Contrasting effects of symbiont inoculation on soil microbiota functionalities in a rehabilitation program of salt-affected lands

doi:10.21203/rs.3.rs-4963746/v1

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Contrasting effects of symbiont inoculation on soil microbiota functionalities in a rehabilitation program of salt-affected lands

https://doi.org/10.21203/rs.3.rs-4963746/v1

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Soil salinization has become a major global ecosystem sustainability issue. In Senegal, promising ecosystem restoration of salt-affected lands has been reached by the implementation of ecological engineering strategies based on beneficial associations between Casuarina species and salt-tolerant symbiotic microorganisms. However, the extent of impacts of symbiont inoculants on the native soil microbiota, and consequently soil functioning is fragmentary. The current study aimed at evaluating the changes in the native soil microbiota associated with the use of a symbiont inoculant in mixed Casuarinaceae plantations in salt-affected lands. The symbiont inoculation lead to a decrease of soil fungal diversity, but not bacteria. However, the whole soil microbiota structure was impacted by symbiont inoculation, as by salinity and Casuarina species. Casuarina species also impacted the diversity of dominant taxa constituting soil microbiota, but only salinity affected bacterial diversity. Important modifications of soil microbiota functionalities were revealed, notably a decrease of bacterial pathogens related to symbiont inoculation and increased abundance of fungal pathogens with salinity. Soil nutrient cycling was also impacted by symbiont inoculation, mostly micronutrient cycling and N fixation, but salinity and Casuarina species were the main factors affecting N cycling in soil.

Environmental Engineering

Casuarinaceae

functional guild

N-cycling

microbiota

soil salinization

Soil salinization is a significant factor of desertification processes, with major food security issues in irrigated lands (Qadir et al., 2014), mainly in arid and semi-arid regions (Sahab et al., 2021). Over one billion hectares of salt-affected land are reported worldwide with a growth rate of more than two million hectares per year (Singh, 2022). In Senegal, soil salinization affects ca. 1,700,000 ha of lands (Diack et al., 2015), particularly in the basins of Casamance, Sine Saloum and Senegal Rivers. The use of salt-tolerant plants has been proposed as an efficient strategy to face land salinization (Barrett-Lennard, 2002; Dagar and Gupta, 2020). However, salinity tolerance in plants varies widely depending on plant genotypes and / or ecotypes within a species (Beauchamp et al., 2009; Ribeiro-Barros et al., 2022), but also on microbial symbionts associated with (Dastogeer et al., 2020; Ngom et al., 2016).

Casuarinaceae are ranked among the most salt-tolerant plants and are recommended for revegetation of degraded sites (National Research Council, 1984). Their ability to colonize degraded environments is notably due to symbiotic interactions with nitrogen-fixing bacteria of the genus Frankia, but also with ecto- and endomycorrhizal fungi promoting phosphate acquisition and water uptake (Diagne et al., 2013). The use of arbuscular mycorrhizal fungi (AMF) and / or nitrogen-fixing bacteria (NFB) as symbiont inoculants was recently shown to improve stress resistance, survival and growth in Casuarina species, with benefits on the understory vegetation diversity (Diagne et al., 2020; Djighaly et al., 2020, 2022).

The use of bioinoculants is growing rapidly due to the need to propose sustainable alternatives to inputs (i.e. synthetic fertilizers, and plant protection agents) in human-managed ecosystems (i.e. agriculture, afforestation, rehabilitation) (Sessitsch et al., 2018). However, a wide range of environmental risks has been highlighted (Hart et al., 2017), notably the modification of native soil microbiota (Cornell et al., 2021; Mawarda et al., 2020). The consequences on plant performance and more globally on ecosystem functioning vary depending on the microbial compartment impacted, bacteria or fungi (Cornell et al., 2021).

Salt-affected ecosystems are characterized by the environmental adaptation of diverse bacteria and fungi playing a key roles, notably in soil nutrient cycling and stress tolerance in plants (Salwan et al., 2019; Yadav et al., 2020; Zhang et al., 2023). Some microbial taxa belonging notably to the Firmicutes, Bacteroidetes, Gemmatimondetes (Guan et al., 2021; Zhang et al., 2023) are widely described for their prevalence in highly saline ecosystems, whereas others, such as Gammaproteobacteria, Alphaproteobacteria, Glomeromycota for their benefits in plant salinity tolerance (Evelin et al., 2019; Mishra et al., 2021). Consequently, the management of this beneficial microbiota is a major issue to mitigate the impact of soil salinization, and the identification of drivers promoting them is central for successful rehabilitation of salt-affected ecosystems.

The current work aimed at characterizing the impact of the use of salt-tolerant symbionts as bioinoculant on the native soil microbiota for a rehabilitation program based on a mixed Casuarinaceae plantation subjected to salt stress. The level of environmental (salinity, bioinoculant, Casuarina species) partitioning of native soil microbiota remains uncertain because salinity is expected to be a major driver of soil microbiota. However, the establishment of the bioinoculant in such disturbed systems may strongly affect the native soil microbiota (Cornell et al., 2021), with beneficial or detrimental impacts on soil functioning.

2.1. Field study site and soil sampling

The experimental plantation (2500 m²) located in the Fatick region (Senegal; ; 14 °01’14 N, 16 °45’23 W) has been implemented in 2013 on a sandy loamy soil, characterized by salinity levels varying from 40 to 500 mM NaCl during the dry season, and subjected to periodic waterlogging during the rainy season (Djighaly et al., 2020). Two Casuarina species, ie. Casuarina glauca and Casuarina equisetifolia were inoculated with a salt-tolerant bioinoculant composed of an arbuscular mycorrhizal strain and an actinorhizal strain, then planted according to a factorial experimental design described in Djighaly et al. (2020).

Soil samples surrounding Casuarinaceae trees were collected from the 3-year old plantation, according to three different environmental factors, (i) the salinity (low and high levels), (ii) the use of symbiont inoculants (inoculated or not not), and (iii) the Casuarina species. For each tree, a composite soil sample was constituted from three soil cores.

2.2. Metataxonomics and data processing

Total DNA was extracted from 500 mg of soil using the FastDNA SPIN kit for soil (MP Biomedicals Europe, Illkirch, France) according to manufacturer’s instructions. Soil microbiota was assessed by MetaHealth expertise (Montpellier, France) using 16S rRNA gene and ITS2 amplification and Illumina Miseq PE250 sequencing, according a metataxonomic approach described in (Khassali et al., 2020).

Illumina sequencing, base calling and demultiplexing were carried out using RTA v1.18.54.0, MCS 2.6.2.1 and bcl2fastq2.20. Paired reads were assembled with vsearch v2.26.1 (Rognes et al., 2016), and primer clipping was performed with cutadapt v4.6 (Martin, 2011). Clustering with SWARM v3.1.14 (Mahé et al., 2021), post-clustering curation with mumu 1.0.2 (https://github.com/frederic-mahe/mumu), chimera detection and taxonomic assignment as detailed in Khassali et al. (2020). The ribosomal database SILVA v138.1 (Quast et al., 2012) and a custom version of the ITS database UNITE v9.0 (Abarenkov et al., 2024) were used for the bacteria and fungi, respectively. Details of data processing steps are provided in Appendix S1. The raw data are available under the bioproject PRJEB72132 (https://www.ebi.ac.uk/ena/data/view/ PRJEB72132).

2.3. Statistics

Data tables were transformed using the R tidyverse package version 1.3.2 (Wickham et al., 2019) and the plots were generated using the R ggplot2 package version 3.4.1 (Wickham, 2016). For soil bacterial and fungal communities analysis, global rarefaction (based on the samples with the smallest sizes) to 21897 and 13479 reads, respectively, were performed with the R vegan package version 2.6–4 (Oksanen et al., 2022) using the rrarefy() function for downstream statistical tests.

Hill numbers (^qD) were used to characterize soil microbiota diversity with the R hilldiv package version 1.5.1 (Alberdi and Gilbert, 2019). \(\:{D}^{q}={\left({\sum\:}_{i=1}^{R}{p}_{i}^{q}\right)}^{1/\left(1-q\right)}\)), where q is the order of the Hill number, R is the total number of categories (i.e., unique numerical values in a numerical matrix), and p is the relative abundance of each category. Soil microbiota structure analysis was performed by nonmetric multi-dimensional scaling (NMDS) with the R package vegan, and significance of differences according environmental variables was assessed by PERMANOVA using adonis2() function and HOMOVA using betadisper() and permutest() functions from the R package vegan. Functional microbial groups were determined based on microbial taxonomy using FAPROTAX (Functional Annotation of Prokaryotic Taxa) (Louca et al., 2016) for bacterial taxa and FungalTraits (Põlme et al., 2020) for fungal taxa.

Differences between environmental factors for soil microbiota diversity or the abundance of functional microbial groups were estimated by three-way ANOVA as implemented in aov() function. The significance of bacterial and fungal OTU association with respect to the salinity level, the use of symbiont inoculant or the Casuarina species was determined using the indicator value (IndVal) index, as implemented in the multipatt() function from the R package indicspecies (version 1.7.6) (De Cáceres and Legendre, 2009). Two different probabilities were calculated, i.e. “A” (specificity), representing the probability of a sample to be defined by a environmental factor (i.e. salinity level, symbiont inoculant, Casuarina species), given that the OTU has been detected, and “B” (sensitivity), representing the probability of finding the OTU in different samples characterized by a given environmental factor. The P values were adjusted for multiple testing based on the Benjamini–Hochberg (BH) correction. Only the OTUs with a P_adjusted value < 0.05 and present in more than half of samples for a given group are considered, i.e. B superior to 0.5.

3.1. Impact of ecological engineering strategies on native soil microbiota

The contribution of symbiont inoculation to changes in the native soil microbiota was compared with that of the salinity level or the Casuarina species transplanted in the rehabilitation site. Salinity was the most significant (P < 0.05) driver of soil bacterial diversity, explaining 9 to 16% of variability (Fig. 1A-C). An increase of soil bacteria diversity was observed in highest salinity levels (Fig. 2A-C). The correlation between soil microbiota diversity and pH, known as a major driver of microbiota diversity, was assessed alongside salinity, revealing negative and positive correlation with bacterial diversity (Fig. 1D-F), respectively. The Casuarina species also significantly contributed to changes of bacterial diversity but mostly for predominant taxa (Fig. 1C; 12% of variability; P = 0.007). The highest level of bacterial diversity were measured for C. glauca (Fig. 2I). In contrast, changes in soil fungal diversity was mainly explained by the use of the symbiont inoculant (Fig. 3B-C; 10 to 12% of variability), and no correlation was observed with salinity and pH (Fig. 3D-F). Symbiont inoculation lead only to a decrease of soil fungal diversity (Fig. 4E-F).

Structure analysis of the soil microbiota revealed a broader impact of rehabilitation program on both bacterial (Figure S1) and fungal (Figure S2) communities, with significant impacts of symbiont inoculation, salinity and plant species. In contrast to the impacts observed on alpha diversity, symbiont inoculation (R² = 0.1042; P = 1e − 04) explained the main difference between bacterial communities whereas it was mainly salinity (R² = 0.0946; P = 1e − 04), for fungal communities. For both communities, significant interactions were observed among the different environmental factors.

3.2. Impact of ecological engineering strategies on major soil functional microbial groups

Changes in functional groups of soil microbiota were assessed using taxonomic-based functional or guild predictions. Bacteria associated with pathotroph lifestyle were significantly (P < 0.05) impacted (Fig. 5), except by salinity for pathogens and parasites. Bacteria classified according their source of energy were either impacted by Casuarina species for chemoheterotroph or symbiont inoculation for H-oxidizing bacteria (Figure S3). In terms of metabolism, symbiont inoculation significantly impacted bacteria involved in methane (CH₄) cycling or Casuarina species in aromatic compound degradation (Figure S4). Nutrient cycling was mostly impacted by Casuarina species (Figure S5), but also by salinity or symbiont inoculation for macronutrients (i.e. N) and micronutrients (i.e. Fe, Mn) (Figure S5), respectively. A deeper investigation of impacts on N cycling revealed positive effect of symbiont inoculation on N inputs, notably global N fixation (Fig. 6), but with a negative effect on the abundance of actinorhizal N-fixers (Fig. 6). Casuarina species differentially impacted the abundance of legume and actinorhizal N-fixers. Nitrogen bioavailability content was also promoted by symbiont inoculation through ureolysis (Fig. 6). Differences in N loss was mainly related to Casuarina species with an increase of bacteria involved in nitrification for C. glauca (Figure S6), and salinity with an increase of bacteria involved in denitrification (Figure S6).

The abundance of fungi involved in soil organic matter degradation was enhanced by symbiont inoculation (Figure S7), whereas plant saprotrophs was impacted by Casuarina species, as well as uncharacterized saprotrophs (Figure S7). Contrary to bacteria, fungal pathogens were mostly promoted by salinity but not Casuarina species (Fig. 7), and no impact was observed on fungal parasites (Fig. 7). Animal and plant symbionts were differentially impacted by environmental factors, salinity and symbiont inoculation or Casuarina species (Fig. 8), respectively. On the other hand, the global plant endophyte community were strongly impacted by symbiont inoculation (Fig. 8).

Salinity is expected to be one of the main factors affecting ecosystem functioning, notably soil microbiota (Lozupone and Knight, 2007; Rath et al., 2019; Yang and Sun, 2020; Zhao et al., 2019). Consequently, ecological strategies based on the use of bioinoculants are developped to counteract negative effects of salt on plant growth and survival. Yet adverse impacts of bioinoculants on soil functioning were observed (Cornell et al., 2021; Gou et al., 2024), with in most cases no resilience of the native microbial community (Mawarda et al., 2020). In the current study, salinity was a major factor of bacterial diversity changes, but symbiont inoculation was the main factor for fungal diversity.

Salinity was described as a key environmental factor driving bacterial community diversity in arid ecosystems (Zhang et al., 2019), but differential impacts are observed depending on salinity ranges (Zhang et al., 2019). Similarly, salinity and pH were shown as strongly correlated to bacterial community diversity, but inversely for pH compared to previous studies (Zhao et al., 2018). Differences in pH gradient might explain these opposite effects, since acid pH gradient (3 < pH < 6) was measured in the current study compared to alkaline pH gradient (7.9 < pH < 8.7) in Zhao et al. (2018). The difference in response between bacteria and fungi along a salinity gradient has been also described in other arid ecosystems (Lin et al., 2023), and contrasting toxicity effects of salt were between bacteria and fungi (Rath et al., 2016). The lack of effect of salinity gradient on the diversity of the fungal community might also be due to other environmental factors outweighing the effect of salinity (Zhao et al., 2019). The presence of vegetation cover, for example, could be one of those since the presence of cover plants can mitigate the effect of salinity on soil microbiota (Dasgupta et al., 2023). The plantation of Casuarinaceae on the current rehabilitation site was indeed shown to enhance the development of herbaceous vegetation (Djighaly et al., 2020). In addition, soil fungal diversity tend to be more closely aligned with vegetation cover both for mutualists (Cassman et al., 2016; Zhang et al., 2021) and saprobes (Francioli et al., 2020), thus less changes are expected in their communities without concurrent changes to vegetation (van der Heijden et al., 1998; Beck et al., 2020).

On the other hand, changes in diversity of the predominant bacterial taxa have been observed between Casuarina species, which might be due to differences in root traits (exudates, genetic, tolerance to stress) between the two species. Bacterial community have been indeed shown as more strongly affected by differences in root traits than fungi (Alahmad et al., 2024; Merino-Martín et al., 2020). The changes in vegetation cover (richness diversity, composition) due to the different environmental factors (salinity, symbiont inoculation, Casuarina species) remains unknown until now, but constitute undoubtedly an important explaining factors to explain variations in soil microbiota (Djighaly et al., 2020). However, the composition of each microbial compartment was globally influenced by the various environmental factors, suggesting that the use of bioinoculant, as well as the type of Casuarina species determine the characteristics of community assembly in addition to salt stress. The consequences in terms of microbial functionalities and ecosystem services could be important (Catano et al., 2023), and have to be taken into account for rehabilitation programs.

Bacteria and fungi play indeed major roles in soil biogeochemical cycles, supporting soil fertility and plant health (Banerjee and van der Heijden, 2022; Saleem et al., 2019; Trivedi et al., 2020). Modifications of biotic and abiotic parameters can have a serious and lasting impact on soil functioning by altering general and specialized microbial functional groups (Griffiths and Philippot, 2013; Xun et al., 2021). For example, salinity, pH, soil moisture, or nutrients were shown to contribute to changes in fungal trophic guilds in saline soils (Zhao et al., 2019). Current results revealed significant impacts on several microbial functional groups or guilds dependent on single or dual environmental factors.

Saprotrophs, symbiotrophs and pathotrophs were mostly impacted by symbiont inoculation and / or Casuarina species. Nevertheless, salinity affected the abundance of fungal plant pathogens, but not bacterial pathogens. Salt stress has also been shown to increase aggressiveness of certain fungal pathogens (Haddoudi et al., 2021). Opposite effects were observed on the abundance of actinorhizal N-fixers and free N-fixers in soils, highlighting complex plant-soil feedbacks, potentially due to priority effects between the competitive actinorhizal symbiont pre-inoculated on Casuarina seedlings and native actinorhizal community on the rehabilitation site, and plant-mediated promoting effect on other N-fixers. Nevertheless, N fixation in soils was reduced for high levels of salinity.

The use of AM fungal inoculant combined with actinorhizal inoculant could also impact the soil microbial community through plant-soil feedbacks by enhancement of host plant growth and modification of the amount, timing, and form of carbon inputs into soil (De Gruyter et al., 2022). AM fungal inoculation was hypothesized to modify the native AM fungal community, as frequently observed (Islam et al., 2021; Thioye et al., 2021, 2019), but the extent of changes are related to properties of AM fungal inoculant and environmental factors (Hart et al., 2017; Islam et al., 2021; Martignoni et al., 2020). In the current study, Casuarina species was the explicative factor of changes, with higher abundance of soil AM fungi associated with C. glauca compared to C. equisetifolia. Similar observation was obtained for native soil rhizobia. These results emphasize the necessity to set up trial with mixed plantation to evaluate the benefits of a range of Casuarina species on soil functioning beyond the effects on tree growth.

Changes in saprotroph abundance might be due to differences in vegetation cover promoted by Casuarina plantation and consequently differences in organic matter composition or abundance. In addition, differential impacts of Casuarina species were observed on bacterial and fungal saprotrophs. Symbiont inoculation impacted also differentially bacterial and fungal saprotrophs, favoring soil fungal saprotrophs and limiting plant bacterial saprotrophs.

By digging deeper into the functions involved in biogeochemical cycles, significant impacts of environmental factors were observed on nutrient cycling and energy flows. Soil carbon content, notably CH₄ cycling and aromatic compound degradation, was affected by symbiont inoculant and Casuarina species, respectively. Multiple microbial guilds are responsible of CH₄ fluxes in soils (Hartman et al., 2024), and the strong changes of soil bacterial community due to symbiont inoculation might have particularly impacted some of the guilds involved in CH₄ cycling. Differences in the abundance of bacteria involved in aromatic compound degradation is probably due to differences in root exudate composition and rates between the two Casuarina o species (Dennis et al., 2010; Staszel-Szlachta et al., 2024). On the other hand, salinity was the main factor negatively impacting soil bacterial community mediating N cycling confirming previous studies in highly salt-affected soils (Li et al., 2021). Nevertheless, impacts on N turnover is dependent on salinity levels, which can lead to inconsistency in conclusions (Tao et al., 2024). Moreover, changes in H-oxidizing bacteria abundance, which are generally associated with N fixation variations (Maimaiti et al., 2007), was not related to salinity levels, but to symbiont inoculation with opposite effects on N fixation. Bioavailability of soil N content for plant was also promoted by symbiont inoculation through ureolytic bacterial community, with probable major benefits on soil fertility (Hasan, 2000). Interestingly, the use of C. glauca promoted bacterial community involved in N cycling compared to C. equisetifolia, from N fixation to nitrification, whereas increasing salinity promoted mainly denitrification.

The development of ecological engineering strategies based on the inoculation of symbionts has to be deployed with caution. Important impacts beyond salt stress were observed, with positive and negative effects on soil microbial functionalities. These results highlights the importance of combining above- and below-ground monitoring of agronomic and environmental traits to evaluate the performance of rehabilitation strategies in salt-affected soils. To go further, the set-up of preliminary monitoring of soil microbiota in sites to be rehabilitated could be a crucial pre-requisites in ecological engineering strategies since the initial status of native soil microbiota rather than the changes due to inoculants has been reported as a promising indicator to predict the benefits of inoculation strategies (Lutz et al., 2023).

Acknowledgement

We acknowledge the “Laboratoire Mixte International Adaptation des Plantes et Microorganismes Associés Aux Stress Environnementaux (LAPSE)” for providing supporting funds to this project. We thank the Palmarin town, its authorities and inhabitants for their welcome and for supporting and facilitating our trials. We also thank Camille Faye and Gilbert Ndong for their support in the field and for monitoring the trials. This work would not have been possible without them.

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The authors declare no competing interests.

FigureS1.png
Figure S1. Non-metric scale analysis (NMDS) of bacterial community structure (based on the Horn–Morisita dissimilarity index) associated two Casuarina species inoculated or not with symbionts in saline soils (Low salinity, 40 to 200 mM NaCl; High salinity 200 to 500 mM NaCl). The top and right marginal density plots represent the density of samples according to low (pale blue) to high (dark blue) salinity levels.
FigureS2.png
Figure S2. Figure 5. Non-metric scale analysis (NMDS) of fungal community structure (based on the Horn–Morisita dissimilarity index) associated two Casuarinaspecies inoculated or not with symbionts in saline soils (Low salinity, 40 to 200 mM NaCl; High salinity 200 to 500 mM NaCl). The top and right marginal density plots represent the density of samples according to low (pale blue) to high (dark blue) salinity levels.
FigureS3.png
Figure S3. Effect of environmental factors (salinity, inoculation, Casuarinaspecies) on the relative abundance of bacterial groups according the energy source. Low salinity, 40 to 200 mM NaCl. Statistics are based on log (1+x) transformed abundance, subjected to three-way ANOVA, and variance distribution for each comparison is represented with the corresponding p-values (P). High salinity 200 to 500 mM NaCl. (-), no symbiont inoculation. (+) symbiont inoculation.
FigureS4.png
Figure S4. Effect of environmental factors (salinity, inoculation, Casuarinaspecies) on the relative abundance of bacterial groups involved in C cycling. Low salinity, 40 to 200 mM NaCl. Statistics are based on log (1+x) transformed abundance, subjected to three-way ANOVA, and variance distribution for each comparison is represented with the corresponding p-values (P). High salinity 200 to 500 mM NaCl. (-), no symbiont inoculation. (+) symbiont inoculation.
FigureS5.png
Figure S5. Effect of environmental factors (salinity, inoculation, Casuarinaspecies) on the relative abundance of bacterial groups involved in nutrient and micronutrient cycling. Low salinity, 40 to 200 mM NaCl. Statistics are based on log (1+x) transformed abundance, subjected to three-way ANOVA, and variance distribution for each comparison is represented with the corresponding p-values (P). High salinity 200 to 500 mM NaCl. (-), no symbiont inoculation. (+) symbiont inoculation.
FigureS6.png
Figure S6. Effect of environmental factors (salinity, inoculation, Casuarinaspecies) on the relative abundance of bacterial groups involved in N loss. Low salinity, 40 to 200 mM NaCl. Statistics are based on log (1+x) transformed abundance, subjected to three-way ANOVA, and variance distribution for each comparison is represented with the corresponding p-values (P). High salinity 200 to 500 mM NaCl. (-), no symbiont inoculation. (+) symbiont inoculation.
FigureS7.png
Figure S7. Effect of environmental factors (salinity, inoculation, Casuarinaspecies) on the relative abundance of fungal guilds with saprotroph trophic mode. Low salinity, 40 to 200 mM NaCl. Statistics are based on log (1+x) transformed abundance, subjected to three-way ANOVA, and variance distribution for each comparison is represented with the corresponding p-values (P). High salinity 200 to 500 mM NaCl. (-), no symbiont inoculation. (+) symbiont inoculation.

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Contrasting effects of symbiont inoculation on soil microbiota functionalities in a rehabilitation program of salt-affected lands

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Abstract

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1. Introduction

2. Materials and Methods

2.1. Field study site and soil sampling

2.2. Metataxonomics and data processing

2.3. Statistics

3. Results

3.1. Impact of ecological engineering strategies on native soil microbiota

3.2. Impact of ecological engineering strategies on major soil functional microbial groups

4. Discussion

5. Conclusion

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Acknowledgement

References

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