Ecosystem restoration in post-agricultural landscapes is a critical response to agricultural land abandonment, climate change, and the escalating biodiversity crisis. However, effective restoration of these landscapes can be hampered by land-use legacies that create biotic and abiotic barriers to ecosystem recovery, particularly in ancient Tertiary landscapes where vegetation is adapted to nutrient deficient soils. While our understanding of how to overcome these barriers when restoring plant communities is improving, there is limited knowledge of how these legacies impact on recovery of soil microbiota - the biodiverse and functionally-important communities of soil microbes. Here, we used amplicon sequencing of the bacterial 16S rRNA gene extracted from soils across a restoration project in southwest Western Australia, a global biodiversity hotspot, to examine recovery of soil microbiota following post-agricultural restoration. We sampled soils at six sites under four land conditions - degraded post-agriculture, actively revegetated post-agriculture, passively regenerated, and remnant bushland - generating 1,609,618 sequences corresponding to 15,009 unique bacterial taxa. We show that soil bacterial communities in revegetated and degraded samples were similar across sites but strongly dissimilar to adjoining remnant samples. We show that limited recovery of bacterial communities was linked to elevated soil phosphorus levels. Together, our results indicate soil microbiota have not recovered despite revegetation taking place up to 17 years ago, and this lack of recovery is likely driven by soil nutrient legacies from past agricultural practices. Our study highlights a key challenge faced by conservation practitioners when integrating soil microbiota into ecosystem restoration in post-agricultural landscapes in ancient, nutrient-poor landscapes.

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 the resilience of whole ecosystems. Fundamentally, restoration planning 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.

Understanding belowground plant-microbial interactions is fundamental to predicting how plant species respond to climate change, particularly in global drylands. However, these interactions are poorly understood, especially for keystone grass species like the pan-palaeotropical Themeda triandra. Here, we used 16S rRNA amplicon sequencing to characterise microbiota in rhizospheres and bulk soils associated with T. triandra. We applied this method to eight native sites across a 3-fold aridity gradient (aridity index range = 0.318 to 0.903 = 87 % global aridity distribution) in southern Australia. By examining the relative contributions of climatic, edaphic, ecological, and host specific phenotypic traits, we identified the ecological drivers of core T. triandra-associated microbiota. We show that aridity had the strongest effect on shaping these core microbiotas, and report that a greater proportion of bacterial taxa that were from the core rhizosphere microbiomes were also differentially abundant in more arid T. triandra regions. These results suggest that T. triandra naturally growing in soils under more arid conditions have greater reliance on rhizosphere core taxa than plants growing under wetter conditions. Our study underscores the likely importance of targeted recruitment of bacteria into the rhizosphere by grassland keystone species, such as T. triandra, when growing in arid conditions. This bacterial soil recruitment is expected to become even more important under climate change.

Microbes support all life forms of the biosphere, contributing to nutrient cycling and climate regulation, with crucial roles in primary production, food production, and planetary health (Anthony et al., 2023). By integrating microbial processes into current ecological paradigms, we have the opportunity to enhance the resilience of the biosphere to environmental change (Averill et al., 2022). A joint conference between the Ecological Society of Australia (ESA) and the Society of Conservation Biology Oceania (SCBO) in Wollongong, Australia, provided an excellent platform to hold a plant–soil ecology symposium that focussed on three key issues. First, to summarise the latest advances in documenting the microbial diversity from Australian ecosystems; second, to establish the ecological interactions that influence microbial diversity and functioning; and third, to harness soil microbial communities to inform fundamental plant ecology, as well as conservation and restoration practices. The field of soil microbial ecology has grown exponentially since the early 2000s, particularly with the onset of high-throughput sequencing (HTS) technologies (Zimmerman et al., 2014). These tools have allowed ecologists to explore microbial diversity across the globe (Tedersoo et al., 2014), to predict the effects of global change factors such as drought and warming on soil microbial communities (Tedersoo et al., 2022), and to address challenges in ecosystem restoration by harnessing beneficial plant–microbe interactions (Averill et al., 2022). Despite such progress, significant gaps in knowledge persist across different geographic regions and biomes. For instance, only 5% of fungal HTS samples in the GlobalFungi Database currently originate from Australia. The majority of samples come from Asia (34%), Europe (31%), and North America (21%; Větrovský et al., 2020). Australia is considered a global centre of diversity for multifunctional root types, such as dual mycorrhizas (roots that associate with both arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi; Teste et al., 2020). Multifunctional root types have allowed members of Acacia (Fabaceae) and Eucalyptus (Myrtaceae) to thrive in the shallow, nutrient-poor soils of Australia (Brundrett, 2017). Additionally, Australia has globally distinct geographic patterns of mycorrhizal type dominance (AM or EM) in forests (Steidinger et al., 2019) and is also a diversity hub for ectomycorrhizal plants (Brundrett & Tedersoo, 2018). The harsh climatic conditions of Australia, including scarce water and extreme events such as fire and drought, play a central role in shaping the composition of both plants and microbial communities above and belowground (Eldridge et al., 2018; Egidi et al., 2019; Legge et al., 2023). These peculiar features make comparison with ecosystems from other ecoregions of the world difficult, highlighting the need for studies addressing challenges specific to the Australian continent. A recent review on the loss of terrestrial biodiversity in Australia found that species extinction rates are predicted to escalate across all taxa assessed (Legge et al., 2023); however, similar assessments are lacking for soil microbial diversity (Birnbaum & Dearnaley, 2023). ESA is the main body for ecologists in Australia and has hosted plant–soil ecology symposia at its annual conference since 2016 (Birnbaum, 2022). These symposia provide a platform to showcase the latest advancements in plant–soil ecology, with a focus on the unique plant–microbial diversity and environmental conditions of Australia. The 2022 ESA plant–soil ecology symposium brought together the largest meeting of plant–soil ecologists in ESA history and was well attended by leading experts in the field and next-generation early career ecologists interested in soil microbial ecology (Fig. 1). Here, we highlight the latest research from the 2022 ESA plant–soil ecology symposium as it pertains to native Australian mycorrhizal fungi; ecological interactions that influence plant–microbial diversity and functioning; and state-of-the-art approaches for utilising soil microbes in ecosystem conservation and restoration (Fig. 2). Arbuscular mycorrhizal (AM) fungi are a widespread group of symbiotic microbes that play a critical role in fostering plant growth and success (Brundrett, 2017). By forming associations with most terrestrial plants, AM fungi can boost plant uptake of key nutrients and water while the plants provide carbon (Smith & Read, 2008). These fungi also have the capacity to offer various other benefits to their host plants such as improved resistance to disease, herbivory, and various abiotic stresses, including drought, salinity, and heavy metals (Norman et al., 1996; Hajiboland et al., 2010; Huang et al., 2020; Riaz et al., 2021). The use of mycorrhizal fungi in agriculture holds enormous promise for promoting sustainable agriculture as these microbes can help to reduce the need for environmentally detrimental fertilisers and pesticides and can also help to protect crops from the effects of climate change (Heuck et al., 2023). While there have been ongoing efforts to explore the diversity and ecological roles of AM fungi in an Australian context, there remains a paucity of knowledge on the drivers of AM fungal community assembly in both natural and agricultural environments of Australia (Fig. 2a). Additionally, the impacts of agricultural practices on resident AM fungal populations have been seldom explored (but see Albornoz et al., 2022). A new initiative at the Western Sydney University led by Adam Frew is seeking to address these issues. 'Dig Up Dirt' (https://www.digupdirt.net/) is a national research project that aims to survey AM fungal communities in agroecosystems via DNA metabarcoding of soil samples to assess how agricultural practices impact soil AM fungal communities and how this varies across Australian climates and soils (Fig. 2b). The project will expand understanding of Australian AM fungal diversity and facilitate more effective management of these key biota to the benefit of sustainable agriculture. Another avenue for explorative research is cataloguing the diversity of rainforest soil fungi in Australia (Fig. 2c). Extensive areas of rainforest were burnt within the Gondwana Rainforests of Australia during catastrophic wildfires in 2019–2020 (Nolan et al., 2020; Baker et al., 2022). Large parts of the rainforest were damaged as part of the 3.8 million ha of forest burnt in the state of New South Wales (Nolan et al., 2020). Having no previous fire history, these ecosystems were severely impacted (Nolan et al., 2020). A recent study assessed fire resilience traits of 228 taxa that were affected by this unprecedented fire event and found over 63% of taxa resprouting and recruitment occurring in 62% of taxa postfire (Baker et al., 2022). However, how the fires may have affected resident mycoflora is unclear, as there is a paucity of knowledge about the diversity of Australian rainforest fungi. Luke Florence (Latrobe University) is seeking to analyse the diversity of rainforest soil fungi in a meta-analysis using HTS data collected from Australian Microbiome (www.australianmicrobiome.com/) and Global Soil Mycobiome (doi: 10.15156/BIO/2263453) datasets (Fig. 2c). Differential abundance analysis has indicated key fungal genera from tropical, subtropical, and temperate rainforests. This research will establish an important baseline understanding of Australian rainforest fungi and elucidate the effects of climate on fungal community composition. This information will be crucial to the future conservation and restoration of these unique ecosystems, especially with changing climate and fire frequencies (Baker et al., 2022). Australia appears to be a global hotspot of Serendipita mycorrhizal fungi with nine new species (of 22 species known world-wide) recently described from the continent (Crous et al., 2020, 2022; Oktalira et al., 2021). These culturable basidiomycetes occur in the underground tissues of a range of plant species, including orchids, grasses, ericads, and liverworts (Weiss et al., 2016). The Dearnaley research group (University of Southern Queensland) is focussed on the discovery of novel Australian Serendipita species and testing these fungi for the capacity to improve agricultural plant growth and disease resistance (Fig. 2b). A recently described species, Serendipita whamiae has been grown in flasks with tomato seedlings (John Dearnaley, University of Southern Queensland). The symbiont increased the growth of plants and protected them against disease caused by powdery mildew fungi. These results show that Australian Serendipita fungi may be of value to sustainable agricultural approaches in future. This clearly warrants more research. We are only beginning to understand how multiple interacting soil organisms impact the associations of mycorrhizal fungi with native plant roots (Frew, 2022; Frew et al., 2022; Ng et al., 2023). Most terrestrial plants are AM, and almost all of these will deal with insect herbivory in one form or another (Frew et al., 2022). Despite the ubiquity of these tripartite interactions, the data on how herbivory can shape communities of AM fungi in plant roots is scant. Using a factorial pot experiment, Adam Frew (Western Sydney University) explored how insect root herbivory impacted the diversity and community structure of AM fungi in the roots of Dicanthium sericium (Frew, 2022). Root herbivory caused reduction in fungal species richness and a shift in community structure, characterised by an increase in certain taxa (e.g. Paraglomus) and a decrease in others (e.g. Archaeospora). Additionally, root herbivory was associated with a decline in plant phosphorus levels, suggesting a potential link between changes in AM fungal communities and plant nutrient status. These findings highlight the critical role that belowground insect herbivory can play in shaping root fungal communities and its potential implications for plant productivity in agricultural settings. Tom Mansfield (Murdoch University) has investigated the effects of Phytophthora cinnamomi dieback on resident fungal communities (Fig. 2c). Phytophthora cinnamomi is an invasive soilborne plant pathogen threatening the persistence of native Australian plant communities, a considerable ecological issue in the Southwest Botanical Province of Western Australia (Shearer et al., 2004, 2007). Seedlings of an ectomycorrhizal tree and AM clover were cultivated in P. cinnamomi-infested and -noninfested soil in a glasshouse. HTS of associated root fungi showed that the host plants maintained their respective mycorrhizal fungal associations in the presence of P. cinnamomi. These findings are significant from a restoration perspective because native fungal communities could improve seedling survival in the presence of the Phytophthora pathogen. Carlos Aguilar-Trigueros (Western Sydney University/University of Jyväskylä) discussed the traits of fungal spores and the importance of a trait-based approach in fungal ecology (Aguilar-Trigueros et al., 2023). These authors found that spore size variation among sexual and asexual spores was strongly linked to species' evolutionary history. This work advances life-history theory by highlighting that the evolution of plant–fungal symbiosis has been the key driver in influencing fungal spore morphology (Aguilar-Trigueros et al., 2023). This work has the potential to provide information about fungal traits in Australia (Fig. 2b). More is being learnt about the impacts of anthropogenic ecosystem degradation on Australian soil microbial communities. In a study conducted by Kumari Rajapaksha (Western Sydney University) looking at three threatened plant community types near Sydney, it was shown that richness and cover of exotic plant species were associated with increased nutrient availability and an abundance of gram-negative bacteria, protozoa, and specific fungal taxa, including AM fungi (Fig. 2c). These relationships differed between the plant community types highlighting the importance of having appropriate plans for monitoring and rehabilitating specific plant community types. Felipe Albornoz (CSIRO) has also recently shown that AM fungal richness was generally higher in degraded sites than in reference native eucalypt woodland sites. Degradation of eucalypt woodlands was associated with the introduction of 'weedy' AM fungi, which might promote or maintain disturbed ecosystems (Fig. 2e). Removing soil nutrients (N and P) and exotic plants may shift AM fungal communities towards reference native eucalypt woodland sites. This poses the question of how do we best manage 'weedy' AM fungi. In a separate study, Pankaj Singh (Western Sydney University) showed that land management practices, irrigation and fertilisation, had contrasting effects on the microbiome assembly of Eucalyptus saligna. Irrigation increased microbial alpha diversity while fertilisation had the opposite effect. Irrigation and fertilisation affected soil edaphic properties, bulk soil, and rhizosphere microbiome significantly. Though there was some effect on the root microbiome, the leaf traits and leaf microbiome assembly were not affected significantly by irrigation or fertilisation. A host's selection and filtering mechanism was the main driver of leaf microbiome assembly, while land management practices were pivotal in bulk soil and rhizosphere microbiome assembly. Digging animals, for example echidna (Tachyglossus aculeatus), quenda (Isoodon fusciventer) and woylie (Bettongia penicillata ogilbyi), are important ecosystem engineers, turning over soil and facilitating the spread of soil fungi, especially mycorrhizal fungi in Australia (Dundas et al., 2018; Tay et al., 2018; Decker et al., 2023). In undisturbed Australian ecosystems, aridity decreases the diversity of microbial carbohydrate-degrading enzymes (CAZYmes). Little is known about how aridity and digging mammals interact to affect the microbial genetic potential for C cycling. Eleonora Egidi from Western Sydney University found that the effects of mammal digging on microbial C cycling were most pronounced under xeric conditions, and that aridity shaped the diversity and structure of CAZYmes, while also driving the effect of mammal bioturbation on the microbial potential for C cycling across Australia. In another study, Anna Hopkins (Edith Cowan University) analysed mammal diggings in Western Australia and found that fungal composition was affected by urban remnant size, type and condition of vegetation, and soil type. These results indicated that digging mammal populations within urban landscapes perform important ecosystem functions by dispersing fungi that facilitate fungal–plant interactions, contributing to ecosystem health (Hopkins et al., 2021). Taken together, these findings highlight the important roles digging animals play in soil fungal ecology and dispersal in both arid and urban ecosystems in Australia and the need for further research. The United Nations Decade of Ecosystem Restoration was announced in 2021 with the goal of highlighting the need for collective efforts to revive degraded ecosystems. Above- and belowground plant microbiomes are fundamental to plant health and ecosystem functioning (Averill et al., 2022). Plant aboveground microbiomes have received considerably more attention; thus, there is considerable research required in the belowground space. Australia provides an opportunity for such investigations, particularly given the extensive vegetation clearing that has occurred over the past two centuries since European colonisation (Legge et al., 2023). Australian trees and shrubs are unusual in that they can often associate with both ectomycorrhizal and arbuscular fungi. In a study of the fungal communities of restored montane forests and former open-cut coal mines (Jeff Powell, Western Sydney University), ectomycorrhizal (EM) fungi were underrepresented while AM fungi were overrepresented, compared with reference sites. The degree of under/overrepresentation was associated with vegetation and soil attributes that are important for the recovery of ecosystem structure and function. This suggests that the occurrence of AM and EM fungi is driven more by the health of the broader ecosystem than by the functional requirements of woody plants. It also implies that AM fungi are an indicator of processes that may negatively affect the recovery of EM fungi. Significantly, patterns were observed suggesting that AM fungi may prevent the recovery of EM fungi and serve as a barrier to restoration; thus, management practices that suppress AM fungi may be required to support ecosystem recovery and restoration. Soil microbiota are fundamentally linked to the restoration of degraded ecosystems; therefore, DNA-based methods to characterise shifts in soil bacterial community composition have been developed to track restoration progress. Successful restoration of postagricultural landscapes back to biodiverse, functional, and self-sustaining ecosystems is often hampered by legacies that linger from decades of unsustainable land use (Lem et al., 2022; Peddle et al., 2023). Developed from restoration chronosequence study datasets, a new 'rehabilitation trajectory assessment' (Liddicoat et al., 2022) offers promise to help monitor and inform adaptive management practices and predict the ecological progress of restoration sites towards reference states (Craig Liddicoat, Flinders University). While our understanding of how plant communities recover is improving, we still lack a sound understanding of how soil microbiota respond postagricultural restoration. Shawn Peddle from Flinders University used an amplicon sequencing of the bacterial 16s DNA extracted from soil to examine how bacterial communities differ between different types of land conditions postrestoration in southwest Western Australia. They used four spatially paired land conditions, degraded, passively restored, actively revegetated, and remnant bushland, either with or without a history of intensive agricultural use. His results show an absence of recovery in soil bacterial communities in postagricultural sites, with degraded and revegetated sites comparable to each other but dissimilar to remnant sites without any history of agricultural land use. These results indicate that revegetation up to 17 yr earlier has not initiated a clear recovery of soil microbiota and a legacy of altered soil nutrient levels is likely impeding recovery. This study highlights the challenges facing the restoration of soil microbiota in postagricultural settings and the implications for whole ecosystem recovery. Although drylands support 40% of the earth's population, with naturally low and infrequent rainfall these systems are particularly vulnerable to the impact of climate change over longer and more severe droughts (James et al., 2013). This is of concern for Australia where drylands support most grazing, and sustain unique and diverse flora and fauna, which have already suffered great biodiversity loss (Zika & Erb, 2009). Research has been conducted to identify key microbes influencing seedling establishment of native plants in grazed drylands under drought scenarios (Jana Stewart, University of New South Wales). Soil-associated microbes were examined in three functionally different key dryland plant species, Acacia aneura, Atriplex nummularia, and Astrebla lappacea, to understand compositional or functional changes that may occur in soil biota under drought stress. Microbes were examined at germination and again after a drought simulation to assess key microbial communities for seedling establishment and resilience to drought. This research indicated the microbial functional groups that are most vulnerable to drought and also those most resilient and resistant to changes in climate. The results of this study offer powerful baseline information to understand ecosystem responses to changes in climate and assist in establishing priorities in land management strategies. The use of microbial inoculants as an ecologically friendly approach to enhance plant growth and establishment in dryland ecosystems has been recommended (Frederick Dadzie, University of New South Wales; Dadzie et al., 2022, Machado-de-Lima et al., 2023). To address these knowledge gaps, a meta-analysis of 62 globally distributed dryland studies, including from Australia, was conducted to test the effect sizes of microbial origin and species type on plant growth. Results presented at the conference showed that microbial inoculation generally increased plant growth in dryland ecosystems compared with their uninoculated counterparts. However, there was no significant difference in plant growth between native microbial inoculants and commercial microbial inoculants (Dadzie et al., 2023). Therefore, inoculation of beneficial microorganisms may yield a positive outcome on plant growth, and factors such as microbial origin and microbial species type might play a limited role in influencing plant performance in dryland ecosystems. Plant–microorganism interactions (i.e. plant–soil feedbacks (PSF)) have been used in soil ecology to study soil and plant reciprocal interactions and inform plant invasions. However, how specific PSF are within and among species has been less studied. Paola Rallo from the Netherlands Institute of Ecology presented her latest research on the specificity of PSF within and among widely distributed grass species (Lolium perenne, Poa pratensis, and Schedonorus arundinaceus), of which all are naturalised in Australia. The results from this work showed differences in PSF effects on plant growth between the three species but not between cultivars within species (Rallo et al., 2023). Furthermore, microbial fungal and bacterial taxa were identified as potential candidates responsible for these effects (Rallo et al., 2023). Giancarlo M. Chiarenza (UNSW Sydney) used the PSF approach with the study species Plantago lanceolata, a cosmopolitan introduced herb (Kloot, 1980). In this unpublished study, seeds from two native (Europe) and two introduced (Australia) populations were planted in a glasshouse in sterile and live inoculated soils coming from 30 Australian sites. There was no evidence of a relationship between seed germination percentage and soil treatment, suggesting that plant–soil feedbacks are not enough to control the seed stage in Plantago lanceolata. As a consequence, biocontrol strategies should focus on a different plant life stage to reduce the risk of plant invasions (Chiarenza, pers. comm). Australia has the second largest area of mangroves and saltmarshes in the world due to its extensive coastline (Giri et al., 2011; Mcowen et al., 2017). These vegetated coastal ecosystems provide critical ecosystem services such as carbon sequestration and storm surge protection (Farrer et al., 2022). However, we know surprisingly little about the role of soil and root microbial communities in these ecosystems (Fig. 2f). Furthermore, there is a fundamental gap in our understanding of the role belowground microbiomes play in restoration success in saltmarsh and mangrove ecosystems. Potential pathways and challenges associated with including the belowground microbiome in mangrove and saltmarsh restoration via a conceptual framework, with a focus on Australia were presented (Christina Birnbaum, University of Southern Queensland). There is a large gap of knowledge that exists in plant–microbe literature as it pertains to coastal restoration warranting future research (Birnbaum & Trevathan-Tackett, 2022). More research is needed to significantly advance both fundamental and applied knowledge through better understanding of the importance of coastal microbiomes to restoration success by stakeholders and funding bodies. Australia's soils are characterised as old, highly weathered, and stripped of nutrients (Fig. 2). At the same time, Australia has high endemism of native plants that rely on belowground fungal and bacterial communities to acquire nutrients and water they need. Australia's native plant diversity is rapidly decreasing due to competition with invasive plants and global climate change (Legge et al., 2023). Our ability to translate fundamental ecological plant–soil research into applied solutions for conservation, restoration, and management of Australia's unique ecosystems as well as agroecosystems has never been more urgent. As we are learning more about plant-associated microbial diversity, we need to work collaboratively to develop cost-effective approaches that best harness soil microbial communities and inform conservation and restoration practices in a changing climate. In summary, we need more targeted inventories to describe soil microbial diversity and function in Australia (similar to Bissett et al., 2016) if we are to tap into its full potential to restore and conserve ecosystems (Fig. 2b,d,f). The crucial next step is to start including soil biota in policy frameworks similar to plants and wildlife (Birnbaum & Dearnaley, 2023). This would ensure more investment into soil research and provide accountability in soil conservation. The authors are grateful to the organisers of the ESA 2022 conference in Wollongong, Australia. CB, EE, AF and AH organised the plant–soil ecology symposium at ESA 2022 as part of the ESA Plant–Soil Ecology Research Chapter. CB, JD, EE, AF, AH, JP, CA-T, CL, FA, MKH, FAD, LF, PS, TM, KR, JS, PR, SDP and GC participated in the symposium and in an extended discussion that led to this manuscript. CB led the writing with all authors contributing to the draft or revising the final version.

Microbiomics is the science of characterizing microbial community structure, function, and dynamics. It has great potential to advance our understanding of plant-soil-microbe processes and interaction networks which can be applied to improve ecosystem restoration. However, microbiomics may be perceived as complex and the technology is not accessible to all. The opportunities of microbiomics in restoration ecology are considerable, but so are the practical challenges. Applying microbiomics in restoration must move beyond compositional assessments to incorporate tools to study the complexity of ecosystem recovery. Advances in metaomic tools provide unprecedented possibilities to aid restoration interventions. Moreover, complementary non-omic applications, such as microbial inoculants and biopriming, have the potential to improve restoration objectives by enhancing the establishment and health of vegetation communities.

Environmental microbiota are becoming more conventional components of restoration ecology studies due to their functional importance in ecosystems. Studying these microbiota offers insight into how they respond to, and potentially drive, ecosystem restoration. However, microbes are everywhere and therefore they pose a risk to sample integrity via uncontrolled contamination, and many of these risks are introduced before entering a molecular facility. Field ecologists who have limited experience in microbial and/or molecular studies may lack the knowledge on how to mitigate microbial contamination risks and, accordingly, may find rigorous collection of microbial samples a daunting task. Here, we present a practical guide that builds on our previous paper to help manage the risks of microbial contamination when undertaking a microbiota restoration study prior to entering a molecular facility. We cover study design and planning, undertaking field sampling, and sample transport and storage. We hope to provide a useful and practical guide to restoration ecologists who wish to include a microbiota component in their studies. If done well, this inclusion offers improved research quality and ultimately enhanced restoration outcomes.

Mining activities modify both aboveground and belowground ecological communities, presenting substantial challenges for restoration. The soil microbiome is one of these impacted communities and performs important ecosystem functions but receives limited focus in restoration. Sequencing soil DNA enables accurate and cost‐effective assessment of soil microbiota, allowing for comparisons across land use, environmental, and temporal gradients. We used amplicon sequencing of the bacterial 16s rRNA gene extracted from soil samples across a 28‐year post‐mining rehabilitation chronosequence to assess soil bacterial composition and diversity following rehabilitation at a bauxite mine in Western Australia's jarrah forest. We show that while bacterial alpha diversity did not differ between reference and rehabilitated sites, bacterial community composition changed dramatically across the chronosequence, suggesting strong impacts by mining and rehabilitation activities. Bacterial communities generally became increasingly similar to unmined reference sites with time since rehabilitation. Soil from sites rehabilitated as recently as 14 years ago did not have significantly different communities to reference sites. Overall, our study provides evidence indicating the recovery of soil bacterial communities toward reference states following rehabilitation. Including several ecological reference sites revealed substantial natural variability in bacterial communities from within a single mine site. We urge future restoration chronosequence studies to sample reference sites that geographically span the restored sites and/or are spatially paired with restored sites to ensure this variability is captured and to improve any inferences on recovery.

The global declines in biodiversity and ecosystem integrity demand effective restoration. Soil microbiota are fundamentally linked to biodiversity and ecosystem restoration, as they are central to important ecological functions (e.g., nutrient cycling) and are extremely species rich. Their importance, plus the growing affordability of high-throughput sequencing, has resulted in rapid growth of studies that associate soil microbiota recovery and ecosystem restoration via native plant revegetation. Here we provide a systematic literature search and meta-analysis of the association between native plant revegetation and recovery of soil microbiota, identifying 26 datasets. We show that the soil microbial composition in revegetated sites was more similar to reference sites than degraded sites, indicating the recovery of soil microbiota with revegetation. However, a significant difference in composition between revegetated and reference sites indicates that a restoration gap remains. Bacteria showed greater recovery than fungi, which is consistent with bacteria having shorter generation times and being less dispersal-limited than fungi. We observed no general effect of revegetation on soil microbial richness. Showing that soil biodiversity is generally being returned via native plant revegetation should help conservation policymakers and practitioners that utilise this approach be more confident that their efforts are helping to combat global ecological and biodiversity declines. However, more research is required into the drivers of – and solutions to – the restoration gap, such as long-term monitoring of soil microbiota recovery, as this gap may present a long-term legacy that cannot be overcome with current-day revegetation practices.

The global biodiversity and land degradation crises have brought about an urgent need and great demand for restoration actions. However, restoration outcomes are often less than ideal, indicating a need for improved restoration practices. Soil microbiota are extremely diverse and functionally important and should be further considered in restoration. However, despite their importance, there remains a gap in understanding of how soil microbiota respond following native plant revegetation. Several studies have used cross‐sectional study designs of restoration chronosequences to infer that revegetation causes the recovery of soil microbiota, but it is near‐impossible to determine cause and effect relationships with cross‐sectional study designs. Here we used high‐throughput amplicon sequencing of the bacterial 16s rRNA gene from soil samples collected at two timepoints, 6 years apart, at a revegetation chronosequence in South Australia. Our results show some indications of recovery but not the additional recovery in bacterial community composition toward the reference sites as expected after this 6‐year period—a result that appears at odds to the expected patterns of revegetation causing recovery of soil microbiota. Spatially dependent factors (e.g. soil chemistry), biotic and abiotic barriers, seasonal differences in sampling, and variability among the ecological reference sites could each help explain this apparent lack of additional microbial recovery. More detailed longitudinal and/or experimental manipulation work is required to further examine the cause‐effect relationships. Our study contributes important new information and highlights knowledge gaps in how soil microbiota respond to revegetation, and we urge caution when attempting to infer causation from cross‐sectional chronosequence studies.

In post-mining rehabilitation, successful mine closure planning requires specific, measurable, achievable, relevant and time-bound (SMART) completion criteria, such as returning ecological communities to match a target level of similarity to reference sites. Soil microbiota are fundamentally linked to the restoration of degraded ecosystems, helping to underpin ecological functions and plant communities. High-throughput sequencing of soil eDNA to characterise these communities offers promise to help monitor and predict ecological progress towards reference states. Here we demonstrate a novel methodology for monitoring and evaluating ecological restoration using three long-term (>25 year) case study post-mining rehabilitation soil eDNA-based bacterial community datasets. Specifically, we developed rehabilitation trajectory assessments based on similarity to reference data from restoration chronosequence datasets. Recognising that numerous alternative options for microbiota data processing have potential to influence these assessments, we comprehensively examined the influence of standard versus compositional data analyses, different ecological distance measures, sequence grouping approaches, eliminating rare taxa, and the potential for excessive spatial autocorrelation to impact on results. Our approach reduces the complexity of information that often overwhelms ecologically-relevant patterns in microbiota studies, and enables prediction of recovery time, with explicit inclusion of uncertainty in assessments. We offer a step change in the development of quantitative microbiota-based SMART metrics for measuring rehabilitation success. Our approach may also have wider applications where restorative processes facilitate the shift of microbiota towards reference states.