D1.5_AGROECOseqC_WP1_WP2_WP4_WP6_10.5281zenodo.14167748

Towards climate-smart sustainable management of agricultural soils AGROECOlogical strategies for an efficient functioning of plant - soil biota interactions to increase SOC sequestration AGROECOseqC Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites Due date of deliverable: M54 Actual submission date: 15.11.2024 Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 2 GENERAL DATA Grant Agreement: 862695 Project acronym: EJP SOIL Programme title: Towards climate-smart sustainable management of agricultural soils Programme website: www.ejpsoil.eu Project title: Project website: Start date of the project: February 1st, 2020 Project duration: 60 months Name of lead contractor: INRAE Funding source: H2020-SFS-2018-2020 / H2020-SFS-2019-1 Type of action: European Joint Project COFUND DELIVERABLE NUMBER: D1.5 DELIVERABLE TITLE: Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites DELIVERABLE TYPE: Report WORK PACKAGE N: WP1 and WP2, WP4, WP6 WORK PACKAGE TITLE: Project management and coordination DELIVERABLE LEADER: Alessandra Trinchera, CREA (Italy) AUTHOR: DOI: Alessandra Trinchera, Dylan Warren Raffa, Skaidre Suproniene, Grazina Kadziene, Alvyra Slepetiene, Aida Skersiene, Modupe Olufemi Doyeni, Marga Ros, Akin Un, Valentina Baratella, Elena Testani, Sara SanchezMoreno, Marjoleine Hanegraaf, Simon Sail, Jim Rasmussen, Sebastien Fontaine 10.5281/zenodo.14167748 LICENSE DISSEMINATION LEVEL: CC BY 4.0 CO Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 3 ABSTRACT The Soil Health Directive pushes towards the application of agricultural management practices based on agroecological intensification in farming systems, to contrast the loss of organic carbon and the reduction of soil biodiversity in the long term. In six long-term and two short-term European experiments, representative of different pedoclimatic regions, some soil physicochemical parameters (soil density, pH, texture, stability of soil aggregates, NO3, NH4, available P (as P2O5)), soil C pools (SOC, water-extractable C, microbial C), bacterial and fungal richness (CHAO index), bacterial and fungal diversity (Shannon index), and mycorrhizal colonization of plant roots (M) were measured as responsive to a gradient of agroecological intensification, applied by introducing conservative management practices such as no-tillage, service crops, introduction of legumes, organic amendments and/or residues restitution, fungi inoculant. The PCA, applied on the eight experimental sites, evidenced the role of pedoclimate conditions on affecting the selected soil indicators. The PCA run on each one experimental site was instead able to very well separate the obtained clusters in function of the agroecological intensification, associating some soil indicators to the applied management practice. A good correlation between SOC and Cmic was found in the experimental sites tested, confirming that Cmic is a fast, sensitive indicator of soil C accumulation. No-tillage emerged as the most relevant factor affecting SOC accrual, able to increase it of 20% after 10 years of experiment in Mediterranean cropping systems, favouring also plant root mycorrhization in field. In acid soils, the no-tillage should be associated to organic amendment to enhance its positive effect on C stock, limiting the reduction of bacteria richness and diversity. The cover crop species choice is key in terms of expected ecosystem service: Poaceae, for increasing SOC accumulation, while Fabaceae and Brassicaceae for supporting the soil N cycle the first ones, or for plant protection the second ones. To correctly apply the agroecological transition in Europe, once again it is necessary to propose the introduction of agronomic practices in a context of regionalization of the environmental measures of the CAP, appropriately designed and implemented on each agroecosystem, considering the pedoclimate, the physical, chemical, and biological characteristics of the soil, as well as the production system. This approach can ensure an adequate and effective application of the Soil Health Directive and face the soil C and biodiversity loss on long term. Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 4 Table of Contents List of Tables ............................................................................................................................................ 4 List of Figures ........................................................................................................................................... 4 List of acronyms and abbreviations ......................................................................................................... 4 1. Introduction ..................................................................................................................................... 5 2 Material and methods……………………………………………………………………………………………………….….……6 3. Main results…………………………………………………………………………………………………………………………….…9 Conclusions………………………………………………………………………………………………………………………………………11 List of references…………………………………………………………………………………………………………………………….. 12 List of Tables Table 1. Description of AGROECOseqC experimental sites………………………………………………………………..7 List of Figures Figure 1. Figure 1. Position map of AGROECOseqC experimental sites. Figure 2. Principal components analysis (PCA) biplot and confidence ellipses ordering the AGROECOseqC experimental sites related to selected soil parameters and bioindicators (SD, SOC, WEOC, WSA, NH4+, NO3-, available P (as P2O5), Cmic, Shan_BC, Shan_FN, Chao_BC, Chao_FN, M). Figure 3. Principal components analysis (PCA) biplots ordering the T1, T2 and T3 treatments in S1, S2, S3, S4, S5, S6, S7 and S8 experimental sites related to selected soil parameters and bioindicators. Figure 4. Principal components analysis (PCA) biplots and confidence polygons ordering the T1, T2 and T3 treatments in S1, S2, S3, S4, S5, S6, S7 and S8 experimental sites related to selected soil parameters and bioindicators. List of acronyms and abbreviations WP Work Package EU European Union SD Soil density SOC Soil Organic Carbon WEOC Water Extractable Organic Carbon WSA Water Stable Aggregates Cmic Microbial C Shan_BC Bacteria Shannon diversity index Shan_FN Fungi Shannon diversity index Chao_BC Bacteria richness Chao_FN Fungi richness M Mycorrhizal colonization intensity PCA Principal Component Analysis Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 5 1. Introduction The Soil Health Directive (EU COM, 2023), as legislative act promoted by European Union (EU), aims at protecting and restoring soil health across its Member States. On July 5, 2023, the new Soil Monitoring Law was enacted, as part of its broader European Union soil strategy for 2030. This law is designed to address key soil threats such as erosion, contamination, and loss of soil biodiversity, to achieve healthy soils by 2050. The directive will establish a comprehensive monitoring framework to ensure sustainable soil use and to regenerate degraded soils to preserve future productivity. It is a response to the alarming situation that “...over 60% of European soils are currently unhealthy, which has significant implications for ecosystem services, food security, and climate resilience” (EU COM Soil Health Directive, 2023). Member States will be required to implement practices that promote soil health and to identify and remediate contaminated sites. This initiative is also a key deliverable of the EU biodiversity strategy and contributes to the objectives of the European Green Deal (Fetting, 2020). In this framework, European policy makers are pushing towards the application of agricultural management practices based on agroecological intensification, both to integrate ecological principles and soil biodiversity management, and to maintain or increase farm productivity, reducing dependency on external inputs and enhancing ecosystem services on long term (Wezel et al., 2015). The soil organic carbon (SOC) loss and the reduction of soil biodiversity are among the other key challenges that European agriculture must face to enhance agricultural productivity while enhancing the ecosystem services from agroecosystems. Soil C pools monitoring is generally used to infer SOC accrual, whose contribution depends on a range of soil physicochemical properties, including the water retention, the carbon and nutrient storage, and the microbial activity (Kopittke et al., 2022). Using multiple parameters, directly or indirectly linked with SOC accrual, can instead improve our ability to assess the soil drivers of carbon pool regulation (Bardgett and van der Putten, 2014). Since microbial diversity strongly drives plant-soil interactions by modulating nutrient availability for plants (Weidner et al., 2015), the resulting agroecosystem functioning will influence plant residue decomposition (van der Heijden and Wagg, 2013), N cycling such as nitrification and denitrification processes (Nardi et al., 2021, Wagg et al., 2021), SOC stabilization and accumulation (Manici et al., 2019), and finally agricultural productivity (Garbach et al., 2016). Considering the soil microbiota, fungal species and, particularly, the mycorrhizal fungi, play a socio-ecological role among neighbouring plants in field by forming a common fungal hyphal mycelium, modulating water, nutrient and energy fluxes (Simard and Durall, 2004), influencing also the C persistence in agricultural soil (Parihar et al., 2020; Cania et al., 2020). A deeper understanding of how these biological communities respond to sustainable farming techniques could highlight their importance in maintaining soil functionality, ultimately supporting resilient agroecosystems. By bridging this knowledge gap, we could better evaluate and enhance soil health in ways that support biodiversity and long-term agricultural productivity (Sharma et al., 2024). Starting from this assumption, the AGROECOseqC project, funded by EJPSOIL program, tested the effect of some agroecological practices on SOC sequestration, considering a set of soil physicochemical parameters and bioindicators, potentially applicable in soil monitoring within the recent Soil Health Directive. Agroecological intensification, based on reducing soil disturbance, increasing plant diversity, and applying organic inputs, were introduced in six European long-term experiments and two short-term ones, to identify the indicators mostly sensitive to the applied management practices and contextualize the obtained feedbacks within the different European pedoclimate regions. Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 6 2. Materials and Methods The AGROECOseqC project operated by applying a multifunctional approach, based on a multi-site study, representative of different European cropping systems under different pedoclimate regions. 2.1 Description of experimental sites Seven European experimental sites, located in Italy (IT, S1), France (FR, S2), Belgium (BE, S3), The Netherland (NL, S4), Lithuania (LT, S5), Spain (ES, S6), Denmark (DK, S7), and an extra-European one in Turkey (TK, S8,) were considered. In Figure 1, a map of the eight experimental sites location is reported. Figure 1. Position map of n. 8 AGROECOseqC experimental sites. Six sites were long-term experiments (S1, S2, S3, S4, S5, S6), while the last two ones were recently established (S7 and S8). Only S1 was organically managed from 2017 (Table 1). In each experimental site, three treatments were compared: the first two at increasing level of ecological intensification (T1<T2) and a third one, taken as control (T3), where no agroecological intensification was introduced (e.g., managed as usual). The tested T1 and T2 treatments consisted in one or more agroecological practices applied in field (no-tillage, cover crop introduction, crop rotation, soil amendment or farmyard manure application, crop residues restitution), alone or in combination: we evaluated the “treatment” as applied agroecological intensification. In Table 1, the eight EU experimental sites were described by reporting main relevant information and the applied agroecological practice/s in each cropping system. Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 7 Table 1. Description of AGROECOseqC experimental sites. Country, site number, institution, geographical coordinates, pedoclimate region, site code, EU country, institution, site location, pedoclimatic zone, mean annual temperature (°C) and rainfall (mm y-1), year of experiment set up, soil texture, soil pH, treatment code, main crop/s, applied management practices, and fertilization. 2.2 Soil /plant root sampling and methodology In each site, T1, T2 and T3 treatment were tested in four replicates (as randomized blocks), for a total of 3 treatments x 4 blocks = 12 plots/site and n. 96 plots in total. Four soil samples per plot were collected in each experimental site at maximum plant demand and then mixed to form n.1 soil composite sample per plot (four replicates per treatment). Also, roots were collected following the same sampling procedures. All the soil parameters and bioindicators were determined on the composite soil or root samples collected in each block. Two groups of soil parameters and microbial indicators were selected: I. Soil parameters: soil bulk density (SD, Mg×m-3), water stable aggregates at 1÷0.25 mm (WSA, %), soil organic carbon (SOC, %), soil organic carbon extractable in cold water (WEOC, g×kg-1) soil ammonia (NH4+, mg×kg-1), soil nitrate (NO3-, mg×kg-1), soil available P (expressed as P2O5, in mg×kg-1). II. Bioindicators: soil microbial biomass C (Cmic, mg×kg-1), Shannon index of soil bacteria (Shan_BC), Shannon index of soil fungi (Shan_FN), Chao index of soil bacteria (Chao_BC), Chao index of soil fungi (Chao_FN), colonization intensity of plant roots (M, %). The selection of the soil parameters and bioindicators was carried out to understand how the applied agroecological practices may have affected the physico-chemical and nutritional status of the agroecosystem, the soil C-pools and nutrients, the soil bacteria and fungi richness and diversity, and the root mycorrhizal fungi colonization (on collected root samples). Sampling and analytical methods applied for soil indicators determinations are described in detail into AGROECOseqC Handbook (D1.1.1_ D.2.3_ D4.1_D4.2.1_D6.1.1_ D6.1.2). 2.3. Statistical evaluation Principal component analysis (PCA) was applied on generated dataset using Past Software Package (Past Version 2.17c, yvind Hammer, D.A.T. Harper, February 2013), considering the effect of: Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 8 1. the European experimental sites, regardless the tested agroecological management practices, 2. the intensity of agroecological management practices (T1, T2 and T3), regardless the experimental site, 3. the agroecological management practices, applied in each core-site. The PCA eigenvalues and related percentages of variance explained by the component 1 (C1) and 2 (C2) were registered. Regression models were used to fit the data, considering predicted R-squared values (Excel, ver. 1908—Microsoft Office 365 software package 2020). All tested parameters were statistically analysed by ANOVA, considering the treatment as fixed factor. Mean comparison was carried out according to a post hoc Tukey’s honestly significant difference (HSD) test at p≤ 0.05 probability level. 3. Main results 4.1. AGROECOseqC experimental sites – Figure 2 reports the results of the PCA applied to the AGROECOseqC dataset of both soil parameters and bioindicators recorded in the eight experimental sites, regardless the applied agroecological management. Figure 2. Principal component analysis (PCA) biplot and confidence ellipses ordering the AGROECOseqC experimental sites related to selected soil parameters and bioindicators (SD, SOC, WEOC, WSA, NH4+, NO3-, available P (as P2O5), Cmic, Shan_BC, Shan_FN, Chao_BC, Chao_FN, M). The first component explains about 27.9% of the soil parameters/bioindicators variance while the second component counted 19.9%, for a total of 47.8 % of the explained variance recorded in the experimental sites. Overall, PCA gave three groups of experimental sites: North-Med-Continental (right side), South-Med-Anatolic (left side), and Atlantic-continental-Nemoral (in the middle). The first component completely separated S8 (TK) from all the other experimental sites, while S1 (IT) and S2 Component 2 –19.9% Component 1 –27.9% Deliverable D1.5 Effect of agroecological intensification on soil C stock and microbial diversity in EU and non-EU experimental sites This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 9 (FR) were partially overlapped but clearly distinguishable from the other European sites. The S3 (BE), S4 (NL), S5 (LT), and S7 (DK) experimental sites instead were not separated, although divergent from S1 (IT), S2 (FR), and S8 (TK). In the end, the S6 (ES) site partially overlapped with S3 (BE) only. Along the positive value of C1, S1 was associated with C-mic, WEOC, and WSA, while S2 site to soil soluble NH4+. Contrastingly, along the negative value of C1, S3 was associated to SD, available P, fungi diversity and richness indexes, while in S6 the soil available P, fungi CHAO index, and mycorrhizal colonization intensity were the main discriminants. The S8 extra-Europe site was instead mainly affected by fungi richness and root mycorrhization. In the middle of C1, the soil fungi Shannon index mainly contributed to S7 asset, the bacteria richness and diversity to S4, while S5 was mostly affected by soil organic carbon, available nitrate, and microbial biomass C. II) AGROECOseqC agroecological intensification - The second step of evaluation consisted in applying principal component analysis to verify how the tested management practices, namely T1, T2, and T3, affected the explained variance of selected soil parameters and bioindicators, regardless or in function of the experimental site. Figure 3 reports the PCA results biplots related to the effect of the T1, T2 and T3 treatments, regardless the effect of AGROECOseqC experimental site. Figure 3. Principal component analysis (PCA) biplots ordering the T1, T2 and T3 treatments in all the S1, S2, S3, S4, S5, S6, S7 and S8 experimental sites related to selected soil parameters and bioindicators (SD, SOC, WEOC, WSA, NH4+, NO3-, P2O5, Cmic, Shan_BC, Shan_FN, Chao_BC, Chao_FN, M). The PCA of tested soil parameters and indicators explained the 49.5% of the total variance, although both the components were not able to discriminate the effect of the treatments, independently from the experimental site. Figure 4 reports the PCA biplots applied to each one AGROECOseqC experimental site for evidencing the effect of T1, T2 and T3 treatments.