I.     Proposal’s context, positioning and objective(s[l1] )

Overall context of the proposal:

vInoculation of beneficial microorganisms: a practice aiming at improving the performance and sustainability of agricultural and horticultural systems. Plants host a diverse community of tightly associated microbes – their microbiome – which increases their access to nutrients and enhances their growth, facilitates tolerance of stress such as drought, and increases disease resistance [1-3]. Manipulation of plant-associated microorganisms has thus considerable potential to improve the performance of cropping and horticultural systems [4, 5]. The plant-associated microorganisms inducing beneficial effects are often called ‘plant probiotics’ by private sector actors working on fruits and horticultural crops as inoculation can improve food nutritional quality [6]. More precisely, inoculation of plant-beneficial microorganisms to soil can allow phytostimulation through several microbial processes including stimulation of root growth [7], biological N₂ fixation [8], phosphate solubilization [7] and protection against plant pathogens [9]. For instance, Partner #1 of this proposal has participated in the ANR project AZODURE (2013-2017) analysing how inoculation of maize by an Azospirillum strain influences the soil microbiota and functioning [10] and the economic and environmental performance of this crop [11]. Although not yet fully explored, microbial inoculation can also improve other ecosystem services delivered by soils such as mitigation of greenhouse gas (GHG) emissions [12]. For instance, inoculating bacteria with N₂O-reducing capacity to pasture [13] or crop soils [14] can mitigate N₂O emissions under greenhouse conditions, and this potential is particularly high for non-denitrifying N₂O-reducing strains as shown by IMMINENT Partner #2. Such N₂O sink capacity may be invaluable microbiological property to develop inoculants delivering new ecosystem services.

To overcome the challenge of increasing food production with a significant reduction of agrochemical use and environmental pollution, many private companies are actively exploiting the potential of microbial inoculants and associated formulations that support inoculant survival. It is predicted that the global market for biofertilizers could reach USD 1.66 billion by 2022 [15]. For example, the private Partner #3 of IMMINENT (PT GHA) sells mixtures of Bacillus strains and substrates as inoculants that allow better growth of crop and horticultural plants. Given the demand for more sustainable agricultural and horticultural systems and mistrust of genetically modified organisms in Europe, improving the efficiency of inoculation approaches using microorganisms naturally occurring in soil is thus of major importance.

vThe need to study biotic interactions for understanding inoculum ability to maintain itself in soil and improving the efficiency and reliability of inoculation. A common difficulty when using microbial inoculants is the inconsistency of their beneficial effects in field conditions and large variability of inoculation outcome [16]. Meta-analyses have shown that this variability is partly due to abiotic conditions (in particular water regime, organic matter, pH and nutrient availability) [5]. However, a large part of the variability in the observed inoculation effects on the performance of agricultural/horticultural systems remains poorly identified. A major assumption of the IMMININENT project is that the role of biotic interactions, i.e. interactions between the inoculum and the native soil microbiota, for inocula survival has been largely overlooked. Actually, many studies have reported that most biostimulants poorly survive in soil upon inoculation because the inoculated microbes are often outcompeted by the native community. For instance, following sowing of inoculated maize seeds, Azospirillum lipoferum CRT1 is not detected by quantitative PCR already after the 6-leaves stage [10]. Private companies in this sector generally invest a great deal in the development of matrices supporting their inoculants in order to extend their survival in soil and efficacy, as it represents a major factor restricting the efficiency of biostimulants on soil functioning and plant performance. In contrast, guiding inoculation approaches based on a better knowledge of biotic interactions requires development of synergies between research capacities of private and public actors.

 [l1]This paragraph refers to the evaluation criteria “Quality and scientific aim”

I.     Proposal’s context, positioning and objective(s[l1] )

Overall context of the proposal:

vInoculation of beneficial microorganisms: a practice aiming at improving the performance and sustainability of agricultural and horticultural systems. Plants host a diverse community of tightly associated microbes – their microbiome – which increases their access to nutrients and enhances their growth, facilitates tolerance of stress such as drought, and increases disease resistance [1-3]. Manipulation of plant-associated microorganisms has thus considerable potential to improve the performance of cropping and horticultural systems [4, 5]. The plant-associated microorganisms inducing beneficial effects are often called ‘plant probiotics’ by private sector actors working on fruits and horticultural crops as inoculation can improve food nutritional quality [6]. More precisely, inoculation of plant-beneficial microorganisms to soil can allow phytostimulation through several microbial processes including stimulation of root growth [7], biological N₂ fixation [8], phosphate solubilization [7] and protection against plant pathogens [9]. For instance, Partner #1 of this proposal has participated in the ANR project AZODURE (2013-2017) analysing how inoculation of maize by an Azospirillum strain influences the soil microbiota and functioning [10] and the economic and environmental performance of this crop [11]. Although not yet fully explored, microbial inoculation can also improve other ecosystem services delivered by soils such as mitigation of greenhouse gas (GHG) emissions [12]. For instance, inoculating bacteria with N₂O-reducing capacity to pasture [13] or crop soils [14] can mitigate N₂O emissions under greenhouse conditions, and this potential is particularly high for non-denitrifying N₂O-reducing strains as shown by IMMINENT Partner #2. Such N₂O sink capacity may be invaluable microbiological property to develop inoculants delivering new ecosystem services.

To overcome the challenge of increasing food production with a significant reduction of agrochemical use and environmental pollution, many private companies are actively exploiting the potential of microbial inoculants and associated formulations that support inoculant survival. It is predicted that the global market for biofertilizers could reach USD 1.66 billion by 2022 [15]. For example, the private Partner #3 of IMMINENT (PT GHA) sells mixtures of Bacillus strains and substrates as inoculants that allow better growth of crop and horticultural plants. Given the demand for more sustainable agricultural and horticultural systems and mistrust of genetically modified organisms in Europe, improving the efficiency of inoculation approaches using microorganisms naturally occurring in soil is thus of major importance.

vThe need to study biotic interactions for understanding inoculum ability to maintain itself in soil and improving the efficiency and reliability of inoculation. A common difficulty when using microbial inoculants is the inconsistency of their beneficial effects in field conditions and large variability of inoculation outcome [16]. Meta-analyses have shown that this variability is partly due to abiotic conditions (in particular water regime, organic matter, pH and nutrient availability) [5]. However, a large part of the variability in the observed inoculation effects on the performance of agricultural/horticultural systems remains poorly identified. A major assumption of the IMMININENT project is that the role of biotic interactions, i.e. interactions between the inoculum and the native soil microbiota, for inocula survival has been largely overlooked. Actually, many studies have reported that most biostimulants poorly survive in soil upon inoculation because the inoculated microbes are often outcompeted by the native community. For instance, following sowing of inoculated maize seeds, Azospirillum lipoferum CRT1 is not detected by quantitative PCR already after the 6-leaves stage [10]. Private companies in this sector generally invest a great deal in the development of matrices supporting their inoculants in order to extend their survival in soil and efficacy, as it represents a major factor restricting the efficiency of biostimulants on soil functioning and plant performance. In contrast, guiding inoculation approaches based on a better knowledge of biotic interactions requires development of synergies between research capacities of private and public actors.

a.      Objectives and research hypothesis[l2] 

vObjectives of the project and project relevance with regard to the state-of-the-art. During the past years, Partners #1 & 4 of IMMINENT have demonstrated that the diversity of the soil microbial community influences the capacity of a bacterial inoculum to maintain a high abundance in soil [17]. More precisely, the level of overlap that exists between the niche of the soil community (community niche) and the niche of the inoculum determines how quickly the latter disappears [16]. Moreover, we have shown that after inoculation of a bacterial strain to soil, and even if this strain is outcompeted and disappears, the inoculation event has a legacy effect (Fig. 1; [18]): a few weeks after inoculation, the resulting soil microbial community becomes dominated by taxa using resources unusable by the inoculum, whereas taxa using the same resources than the inoculum become rare. An inoculation event thus modifies the soil community, reducing the level of overlap between the community niche and niche of the inoculum. Given the importance of niche overlap for inoculum survival, this opens a window of opportunity for future inoculations.

Based on these results, IMMINENT aims at developing strategies to improve the performance of biostimulants by understanding how they interact with the soil native communities and guiding inoculation practice accordingly. For that purpose, three academic groups (Partners #1, 2 & 4) and one firm developing researches on biostimulants and on their formulation (Partner #3) have joined forces to co-construct this proposal during the past 10 months, addressing the following questions: (Q1) Does the overlap between the niche of the native soil community and the niche of the inoculated bacteria determine how quickly the inoculum disappears? (Q2) Can a recurrent inoculation approach using the ‘legacy effect’ (Fig. 1) improve the ability of survival of the inocula after inoculation? and (Q3) To what extent are the outcomes of the recurrent inoculation approach robust across a range of soil conditions? These questions will be addressed for 2 model bacterial inocula: (i) a mixture of Bacillus strains used by PT GHA as plant growth stimulants; and (ii) a new class of inoculants aiming at reducing the environmental footprint of agricultural production composed by Dyadobacter and Pedobacter (bacteroides) strains. These are able to reduce N₂O into N₂ without producing N₂O, thus contributing to the abatement of N₂O emissions from soils. Finally, (Q4) Can we provide a first proof of concept concerning the use of the recurrent inoculation approach based on the niche concept to increase the survival of biostimulants and their effects in terms of plant performance and GHG emissions under greenhouse and field conditions? Q1-2 will address the fundamentals to design innovative and knowledge-based inoculation practices, while Q3-4 will evaluate the feasibility, robustness and performance of such practices across contrasted soil conditions and under field conditions. The main hypotheses of this project are that using recurrent inoculation events can help shaping the soil microbial community and improving the maintenance of inocula in soil, and that the identification of inoculant-specific niches can guide the selection of prebiotics/formulations that support their survival, ultimately increasing the effect of the inocula. Our specific hypotheses are given in the description of each workpackage below.

b.      Position of the project as it relates to the state of the art[l3] 

v Innovative nature of the project, ambitiousness and originality of the objectives and the methodology. Microbial inoculants in agriculture can provide manifold ecosystem services to society that may not only be related to the provision of food. For instance, agriculture is the main source of terrestrial N₂O emissions, a potent greenhouse gas with a global warming potential over 100 years of about 298 and 11.9 times that of CO₂ and CH₄, respectively. Efficient inoculation of microorganisms to reduce N₂O emissions by agricultural soils would represent a novel N₂O mitigation strategy which could further boost the market of inoculants. The project IMMINENT will lead to major breakthroughs: (1) the development of more efficient inoculation strategies based on ecological niche theory – through the use of repeated inoculations or selection of prebiotics; (2) the development of inoculants and inoculation strategies aiming at N₂O mitigation; and (3) the development of a multi-functional product that concomitantly and efficiently improves plant production while reducing the environmental footprint of agriculture. Since the scientific issues addressed here are original, our results could be published in high ranking journals and the project breakthroughs should impact scientific domains including microbial ecology, general ecology (results relevant for biological invasions issues) and agroecology. The planned work is expected to lead to a new vision of factors driving the fate of inocula in soil and the efficiency of inoculation strategies.

c.      Methodology and risk management[l4] 

vOverall approach and methodology used to reach our objectives. The experimental set-up of the project consists of 4 sets of experiments corresponding to 4 WPs (see Figure 2), i.e. (WP1) a lab experiment in very simplified conditions, aiming at analyzing the mechanisms underlying the inocula-soil community interactions and more particularly testing the importance of the niche-based theory processes for xxxx. This will lead us to select the 2 inocula to be used in WP2; (WP2) a lab experiment with unplanted soil microcosms, for testing the efficiency of different recurrent inoculation strategies to improve inoculum’s ability to better maintain high abundances as compared to a single inoculation strategy, while also assessing underlying mechanisms. This will lead to choose the most promising recurrent inoculation strategy for WP3; (WP3) a greenhouse experiment with planted soil mesocosms, aiming at testing the relevance of the mechanisms highlighted in WP1&2 and the robustness of the benefits of the recurrent inoculation strategy across different types of planted soils; (WP4) a field trial which will allow a proof of concept for the feasibility and actual benefits of the recurrent inoculation strategy in real conditions. The overall project structure consists of 5 WPs including the transversal WP0 coping with coordination and management issues (Figure 2), and the contribution of each partner is presented further below.

Fig. 2: General organization of the IMMINENT project[l5] .

WP0 - Project management. Principal investigator, PI: X Le Roux (LEM), assisted by L Philippot (AgroEcol) and C Lapadatescu (PT GHA) for forming an executive committee

The PI will be in charge of the coordination of the project. He will cope with the administrative duties and the links with the ANR. He will be assisted by an executive committee, EC. At least every trimester and more often as needed, the EC will check the consistency of the workplan for the different experiments across WP1-4, in particular the inocula, successful recurrent inoculation approaches and prebiotics identified in WP1-2 to be used in WP3 and WP4. The EC will also be in charge of the elaboration of the consortium agreement and will deal with Intellectual Property issues and the policy applied for results use and publication.

WP1[l6] [l7]  - Testing best couples (soils x strains). Leaders: Ayme Spor (Agroecology, LEM). Contributors: LEM, AgroEcology, PT GHA & UniGro

Objectives: This methodological WP aims at providing (i) a method to monitor the survival of the different strains used inocula and (ii) a selection of soils and strains to be used in the following WPs.

Task 1.1 : Set up an assay to quantify the inoculated strains

To quantify the inoculated strains, we will generate antibiotic-resistant (rifampicin) mutants, by inoculating them on solid media supplemented with the antibiotic. Fitness of the mutants will be assessed by comparing their growth curve in liquid medium, in the presence of the antibiotics, with the growth curve from the original strain[l8] .

Task 1.2 : Screening (soil X strain) candidates

We will conduct a screening of 10 [l9] French agricultural soils with contrasted physico-chemical properties genotypes to identify 3 candidate soils for the other WPs. For this purpose, replicated soil microcosms of 50 g will be established. The different candidate strains (Pedobacter saltans, Dyadobacter fermentans, Bacillus 1 ©Premier Tech, Bacillus 2©Premier Tech, Bacillus 3©Premier Tech, Bacillus 4©Premier Tech) will be grown in liquid culture and, after washing the cells, inoculated separately in each soil microcosm. The survival of the six strains in the 10 soils will then be monitored after two weeks. This will be achieved thanks to the approach set up above. The best combinations strain X soil will be used in the subsequent workpackages.

Risk management:

The plate counting approach using a selective media has the advantage of having a very low cost but the level of risk is medium due to (i) variable frequency at which detectable mutants arise in a bacterial population and to (ii) relatively high number of naturally resistant strains in the tested soils. The mitigation strategy will consist in designing strain-specific primers and probes to quantify the inoculum by real time PCR with a Taqman assay. The level of risk of this alternative approach is low since the partners have a strong expertise in qPCR and the complete genomes of the targeted strains are available, which will facilitate the design of such primers and probes. The analysis of the complete genomes of the different candidate strains for designing primers and probes is already under way just in case.

WP2 - Testing the usefulness of the Ecological Niche Theory to predict the outcome of the competition between the model inocula and assembled communities under lab conditions. Leaders: T Pommier (LEM) & F Barraud (PT GHA). Contributors: LEM, AgroEcology, PT GHA & UniGro

Objectives & hypotheses: test the hypotheses that the survival of each inoculum studied increases when the overlap between its niche and the community niche decreases. Based on previous results, we assume that the use of organic C sources is a key dimension of niches in this context.

Approach: In sterile soil, use assemblages of heterotrophs (including denitrifiers) isolated from plant rhizosphere. These assemblages will be selected not to modulate strain richness but rather functional diversity (i.e. community niche – see Salles et al. 2009). Assemblages will be grown in liquid medium and sterile soils and subsequently subjected to inoculation by each inoculum strain, whose survival will be monitored; for each assemblage, community niche will be calculated based on resource use profiles [16].

Task2.1: Characterizing the niche of bacterial strains and inocula and identification of potential prebiotics (specific to each inoculum)

We will screen bacterial culture collections available at LEM, AgroEcol and RUG for bacterial strains isolated from plant rhizosphere, belonging to a large phylogenetic distribution and with the ability or not to produce and/or reduce N₂O. A selection of 200 isolates plus the different inocula will be further characterized by inoculating them on BIOLOG GEN IIIA and BIOLOG AN plates under aerobic and anaerobic (denitrifying) conditions, respectively (Mallon et al., 2015; Salles et al 2009). BIOLOG measurements will be performed using OMNILOG, available at LEM. We will thus generate, for all the strains of the pool and each inoculum, an utilization pattern of carbon sources under aerobic or anaerobic conditions, which represents strain niche in the presence or absence of oxygen.

Task2.2: Creating bacterial assemblages with contrasted niches and different overlaps with the niche of the inocula; characterizing the kinetics of inocula survival as a function of the overlap between community niche and inocula niche

From the pool of 200 species screened, ca. 100 encompassing a broad distribution of niches will be used to create bacterial assemblages of 8 species in sterile soil. For each inoculum, we will use a slightly different approach to create niche overlap.

N₂O reducing inocula: Assembled communities used here will be selected based on the predicted[l10]  overlap between their niche and the inoculum niche and their net N₂O production, generating a total of 36 assemblages (table xx).

Table xx: Classification of the assemblages used for testing each N₂O-reducing inoculum. The ‘overlap’ refers to the amount of carbon sources shared between community and each inocula. For each combination of (i) overlap and (ii) net N₂O production level, we will use 4 compositional replicates (i.e. each assemblage corresponds to a specific community composition). Compositional replicates will be drawn from the pool of 100 species, including the species used for PGPR inoculum, allowing us to determine their potential interactions with the DENIT inoculum.

Assemblages will be inoculated with one of the four following inocula, with or without oxygen (i.e. 36 assemblages x 4 inocula x 2 conditions = 288 conditions): Pedobacter, Dyadobacter, mixture of both strains (thus twofold density in total as compared to the two first situations), and mixture of both strains (half density of each). The assemblages will be evaluated in the presence and absence of oxygen (with nitrate). For each assemblage we will quantify the survival of the inoculum through time by plating on selective media plus antibiotics. We will also quantify total respiration (presence of oxygen) and net N₂O production (absence of oxygen). We will infer whether the ability of the inocula to survive and consume N₂O are correlated

PGPR inocula: 36 assemblages of 8 species will be drawn randomly form the pool of 100 characterized bacterial species plus the two DENIT inocula. Niche overlap will be a posterior calculated according with BIOLOG data. Assemblages will be inoculated with the following inoculate: PGPR1, PGPR2, PGPR3, PGPR4, PGPR1à4 half, PGPR1à4 full. For the first 5, inoculants will have identical final cell density, whereas for PGPR1à4 full each bacterium will have the same density as when introduced alone (final density = 2X density PGPR1, PGPR2, PGPR3, PGPR4, PGPR1à4 half). The assemblages will be evaluated only in the presence of oxygen (in the presence of nitrate). For each assemblage we will quantify the survival of the inoculum through time by plating on selective media plus antibiotics, as well as respiration (presence of oxygen). Total number of samples: 36 assemblages x 6 inocula x 1 condition (O2) = 216 flasks.

Soil assemblages: A selection of half of the assemblages used in liquid media, selected according to their results (best survival and lowest net N2O production) will be recreated again and introduced in sterile soil (Gamma radiated) to verify their effectiveness in a heterogeneous environment. IN order to do so, assemblages will be inoculated in flasks containing 50g of soil [JFS11] at 70% water holding capacity and incubated for 2 months to ensure full soil colonization (Mallon et al 2015). After this period, the inocula will be introduced (as in for the assemblages in liquid media) and their fate followed over time, using destructive sampling (4 time points). Part of the soil will be used for respiration measurements whereas the other will be incubated under anaerobic conditions to measure net N2O production.

Total number of samples denitrifying inocula: 18 assemblages x 4 inocula x 1 condition (O2) x 4 time points = 288 flasks

Total number of samples PGPR inocula: 18 assemblages x 6 inocula x 1 condition (O2) x 4 time points = 432 flasks

Our hypotheses are represented in figure 3. For all scenarios (DENIT and PGPR), we expect survival to be negatively correlated with niche overlap and community niche. However, the survival of DENIT inocula will be also driven by the amount of N2O produced by the assemblage, i.e. assemblages with high N2O production will promote survival for longer periods.

Note: If Successful è  survival could be improved by formula using prebiotics and/or substrates defined according to the inoculum’s niche.

Risk management for WP2:

xxxxx

WP3 - Evaluating the effects of different inoculation strategies on the fate of inocula in soil microcosms. Leaders: X  Le  Roux (LEM) & L Philippot (AgroEcol). Contributors: LEM; AgroEcol, PT GHA & UniGro

Objective & hypothesis: evaluate several recurrent inoculation strategies for different inocula densities in term of inoculum survival ; test whether repeated inoculations displace soil community niche in such way that survival improves with recurrent inoculation events.

Method: evaluate the effects of number and frequency of inoculation events in a model soil.

Task 3.1: Surveying temporal changes in inocula abundances following single versus recurrent inoculation event(s)

To be done for (i) the N₂O-reducing inoculum, (ii) the PGPR inoculum, and (iii) an inoculum composed of the N₂O-reducing inoculum plus the PGPR inoculum.

3 inocula types X 5 repl X weekly sampling

2.1.b:

Experimental design:

Task 3.2: Characterizing the progressive shaping of community niche by recurrent inoculation events; generating knowledge-based guidance for successful recurrent inoculation strategies

Risk management for WP3:

xxxxx

WP4 - Applicability of the recurrent inoculation approach across different soils. Leaders: L Philippot (AgroEcol) & C Lapadatescu (PT GHA). Contributors: LEM; AgroEcol, PT GHA & UniGro

Objective: To assess whether the survival of the two inocula (selected in WP1&2) can be improved in planted soils with contrasted properties by the recurrent as compared to unique inoculation approach, and assess the consequences for selected soil functions and agricultural/horticultural outputs

Hypothesis: Niche-based interactions between the inoculum and the native soil community have an important role in determining the fate of the inoculum and post-inoculation shaping of the soil community niche across a broad range of soil properties ; therefore a recurrent inoculation strategy can be successful in improving the maintenance of the inoculum, key soil functions and agricultural/horticultural outputs across different soil types

Task 4.1: Evaluating across multiple soils the effects of the recurrent inoculation approach on the fate of two types of inocula

Three French agricultural soils with contrasted physico-chemical properties identified in WP1will ne used to prepare planted soil mesocosms. The mesocosms will be inoculated during x weeks under the following conditions for each soil type (see Table X): (a) best recurrent inoculation strategy identified in WP2, e.g. with inoculation at T1, T2, T3 and T4; (b) only one inoculation at T1;  (c) only xaone inoculation at T4; and (d) No inoculation. The same inoculum concentration will be used for conditions (a), (b) and (c) while the corresponding volume of sterile water will be used for the non-inoculated control (d).  Both types of inocula used either for decreasing greenhouse gas emissions (Dyadobacter and/or Pedobacter) or for promoting plant growth (mixture of Bacillus strains) will be tested in this WP under the conditions described above and in Table X.

In all soil mesocosms, we will growth X as model crop [l12] . Plants will be grown in a High-Throughput Phenotyping Platform at the INRA Dijon(http://www.dijon.inra.fr/en/Tools-and-Resources/Tools_resources_dijon_inra/PPHD) ? All soil mesocosms will regularly be fertilized with a mixture of NH₄⁺ and NO₃^- using the automatic watering system of the plateform. The survival of both types of inoculum in the different soils will be estimated one week after each inoculation (T1 to T4) using the approach developed in WP2.

Task 4.2: Assessing the efficiency of the recurrent as compared to unique inoculation strategy on key functions (in particular abatement of N₂O emission) and plant growth during the early growing period

In the 60 soil mesocosms inoculated with the N₂O reducing inoculum, we will monitor the N₂O emissions by the non-destructive, flux chamber technique. The chambers will consist of cylindrical polyvinyl chloride plastic pipes of 8 cm inner diameter and 20 cm height with a rubber septum inserted into the PVC lid. At the time of measurement, the PVC pipe will be sealed onto a collar previously inserted into the soil and the headspace gas of the pipes will be collected for each microcosms for 2 h using syringes and glass vials. N₂O concentration in the headspace will be determined by gas chromatography using a GC equipped with an EC-detector. These N₂O emissions will be quantified x days after each inoculation (T1 to T4[l13] ) i.e. when the survival of the inoculum will also be assessed in task 31. In addition, at T4, we will quantify key soil microbial processes (N mineralization, potential nitrification, and potential gross N₂O production and N₂O reduction rates; see methods in Domeignoz-Horta et al 2015 and Assemien et al. 2019) as well as the abundance of the corresponding microbial guilds (nitrifiers, N₂O producers, N₂O reducers) by real time PCR as previously described (Bru et al. 2011). Finally we will also assess the inorganic N pools (nitrate and ammonium) by colorimetry using a photometer.

In all soil mesocosms, we will assess plants traits such total biomass, XXXXXX[d14] ,. In the High-Throughput Phenotyping Platform, robots and cameras are filming the aerial and root sections of thousands of plants, on various wavelengths, which will be used to determine plant traits such as greenness, etc…[l15] 

Table X: Overview of the design planned for WP4

Risk management for WP4:

This WP will use the best recurrent inoculation strategy identified in WP2. This is why we will begin the work for WP3 (and 4) 12 months later than the work of WP1 & 2. For the experimental setup, there is a risk is related to the inoculation of the strains, which may not thrive to survive long enough in one or more of the selected soils. This risk has been minimized by screening a larger selection of bare soils in WP1 and identification of candidate soils in which the amount of inoculated strains was above the detection limit during at least 15 weeks. Another risk is related to the cultivation of plants, which is always associated with potential plant growth disorders or even mortality. That said, the risk is relatively low due to the large experience of our colleagues at the High-Throughput Phenotyping Platform and we have schedule enough time for this WP to allow starting over the plant culture in case of failure. Otherwise the methodological risk is low due to the strong experience of the partners in the proposed methods.[X16] [l17] 

WP5 - Proof of concept: efficiency of the recurrent inoculation practice under field conditions. Leaders: B Solnais (PT GHA) & X Le Roux (LEM). Contributors: LEM; AgroEcology, PT GHA & UniGro

Objective & hypothesis: Test to which extent the inoculation strategies selected in WP3 can improve inocula survival and efficiency under natural conditions. Approach: Quantify inocula survival, soil functions and plant production/yield for a complete growing season.

Task 5.1: Proof of concept for N₂O-reducing bacterial inocula only

Task 5.2: Proof of concept for biostimulant inocula only

Task 5.3: First test for sequentially applied / mixed inocula (biostimulant + N₂O reducing bacteria)

Not solid scientific basis here but potential for application - Only a test but go beyond the scope

The main deliverables of IMMINENT are as follows:

xxx

Table XX: Gant diagram of the IMMINENT project

xxxx

Table XX: Risk management planned for the IMMINENT project

xxxxx

II.  Organisation and implementation of the project[l18] 

a.      Scientific coordinator and its consortium / its team[l19] 

The Scientific Coordinator of IMMINENT, X. Le Roux (see CV in Annex 1), has been trained as an ecosystem and plant ecologist (PhD plus 5 years experience at INRA Clermont Ferrand, until 2000) and soil microbial ecologist since 2001 where he leads a research group. He has already published 113 papers including in Global Change Biology, ISME J., Nature, Trends Ecol Evol and has a h-index of 44 (WoS). He has a long experience of project management (including 4 European projects as coordinator in the past 10 years; one active when IMMINENT would begin). Further, he has experience of private-public relationships, as he has been the Director of FRB, a French structure promoting this type of relationships. He has participated (main contact for LEM) to 2 research projects with the French company Veolia/Anjou Recherche on strategies to improve the management of the microbial resource in biofilters [19].

The IMMINENT consortium gathers the complementary expertises required to cover all our objectives, i.e. it integrates skills in the following domains: soil microbial ecology and N-cycling (Partner #1); plants-microorganisms interactions (Partner #2); R&D on inocula and pre-biotics (Partner #3); and ecological niche theory and modelling (Partner #4). The distribution of tasks and roles is tailor-made, suiting the expertise and means of each partner. The academic partners have already collaborated fruitfully during the past years (xx publications between at least 2 of the 3 academic  partners since 20XX). Further, Partner #3 has a long experience in conducting R&D activities on xxx and the PI of IMMINENT has successful experiences of Public-Private projects.

Partner #1: LEM - Laboratoire d’Ecologie Microbienne ; Team Microbial functional diversity and nitrogen cycling ; UMR1418 – INRA / Université Lyon 1 / CNRS

Partner#1 has an internationally recognized expertise for studying (1) the functional diversity and ecology of soil microorganisms involved in N cycling, and (2) the links between the diversity, abundances and activities of N-related microbial groups and ecosystem functioning and services, including GHG emission. This partner will characterize N cycling microbial activities (biochemical assays using the AME ‘Microbial Activities in Environmental samples’ facility at LEM), nitrifier abundances (qPCR assays using the DTAMB FR facility), nitrifier diversity (using the iBio bioinformatics facility of LEM), and N₂O production and emission rates (including with chambers[l20] ). It will use highly specific digital droplet PCR thanks to the DTAMB platform to finely survey inocula survival. X. Le Roux (involvement: 40%) will lead WP0 (Coordination and management) and will co-lead WP2 and WP4. He will also be part of the IMMINENT executive board, having experience of the coordination and management of projects and programmes, including the coordination of four European projects so far. T. Pommier (involvement 20%) will co-lead WP1. He is a junior INRA scientist with a h factor of 19 (37 papers, including in Ecology Letters, Global Change Ecology, Nature, PNAS). More generally, the LEM members involved in IMMINENT have skills on the ecosphysiology, ecology and diversity of the major microbial functional groups involved in N cycling, and employ state-of-the-art biochemical assays and molecular tools.

3 recent relevant publications:

·  Mallon CA, Le Roux X*, Dini-Andreote F, Poly F & Salles JF* (2018) An alien’s legacy: bacterial invasions steer the soil microbial community away from the invader’s niche. ISME J. 12: 728–741. (*shared leadership on this work)

·  Li M (…) & Pommier T (2019) Facilitation promotes invasions in plant-associated microbial communities. Ecol. Lett. 22, 149-158.

·  Ma B (…) & Le Roux X (2019) How do soil microorganisms and plants concurrently respond to N, P and NP additions? Application of the ecological framework of (co-)limitation by multiple resources at community and taxa levels. J. Ecol. (in press).

Partner #2 - UMR INRA 1347 Agroécologie (L Philippot, F. Bizouard, D. Bru, A. Spor) conducts research on the ecology of microorganisms that transform nitrogen fertilizers for limiting the loss of nutrients and the production of greenhouse gases with a strong interest in i) the diversity and ecology of denitrifiers and (ii) plant-microbe interactions. This partner will bring its expertise on the genomics, diversity and ecology of N₂O reducing bacteria. Partner #2 will assess potential nitrification rates as well as N2O fluxes. This partner will also quantify the abundance of N₂O producers and N₂O reducers and will be responsible for the planted soil mecocosm experiment conducted in the high throughput phenotyping plateform at the INRA Dijon. L. Philippot (involvement 20%) will lead WP4 and co-lead WP2 and WP3 . He is a microbial ecologist who has a h-index of 47 and has published more than 130 papers (including in Global Change Biology, ISME J., Nature Climate Change, Nature Microbiology Reviews). A. Spor is an INRA researcher with a h index of 16 (32 papers including in Cell, Nature Biotechnology, Nature Microbiology Reviews, PNAS) and a strong expertise in ecology, bioinformatics and biostatistics. A. Spor will lead WP1 [l21] 

3 recent relevant publications:

·  Jones C.M., Spor A., Brennan F.P, Breuil M.C., Bru D., Lemanceau P ., Griffiths B., Hallin S., Philippot L. 2014. Recently identified microbial guild mediates soil N₂O sink capacity. Nature Climate Change. 4: 801-805

·  Domeignoz-Horta L.A., Philippot L., Peyrard C., Bru D., Breuil M.C., Bizouard F., Justes E. Mary B., Léonard J., Spor A. 2018. Peaks of in situ N₂O emissions are influenced by N₂O producing and reducing microbial communities across arable soils. Global Change Biology. 24: 360-370

·  Domeignoz Horta L., Putz M., Spor A. Breuil M-C. Bru D., Bizouard F., Hallin S; Philippot L. 2016. Non-denitrifying nitrous oxide-reducing bacteria - An effective N₂O sink in soil. Soil Biology Biochemistry 103: 376-379

Partner #3[l22]  - PREMIER TECH GHA, Vivy centre (C Lapadatescu, F Barraud, B Solnais, C Gruau) is located in Anjou (49) since 1978, now part of Végépolys (‘pôle de compétitivité’ on plant sciences). It performs R&D activities related to inocula formulation and prebiotics in particular. C. Lapadatescu, PT GHA Scientific Director and based in Vivy, is an engineer (PhD in Microbiology) who has 20 years of experience in innovation and active compounds. She has co-designed this proposal and will co-lead its WP3.  F. Barraud is R&D Director, with a speciality in the biology and properties of inocula and culture supports. B. Solnais, Responsable technique Projets and engineer (M2 Plant health & fungi biotechnology), will co-lead WP4. They will work with C. Gruau (Coordinatrice Technique Projets -  M2 Plant Biology & Horticulture).

3 recent relevant publications:

·  x

·  x.

·  x.

Self-funded Partner #4[l23]  - University of Groningen, The Netherlands (JF Salles, CA Mallon, JH Veldsink) has largely contributed to the field of soil microbial invasions by using modelling and community niche approach to investigate the mechanisms leading to successful invasions and their potential impact for the functioning of the native soil communities. JF Salles is associate professor in Microbial Community Ecology with h-index of 23 and 78 publications including in ISME J., Microbiome, PNAS and Trends in Microbiology. CA Mallon is a younger researcher with large experience in soil microbial invasions.

3 recent relevant publications:

·  x

·  x.

·  x.

Note: The CV of the scientific coordinator and of each partner’s scientific leader is included in Annex #1.

Implication of the scientific coordinator and partner’s scientific leader in on-going project(s)

In grey: ongoing projects that will end before IMMINENT starts

b.      Implemented and requested resources to reach the objectives[l24] 

Project budget: total cost= 783k€ ; total required to ANR: 462k€ (192 k€ for functioning; 49k€ for equipment; 21k€ for missions; 200k€ for two PhDs; permanent positions: 0k€)

Partner 1: LEM

Staff expenses

Costs linked to the researchers, engineers, technicians and other scientific staff affected to the project; in the case of a JCJC project: cost of partially releasing the young researcher from teaching duties. Justification in relation to the scientific objectives.

Instruments and material costs

Acquisition, depreciation or rental costs of instruments or material and the scientific consumables specifically used for the achievement of the project. Justification in relation to the scientific objectives.

Building and ground costs

Rental costs of new premises and lands or the fitting of premises or pre-existing lands for the use of the project. Justification in relation to the scientific objectives.

Outsourcing / subcontracting

Acquisition costs of (1) Licences, patent, brand, software, database, copyrights etc.; (2) Subcontracting costs; for the achievement of the project. Justification in relation to the scientific objectives.

General and administrative costs & other operating expenses

Missions expenses and travel costs of the permanent and temporary staff affected to the project; conferences organisation costs. Justification in relation to the scientific objectives.

General and administrative costs & other operating expenses

Partner 2: AgroEcology

Staff expenses

Instruments and material costs

Building and ground costs

Outsourcing / subcontracting

General and administrative costs & other operating expenses

Partner 3: Premier Tech

Staff expenses

Instruments and material costs

Building and ground costs

Outsourcing / subcontracting

General and administrative costs & other operating expenses

Requested means by item of expenditure and by partner*

* The amounts indicated here must be strictly identical to those entered on the website. If both information are not consistent, if they were badly filled in or lacking, the information entered online will prevail on those reported in the submission form / scientific document.

** For marginal cost beneficiaries, these costs will be a package of 8% of the eligible expenses. For full cost beneficiaries, these costs will be a sum of max. 68% of staff expenses and max. 7% of other expenses.

III.         Impact and benefits of the project[l25] [l26] 

The forest ecosystems of the Congo basin span across much of Central Africa. It constitutes the second largest area of contiguous moist tropical forest left in the world and represent approximately one fifth of the world's remaining closed canopy tropical forest (ref). Global change is expected to strongly affect the distribution of termite species in these tropical ecosystems in relation to fragmentation of habitats and land-use changes [23]. Despite its ecological importance, the Mayombe forest –which forms the southern-western margin of the Congo Basin’s tropical rainforest– was subjected to decades of unsustainable utilization of its natural resources [24]. Due to this anthropogenic pressure, it is a relevant site for the field experiments and scenario development envisaged in this project. By addressing the contribution of termites to N₂O emission at landscape scale in the Mayombe forest, along with the abiotic and biotic (microbial) factors governing these emissions by termites, the present project will contribute to tackle the ANR Action Plan Challenge 1. It will also help documenting major international initiatives, in particular the future IPCC and IPBES assessment. Three major outcomes of the project are expected:

ACADEMIC IMPACTS:

* Demonstrating to what extent N₂O emissions/uptake rates vary according to termite feeding guilds. This will be evaluated for the first time for the main different guilds of termites (soil-feeders, wood-feeders, grass-feeders and fungus-cultivating termites) using both living termites and their guts.

* Renewing our understanding of the microbial determinants of N₂O emission/uptake from termites. The project will assess for the first time the potential role of different groups of nitrifiers and denitrifiers, along with the possible interactive effect of methanotrophs, for N cycling and N₂O emissions by termites. We will also distinguish gross and net N₂O production and will account, again for the first time for the roles of the two known clades of N₂O-reducers.

* Scaling the amounts of N₂O emitted/uptaken by different termite species feeding guilds up to the landscape scale in the Mayombe forest. Based on an extensive field campaign (actually 5 field campaigns covering different seasons and years), this will be a premiere for quantification of N₂O emissions of and land uses in tropical humid forest.

* Exploring to which extent termite species assemblages and associated N₂O fluxes might change under plausible land-use scenarios. This is a critical step in estimating the global contribution of termites to the N₂O emission budget in tropical forests.

In addition, several Partners of TERSNO are involved in academic teaching duties in Créteil, Montpellier and Lyon in particular. Therefore, the results of the project will feed the courses dedicated to the students enrolled in microbial ecology, conservation biology or agroforestry. 

Scientific publications will be used to communicate the results of TERSNO. Publications will target high-rank international journals. The results of TERSNO may also be published in book chapters, and we envisage to write a synthesis about GHG emission by termites across different tropical biomes.

SOCIO-ECONOMIC IMPACTS:

* Dissemination of results to local stakeholders. Based on the predicted N₂O fluxes according to land-use scenarios in the Mayombe forest area, we plan to develop a policy brief (maximum 4 pages) suited to relevant local decision makers, in particular public authorities involved in the management of forests in the Mayombe and more generally in the tropics, to raise awareness about the risk that some changes in land-use can have in term of greenhouse gas emission through altered termite abundances and diversity in these environments. The National Institute for Forestry of Congo (IRF) will host a two-day workshop on “Impacts of land-use changes on soil macrofauna and GHG emission in the Mayombe ecosystems” dedicated to master students, officers of Ministry of Forestry involved in forest management programmes and stakeholder leaders from local communities of the Mayombe forest.

* Dissemination of results to international stakeholders. We will synthesize the plausible temporal trends in termite assemblages and associated N₂O emissions based on land-use change scenarios for this tropical forest area over the next decades. The synthesis will be shaped in a palatable way for the TSU and experts working for the future IPCC and IPBES regional assessments. Through his function in BiodivERsA and in IPBES regional assessment, X Le Roux has experience and contacts for developing this kind of policy brief and synthesis.

IV.        References related to the project[l27] [l28] 

. 1 - Philippot L. et al. (2013) Nature Reviews Microbiology. 11:789-799. u 2 - de Vrieze J. (2015) Science 349: 680-683. u 3 - Rho H. et al. (2017) Microb. Ecol. 75: 407-418. u 4 - Finkel O.M. et al. (2017) Curr. Opin. Plant Biol. 38: 155-163.  u 5 - Schütz L et al. (2018) Front. Plant Sci. 8:2204. doi: 10.3389/fpls.2017.02204 u  6 - Jiménez-Gómez A. et al. (2017) AIMS Microbiology, 3(3): 483-501. DOI: 10.3934/microbiol.2017.3.483.  u  7 -  Bashan Y. & de-Bashan L.E. (2010) Adv. Agron. 108: 77-135.  u  8 - Dobbelaere S. et al. (2001) Aust. J. Plant Physiol. 28: 871-879.  u  9 – Paredes-Paliz K. et al. (2018) Plant Biol. 20: 497-506.  u  10 - Florio A. et al. (2017) Sci. Reports 7: 8411  u  11 - Bounaffaa M. et al. (2018) Ecol. Econ. 146: 334-346.  u  12 – Jones C.M. (2014) Nature Climate Change. 4: 801-805 u  13 - Gao N. et al. (2016) Soil Biol Biochem 97: 83-91.  u  14 - Domeignoz-Horta L. et al. (2016) Soil Biol Biochem. 103: 376-379. u  15 – Timmusk S. et al. (2017) Front Plant Sci. 8: 49. u  16 - Ruzzi M. & Aroca R. (2015) Scientia Horticulturae 196: 124–134. u  17 – Mallon C. et al. (2015) Ecology 96: 915-926. u  18 – Mallon C. et al. (2018) ISME J. 12: 728–741.   u  19 – Cabrol L. … & Le Roux X. (2016) Environ. Sci. Technol. 50: 338–348.

 [l1]This paragraph refers to the evaluation criteria “Quality and scientific aim”

 [l2]Present the objectives and the research hypothesis; present the scientific and technical barriers to be lifted; present the expected results; if applicable describe any final products developed

 [l3]Emphasise the originality and the novelty of the proposal - concerning its objectives and its methodology – and its position in relation to the state of the art; show the contributions of the project partners to this state of the art; present any preliminary results. In the case of a project proposal following up on previous project(s) already funded by ANR or by another body, provide a summary of the results achieved and clearly describe the new issues raised and the new objectives set out in the light of the earlier project

 [l4]Describe the methodology and its relevance to reach the objectives, detail the scientific risks and fall-back solutions envisaged.

Set out the scientific programme and justify the work programme's task breakdown with regard to the objectives being pursued.

-        For each task, describe the objectives, the work programme, deliverables, partners' contributions, methods and technical decisions, risks, and fall-back solutions. Illustrate with a Gantt chart.

-        For research projects dealing with subjects that may harm humans, animals, or the environment, discuss the ethical aspects of the project.

-        If applicable, indicate the conditions of access to a research infrastructure (IR) or a very large research infrastructure (TGIR)

⚠ Concerning PRCI proposal, it is mandatory for applicants to provide the scientific contribution of the French and foreign teams

 [l5]will be updated

 [l6]Laurent

 [l7]To be confirmed with Ayme

 [l8]Any ref Joana for this approach?

 [l9]10*3*6=180 microcosms!

 [l10]We have to predict a priori both overlap and emission from monocultures è to be said

 [JFS11]One soil??? Which one?

 [l12]Which plant?

 [l13]I am not sure whether or not it is feasible. At least not if doing the classical approach consisting in samling the head space every 30 min for 2h…in 60 mesocosms…placed on conveyors!

 [d14]To be discussed with Carmen

 [l15]I asked for more information. I might expand the text in relation to the PPHD when I have it.

 [X16]Description du risqué ci dessus ok; mais moyen de limiter ce risqué ou de se retourner trop faible pour l’instant

 [l17]Juste, any suggestion? Difficile cependant de ne pas evoquer ce risqué et d’en proposer un autre a la place.

 [l18] evaluation criteria “Organisation and implementation of the project”

·        [l19]In the case of a collaborative research project (PRC, PRCE, PRCI),

-        Present the scientific coordinator, his/her experience as a scientific coordinator or a project manager, his/her experience in the scientific field (including the foreign scientific coordinator in a PRCI proposal)

-        Present the consortium and its complementarity: demonstrate the quality and complementary nature of the consortium specifying the identity of the scientists involved and their institution and all other items providing a framework for judging the quality and complementarity of partners and consortia

-        Complete the following table including information concerning the involvement of the scientific coordinator and partner’s scientific leader in regional, national and international on-going projects.

 [l20]Not if the plants are grown in Dijon

 [l21]Ok Ayme?

 [l22]Expend see LEM for instance

 [l23]Expend see LEM for instance

 [l24]Describe the means – those previously available and those requested – to achieve the objectives.

-        Scientific and technical justification of the requested means – per item of expenditure and by partner -–, linked to the objectives of the proposal.

-        Summarise the requested funds in the table below in accordance with the information filled out on the website and with ANR’s grant allocation rules (règlement relatif aux modalités d’attribution des aides de l’ANR ).

-        Description of the context in terms of human and financial resources available thanks to previous or ongoing projects.

-        If a partner is relying on its own funds, justify the available means to realise its tasks.

⚠ The sub-criteria “Appropriateness of implemented and requested resources to the project’s objectives” is as important as the other sub-criteria. The reviewers will wait for a high level of detail in the calculation and its scientific justification.

Examples: What kind of contract for the temporary staff, duration, for which task? What kind of instrument, for which task, why buying instead of renting? What kind of mission (conferences, meeting, data collection, etc.), national / international, for how many people, how much time/how many times?

 [l25]refers to the evaluation criteria “Impact and benefits of the project”

 [l26]For every funding instruments:

Describe in what scientific fields and eventually economic, social or cultural field project results may have an impact, in the short, medium or long term.

For a PRCE project,

-        Describe actions to transfer technology and innovation to the social and economic world.

 [l27]refers to the evaluation criteria « “Quality and scientific aim”

 [l28]List the bibliographical references used for the proposal.

The bibliography must be included in the 20-page limit. The bibliography may include preprints that are yet to be published in a peer-reviewed journal, especially those referencing preliminary data. If available, please indicate the “open access” link to improve accessibility for the reviewers. Highlight the references for which one or more of the authors are involved in the project