Practical applications of soil microbiota to improve ecosystem restoration: current knowledge and future directions

Practical applications of soil microbiota to improve ecosystem restoration: current knowledge and future directions Shawn D. Peddle 1, *,Riley J. Hodgson 1 ,Ryan J. Borrett 2 ,Stella Brachmann 3 , Tarryn C. Davies 1 ,Todd E. Erickson 4,5 ,Craig Liddicoat 1 , Miriam Muñoz-Rojas 6,7 ,Jake M. Robinson 1 ,Carl D. Watson 1 , Siegfried L. Krauss 4,8 and Martin F. Breed 1 1 College of Science and Engineering, Flinders University, Sturt Road, Bedford Park, South Australia 5042, Australia 2 SoilsWest, Centre for Sustainable Farming Systems, Food Futures Institute, Murdoch University, 90 South Street, Murdoch, Western Australia 6150, Australia 3 University of Waikato Te Whare Wananga o Waikato Gate 1, Knighton Road, Hamilton 3240, New Zealand 4 Department of Biodiversity, Conservation and Attractions, Kings Park Science, Kattidj Close, Kings Park, Western Australia 6005, Australia 5 Centre for Engineering Innovation, School of Agriculture and Environment, The University of Western Australia, Stirling Highway, Crawley, Western Australia 6009, Australia 6 Department of Plant Biology and Ecology, University of Seville, C. San Fernando, Sevilla, Spain 7 School of Biological, Earth and Environmental Sciences, Centre for Ecosystem Science, University of New South Wales, Sydney, New South Wales 2052, Australia 8 School of Biological Sciences, The University of Western Australia, Stirling Highway, Crawley, Western Australia 6009, Australia ABSTRACT Soil microbiota are important components of healthy ecosystems. Greater consideration of soil microbiota in the restoration of biodiverse, functional, and resilient ecosystems is required to address the twin global crises of biodiversity decline and climate change. In this review, we discuss available and emerging practical applications of soil microbiota into (i) restoration planning, (ii) direct interventions for shaping soil biodiversity, and (iii) strategies for monitoring and predicting restoration trajectories. We show how better planning of restoration activities to account for soil microbiota can help improve progress towards restoration targets. We show how planning to embed soil microbiota experiments into restoration projects will permit a more rigorous assessment of the effectiveness of different restoration methods, especially when complemented by statistical modelling approaches that capitalise on existing data sets to improve causal understandings and prioritise research strategies where appropriate. In addition to recovering belowground microbiota, restoration strategies that include soil microbiota can improve theresilienceofwholeecosystems.Fundamentally,restorationplanning should identify appropriate reference target ecosystem attributes and –from the perspective of soil microbiota –comprehensibly consider potential physical, chemical and biological influences on recovery. We identify that inoculating ecologically appropriate soil microbiota into degraded environments can support a range of restoration interventions (e.g. targeted, broad-spectrum and cultured inoculations) with promising results. Such inoculations however are currently underutilised and knowledge gaps persist surrounding successful establishment in light of community dynamics, including priority effects and community coalescence. We show how the ecological trajectories of restoration sites can be assessed by characterising microbial diversity, composition, and functions in the soil. Ultimately, we highlight practical ways to apply the soil microbiota toolbox across the planning, intervention, and monitoring stages of ecosystem restoration and address persistent open questions at each stage. With continued collaborations between researchers and practitioners to address knowledge gaps, these approaches can improve current restoration practices and ecological outcomes. Key words: ecosystem restoration, improved ecological outcomes, positive soil legacy, recovery trajectory, restoration genomics, restoration methods. *Author for correspondence (Tel.: +61 8 8201 2113; E-mail: shawn.peddle@flinders.edu.au). Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Biol. Rev. (2024), pp. 000–000. 1 doi: 10.1111/brv.13124 CONTENTS I. Introduction .........................................................................2 II. Soil microbiota in restoration planning .....................................................3 (1) Considering soil microbiota and restoration goal setting ................................... 4 (2) When to prioritise investment in soil microbiota ......................................... 5 (3) Improving conclusions on causation in soil microbiota restoration ........................... 5 (4) Embedding soil microbiota experiments ............................................... 6 (5) Modelling approaches to ascertain causation ............................................ 6 III. Restoration interventions that directly target soil microbiota ....................................7 (1) Whole-soil translocations and microbial inoculations ..................................... 8 (2) Seed enhancements that contain microbial additives ...................................... 9 (3) Microbial cultures and suspensions .................................................. 10 (4) Targeting specific microbiota ...................................................... 10 (5) Promoting positive soil legacies ..................................................... 11 IV. Monitoring soil microbiota for restoration .................................................11 (1) Monitoring microbial diversity, composition, and function ................................ 11 (2) Monitoring restoration trajectories with soil microbiota .................................. 13 V. Conclusions .........................................................................14 VI. Acknowledgements ...................................................................14 VII. Author contributions ..................................................................14 VIII. References ..........................................................................14 I. INTRODUCTION Overexploitation of natural systems has led to the biodiversity crisis (Ceballos et al., 2015; Dirzo & Raven, 2003) and vast areas of degraded ecosystems (Gibbs & Salmon, 2015). While conserving remnant ecosystems is a priority, there is also a need to restore degraded areas to biodiverse and functioning ecosystems (Higgs et al., 2018; Perring, Erickson & Brancalion, 2018; Moreno-Mateos et al., 2020). Accordingly, there is a marked increase in ecosystem restoration globally, with targets to restore more than 350 million hectares under The Bonn Challenge and the United Nations declaring 2021–2030 the Decade on Ecosystem Restoration. However, there is considerable room to improve the success of restoration projects (Crouzeilles et al., 2016; Wortley, Hero & Howes, 2013). The essential role of soil in ecosystem restoration is recognised, mainly by considering soil physical and chemical processes in ecosystem recovery (Costantini et al., 2016; Muñoz-Rojas, 2018; Perring et al., 2015). Over the last 15 years however, increased attention has been given to soil microbiota –the communities of bacteria, archaea, fungi, viruses and protists within soils –and their interactions in the soil system and with aboveground biota due to their essential functional roles (Harris, 2009; McKinley, 2019; Eisenhauer et al., 2017). Soil microbiota are among the most biodiverse and functionally important ecosystem components and are essential to many biogeochemical processes. For example, biological nitrogen fixation by diazotrophs, nitrogen-fixing bacteria and archaea forms the foundation of Earth’s terrestrial productivity (Zhu et al., 2022; Vitousek et al., 2013) and cyanobacteria (carbon and nitrogen fixers) combine with fungi, bacteria, lichens, and other organisms to form biological soil crusts (“biocrusts”) which can stabilise soil landscapes and enhance water availability (Weber et al., 2022; Yan-Gui et al., 2013). Furthermore, soils are home to over half of Earth’sbiodiversity(Anthony, Bender & van der Heijden, 2023) and belowground microbial biomass is often comparable in scale to aboveground plant or animal biomass (Fierer, 2017). Soil microbiota also interact with aboveground ecosystem components and are intimately involved in plant and animal health, and vice versa. For example, the relationship between plants and arbuscular mycorrhizal fungi is one of the oldest terrestrial symbiotic interactions (Field & Pressel, 2018;Tisserantet al., 2013) where plants depend on fungi to gather essential nutrients in exchange for carbohydrates. Consequently, we can expect reciprocal shifts in above- and belowground ecosystem components (Kardol & Wardle, 2010;Proberet al., 2015). Therefore, improving the integration of soil microbiota and associated microbial ecology into ecosystem restoration will have considerable benefits across restoration planning, intervention, and monitoring phases (Fig. 1). Historically, scientists faced technological challenges in quantifying and grasping the diversity and composition of soil microbiota, as traditional culture-dependent methods were only able to grow <1% of microbial taxa (Alivisatos et al., 2015; Vartoukian, Palmer & Wade, 2010). However, modern sequencing technologies enable a detailed taxonomic and functional understanding of soil microbiota. For example, the now routine high-throughput amplicon sequencing of DNA extracted from soil samples can provide a detailed taxonomic view of the microbiota within a given sample (Berg et al., 2020; Fierer, 2017). These amplicon data sets can then be associated with spatial, land-use, Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. 2Shawn D. Peddle and others 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License environmental condition and/or restoration-intervention data to answer ecological questions (Tedersoo et al., 2019; Thomsen & Willerslev, 2015; Breed et al., 2019). Advances in DNA-based technologies and improvements in our understanding of plant–soil–ecosystem interactions are enhancing our ability to use soil microbiota in restoration (Mohr et al., 2022). Indeed, there are several reviews on soil microbiota in a restoration context, and most have focused on theoretical aspects of including soil microbiota in restoration or relevant technological advancements (Coban, De Deyn & van der Ploeg, 2022; Contos et al., 2021; Rawat et al., 2022). Here, we complement these previous reviews by focussing on the practical interface of soil microbiota and ecosystem restoration and highlight key knowledge gaps that are limiting effective integration of soil microbiota into restoration. We highlight where and how the integration of soil microbiota has successfully occurred and identify opportunities and challenges for improved integration to enhance restoration outcomes. II. SOIL MICROBIOTA IN RESTORATION PLANNING Planning a restoration project requires setting realistic goals, making informed choices of interventions, and deciding on indicators to monitor progress towards stated goals (Hobbs & Norton, 1996; Collen & Nicholson, 2014; Suding et al., 2015). Unfortunately, despite enormous growth in the scope and scale of restoration globally, many projects fail to achieve their stated goals despite the growing scientific rigour of restoration practice (Crouzeilles et al., 2016; Wortley et al., 2013; Sun et al., 2017). The reasons for these shortfalls are numerous and include insufficient consideration of soil microbiota (Heneghan et al., 2008; Kardol & Wardle, 2010; Farrell et al., 2020). Accordingly, restoration projects should routinely consider soil microbiota early as part of their modus operandi –together with more traditional targets and assessments for flora and fauna. These projects will then be in a better position to determine their ecological starting place, Fig. 1. The Society for Ecological Restoration (SER) recovery wheel (Gann et al., 2019) and how improved integration of soil microbiota into the planning, intervention, and monitoring phases of ecosystem restoration projects could contribute to each of the six recovery outcome themes. Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. Soil microbiota and ecosystem restoration 3 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License trajectory and target –all components of best-practice restoration (Kardol & Wardle, 2010; Heneghan et al., 2008; Gann et al., 2019). Here, we outline how and when restoration projects should plan to incorporate soil microbiota from the outset to maximise benefits to ecological outcomes while avoiding wasted resources. We also highlight that improving our understanding of how specific restoration interventions affect soil microbiota is needed to plan restoration effectively. (1) Considering soil microbiota and restoration goal setting Quantifying the severity of the degradation of an ecosystem is crucial in determining the level of intervention required to meet targets (Heneghan et al., 2008; Chazdon, 2008). For example, when a restoration site is depleted of mycorrhizal fungi required by a target plant species (e.g. mixotrophic orchid species are entirely dependent on orchid mycorrhiza for germination), there is little sense in investing resources to establish the plant without simultaneously addressing the lack of symbiotic fungi (Koziol, Crews & Bever, 2020). Furthermore, invasive plant species in a degraded landscape can modify soil microbiota to the point that the soil environment is in an alternate state of dynamic equilibrium (Suding, Gross & Houseman, 2004; Gornish et al., 2020). Here, removing the invasive plants and revegetating the landscape relies on the soil microbiota to move towards a state that is more supportive of the recovering native plant community which is by no means guaranteed (Harris, 2009). A revegetation-only approach may not overcome persistent soil legacies (i.e. altered nutrient levels from fertiliser use, altered soil structure from compaction, invasive species, undesirable biological communities) and risks perpetual states of ecological invasion (Anthony et al., 2019; Bell, Siciliano & Lamb, 2020). As a result, specific interventions that address invasive plants and altered soil microbiota (see Section III) need to be part of the restoration planning phase. Moreover, major disturbance to soil physical and chemical conditions (e.g. from mining, erosion, compaction, excess nutrients) will alter the foundational habitat for soil microbiota, so addressing limiting abiotic factors also represents a key priority in restoration planning (Robinson et al., 2024). There is immense value in setting early goals to understand soil microbial ecology at the initial stages of a restoration project. This goalsetting process will help the restoration practitioner to quantify and pre-empt biotic and abiotic constraints or opportunities (e.g. a lack of mycorrhizal fungi, plant-associated pathogens for structuring plant communities, altered soil physical or chemical properties). If barriers to recovery are not identified as part of the planning stage, ecosystem recovery will likely be inhibited (Hobbs & Norton, 2004). Practitioners should address these constraints in a restoration project by, for example, using knowledge of plant–soil feedbacks in the planning phase. Restoration projects could promote negative feedbacks between plant and soil communities by, for example, inoculating sites with late-succession soil microbiota that encourages vegetation diversity in the early recovery phase (Carbajo et al., 2011; Kardol, Martijn Bezemer & Van Der Putten, 2006). This can lead to mycorrhizal fungi outpacing bacterial pathogens, potentially promoting community evenness in late-succession plants (Fierer, 2017; Kardol & Wardle, 2010). Integrating soil microbial ecology knowledge into predictive ecological frameworks (e.g. modelling different environmental change scenarios, including microbiota assembly and functional dynamics) could further allow targeted site-specific restoration plans (Eviner & Hawkes, 2008). Reference site selection and assessments are central elements of planning and defining goals in a restoration project (Gann et al., 2019). Soil physical and chemical conditions, together with plant diversity and other factors in reference sites, shape microbiota development (Fierer, 2017). While reference site soil microbiota are increasingly used in restoration monitoring (see Section IV), they are not routinely assessed during the planning phase. Gaining information on the composition, and even functional characteristics, of microbiota in both degraded and target reference sites will position projects better to tailor their interventions to address varied levels of degradation in the whole ecosystem. Soil microbiota are highly heterogeneous across even small (<1cm 2 ) spatial scales (Fierer, 2017) and therefore reference site selection and sampling design are crucial to capture variation adequately (van der Heyde, Bunce & Nevill, 2022; Liddicoat et al., 2022). This high level of spatial variation can impact assessments of community composition and function and distort interpretations of the reference community (Peddle et al., 2022). The numbers and locations of reference site samples should account for vegetation and soil heterogeneity to provide the best possible picture of microbiota targets (Peddle et al., 2022; van der Heyde et al., 2022). One option is to implement a stratified random sampling scheme. This approach involves dividing the study area into distinct strata based on relevant factors influencing biodiversity distribution, such as vegetation and soil types or topographical features. Within each stratum, random sampling points are selected to ensure representative coverage of the area while minimising bias and distortions caused by heterogeneity. Additionally, employing systematic sampling techniques, such as grid or transect sampling or pooling samples to account for landscape heterogeneity (Bissett et al., 2016) can further enhance spatial representativeness and accuracy of biodiversity assessments. A key open question in integrating soil microbiota into restoration is: what do “good”soil microbial communities look like in terms of species composition and/or functionality? The composition of soil microbiota will vary greatly in different contexts and environments with no single “ideal”microbial community (Fierer, Wood & de Mesquita, 2021). Generally speaking, the microbial community composition most suited for any given restoration site should be informed by suitable reference sites. However, understanding the specific elements of microbial communities and drivers of microbial diversity, composition and function that can be Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. 4Shawn D. Peddle and others 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License generalised across environments will improve how and where we integrate microbiota into restoration (Liddicoat et al., 2024). In some cases, desirable microbiota characteristics might be informed by higher-level functional outcomes (e.g. establishment of sensitive plants, nutrient cycling, disease suppression). Various microbial taxa have seen increased research focus on their uses for restoring particular ecosystem processes or connections. For example, plant growthpromoting rhizobacteria have potential for their ability to improve plant growth (Radhapriya, Ramachandran & Palani, 2018; Solans, Pelliza & Tadey, 2022) and enhance germination (Domínguez-Castillo et al., 2021), and arbuscular mycorrhizal fungi can promote recovery of native vegetation via mechanisms that enhance phosphorus uptake in plants (Koziol et al., 2018) and improve soil physicochemical properties (Willis, Rodrigues & Harris, 2013). While there are “good”members of microbial communities, there are also pathogens that can be harmful to microbial communities and other ecosystem components (e.g. Phytophthora cinnamomi is a soil-borne plant pathogen) (Mansfield et al., 2024) and restoration plans need carefully to consider the risks of inadvertently spreading harmful pathogens. Importantly however, plant–pathogen interactions may be beneficial in restoration as they also play a significant role in shaping plant diversity and community dynamics. Pathogens can influence plant diversity through various mechanisms, including selection pressure on host species and facilitation of competitive interactions (Bever, Mangan & Alexander, 2015), which may impact plant and soil community stability. (2) When to prioritise investment in soil microbiota Another key question that needs to be addressed to ensure restoration is as efficient and effective as practically possible is: when will the inclusion of soil microbial data improve restoration success? While the explicit consideration of soil microbiota in restoration could arguably provide benefits to all projects, it does come with additional costs (e.g. soil sampling, DNA extraction and sequencing, complex bioinformatics) and potential risks (e.g. introduction of harmful pathogens, public or policymaker scepticism from undesirable outcomes) that need to be considered to maximise positive restoration outcomes and avoid wasted resources. Soil ecosystems are complex and highly variable both within and across sites which means a one-size-fits-all recommendation is problematic. Furthermore, soil microbiota are unlikely to be the only factor hindering restoration outcomes. Restoration projects should therefore include risk assessments and cost–benefit analyses on a case-by-case basis to determine if, and to what extent, soil microbiota should be included. The inclusion of soil microbiota in any given restoration project and any determination on the likelihood of that inclusion translating into cost-effective improved restoration outcomes will be largely dependent on the project’s goals and level of degradation or disturbance of soil physical, chemical, and biological properties. Decisions on including soil microbiota in restoration plans and interventions should be informed largely by the impact that degrading processes have had on soils and the level of investment that is available. Even short-term disturbances to vegetation communities with minimal disturbance to soils can cause shifts in soil microbial diversity and composition (Navarrete et al., 2015;Quet al., 2024). However, if soil physical and chemical properties remain similar to an undisturbed state, a focus on restoring vegetation communities alone may be sufficient to see the recovery of soil microbiota. On the contrary, if degrading processes have substantially modified soil physical or chemical properties, then soil biological properties will most likely be impacted as well. For example, restoration sites that were previously used for agriculture with extensive fertiliser applications can have longlasting nutrient legacies that persist for decades to millennia (Turley et al., 2020; Parkhurst, Standish & Prober, 2022). These persistent land-use legacies can then act as an abiotic barrier and impede the recovery of soil microbiota and present situations where restoration should plan interventions that specifically seek to overcome these abiotic constraints (Peddle et al., 2024). Additionally, alterations in soil pH, moisture, and structure resulting from degradation can also influence microbial community composition and activity, further emphasising the relevance of soil physicochemical assessments in guiding the inclusion of soil microbiota in restoration initiatives. Physical and chemical conditions are generally easier to observe and test than soil microbiota and should be considered to provide as near-optimal conditions as possible with reference sites as a guide. This sets the foundation for development of biological communities (Robinson et al., 2024). Restoration planning can, of course, consider “in-principle”influences on (and via) microbiota, however, practitioners will be blind to actual effects and recovery if relevant attributes of microbiota remain uncharacterised. By integrating assessments of soil physical, chemical, and biological properties, restoration practitioners can tailor decisions on the inclusion of microbiota-based interventions (see Section III) to the specific needs of degraded ecosystems, facilitating more effective restoration outcomes. (3) Improving conclusions on causation in soil microbiota restoration To determine better the level of effort required to affect recovery of soil microbiota it is important to improve our understanding of how soil microbiota responds following traditional restoration interventions such as revegetation. If revegetation alone largely leads to recovery of soil microbiota, then costly assessments and interventions focussed on microbiota are probably not needed. However, attributing soil microbial recovery solely to revegetation without properly ascertaining causation will lead to soil microbiota being overlooked and risks missing opportunities either to address this crucial ecosystem component directly (Lem et al., 2022) or to utilise soil microbiota more as drivers of change as opposed to solely passengers (Harris, 2009). Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. Soil microbiota and ecosystem restoration 5 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Observational studies of soil microbiota following revegetation often indicate that soil microbiota in restoration sites resemble reference sites more closely with increasing time since restoration (Barber et al., 2017; Gellie et al., 2017; Klopf et al., 2017; Ngugi et al., 2018; Parsons et al., 2020; Sun et al., 2017; Yan et al., 2019; Banning et al., 2011). These studies are often used to infer that the restoration intervention (e.g. native plant revegetation) is causing the restoration of soil microbiota but further rigour is needed to improve our knowledge of causal mechanisms affecting the recovery of soil microbiota. Observational chronosequence-based studies often suffer from unmeasured or unaccounted factors that can confound results and cloud conclusions. Other soil characteristics (both biotic and abiotic), climate, aboveground biological influences (e.g. vegetation, land-management history), topographic relief, parent geological materials, age of development, and spatial location (e.g. proximity to external influences) will influence soil microbiota composition (Delgado-Baquerizo et al., 2020; McBratney, Santos & Minasny, 2003; Pino et al., 2019) and may vary independently of a restoration intervention. Furthermore, restoration methods may change over time; for example, an unpredictable supply of seed resources may cause temporal variation in revegetation (Broadhurst et al., 2016; Ladouceur et al., 2018), or there may be interseasonal changes in climate, or changes in revegetation practices or planting crew. These time-dependent changes to restoration practice can introduce uncontrolled variation across the chronosequence and must be considered during chronosequence studies. In many situations, collecting sufficient covariate data to explain fully (or develop models to account for) soil microbiota spatial autocorrelations is impractical. Therefore, ensuring that these unmeasured or unaccounted influences do not compromise experimental designs and sampling plans by having appropriately designed studies is necessary. Despite their limitations, chronosequence designs are useful for inferring ecological responses to restoration interventions through time without long-term sampling or controlled experiments (Walker et al., 2010). However, explicitly planning to embed good quality experiments –such as those with adequate replication, controls and randomisation –into restoration projects will help to alleviate issues with spatial autocorrelation (van der Heyde et al., 2022) or pseudo-replication (i.e. treatment N=groups of 1) and assist in minimising the effects of confounding factors (e.g. changes in restoration planting methods, seed supply, climate variation, spatial location). However, it should be noted that truly longitudinal and/or manipulative studies are needed to produce high-quality evidence and conclusive support on causation (Lem et al., 2022) (see Section II.5). (4) Embedding soil microbiota experiments By planning to embed well-designed experiments into restoration projects, practitioners and researchers could form partnerships to address many of the limitations of chronosequence (i.e. space-for-time) designs (Broadhurst et al., 2023). Embedded experiments could nest replicated soil microbiota interventions (e.g. different soil inoculation methods or revegetation techniques) within reference and restoration sites or include spatially independent and replicated restoration interventions across a project (Fig. 2). Such an approach will improve the evidence base of the effect of specific restoration interventions on the recovery of soil microbiota and their associated functions. Adequately replicated, randomised, controlled and comparable restoration sites are not often routinely present in restoration projects unless planned for from the outset. This lack of core scientific design principles in systems that are often used in observational studies limits conclusions that can be drawn from such research. For this reason, restoration projects could improve our evidence base by collaborating with researchers and embedding microbiota-focussed experiments into restoration projects. Well-designed longitudinal studies that repeatedly sample the same restoration sites through time will provide more robust evidence on cause– effect relationships than cross-sectional chronosequence studies alone (Christie et al., 2019; Lem et al., 2022). Embedding experiments would also help close critical restoration knowledge gaps, such as knowing when a focus on soil microbiota will substantially improve restoration success. However, by their very nature, longitudinal studies require years of research and given the urgency required to address the biodiversity crisis, statistical modelling methods (e.g. structural–causal modelling, see Section II.5) will be useful to help understand the key knowledge gaps that need to be addressed with on-site long-term experiments and what can be solved with observational study designs alone. (5) Modelling approaches to ascertain causation Where particular microbiota-oriented outcomes are desired, but restoration activity cannot wait for definitive experimentally derived knowledge on cause–effect relationships, certain modelling approaches may help to distil information from relevant existing microbiota-restoration data sets. Techniques such as structural–causal modelling, structural equation modelling and path analysis can be applied to test hypotheses using observational cross-sectional data (Arif & MacNeil, 2023; Grace & Irvine, 2020). These approaches involve specifying a theoretical model that reflects likely causal relationships among variables of interest, including both observed and latent (i.e. unmeasured) variables. Then, the hypotheses are tested by specifying directional paths that represent the assumed causal relationship between variables in the model. Using the observed data, parameters (i.e. coefficients) of the specified model are estimated. These estimates assess the strength of the hypothesised causal/directional relationships and goodness of fit metrics indicate how well the model aligns with the data, even in the absence of experimental evidence (Eisenhauer et al., 2015), facilitating informed decision making in restoration planning. Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. 6Shawn D. Peddle and others 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License As a specific example, we could consider a scenario where a restoration intervention aims to enhance soil fertility and plant growth by introducing specific microbial species or communities. By using structural causal modelling, researchers can construct a theoretical model that includes variables related to soil microbiota composition and function, soil fertility, and plant performance. They can hypothesise directional paths between these variables representing the assumed causal relationships. Through analysis of observational cross-sectional data from similar restoration projects and controlling for covariates, researchers can estimate the parameters of the model and assess the strength and direction of the hypothesised causal relationships. For instance, it might be that certain microbial taxa or functional genes are strongly associated with increased soil fertility, which in turn positively impacts plant growth. While structural–causal modelling with cross-sectional data is powerful, it has limitations. It cannot establish causality as definitively as controlled or longitudinal experiments, and causality may be more challenging to infer in the presence of unobserved confounders especially in systems such as soil with thousands of distinct taxa and many functional groups (Eisenhauer et al., 2022). However, it allows researchers to specify, estimate and evaluate complex causal models providing insights into causal relationships (e.g. the relative roles of physical, chemical and biological properties of soil in limiting soil ecosystem recovery; the facilitation or following roles of soil biota and aboveground plant and animal communities during restoration; the influence of climate change and other major global degrading forces on limiting the recovery of soil ecosystems) without the need for experimental or longitudinal designs. Furthermore, the strength of causal claims should always be considered in the context of the study’s design and the potential presence of unobserved confounding variables. III. RESTORATION INTERVENTIONS THAT DIRECTLY TARGET SOIL MICROBIOTA It is possible to manipulate soil microbiota to assist in the recovery of degraded ecosystems by reinforcing beneficial interactions between plant species and soil microbiota lost Fig. 2. Embedding soil microbiota experiments into restoration sites. (A) A soil microbiota translocation field experiment embedded into an ongoing restoration project in Western Australia (photograph credit: Shawn Peddle). (B) Embedded soil microbiota experiments in a restoration site in the Mt Lofty Ranges, South Australia (photograph credit: Tarryn Davies). Designing and embedding experiments into restoration projects will allow for improved causal conclusions in testing microbiota-focussed hypotheses. Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. Soil microbiota and ecosystem restoration 7 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License through degradation (Aghili et al., 2014; van der Putten et al., 2016; Albornoz et al., 2022). Soil microbiota-focussed interventions can improve plant species prospects by improving plant growth, and depending on the ecological system, diverse soil microbiota have also been shown to mediate vegetation community diversity and improve ecosystem productivity (Naeem et al., 1994; Yang et al., 2021). Informed by mechanisms of ecosystem recovery and species-specific responses, soil microbiota interventions can advance restoration objectives and restore the diminished capacity of impacted ecosystems to recover naturally. In this section, we review more established (e.g. soil inoculations) and less well-established (e.g. specific microbial cultures, seed enhancements) ways to manipulate soil microbiota to improve restoration outcomes. We note that obvious abiotic barriers to the development of site-specific favourable soil microbial communities (e.g. soil substrate problems, excess nutrients, low pH, high salinity) should be identified and addressed before attempting direct manipulation of soil microbiota. (1) Whole-soil translocations and microbial inoculations Translocating whole soil communities –whether in the form of intact turfs or homogenised bulk soil –is one way of inoculating soil microbiota into degraded ecosystems to shift the microbial community towards one that is more representative of a target ecosystem. This essentially involves collecting soil from a reference ecosystem and translocating it directly into a restoration site (Koziol et al., 2018; Wubs et al., 2016; Carbajo et al., 2011). Inoculating degraded sites with reference ecosystem soil and associated biota has been shown to improve the growth and establishment of desirable native plants and exclude weeds in both greenhouse and field conditions (Koziol et al., 2018; Wubs et al., 2016; Fahey & Flory, 2022). For example, Wubs et al.(2019a) showed that soil inoculations can have ecosystem legacy effects that steer successional changes and can last for at least two decades. Importantly, however, Gerrits et al.( 2023) highlight how the directionality of this legacy effect depends on the suitability or fit of translocated soil to the recipient site, with mismatches steering communities in the wrong direction. Similar interventions can also shift the direction of the development of vegetation communities (Wubs et al., 2016) and improve prospects for native vegetation success (Wubs et al., 2019b). However, while research has shown a benefit for the restoration of vegetation, few studies have focussed on the efficacy of soil translocations to shifting whole microbial communities themselves. Substantial knowledge gaps remain on the effectiveness of soil translocations, including: what methods are most effective (e.g. bulk soil, intact turfs, volumes required), to what extent do soil physical and chemical properties in recipient sites impact establishment, how do priority effects impact on microbial community recovery (i.e. establishment may be dependent on the order of arrival of specific taxa), and, how does the coalescence of distinctly different soil communities impact successful establishment? As such, further research on whole-soil translocations and inoculations should focus on addressing these knowledge gaps via embedded experiments to understand better how soil volume, translocation method, and community coalescence dynamics affect microbial community assembly across varied ecosystems and soil types. Addressing these knowledge gaps will then enable the research community to develop decision-support frameworks to help determine when whole-soil translocations will provide restoration benefits that are commensurate with cost. Another critical open question relating to soil translocation is: how can we minimise the impacts soil translocations have on donor ecosystems? While soil translocations may be effective, soil collection can impact remnant habitats and consideration is needed to limit impacts to remnant sites while providing a benefit to degraded sites. Solutions are needed to scale up soil translocations outside situations where soil can be harvested because existing remnant habitat is already being cleared. As such, decisions on interventions impacting remnant habitat will need to weigh factors such as the contribution of remnant habitat to support the integrity and viability of restoration or conservation efforts (Tulloch et al., 2016; Wintle et al., 2019), or if a degree of destructive harvesting of soil resources from remnant sites can provide restoration benefits that outweigh impacts to remnant habitat. To address the need for reliable seed sourcing in restoration or revegetation, seed-production areas are being established instead of relying on sourcing seeds from remnant habitats (i.e. target plants are grown ex-situ “en masse”to produce seed stock) (Zinnen et al., 2021). This concept could potentially be applied to soil microbiota with soil microbiota production areas, although various open questions (i.e. how do we cultivate whole target microbial communities, can we subset communities to focus on particular taxa, and what is the “ideal”composition of these communities) need to be addressed before soil microbiota production areas can be effectively implemented at scale. Despite these knowledge gaps, whole-soil translocations are increasingly used in large-scale restoration projects where topsoil is salvaged as part of the initial disturbance (e.g. surface strip mining) and then reinstated during restoration (Tibbett, 2010; Schmid et al., 2020; Liddicoat et al., 2022). The objective of topsoil transfer is to preserve the soil-stored seedbank rather than the soil microbiota per se. Still, benefits from the reservoir of microbiota contained in these topsoils present an opportunity to improve restoration outcomes. Limiting the amount of time for which soils are stockpiled before translocation is crucial as stockpiling can disrupt biological integrity and impact microbial diversity and composition (Hernandez et al., 2024; Valliere et al., 2022). In bestpractice cases, the direct return of harvested topsoil to nearby restoration sites will limit the physical and biological degradation of soil from long-term stockpiling (Rokich et al., 2000; Peddle et al., 2022). However, the impact of the collection and homogenisation of vertical soil profiles during the transfer process on soil microbiota is likely detrimental but still poorly understood. Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. 8Shawn D. Peddle and others 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License An emerging approach that avoids broadacre spreading of whole soil is the targeted use of microbiota from local soils via extruded pellets or coatings as a vessel for seed delivery (Gornish, Arnold & Fehmi, 2019; Madsen et al., 2016) (Fig. 3). This method is designed to improve the precision of seed delivery in large-scale restoration efforts that simultaneously provide beneficial soil microbiota and the target seeds. Such an approach can reduce the demand for soil by 100-fold (Stock et al., 2020). However, similar questions to those identified earlier in relation to whole-soil inoculations are still unresolved and applicable here. For example: how do the mechanical and chemical disturbances of creating the pellets affect microbial community composition, and how well can targeted microbial communities in extruded pellets be established within donor soils with vastly different microbial communities or physicochemical properties? (2) Seed enhancements that contain microbial additives Seed enhancements that add specific microbial inoculants in a restoration context can improve the germination and growth of desirable plant species (Chua et al., 2019; Dadzie et al., 2023;O’Callaghan, 2016). Seed coating involves the precise application of binders and mineral powders to seeds to create a thin artificial layer of material capable of altering the physical shape of seeds and/or carrying beneficial products such as microbiota (Fig. 3A–C) (Brown et al., 2021; Erickson et al., 2021). Similarly, extruded pellets made via extrusion or moulding technologies can make larger seed-soil matrices while offering the same microbial inoculation opportunities. For instance, microbiota can be added to the seed coat and/or into extruded pellet ingredients either dry within the powder or wet via the binder (Alfonzetti et al., 2023; Dadzie et al., 2023;Munroet al., 2024). Alternatively, seed priming involves immersing seeds in water-based (i.e. hydro-prim- ing) or osmotically controlled (i.e. osmo-priming) solutions or soil matrix (i.e. matrix-priming) to commence the process of germination under controlled conditions, followed by a redrying step (Brown et al., 2021; Madsen et al., 2018). Once sown, primed seeds show a much higher and more rapid germination potential, recruitment synchronicity and seedling vigour. Priming with additives like microbial suspensions can facilitate the uptake of beneficial microbiota directly onto and potentially into the seeds (O’Callaghan, 2016; Muñoz- Rojas, 2018). Consequently, the targeted microbiota will be established directly in the soil near the germinating seed or within the seedling tissue itself (O’Callaghan, 2016; Chua et al., 2019). There have been successful implementations of naturally obtained microbiota that benefit key restoration plant species by seed enhancements (including extruded seed pelleting and/or hydro-priming with microbial additives) (Muñoz-Rojas et al., 2018;Dadzieet al., 2022). The improved accessibility and effectiveness of these techniques represent a valuable opportunity for restoration. C B # F % A DE Fig. 3. Manipulating soil microbiota as part of restoration interventions. Eucalyptus seeds (A) before and (B) after coating with mycorrhizae inoculants (photograph credit: Todd Erickson). (C) Seedlings germinated from extruded seed pellets containing live soil (photograph credit: Todd Erickson). (D) Control (sand +bentonite, left) and cyanobacteria encapsulated pellets (right) (photograph credit: Miriam Muñoz-Rojas). (E, F) Whole soil translocation experiment with (E) translocated intact soil core and (F), resampling 1 year after translocation to assess establishment and dispersal of soil microbiota (photograph credit: Shawn Peddle). Biological Reviews (2024) 000–000 © 2024 The Author(s). Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society. Soil microbiota and ecosystem restoration 9 1469185x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/brv.13124 by Readcube (Labtiva Inc.), Wiley Online Library on [27/11/2024]. 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