PC00

[0271] Table X - I: Core strains consistently recruited by all three plants from each SynCom (SSC, AtSC, LjSC, HvSC). The number in parenthesis in the column “Closest species” indicates the sequence identity percentage of the isolated strain compared to the closest match in the database, which was inferred using the GTDB database (v2.3.0, Chaumeil et al., 2020). P7198PC00

[0272] The core strains from the SSC are highly root competent in multiple SynCom contexts. Root colonization strategies of bacteria might be more similar for Arabidopsis and Barley as compared to Lotus.

[0273] Some isolates are consistently efficient root colonizers, though others display differential competitiveness, depending on the host and community context.

[0274] Conclusions Interestingly, approximately 10 strains, see Table X - 1, are found in each SynCom to be consistently recruited by all three plants, supporting the existence of general root colonizers.

[0275] Example 8 - Assessment of gene clusters that are associated with root colonization in the core set of each inoculum. P7198PC00

[0276] Materials and Methods

[0277] Comparative genomics was used to compare the genome sequences of general core competitive root colonizers. Firstly, bacterial genes of all SynCom members were grouped into 113,706 groups of orthologous sequences (orthogroups) using Orthofinder (version 2.5.4; default parameters), encompassing 97.8% of all bacterial genes in the SSC. Using these orthogroups, continuous metagenome-wide association analysis (MWAS) was conducted using the change in relative abundance between inoculum and root microbiomes (Iog2foldchange values by ANCOM-BC differential abundance analysis; see example 7 and Figure 15) as input. This approach yielded 9,684 orthogroups associated with root competence across hosts and SynComs. Next, significant orthogroups among the 29 general core strains listed in Table X - I, (11 in AtSC, 7 in HvSC, 5 in LjSC, 1 in SSC, and 5 in LjSC and SSC) were selected and further filtered for those orthologous gene sequences that are part of gene clusters. Herein, gene clusters were defined by the presence of at least five consecutive significantly associated genes.

[0278] Results

[0279] 20 out of 29 general core strains encoded gene clusters with significant association to high fold change in their relative abundance between inoculum and final root microbiome (Figure 16 and Table Y).

[0280] Table Y: Unique gene clusters and / or gene clusters without predicted function. Table Y displays all the gene clusters that either lack a functional annotation or that are found in a single core strain and were therefore omitted from Figure 16. P7198PC00

[0281] Gene clusters that are linked to root colonization irrespective of the community context predominantly encode flagella, monosaccharide transport / metabolism-related proteins (ribose / xylose), and the type IV secretion system (T4SS) (Figure 16)

[0282] In the AtSC, ABC transporters involved in the transport of nickel, oligopeptides, and phosphonate is most often found among general core strains. Other types of root competent ABC transporters in the AtSC include transporters of monosaccharides, nitrate, pyrimidine, branched-chain amino acids, polar amino acids, sugar, and urea. The ability to synthesize cobalamin is also root competent in the AtSC.

[0283] In the HvSC, in turn, type II (T2SS), III (T3SS), and VI (T6SS) secretion systems are associated with root competence, as well as gene clusters involved in the transport of biopolymers, tetratricopeptide gene clusters, and chemotaxis-related gene clusters. P7198PC00

[0284] In the LjSC, some LjSC-specific gene clusters are phage proteins, phosphonate transport, and cytochrome C.

[0285] Aside from gene clusters that overlap across the general core strains, there are also gene clusters restricted to only one general core strain and gene clusters that are unannotated (Table Y). A few of these gene clusters display annotations that could play a role in enhancing competitiveness, such as the biosynthesis of antibiotics, or are associated with root competence, such as the degradation of plant cell wall components.

[0286] Conclusion

[0287] In conclusion, root competent bacterial strains display diverse metabolic capabilities, typically carry a flagellum, and encode multiple secretion systems that are predicted to be involved with host-microbe and microbe-microbe interactions.

[0288] Example 9 - Assessment of unique genes among the most competitive root colonizers and their relation to root colonization.

[0289] Materials and Methods

[0290] To identify unique bacterial traits among the general core strains that make them competitive, the unique genes of each of the seven SSC general core strains (see results, Example 7 and Table X - 1 first 7 listed isolates, corresponding to SynCom: SSC) with respect to all other SSC strains of the same genus that displayed significant association to root competence were selected.

[0291] Results

[0292] Unique (unique in comparison to related SSC strains from the same genus) genes that display significant association to root competence present in more than one SSC general core strain include T4SS genes, bleomycin resistance genes, and aminoglycoside resistance genes (Table B - I (protein sequences of the OGs; Table B - II (gene sequences of the OGs); Figures 17-18). The competence-associated gene sequences identified in this analysis were aligned with the genomes of 3,837 non-plant-associated (NPA), plant-associated (PA), root-associated (RA), and soil bacteria studied by Levy et al. (2018) (Figures 18). This alignment revealed a substantially higher number of T4SS gene occurrences in both PA and RA bacteria. Using a similar approach, the occurrence P7198PC00 of bleomycin and aminoglycoside resistance genes was investigated, showing a similar higher prevalence of these genes among PA and RA bacteria.

[0293] Additional genes that may be associated with high competitiveness and are overrepresented in PA and / or RA bacteria include those related to symbiosis (Carbamoyltransferase), breakdown of plant cell walls (Pectate lyase), resistance to oxidative stress (Mn-containing catalase), or those without any known annotation (Table B, Figures 17-18). Transporter proteins might also be linked to competitiveness, as a sugar transporter, an iron transporter, and a highly significant putative integral membrane protein were found to be overrepresented in PA and RA bacteria.

[0294] Table B - I: Orthogroups (OGs), generated by comparative genomics analysis, that have been identified as competitiveness-associated functions relevant to root colonisation. The SEQ ID Nos of the protein sequences as provided in the sequence listing enclosed are, in order as they appear in the table: SEQ ID NO: 267 to SEQ ID NO: 499. P7198PC00 P7198PC00 P7198PC00 P7198PC00 P7198PC00 P7198PC00

[0295] Table B - II: Nucleotide sequences encoding the protein sequences listed in Table B-l. The SEQ ID NOs of the nucleotide sequences as provided in the sequence listing enclosed are, in order as they appear in the table: SEQ ID NO: 766 to SEQ ID NO: 998. P7198PC00 P7198PC00 P7198PC00

[0296] Conclusions

[0297] In conclusion, the bacterial traits that likely confer a competitive advantage to the SSC general core strains are primarily related to antibiotic resistance and strategies for suppressing the plant’s immune response, such as through T4SS. Competitiveness may also arise from the ability to break down the plant cell wall, resist oxidative stress, and play an essential role in the plant’s iron or nitrogen metabolism.

[0298] Example 10 - Assessment of unique genes among the most competitive host-specific root colonizers.

[0299] Materials and Methods

[0300] To investigate host-specific and context-specific traits, the unique genes of bacterial strains exhibiting context-dependent competitiveness were analysed. This includes strains that show high abundances specifically on the root in host-specific SynComs or on the root of a single host. The focus was on several strains: Pseudomonas strain KB_12 (AtSC-specific and partially Arabidopsis-specific), Rhizobium strain 2TAF27 (Barley-specific), and nitrogen-fixing Mesorhizobium strains LjNodule214, P2_A12, P1_H10, and P2_D6 (Lotus-specific). In this example, however, the focus is only put on Pseudomonas strain KB_12.

[0301] Unique genes were defined as those genes that do not share orthologous sequences in closely related bacteria from the same family (Pseudomonas in this case). They are identified by taking the ortholog families described in Example 8, identifying which are P7198PC00 present in the relevant strain (i.e. KB_12) and display context-dependent competitiveness. Subsequently, the ortholog families that are missing in the closely related strains from the same genus are categorized as unique.

[0302] Results

[0303] Pseudomonas strain KB_12 demonstrates high competitiveness in the AtSC, particularly when inoculated on Arabidopsis, but not in the SSC. Approximately 1.2% of KB_12’s genes are significantly associated with its competitiveness in the AtSC and are unique compared to other AtSC Pseudomonas strains (Figure 20A). The genes that distinguish KB_12 from these strains are involved in chemotaxis, galactose / inositol metabolism, and denitrification (specifically nitrous oxide reduction) (Figures 20B and 21). The gene most strongly associated with competitiveness in KB_12 encodes an alpha / beta hydrolase.

[0304] Conversely, the genes absent in KB_12 but present in all four Lotus-derived Pseudomonas strains that outcompete KB_12 in the SSC are linked to the inhibition of biofilm formation (diguanylate cyclase), transport of succinate, aspartate, and fumarate (TRAP-type C4-dicarboxylate), and post-transcriptional silencing (CCR4-NOT transcriptional regulation) (Figure 20C).

[0305] Conclusion

[0306] The context-dependent competitiveness of Pseudomonas strain KB_12 underscores its specialized adaptation to the Arabidopsis-associated SynCom (AtSC) environment, where unique genes related to chemotaxis, metabolism, and denitrification drive its success. Conversely, its competitiveness is diminished in the SSC due to the absence of specific genes found in Lotus-derived strains. The methodology employed to identify these traits, through comparative analysis of unique gene sets, proves crucial in understanding how host and environmental context shape bacterial competitiveness, offering valuable insights into microbial adaptation and interaction.

[0307] Example 11 - Comparative Assessment of Functional Potential in Different Synthetic Community Designs

[0308] Aim

[0309] To evaluate the extent to which different synthetic community (SynCom) design strategies recover the functional potential of the full root microbiome, by comparing P7198PC00

[0310] KEGG Orthologue (KO) coverage in reduced SynComs versus full-complexity assemblies.

[0311] Materials and Methods

[0312] Simulated synthetic communities were computationally constructed using different isolate selection strategies from the available bacterial culture collections derived from Arabidopsis thaliana, Hordeum vulgare, and Lotus japonicus (AtSC, HvSC, LjSC). KO presence was assessed via annotated genome content using the KEGG database (as described in the preceding examples).

[0313] A reduced-complexity SynCom was assembled by selecting one isolate per bacterial family ("1 -per-family" strategy), mimicking the approach disclosed in Wippel et al. (2021). This was compared to the full SuperSynCom (SSC), which included a comprehensive representation of isolates from all three plant hosts. For each strategy, KO diversity was calculated and visualised based on the total number of unique KEGG Orthologues recovered.

[0314] The simulations were repeated 1 ,000 times for the "1 -per-family" strategy to assess robustness, and confidence intervals were determined for KO coverage estimates.

[0315] Results

[0316] The "1 -per-family" SynComs consistently recovered approximately 6,000 unique KOs (mean across simulations), with minor variance across iterations. In contrast, the SSC community design recovered over 8,000 unique KOs, representing the full functional diversity present in the three host-derived collections.

[0317] This -25% reduction in KO content in the "1 -per-family" designs reflects a significant loss of functional potential. Specifically, the reduced SynComs lacked representation of numerous genes later shown to be critical for successful root colonisation and competitiveness, including those from the 266 general KOs listed in Table A-l.

[0318] Figure 22 illustrates this outcome, showing that the simplified community design of Wippel et al (2021) inherently restricts the functional landscape that can be explored or utilised. P7198PC00

[0319] Conclusion

[0320] The strategy of selecting only one isolate per bacterial family, as implemented in Wippel et al (2021), significantly underrepresents the functional capacity of natural root microbiomes. By contrast, the broader isolate selection approach of the present invention retains the majority of KO diversity, enabling both (i) identification of key colonisation-associated genes, and (ii) construction of microbial consortia with superior functional traits.

[0321] References

[0322] Paloma Duran, Jose Flores-Uribe, Kathrin Wippel, Pengfan Zhang, Rui Guan, Barbara Melkonian, Michael Melkonian & Ruben Garrido-Oter (2022). Shared features and reciprocal complementation of the Chlamydomonas and Arabidopsis microbiota. Nature Communications volume 13, 406

[0323] Levy, A., Salas Gonzalez, I., Mittelviefhaus, M., Clingenpeel, S., Herrera Paredes, S., Miao, J., ... & Dangl, J. L. (2018). Genomic features of bacterial adaptation to plants. Nature genetics, 50(1), 138-150.

[0324] Satjarak et al. 2022. Microbiome and related structural features of Earth’s most archaic plant indicate early plant symbiosis attributes. Scientific Reports volume 12, 6423.

[0325] Shade, A., & Stopnisek, N. (2019). Abundance-occupancy distributions to prioritize plant core microbiome membership. Current opinion in microbiology, 49, 50-58.

[0326] Lin, H., & Peddada, S. D. (2020). Analysis of compositions of microbiomes with bias correction. Nature communications, 11(1), 3514.

[0327] Wippel, K., Tao, K., Niu, Y. et al. Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota. Nat Microbiol 6, 1150-1162 (2021). https: / / do i . org / 10.1038 / s41564-021 -00941-9

[0328] Items

[0329] 1 . A method of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating a bacterial entity from a plant microbiome, P7198PC00 b. Obtaining a genomic sequence of said bacterial entity, c. Determining presence of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, in the obtained genomic sequence, d. Selecting the bacterial entity corresponding to the obtained genomic sequence comprising at least 50% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0330] 2. A method of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - I, and ii. the same biological function as the corresponding sequence in Table A-l, in the obtained genomic sequence, d. Selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0331] 3. The method according to item 2, wherein step (d) comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 65% of the entries listed in Table A-l, as identified in step (c), such as at least 66% of the entries listed in Table A-l, such as at least 67% of the entries listed in Table A-l, such as at least 68% of the entries listed in Table A-l, such as at P7198PC00 least 69% of the entries listed in Table A— I , such as at least 70% of the entries listed in Table A— I, such as at least 75% of the entries listed in Table A— I, such as at least 80% of the entries listed in Table A— I , such as at least 85% of the entries listed in Table A— I , such as at least 90% of the entries listed in Table A- I.

[0332] 4. A method of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining presence of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, in the obtained genomic sequence, wherein presence of at least 50% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, in said genomic sequence indicates that the corresponding bacterial entity can successfully colonize the roots of a plant species.

[0333] 5. A method of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l; wherein the presence of protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

[0334] 6. The method according to any of the preceding items, wherein the method further comprises: P7198PC00 a. Determining presence of the homologous protein sequences as defined in Table B - I, or functional variants of the homologous protein sequences as defined in Table B - I, in the obtained genomic sequence, b. Selecting the bacterial entity corresponding to the obtained genomic sequence comprising at least 50% of the homologous protein sequences as defined in Table B - I, or functional variants of the homologous protein sequences as defined in Table B - I, and at least 50% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I.

[0335] 7. The method according to any of the preceding items, wherein the method further comprises: a. determining, for each bacterial entity, the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table B-l, and having the same biological function as the corresponding sequence in Table B-l, in the obtained genomic sequence; b. selecting a combination of said bacterial entities such that the combination collectively comprises; i) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, and ii) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B-l, as identified in step a).

[0336] 8. The method of any one of the preceding items, wherein the combination of bacterial entities collectively comprises at least 60% of the protein sequences listed in Table A-l or homologous sequences having at least 30% full-length sequence identity thereto, and comprises a biological function equivalent to that described for the corresponding sequence in Table A-l, such as at least 70% of the protein sequences listed in Table A-l, such as at least 85% of the protein sequences listed in Table A-l, such as at least 90% of the protein sequences listed in Table A-l. P7198PC00

[0337] 9. The method of any one of any one of the preceding items, wherein the homologous protein sequences having the biological function equivalent to that described for the corresponding sequence in Table A-l are sequences which are assigned to the same KEGG Orthology identifier as defined in Table A-l.

[0338] 10. A method of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating a bacterial entity from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining presence of the nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II, in the obtained genomic sequence, d. Selecting the bacterial entity corresponding to the obtained genomic sequence comprising at least 50% of the nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II, thereby a obtaining a microbial consortium that successfully colonizes roots of a plant.

[0339] 11 . A method of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - II, and ii. the same biological function as the corresponding sequence in Table A-l I, in the obtained genomic sequence, d. selecting a combination of said bacterial entities such that the combination collectively comprises nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), P7198PC00 thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

[0340] 12. A method of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. providing a bacterial entity isolated from a plant microbiome, b. obtaining a genomic sequence of said bacterial entity, c. determining presence of the nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II, in the obtained genomic sequence, wherein presence of at least 50% of the nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II, in said genomic sequence indicates that the corresponding bacterial entity can successfully colonize the roots of a plant.

[0341] 13. A method of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - II, and ii. the same biological function as the corresponding sequence in Table A-ll, in the obtained genomic sequence; wherein the presence of nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A-ll, as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

[0342] 14. The method according to any of the preceding items, wherein the method further comprises: P7198PC00 a. Determining presence of the nucleotide sequences as defined in Table B - II, or functional variants of the nucleotide sequences as defined in Table B - II, in the obtained genomic sequence, b. Selecting the bacterial entity corresponding to the obtained genomic sequence comprising at least 50% of the nucleotide sequences as defined in Table B - II, or functional variants of the nucleotide sequences as defined in Table B - II, and at least 50% of the nucleotide sequences as defined in Table A - II, or functional variants of the nucleotide sequences as defined in Table A - II.

[0343] 15. The method according to any of the preceding items, wherein the method further comprises: a. Determining, for each bacterial entity, the presence of the nucleotide sequences as defined in Table B - II, and having the same biological function as the corresponding sequence in Table B-l I, in the obtained genomic sequence, and b. Selecting a combination of said bacterial entities such that the combination collectively comprises; i. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B - II, and ii. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II.

[0344] 16. The method of any one of items 10 or 12, wherein the combination of bacterial entities collectively comprises at least 60% of the nucleotide sequences listed in Table A— 11 , or homologous sequences having at least 30% full-length sequence identity thereto, and the presence of a biological function equivalent to that described for the corresponding sequence in Table A— 11 , such as at least 70% of the nucleotide sequences listed in Table A— II, such as at least 85% of the nucleotide sequences listed in Table A— II, such as at least 90% of the nucleotide sequences listed in Table A— 11.

[0345] 17. The method of any one of items 10, 12 or 13, wherein the homologous sequences having the biological function equivalent to that described for the P7198PC00 corresponding sequence in Table A-ll are sequences which are assigned to the same KEGG orthology identifier as defined in Table A-ll.

[0346] 18. The method according to any of the preceding items, wherein the microbial consortium has the capacity to successfully adapt to a plant root environment and / or ensure competitive colonisation of the roots of a plant species.

[0347] 19. The method according to any of the preceding items, wherein the plant microbiome comprises soil, air and / or water surrounding a plant, such as wherein the plant microbiome originates from soil, air and / or water surrounding a plant.

[0348] 20. The method according to any of the preceding items, wherein the plant microbiome comprises the endosphere, phyllosphere and / or rhizosphere of a plant, such as wherein the plant microbiome originates from the endosphere, phyllosphere and / or rhizosphere of a plant.

[0349] 21. The method according to any of the preceding items, wherein the plant microbiome comprises compost.

[0350] 22. The method according to any of the preceding items, wherein the at least one plant species belongs to a viridiplantae, an angiosperm or a gymnosperm.

[0351] 23. The method according to any of the preceding items, wherein the angiosperm is a monocot or a eudicot.

[0352] 24. The method according to any of the preceding items, wherein the at least one plant species belongs to Brassicaceae, Fabaceae or Poaceae.

[0353] 25. The method according to any of the preceding items, wherein the at least one plant species belongs to Arabidopsis, Lotus, Hordeum, Zea, Triticum, Oryza, Solanum, Glycine, Saccharum, Manihot or Musa.

[0354] 26. The method according to any of the preceding items, wherein the at least one plant species is Arabidopsis thaliana, Hordeum vulgare or Lotus japonicus. P7198PC00

[0355] 27. The method according to any of the preceding items, wherein the at least one plant species is a maize species, a wheat species, a rice species, a potato species, a tomato species, a soybean species, a sugarcane species, a cassava species or a banana species.

[0356] 28. The method according to any of the preceding items, wherein the bacterial entity is an individual bacterial strain or a plurality of bacterial strains.

[0357] 29. The method according to any of the preceding items, wherein the bacterial entity is a naturally occurring bacterial consortium or a synthetic bacterial consortium.

[0358] 30. The method according to any one of the preceding items, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A— I , such as at least 60% of the entries listed in Table A— I , such as at least 65% of the entries listed in Table A— I, such as at least 70% of the entries listed in Table A— I , such as at least 75% of the entries listed in Table A— I , such as at least 80% of the entries listed in Table A— I , such as at least 85% of the entries listed in Table A— I , such as at least 86% of the entries listed in Table A— I , such as at least 87% of the entries listed in Table A— I, such as at least 88% of the entries listed in Table A- I, such as at least 89% of the entries listed in Table A-l, such as at least 90% of the entries listed in Table A-l, such as at least 95% of the entries listed in Table A-l, such as at least 99% of the entries listed in Table A-l.

[0359] 31 . The method according to any one of the preceding items, wherein the presence of protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A-l, such as at least 60% of the entries listed in Table A-l, such as at least 65% of the entries listed in Table A-l, such as at least 70% of the entries listed in Table A-l, such as at least 75% of the entries listed in Table A-l, such as at least 80% of the entries listed in Table A-l, such as at least 85% of the entries listed in Table A-l, such as at least 86% of the entries listed in Table A-l, such as at least 87% of the entries listed in Table A-l, such P7198PC00 as at least 88% of the entries listed in Table A— I , such as at least 89% of the entries listed in Table A— I , such as at least 90% of the entries listed in Table A-

[0360] I, such as at least 95% of the entries listed in Table A-l, such as at least 99% of the entries listed in Table A-l, is indicative that the bacterial entity can successfully colonise the roots of a plant species.

[0361] 32. The method according to any one of the preceding items, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A-l I, such as at least 60% of the entries listed in Table A— 11 , such as at least 65% of the entries listed in Table A-l I, such as at least 70% of the entries listed in Table A-l I, such as at least 75% of the entries listed in Table A-l I, such as at least 80% of the entries listed in Table A-l I, such as at least 85% of the entries listed in Table A-l I, such as at least 86% of the entries listed in Table A— 11 , such as at least 87% of the entries listed in Table A— II, such as at least 88% of the entries listed in Table A-

[0362] II, such as at least 89% of the entries listed in Table A— 11, such as at least 90% of the entries listed in Table A— II, such as at least 95% of the entries listed in Table A-l I, such as at least 99% of the entries listed in Table A-l I.

[0363] 33. The method according to any one of the preceding items, wherein the presence of protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A— II, such as at least 60% of the entries listed in Table A— 11, such as at least 65% of the entries listed in Table A— 11, such as at least 70% of the entries listed in Table A-l I, such as at least 75% of the entries listed in Table A-l I, such as at least 80% of the entries listed in Table A-l I, such as at least 85% of the entries listed in Table A— 11 , such as at least 86% of the entries listed in Table A— II, such as at least 87% of the entries listed in Table A- II, such as at least 88% of the entries listed in Table A— II, such as at least 89% of the entries listed in Table A— II, such as at least 90% of the entries listed in Table A— 11, such as at least 95% of the entries listed in Table A— 11, such as at least 99% of the entries listed in Table A-l I, is indicative that the bacterial entity can successfully colonise the roots of a plant species. P7198PC00

[0364] 34. The method according to any one of the preceding items, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table B— I, such as at least 60% of the entries listed in Table B— I , such as at least 65% of the entries listed in Table B— I , such as at least 70% of the entries listed in Table B— I , such as at least 75% of the entries listed in Table B— I , such as at least 80% of the entries listed in Table B— I , such as at least 85% of the entries listed in Table B— I , such as at least 86% of the entries listed in Table B— I , such as at least 87% of the entries listed in Table B— I, such as at least 88% of the entries listed in Table B-

[0365] I, such as at least 89% of the entries listed in Table B-l, such as at least 90% of the entries listed in Table B-l, such as at least 95% of the entries listed in Table B-l, such as at least 99% of the entries listed in Table B-l.

[0366] 35. The method according to any one of the preceding items, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table B— II, such as at least 60% of the entries listed in Table B— 11 , such as at least 65% of the entries listed in Table B-l I, such as at least 70% of the entries listed in Table B-l I, such as at least 75% of the entries listed in Table B-l I, such as at least 80% of the entries listed in Table B-l I, such as at least 85% of the entries listed in Table B-l I, such as at least 86% of the entries listed in Table B— 11 , such as at least 87% of the entries listed in Table B— II, such as at least 88% of the entries listed in Table B-

[0367] II, such as at least 89% of the entries listed in Table B— 11, such as at least 90% of the entries listed in Table B— II, such as at least 95% of the entries listed in Table B— 11, such as at least 99% of the entries listed in Table B— 11.

[0368] 36. The method of any one of the preceding items, wherein the homologous sequences having the same biological function as the corresponding sequence in Table A-l are sequences which are assigned to the same KEGG orthology identifier as defined in Table A-l.

[0369] 37. The method of any one of the preceding items, wherein the homologous sequences having the same biological function as the corresponding sequence P7198PC00 in Table A-ll are sequences which are assigned to the same KEGG orthology identifier as defined in Table A-ll.

[0370] 38. The method of to any one of the preceding items, wherein the homologous sequences having the same biological function as the corresponding sequence in Table B-l are sequences which are assigned to the same Orthogroup identifier as defined in Table B-l.

[0371] 39. The method of any to any one of the preceding items, wherein the homologous sequences having the same biological function as the corresponding sequence in Table B-l I are sequences which are assigned to the same Orthogroup identifier as defined in Table B-l I.

[0372] 40. The method according to any of the previous items, wherein the method comprises selecting the bacterial entity corresponding to the obtained genomic sequence comprising at least 55% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, thereby a obtaining a microbial consortium, such as comprising at least 60% of the homologous protein sequences, such as comprising at least 65% of the homologous protein sequences, such as comprising at least 70% of the homologous protein sequences, such as comprising at least 75% of the homologous protein sequences, such as comprising at least 80% of the homologous protein sequences, such as comprising at least 85% of the homologous protein sequences, such as comprising at least 90% of the homologous protein sequences, such as comprising at least 95% of the homologous protein sequences, such as comprising at least 99% of the homologous protein sequences, such as comprising about 100% of the homologous protein sequences.

[0373] 41. The method according to any of the previous items, wherein the microbial consortium comprises at least 85% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, such as at least 86%, such as at least P7198PC00

[0374] 88%, such as at least 90%, such as at least 92%, such as at least 94%, such as at least 98%, such as at least 99%, such as about 100%.

[0375] 42. The method according to any of the previous items, wherein the microbial consortium comprises at least 85% of the homologous protein sequences as defined in Table B - 1, or functional variants of the homologous protein sequences as defined in Table B - I, such as at least 86%, such as at least 88%, such as at least 90%, such as at least 92%, such as at least 94%, such as at least 98%, such as at least 99%, such as about 100%.

[0376] 43. The method according to any of the preceding items, wherein the microbial consortium is a plurality of bacterial strains, a naturally occurring bacterial consortium or a synthetic bacterial consortium, and wherein: a. each bacterial strain comprised in the microbial consortium comprises at least 50% of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, and the microbial consortium comprises at least 80% of the homologous protein sequences as defined in Table A - 1, or functional variants of the homologous protein sequences as defined in Table A - I, preferably about 100% of said homologous protein sequences or functional variants thereof.

[0377] 44. The method according to any of the preceding items, wherein the bacterial entity is a plurality of bacterial strains, a naturally occurring bacterial consortium or a synthetic bacterial consortium, and wherein: a. each bacterial strain comprised in the microbial consortium comprises at least 50% of the homologous protein sequences as defined in Table A - 1 or functional variants of the homologous protein sequences as defined in Table A - I, and at least 50% of the homologous protein sequences as defined in Table B - 1, or functional variants of the homologous protein sequences as defined in Table B - I, and b. the microbial consortium comprises at least 80% of the homologous protein sequences as defined in Table A - 1, or functional variants of the P7198PC00 homologous protein sequences as defined in Table A - I, preferably about 100% of said homologous protein sequences, or functional variants thereof.

[0378] 45. The method according to any of the previous items wherein determining presence of the homologous protein sequences of the bacterial entity comprises performing proteome analysis, said proteome analysis comprising: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, and f. Obtaining annotated proteins, thereby determining the presence of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, in the bacterial entity.

[0379] 46. The method according to any of the previous items wherein determining presence of the homologous protein sequences of the bacterial entity comprises performing proteome analysis, said proteome analysis comprising: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, and f. Obtaining annotated proteins, thereby determining the presence of the homologous protein sequences as defined in Table A - I, or biological functions of the homologous protein sequences as defined in Table A - I, in the bacterial entity. P7198PC00

[0380] 47. The method according to any of the previous items wherein determining presence of the homologous protein sequences of the bacterial entity comprises performing proteome analysis, said proteome analysis comprising: a. Obtaining a genomic sequence of the bacterial entity b. Identifying protein-coding genes, such as open reading frames (ORFs), c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, and f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences as defined in Table A - I, or functional variants of the homologous protein sequences as defined in Table A - I, in the bacterial entity.

[0381] 48. The method according to any of the previous items wherein determining presence of the homologous protein sequences of the bacterial entity comprises performing proteome analysis, said proteome analysis comprising: a. Obtaining a genomic sequence of the bacterial entity b. Identifying protein-coding genes, such as open reading frames (ORFs), c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMMER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, and P7198PC00 f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences as defined in Table A - I, or biological functions of the homologous protein sequences as defined in Table A - I, in the bacterial entity.

[0382] 49. The method according to any of the previous items wherein determining presence of the homologous protein sequences and / or of the nucleotide sequences of the bacterial entity and / or of an individual bacterial isolate comprises: a. performing metagenomic analysis comprising whole-genome shotgun sequencing in the absence of culturing, b. metagenomic assembly to generate metagenome-assembled genomes (MAGs), c. determining presence of homologous protein sequences according to any one of the preceding items.

[0383] 50. The method according to any one of the previous items wherein determining the biological function of the homologous protein sequences and / or of the nucleotide sequences of the bacterial entity and / or of an individual bacterial isolate comprises functional annotation of the identified genes, such as determination of KEGG-orthologs (KOs) and / or Orthologous Groups (OGs).

[0384] 51. The method according to any one of the preceding items, wherein successfully colonising the root of a plant comprises the presence of a stable and detectable population of the bacterial entity on or within the plant microbiome for a period of at least 14 days post-inoculation with the microbial consortium.

[0385] 52. The method according to any one of the preceding items, wherein successful colonisation of the plant root results in one or more changes to the plant selected from growth, metabolic activity, nutrient uptake, chemical composition, physical properties, resistance to environmental stress and / or behaviour changes. P7198PC00

[0386] 53. A method of improving growth, productivity and / or resilience towards biotic and abiotic stresses of a plant, the method comprising inoculating root(s) of the plant with the microbial communities produced by the methods according to any one of the previous items.

[0387] 54. The method according to item 42, wherein biotic stress are plant pathogens.

[0388] 55. The method according to item 42, wherein abiotic stress is drought, salinity, heat stress, cold stress, oxidative stress, heavy metal toxicity, low soil pH, high soil pH and / or nutrient limitation.

[0389] 56. The method according to item 44, wherein the oxidative stress is characterized by the accumulation of reactive oxygen species in plant cells, measured as increased levels of reactive oxygen species, such as hydrogen peroxide or superoxide anion.

[0390] 57. The method according to any one of the preceding items, wherein the method results in improved plant growth compared to a plant not inoculated with the microbial consortium produced by the methods according to any one of the previous items.

Claims

P7198PC00Claims1. A method of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous protein sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - 1, and ii. the same biological function as the corresponding sequence in Table A- I, in the obtained genomic sequence, d. Selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.

2. A method of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table A-l and having the same biological function as the corresponding sequence in Table A-l; wherein the presence of protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A-l, as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

3. The method according to any one of the preceding claims, wherein the method further comprises:P7198PC00 a. determining, for each bacterial entity, the presence of homologous protein sequences having at least 30% full-length sequence identity with the sequences as defined in Table B— I, and having the same biological function as the corresponding sequence in Table B— I , in the obtained genomic sequence; b. selecting a combination of said bacterial entities such that the combination collectively comprises; i) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— I , and ii) protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B— I , as identified in step a).

4. The method of any one of any one of the preceding claims, wherein the homologous protein sequences having the biological function equivalent to that described for the corresponding sequence in Table A-l are sequences which are assigned to the same KEGG Orthology identifier as defined in Table A-l.

5. A method of producing a microbial consortium that successfully colonizes roots of at least one plant species, the method comprising: a. Isolating bacterial entities from a plant microbiome, b. Obtaining genomic sequences of said bacterial entities, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - II, and ii. the same biological function as the corresponding sequence in Table A- II, in the obtained genomic sequence, d. selecting a combination of said bacterial entities such that the combination collectively comprises nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), thereby obtaining a microbial consortium that successfully colonizes roots of a plant species.104P7198PC006. A method of predicting whether a bacterial entity can successfully colonize the roots of a plant species, the method comprising: a. Providing a bacterial entity isolated from a plant microbiome, b. Obtaining a genomic sequence of said bacterial entity, c. Determining, for each bacterial entity, the presence of homologous nucleotide sequences having: i. at least 30% sequence identity with the sequences as defined in Table A - II, and ii. the same biological function as the corresponding sequence in Table A— 11, in the obtained genomic sequence; wherein the presence of nucleotide sequences and biological functions corresponding to at least 50% of the entries listed in Table A— II, as identified in step (c), is indicative that the bacterial entity can successfully colonise the roots of a plant species.

7. The method according to any one of the preceding claims, wherein the method further comprises: a. Determining, for each bacterial entity, the presence of the nucleotide sequences as defined in Table B - II, and having the same biological function as the corresponding sequence in Table B-l I, in the obtained genomic sequence, b. Selecting a combination of said bacterial entities such that the combination collectively comprises; i. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table B - II, and ii. protein sequences and biological functions corresponding to at least 50% of the entries listed in Table A— 11.

8. The method according to any one of the preceding claims, wherein the microbial consortium has the capacity to successfully adapt to a plant root environment and / or ensure competitive colonisation of the roots of a plant species.

9. The method according to any one of the preceding claims, wherein the plant microbiome comprises soil, air and / or water surrounding a plant, such as105P7198PC00 wherein the plant microbiome originates from soil, air and / or water surrounding a plant.

10. The method according to any one of the preceding claims, wherein the plant microbiome comprises the endosphere, phyllosphere and / or rhizosphere of a plant, such as wherein the plant microbiome originates from the endosphere, phyllosphere and / or rhizosphere of a plant.

11. The method according to any one of the preceding claims, wherein the plant microbiome comprises compost.

12. The method according to any one of the preceding claims, wherein the at least one plant species belongs to a viridiplantae, an angiosperm or a gymnosperm.

13. The method according to any one of the preceding claims, wherein the angiosperm is a monocot or a eudicot.

14. The method according to any one of the preceding claims, wherein the at least one plant species belongs to Brassicaceae, Fabaceae or Poaceae.

15. The method according to any one of the preceding claims, wherein the at least one plant species belongs to Arabidopsis, Lotus, Hordeum, Zea, Triticum, Oryza, Solanum, Glycine, Saccharum, Manihot or Musa.

16. The method according to any one of the preceding claims, wherein the at least one plant species is Arabidopsis thaliana, Hordeum vulgare or Lotus japonicus.

17. The method according to any one of the preceding claims, wherein the at least one plant species is a maize species, a wheat species, a rice species, a potato species, a tomato species, a soybean species, a sugarcane species, a cassava species or a banana species.

18. The method according to any one of the preceding claims, wherein the bacterial entity is an individual bacterial strain or a plurality of bacterial strains.106P7198PC0019. The method according to any one of the preceding claims, wherein the bacterial entity is a naturally occurring bacterial consortium or a synthetic bacterial consortium.

20. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A— I , such as at least 60% of the entries listed in Table A— I , such as at least 65% of the entries listed in Table A— I , such as at least 70% of the entries listed in Table A— I , such as at least 75% of the entries listed in Table A— I , such as at least 80% of the entries listed in Table A— I , such as at least 85% of the entries listed in Table A— I , such as at least 86% of the entries listed in Table A— I , such as at least 87% of the entries listed in Table A— I, such as at least 88% of the entries listed in Table A- I, such as at least 89% of the entries listed in Table A-l, such as at least 90% of the entries listed in Table A-l, such as at least 95% of the entries listed in Table A-l, such as at least 99% of the entries listed in Table A-l.

21. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 70% of the entries listed in Table A-l, such as at least 75% of the entries listed in Table A-l, such as at least 80% of the entries listed in Table A-l, such as at least 85% of the entries listed in Table A-l, such as at least 86% of the entries listed in Table A-l, such as at least 87% of the entries listed in Table A-l, such as at least 88% of the entries listed in Table A-l, such as at least 89% of the entries listed in Table A-l, such as at least 90% of the entries listed in Table A-l, such as at least 95% of the entries listed in Table A- I, such as at least 99% of the entries listed in Table A-l.

22. The method according to any one of the preceding claims, wherein the presence of protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A-l, such as at least 60% of the entries listed in Table A-l, such as at least 65% of the entries listed in Table A-l, such107P7198PC00 as at least 70% of the entries listed in Table A— I , such as at least 75% of the entries listed in Table A— I , such as at least 80% of the entries listed in Table A- I, such as at least 85% of the entries listed in Table A-l, such as at least 86% of the entries listed in Table A-l, such as at least 87% of the entries listed in Table A-l, such as at least 88% of the entries listed in Table A-l, such as at least 89% of the entries listed in Table A-l, such as at least 90% of the entries listed in Table A-l, such as at least 95% of the entries listed in Table A-l, such as at least 99% of the entries listed in Table A-l, is indicative that the bacterial entity can successfully colonise the roots of a plant species.

23. The method according to any one of the preceding claims, wherein the presence of protein sequences and biological functions corresponding to at least 70% of the entries listed in Table A-l, such as at least 75% of the entries listed in Table A-l, such as at least 80% of the entries listed in Table A-l, such as at least 85% of the entries listed in Table A-l, such as at least 86% of the entries listed in Table A-l, such as at least 87% of the entries listed in Table A-I, such as at least 88% of the entries listed in Table A-l, such as at least 89% of the entries listed in Table A-l, such as at least 90% of the entries listed in Table A-l, such as at least 95% of the entries listed in Table A-l, such as at least 99% of the entries listed in Table A-l, is indicative that the bacterial entity can successfully colonise the roots of a plant species.

24. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A-l I, such as at least 60% of the entries listed in Table A— 11 , such as at least 65% of the entries listed in Table A-l I, such as at least 70% of the entries listed in Table A-l I, such as at least 75% of the entries listed in Table A-l I, such as at least 80% of the entries listed in Table A-l I, such as at least 85% of the entries listed in Table A-l I, such as at least 86% of the entries listed in Table A— 11 , such as at least 87% of the entries listed in Table A— II, such as at least 88% of the entries listed in Table A-II, such as at least 89% of the entries listed in Table A— 11, such as at least 90% of the entries listed in Table A— II, such as at least 95% of the entries listed in Table A-l I, such as at least 99% of the entries listed in Table A-l I.108P7198PC0025. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 70% of the entries listed in Table A— 11, such as at least 75% of the entries listed in Table A— 11 , such as at least 80% of the entries listed in Table A— II, such as at least 85% of the entries listed in Table A— II, such as at least 86% of the entries listed in Table A— 11, such as at least 87% of the entries listed in Table A— II, such as at least 88% of the entries listed in Table A— II, such as at least 89% of the entries listed in Table A— 11 , such as at least 90% of the entries listed in Table A— II, such as at least 95% of the entries listed in Table A- II, such as at least 99% of the entries listed in Table A— 11.

26. The method according to any one of the preceding claims, wherein the presence of protein sequences and biological functions corresponding to at least 55% of the entries listed in Table A— 11, such as at least 60% of the entries listed in Table A— II, such as at least 65% of the entries listed in Table A— II, such as at least 70% of the entries listed in Table A— 11 , such as at least 75% of the entries listed in Table A— II, such as at least 80% of the entries listed in Table A- II, such as at least 85% of the entries listed in Table A— 11, such as at least 86% of the entries listed in Table A— II, such as at least 87% of the entries listed in Table A— 11 , such as at least 88% of the entries listed in Table A— 11, such as at least 89% of the entries listed in Table A— 11, such as at least 90% of the entries listed in Table A— II, such as at least 95% of the entries listed in Table A— II, such as at least 99% of the entries listed in Table A— 11, is indicative that the bacterial entity can successfully colonise the roots of a plant species.

27. The method according to any one of the preceding claims, wherein the presence of protein sequences and biological functions corresponding to at least 70% of the entries listed in Table A— 11, such as at least 75% of the entries listed in Table A— II, such as at least 80% of the entries listed in Table A— II, such as at least 85% of the entries listed in Table A— 11 , such as at least 86% of the entries listed in Table A— II, such as at least 87% of the entries listed in Table A- II, such as at least 88% of the entries listed in Table A— II, such as at least 89% of the entries listed in Table A— II, such as at least 90% of the entries listed in109P7198PC00Table A— 11, such as at least 95% of the entries listed in Table A— 11, such as at least 99% of the entries listed in Table A— 11, is indicative that the bacterial entity can successfully colonise the roots of a plant species.

28. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table B— I, such as at least 60% of the entries listed in Table B— I , such as at least 65% of the entries listed in Table B— I , such as at least 70% of the entries listed in Table B— I , such as at least 75% of the entries listed in Table B— I , such as at least 80% of the entries listed in Table B— I , such as at least 85% of the entries listed in Table B— I , such as at least 86% of the entries listed in Table B— I , such as at least 87% of the entries listed in Table B— I, such as at least 88% of the entries listed in Table B- I, such as at least 89% of the entries listed in Table B-l, such as at least 90% of the entries listed in Table B-l, such as at least 95% of the entries listed in Table B-l, such as at least 99% of the entries listed in Table B-l.

29. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 70% of the entries listed in Table B-l, such as at least 75% of the entries listed in Table B-l, such as at least 80% of the entries listed in Table B-l, such as at least 85% of the entries listed in Table B-l, such as at least 86% of the entries listed in Table B-l, such as at least 87% of the entries listed in Table B-l, such as at least 88% of the entries listed in Table B-l, such as at least 89% of the entries listed in Table B-l, such as at least 90% of the entries listed in Table B-l, such as at least 95% of the entries listed in Table B- I, such as at least 99% of the entries listed in Table B-l.

30. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 55% of the entries listed in Table B— II, such as at least 60% of the entries listed in Table B— 11 , such as at least 65% of the entries listed110P7198PC00 in Table B— II, such as at least 70% of the entries listed in Table B— II, such as at least 75% of the entries listed in Table B— 11, such as at least 80% of the entries listed in Table B— II, such as at least 85% of the entries listed in Table B— II, such as at least 86% of the entries listed in Table B— 11 , such as at least 87% of the entries listed in Table B— II, such as at least 88% of the entries listed in Table B- II, such as at least 89% of the entries listed in Table B— 11, such as at least 90% of the entries listed in Table B— II, such as at least 95% of the entries listed in Table B— 11, such as at least 99% of the entries listed in Table B— 11.

31. The method according to any one of the preceding claims, wherein the method comprises selecting a combination of said bacterial entities such that the combination collectively comprises protein sequences and biological functions corresponding to at least 70% of the entries listed in Table B— II, such as at least 75% of the entries listed in Table B— 11 , such as at least 80% of the entries listed in Table B— II, such as at least 85% of the entries listed in Table B— II, such as at least 86% of the entries listed in Table B— 11, such as at least 87% of the entries listed in Table B-ll,such as at least 88% of the entries listed in Table B— II, such as at least 89% of the entries listed in Table B— 11 , such as at least 90% of the entries listed in Table B— II, such as at least 95% of the entries listed in Table B- II, such as at least 99% of the entries listed in Table B— 11.

32. The method of any one of the preceding claims, wherein the homologous sequences having the same biological function as the corresponding sequence in Table A-l are sequences which are assigned to the same KEGG orthology identifier as defined in Table A-l.

33. The method of any one of the preceding claims, wherein the homologous sequences having the same biological function as the corresponding sequence in Table A-l I are sequences which are assigned to the same KEGG orthology identifier as defined in Table A-l I.

34. The method of any one of the preceding claims, wherein the homologous sequences having the same biological function as the corresponding sequence in Table B-l are sequences which are assigned to the same Orthogroup identifier as defined in Table B-l.111P7198PC0035. The method of any one of the preceding claims, wherein the homologous sequences having the same biological function as the corresponding sequence in Table B-ll are sequences which are assigned to the same Orthogroup identifier as defined in Table B-ll.

36. The method according to any one of the preceding claims wherein determining presence of the homologous protein sequences of the bacterial entity comprises performing proteome analysis, said proteome analysis comprising: a. Obtaining a genomic sequence of the bacterial entity, b. Identifying protein-coding genes, c. Functionally annotating said protein-coding genes, d. Comparing said functionally annotated protein-coding genes with publicly available databases, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, and f. Obtaining annotated proteins, thereby determining the presence of the homologous protein sequences as defined in Table A - I, or biological functions of the homologous protein sequences as defined in Table A - I, in the bacterial entity.

37. The method according to any one of the preceding claims wherein determining presence of the homologous protein sequences of the bacterial entity comprises performing proteome analysis, said proteome analysis comprising: a. Obtaining a genomic sequence of the bacterial entity b. Identifying protein-coding genes, such as open reading frames (ORFs), c. Functionally annotating said protein-coding genes, such as via EggNOG annotation and / or InterPro, d. Comparing said functionally annotated protein-coding genes with publicly available databases, such as KEGG, COG, Pfam and InterPro, e. Identifying matches between said functionally annotated protein-coding genes with publicly available databases, via methods such as (DIAMOND) BlastP, HMM ER search, sequence profile searches, Recursive BlastP and FoldSeek, and structural similarity searches such as implemented in FoldSeek, andP7198PC00 f. Obtaining annotated proteins comprising matching gene ontology (GO) and KEGG ontology (KO) terms, thereby determining the presence of the homologous protein sequences as defined in Table A - I, or biological functions of the homologous protein sequences as defined in Table A - I, in the bacterial entity.

38. The method according to any one of the preceding claims wherein determining the biological function of the homologous protein sequences and / or of the nucleotide sequences of the bacterial entity and / or of an individual bacterial isolate comprises functional annotation of the identified genes, such as determination of KEGG-orthologs (KOs) and / or Orthologous Groups (OGs).

39. The method according to any one of the preceding claims, wherein successfully colonising the root of a plant comprises the presence of a stable and detectable population of the bacterial entity on or within the plant microbiome for a period of at least 14 days post-inoculation with the microbial consortium.

40. The method according to any one of the preceding claims, wherein successful colonisation of the plant root results in one or more changes to the plant selected from growth, metabolic activity, nutrient uptake, chemical composition, physical properties, resistance to environmental stress and / or behaviour changes.

41. A method of improving growth, productivity and / or resilience towards biotic and abiotic stresses of a plant, the method comprising inoculating root(s) of the plant with the microbial communities produced by the methods according to any one of the preceding claims.

42. The method according to claim 41 , wherein biotic stress are plant pathogens.

43. The method according to claim 41 , wherein abiotic stress is drought, salinity, heat stress, cold stress, oxidative stress, heavy metal toxicity, low soil pH, high soil pH and / or nutrient limitation.P7198PC0044. The method according to claim 43, wherein the oxidative stress is characterized by the accumulation of reactive oxygen species in plant cells, measured as increased levels of reactive oxygen species, such as hydrogen peroxide or superoxide anion.

45. The method according to any one of the preceding claims, wherein the method results in improved plant growth compared to a plant not inoculated with the microbial consortium produced by the methods according to any one of the preceding claims.

Images