Characterization of ECM fungal community under three different vegetation restorations in iron tailing

Wenxu Zhu (  zhuwenxu.315@163.com ) Shenyang Agricultural University https://orcid.org/0000-0002-7367-3754 Changjun Ding Chinese Academy of Forestry Keye Zhu Shenyang Agricultural University Weixi Zhang Chinese Academy of Forestry Dejun Liang Liaoning Provincial Poplar Institure XiaoJiang Wang Inner Mongolia Academy of Forestry Sciences Aiping Li Inner Mongolia Academy of Forestry Sciences Xiaohua Su Chinese Academy of Forestry


Introduction
China is one of the world's most biodiverse countries and also a large exporter of commodities such as iron ore. Iron mining activities have important economic and social bene ts, despite having a signi cant impact on the landscape (Skirycz et al., 2014), soil basic characteristics and soil microbial community, as it not only dramatically occupy and destroy a large amount of land, produce large amount of mine tailings, in uence soil pH value and soil structure, decrease plant water availability, reduce soil organic matter stock (Oliveira et al., 2014), threat to the biodiversity and result in compromised ecosystem functions, but also pollute air environment and affect human health for a long time (Zhang et al., 2010;Li, 2006). From a long-term perspective of sustainable use of land resources and protection of the ecological environment, there is an urgent need for a feasible way to restore the degraded ecosystems (Wang et al., 2017).
In areas degraded by iron mining, natural regeneration is slow and often impossible (Silva et al., 2006) due to the physical and chemical characteristics of the substrate (González-Alcaraz and van Gestel 2017), such as poor physical structure (Silva et al., 2006), alkaline pH (Wu et al., 20206), low water retention capacity, nutrient (N, P) de ciency , and high metal stress (López-Orenes et al., 2017; Wang et al., 2017). In addition, in degraded iron ore areas with the characteristic of low nutrient concentration and high density of the substrate, arti cial vegetation restoration is a huge challenge (Rios et al., 2021;Wang et al., 2017). In recent years, the recovery of iron-ore mined areas can be achieved through a variety of hard engineering techniques, and phytoremediation has been considered to be a more e cient, economical, and environmentally sustainable remediation strategy compared to physical and chemical methods (Wang et al., 2017;Skirycz et al., 2014), as it could preserve the soil resource, improve soil structure, physico-and bio-chemical properties, biodiversity patterns, ecosystem functioning (Gastauer et al., 2019), soil microbial diversity (Touceda-Gonzalez et al., 2017; Xue et al., 2015), ultimately creating self-sustaining vegetation communities. Given that, the selected species for revegetation purposes must be able to thrive under these multi-stress scenarios (Peng et al., 2019), and the selection of suitable plants species is the rst step for restoration of mine tailings.
In previous studies, many herbaceous  Iron ore in China is widespread and relatively concentrated. At present, there are ve major areas of concentrated distribution of iron ore reserves in China, among which Anshan-Benxi iron mine wasteland in the northeast covers the largest area. Iron ore mining has made a great contribution to the regional economic development (Wilson, 2012), however, serious environmental problems are caused by iron mining in China. Furthermore, with the implementation of national policies related to ecological civilization construction, it is imperative to carry out reasonable mine ecological restoration in Anshan-Benxi iron mined area to ensure the harmonious development of society, economy and environment.
Therefore, the construction of green mines and ecological restoration should be actively promoted in the process of mining development (Sheoran et al., 2010). At present, considerable researches mainly focused on the vegetation restoration measures and technique (Zhang, 2018;Zhang et al., 2018), as well as the effects of ecological restoration on soil macro-animal communities (Liu et al., 2009) and soil microorganisms (Deng et al., 2020a) in iron mining areas of Liaoning Province. However, information as to the ECM fungal communities associated with different revegetation is insu cient and therefore needed. Therefore, the objective of this study was to investigate whether the three native woody plants, including Pinus koraiensis Sieb. et Zucc., Robinia pseudoacacia L, Populus simonii Carr, can improve the soil basic characteristics and soil ECM fungal community after 15 years of aided phytostabilization under eld conditions. It is hypothesized that 1) vegetation restoration could promote the accumulation of soil nutrients; 2) vegetation restoration could clearly shift soil ECM fungal community diversity and composition; 3) along with the restoration of vegetation, the remarkable abiotic changes were the accumulation of soil nutrients, which affect the shifts of ECM fungal communities. The ndings of this study will be bene cial for the selection of suitable vegetation types to accelerate the vegetation restoration process in iron mine tailing.

Site information
The study area is located in Dengta City, Liaoyang City, Liaoning province, China (40.74 N, 122.86 E), which is classi ed as north temperate continental climate with the feature of warm spring, hot summer, cool autumn, cold winter, four distinct seasons, rain hot season, su cient sunshine. The annual average temperature is 8.8℃, and the annual average frost-free period is 171 days. The rainfall is abundant, mainly in summer, with an average annual total rainfall of about 686.0 mm. Pinus tabuliformis, Larix gmelinii, Pinus koraiensis, Robinia pseudoacacia, Populus simonii, and Ulmus pumila are the main vegetation.

Sample collection
The details of study area and plot setting were described in the study from Deng et al.  (20 × 20 m) were randomly established in each site as repetitions, with a distance of approximately 50 m. In each plot, 9 plants with well growth and consistent growth were randomly selected, then large pieces of sand and other debris on the surface were removed. Fine root samples and soil samples were collected at a depth of 0-30 cm, and the rhizosphere soil of 9 plants at the same plot were collected, mixed as one sample, then placed in a ziplock bag and taken back to the laboratory in ice boxes, resulting in 12 samples. The fresh soil samples were divided into two parts. One part removed stone and plant residues was passed through a 2-mm sieve and immediately put into 2 ml centrifugal tube and stored at −80 °C until DNA extraction, and the other part was air-dried and sieved for determination of soil characteristics.

The determination of soil parameters
The soil pH was assayed in soil: water (w/v) of 1:2.5 H 2 O suspensions following shaking of the samples for 30 min, using a pH meter (Mettler Toledo pH (FE20)). The contents of soil total carbon (TC) and total nitrogen (TN) were determined by an elemental analyzer (Euro Vector EA3000). The concentrations of total phosphorus (TP) and available phosphorus (AP) were measured by spectrophotometer (UV-9000S) after digestion with H 2 SO 4 -HClO 4 and extracted with 0.5 mol·L −1 NaHCO 3 , respectively. The available K content was determined by atomic absorption spectrometry using1.0 mol·L −1 NH 4 OAc as extractant. The concentration of available N was measured by the alkali solution diffusion method.

DNA extraction
The DNA was extracted from 0.

Data analysis
Soil characteristics and soil ECM fungal community diversity among different samples were subjected to ANOVA and means were compared by Tukey's test (p < 0.05). Venn diagram was used to analyze the shared and unique OTUs among different samples in Rstudio with the package of vegan. NMDS was used to compare the difference of ECM fungal beta diversity and carried out using R studio with the packages of vegan, permute, and lattice. LEfSe analysis, namely LDA Effect Size analysis, can nd the species with signi cant differences in abundance between groups (i.e. Biomaker). Spearman's correlation coe cients between soil basic characteristics and ECM fungal community diversity and composition were analyzed using SPSS 20.0. The effects of experimental variables on ECM fungal communities in roots were analyzed by canonical correspondence analysis (CCA) using the CANOCO 5.10 software package.

Contribution of soil properties to ectomycorrhizal fungal community composition
For the ectomycorrhizal fungal community at the phylum level, all the eight soil characteristics explained 99.9% of the variance, with axis 1 explaining 82.40% of the variance and axis 2 explaining 16.60% (Fig.   6A). For the fungal community at the genus level, all the eight soil characteristics explained 65.0% of the variance (Fig. 6B), with axis 1 explaining 40.10% of the variance and axis 2 explaining 24.90% (Fig. 6B).
Notably, the concentration of AP in soil was positively correlated with Mortierellomycota (r = 0.68, p < 0.05) and Glomeromycota (r = 0.59, p < 0.05). Aphelidiomycota was signi cantly positive correlation with the concentration of soil TN (r = 0.59, p < 0.05), TP (r = 0.77, p < 0.01), and AP (r = 0.90, p < 0.01). While, Mortierellomycota (r = -0.73, p < 0.01), Glomeromycota (r = 0.65, p < 0.05), and Aphelidiomycota (r = -0.75, p < 0.01) decreased with the increase of soil pH (Table 3).  Vegetation restoration and reconstruction regulate the interaction between microbial community and forest development, which is mainly manifested in the dynamic changes of microbial diversity and structure (Chanthorn et al., 2017). Our ndings generally suggested that soil ectomycorrhizal fungal community diversity varied with vegetation restoration, and RPL hold the highest ectomycorrhizal fungal Chao1 index, Pielou_e index, Shannon index, and Observed_species (Fig. 2). This nding is coherent with the results of Deng et al. (2020a, 2020b). Soil microorganisms participate in a series of soil biochemical processes, which are closely related to the conversion of soil organic carbon (Rallage et al., 2021). In the process of vegetation restoration, a large amount of exogenous carbon entering the soil will be decomposed by soil carbon degrading enzymes to release low-molecular-weight sugars, providing important carbon and energy sources for microbial growth and metabolism (Davidson et al., 2004), thereby increasing soil microbial community diversity.
Soil ectomycorrhizal fungal diversity reveals that the revegetation process plays an important role in the development of the microbial community composition. The results showed that overall ectomycorrhizal fungal community structure differed signi cantly among three different vegetation (Fig. 5), which supported our second hypothesis, con rming previous results which showed that ECM community structures may be directly impacted by their host (Sugiyama et  In our study, the predominant ectomycorrhizal fungal group was Ascomycota, which was consistent with previous study (Guo et al., 2020). Ascomycota were detected in all sites, which degrade cellulose and more complex carbohydrates in the litter (Schoch et al., 2006)  Soil microbes should be considered drivers of productivity diversity in terrestrial ecosystems (van Der Heijden et al., 2008). In the process of vegetation restoration, soil fungal community was signi cantly affected by the changing soil properties (Cline and Zak, 2015), which in turn, were most likely affected by

Conclusion
In conclusions, the current study has uncovered the distinct difference of soil characteristics and ectomycorrhizal fungal community composition in a typical Fe ore tailing in Liaoning. It is noteworthy that soil properties could be improved by different revegetation types, and RPL could signi cantly better improve soil nutrients than PKSZ and PSC. In addition, compared to PKSZ and PSC, RPL could better improve soil ectomycorrhizal fungal community diversity. Soil ectomycorrhizal fungal community composition signi cantly differed depending on revegetation types. Changes of soil nutrients caused by different revegetation types were key factors affecting the ectomycorrhizal fungal community diversity and composition. Thus, these results indicated that RPL might be a more suitable species for the revegetation of iron mine tailings.