Soil Fungal Community Composition and Diversity of Culturable Endophytic Fungi from Plant Roots in the Reclaimed Area of the Eastern Coast of China

As an important resource for screening microbial strains capable of conferring stress tolerance in plants, the fungal community associated with the plants grown in stressful environments has received great attention. In this study, high-throughput sequencing was employed to study the rhizosphere fungal community in the reclaimed area (i.e., sites F, H, and T) of the eastern coast of China. Moreover, endophytic fungi from the root of six plant species colonizing the investigated sites were isolated and identified. The differences in soil physicochemical parameters, fungal diversity, and community structure were detected among the sampling sites and between the seasons. Ectomycorrhizal (ECM) fungi (e.g., genera Tuber and Geopora) were dominant at site F, which was characterized by high soil total carbon (SC) and total nitrogen (SN) contents and low soil electrical conductivity (EC) value. Arbuscular mycorrhizal (AM) fungi, including genera Glomus, Rhizophagus, and Entrophospora were dominant at sites H (winter), H (summer), and T (summer), respectively. The positive relationship between the EC value and the abundance of genus Glomus indicated the ability of this AM fungus to protect plants against the salt stress. Endophytic fungi at sites F (Aspergillus and Tetracladium), H (Nigrospora), and T (Nigrospora, Coniochaeta and Zopfiella) were recognized as the biomarkers or keystone taxa, among which only genus Aspergillus was isolated from the plant roots. The aforementioned AM fungi and endophytic fungi could contribute to the promotion of plant growth in the newly reclaimed land.


Introduction
The coastal area (Yellow Sea) in Jiangsu province of China has a great length of coastline (1039.7 km) and a large mudflat area (6520.6 km 2 ) [1]. Large-scale coastal reclamation activities have been performed in China since 1950 to mitigate the conflict between the growing population and shrinking usable land [2]. Reclamation of coastal areas for agriculture, aquaculture, and forestry was considered as the preferred strategy to increase the food supply and improve the eco-environment [3]. So far, lots of reclamation activities have been done. However, the process of plants development in the reclaimed area is lagging behind due to some abiotic factors, among which saline stress is the most detrimental to plant growth [4]. Therefore, it is difficult to promote the exploitation and utilization of the reclaimed area, and more attention should be paid to improve saline stress tolerance of plants.
In saline habitats, only the halophytes that constitute about 1% of the world's flora can thrive, contributing important eco-functions in the desert and coastal areas [5]. Halophytes are capable of coping with multiple environmental stresses, such as high salinity, tidal flooding, and nutrient deprivation [6,7]. Along with the reclamation processes, the vegetation will gradually shift from halophytes to non-halophytes (salt tolerant grasses, shrubs, and trees) in the old reclaimed regions. In a preliminary investigation in the reclaimed area, Salix was observed as the only tree species growing in all the investigated sites. As a pioneer tree species, willow could naturally grow in harsh soil conditions, which was partly ascribed to the development of dual mycorrhizal symbiosis (i.e., forming both arbuscular mycorrhizas and ectomycorrhizas within the same root system) [8]. The ability of some tree species (e.g., genera Alnus, Eucalyptus, Populus and Salix) to form dual mycorrhizal symbiosis is recognized as a key factor to improve their adaptions to unfavorable habitat conditions (e.g., the fluctuations of soil temperature, water content, salinity, and nutrients) [9,10]. It is necessary to explore the fungal community associated with plants (e.g., Salix) that survived or thrived in the reclaimed area.
Great attention has been put on the soil fungal communities, which maintains the ecosystem balance by contributing to the soil organic matter decomposition and mineralization [11]. It is noteworthy that some types of fungi in soil could penetrate into the plant tissue through the roots and wounds or by horizontal transmission through spores, forming extensive symbiotic relations with the host plant [12]. Being the two major groups of root fungal symbionts, mycorrhizal and endophytic fungi are generally believed to be critically important to improve host fitness, including productivity and abiotic tolerance, especially under stressful conditions [13,14]. There is relatively limited information concerning the role of the ectomycorrhizal (ECM) symbiosis in enhancing salt tolerance of plants. In contrast, considerable evidence indicates that arbuscular mycorrhizal (AM) fungi colonization in plant roots augments water and nutrient uptake capacities and enhances plant resistance to salinity stresses [15]. Unfortunately, AM fungi can hardly be grown in pure culture to obtain a large amount of inoculum, which restrained its large-scale application into integrated management of plants development in harsh environments [16,17]. Instead, endophytic fungi that can easily meet the requirements of mass production are gradually recognized as a highly promising mutualistic partners of plants. Endophytic fungi benefit their hosts by promoting resistance against high salinity stress via acting as elicitors in the process of resistance induction [18]. Nevertheless, endophytic fungi are known to shift their functional role between pathogenicity and mutualism depending on fungal genotype, host, and abiotic conditions [19]. The available information concerning the diversity of endophytic fungi and their potential function is limited, which deserves extensive investigation.
The research on plant-associated fungi in the coastal area has attracted global attention [20][21][22][23]. Most of the studies on fungi in the coastal area of China focused on mangroves ecosystems along the south China coast [24] and the salt marsh ecosystem in the Yellow River Delta [25]. Along the east China coast, limited information was obtained concerning either the root endophytic fungi or the soil fungi community in the reclaimed area. Moreover, the relationship between the soil fungal communities and the endophytic fungi is so complicated [26] that it deserves further investigation. The aims of this study include: (i) investigate the fungal community composition in the reclaimed regions and discover the critical factor mediating fungal genera succession; (ii) explore the potential relationship between the fungi in rhizosphere soil and the root endophytic fungi; (iii) examine the functional microbial taxa of the soil fungal community in the reclaimed regions. Identifying fungal community composition in the rhizosphere of the plants survived in the reclaimed area will be helpful in understanding the development of the host plants and their coevolution with symbionts. Furthermore, collecting the isolated root endophytic fungi and constructing a fungal resource bank can pave the way for the ecosystem reconstruction of the reclaimed area along the east China coast.

Study Sites and Sampling
In two seasons (summer and winter), sites F (32.603 • N, 120.880 • E), H (32.609 • N, 120.933 • E), and T (32.155 • N, 121.475 • E) located in Nantong, Jiangsu province of China were chosen for the investigation of fungal community. The average annual precipitation and temperature were 1399.1 mm and 16.6 • C, respectively. Site T (close to the sea embankment) located in a region that was reclaimed recently, while site F (with a longer age of land reclamation) situated in a coastal shelter forest along the highway. In the middle of sites T and F, site H that was close to a farm was selected. At site H, the plants including Salix matsudana, Tamarix chinensis, Suaeda glauca, Celtis sinensis, and Pyrus betulifolia were distributed. At site T, Salix matsudana and Tamarix chinensis were observed in summer and winter, respectively. At site F, Salix matsudana was the dominant species in the shelter forest. The willows survived at sites F, H, and T for around 5, 3, and 2 years, respectively. Three 10 m × 10 m plots near the willows at each site were randomly selected for soil sampling. In each plot, five subsamples were collected and then mixed to make a composite soil sample. The rhizosphere soil was sampled using a shovel, and the plant roots below the ground was collected. The plant material and soil samples were transported to the laboratory and stored in a refrigerator for further analysis.

Physicochemical Analysis of the Soil Samples
The physicochemical parameters of the soil samples, including total carbon (SC), total nitrogen (SN), pH, and electrical conductivity (EC) were measured. Soil SC and SN were identified by combustion using a Vario EL Cube elemental analyzer (Elementar Ltd., Frankfurt, Germany). Soil pH and EC values was measured in 1:5 (v/v) soil/water extracts by a portable meter (Thermo-Orion Inc., Waltham, MA, USA).

Isolation of Endophytic Fungi
Endophytic fungi were isolated from fresh plant roots, which were disinfected by soaking in 75% ethanol (0.5 min), 3% sodium hypochlorite (5-8 min) and 75% ethanol (1 min) and were then rinsed twice for 1 min in sterile water. The plant roots were cut into small pieces (0.5 cm) using a sterilized scalpel. Segments from each plant tissue were randomly chosen and placed in Petri dishes containing potato dextrose agar (PDA) supplemented with 50 µg·mL −1 streptomycin and 50 µg·mL −1 chloramphenicol. These plates were incubated at 28 • C until fungal growth appeared. The colonies were counted and grouped by their morphologic characteristics, and representative isolates of fungal diversity were collected, purified, and preserved for future analysis.

Identification of Isolated Fungi
The isolated fungi were grown for 7 to 10 days on plates of potato-dextrose agar (PDA) in an incubator at 25 • C. After incubation, cultures were examined for color, margin, and texture of colonies in the agar medium, and were preliminarily identified based on morphological characteristics. For DNA extraction, mycelia were transferred from PDA into 250 mL Erlenmeyer's flasks containing potato-dextrose broth without shaking. After 5 days of growth at 28 ± 2 • C, approximately 100 mg of the mycelial biomass was harvested. The genomic DNA was isolated using the Qiagen DNeasy Mini Kit according to the manufacturer's instructions. The internal transcribed spacer (ITS) regions of rDNA were amplified with universal primers ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) and ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3 ). The PCR products were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd., China. The resulting ITS sequences were searched for homology with other genera and species deposited in the GenBank database using NCBI BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 16 October 2021)). The phylogenetic reconstruction was calculated using the neighborjoining (NJ) algorithm and the maximum-parsimony (MP) method. NJ analysis was conducted using the MEGA version 6.0 software with bootstrap values calculated from 1000 replicate runs. MP analysis was performed using the PAUP version 4.0 beta 10 program with bootstrap values (1000 replicate runs).

High-Throughput Sequencing and Data Processing
The genomic DNA of the soil samples (in summer and winter) was extracted using the Mag-Bind Soil DNA Kit Protocol (OMEGA, Norcross, GA, USA). The rDNA region was amplified by polymerase chain reaction (PCR) using the ITS1F (5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ). Single-molecule real-time (SMRT) sequencing on PacBio Sequel II platforms was employed to generate circular consensus sequences (CCS) according to the standard protocols by Biomarker Technologies Corporation Co., Ltd. (Beijing, China). CCS reads are filtered, clustered, and de-noised to generate full-length amplicon tags for species annotation and abundance analysis.
Further analysis including diversity analysis, differential analysis between groups, correlation analysis, function prediction, etc. were carried out using the tools on the platform BMKCloud (www.biocloud.net (accessed on 12 October 2021)). Fungi Functional Guild (FUNGuild) was used to analyze fungal OTU by taxonomy of ecological guilds, and classify large sequence libraries into categories with ecological significance. Non-Metric Multi-Dimensional Scaling Ordination (NMDS) was used to visualize the potential differences (based on the Jaccard matrix) in the soil fungal community structure between groups or within groups. Significance of differences between sample clusters was assessed with Analysis of Similarity (ANOSIM). Random Forest (RF) models were generated, which provides the initial ranking of the genera according to its importance by the mean decrease Gini. Then, the top 30 genera were selected and their abundance was presented in the heatmap. Linear discriminant analysis Effect Size (LEfSe) analysis was conducted to identify and compare unique fungal taxa significantly related to each site at the different seasons. The biomarkers fungal groups were depicted in cladograms, and linear discriminant analysis (LDA) scores ≥4.0 were then performed. Spearman rank correlation analysis was processed based on the abundance of each species in the investigated sites. The correlation network was constructed with the ones with correlation larger than 0.1 and p-value smaller than 0.05. Canonical Correspondence analysis (CCA) was used to reflect the relationship between the fungi (top 10 genera and the key genera identified by random forest, LefSe, and co-occurrence analysis) at the genus level and environmental variables. Environmental variables were chosen only when their addition did not cause an inflation factor of variance >20.

Statistical Analysis
Two-way analysis of variance (ANOVA) was used to determine the individual and combined effects of sites distribution and sampling seasons on the soil physicochemical properties using IBM SPSS 25.0 (IBM Inc., Chicago, IL, USA).

Soil Physicochemical Properties
A two-way ANOVA indicated that the physicochemical parameters (i.e., the EC values and contents of SN and SC) of the soil samples differed significantly (p < 0.05) among the three sampling sites (i.e., sites F, H, and T) and between the two seasons (summer and winter 2020) ( Table 1). There was a significant difference in pH values of the soil samples between two seasons (p < 0.05). A significant interaction term (i.e., sampling sites × seasons) for EC value and SC content (p < 0.05) was also detected. In both seasons, the average percentages of TN and SC content in the rhizosphere soil samples were gradually decreased in the order of sites F, H, and T. The salinity level (EC value) in the rhizosphere soils was the highest at site H, while it was not so high as expected at site T. The investigated sites were located in the coastal area of Nantong, Jiangsu Province of China. There is a long history of the coastal mudflats' reclamation in this area. Although the detailed reclamation ages of the sampling sites were unknown, the vertical distance from the location of the sites (in the order of sites F, H, and T) to the coastline could indicate a chronosequence reclamation of coastal mudflat. If the coastal areas were reclaimed at different stages, the impact of reclamation on soil properties could be varied with each other [2,27]. In general, the average percentages of soil SN and SC contents increased with increasing reclamation ages. At site T, the low EC value could be due to the desalination measures employed in the newly reclaimed land.

Soil Fungal Diversity and Community Structure
In the soil samples, the species diversity was evaluated using the Shannon and Simpson index, while ACE and Chao1 indexes were used to reflect the species richness of fungi communities ( Table 2). In terms of the fungal diversity and richness in the investigated area, the diversity of fungi was highest at site F, followed by the sites H and T, and the index values were higher in winter compared with those in summer. In this study, six major phyla were identified in all collected soil samples, including the phyla Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Rozellomycota, and Glomeromycota (Figure 1a). Except for the sample HS, the majority of OTUs in the investigated sites were belonging to phylum Ascomycota. In the sample HS, the highest abundance of phylum Basidiomycota was found, and its abundance was even higher than phylum Ascomycota. The findings that Ascomycota and Basidiomycota were predominant phyla in the sampling sites were consistent with an investigation on the reclaimed land in the Sanjiang plain, China [11].
to a farm along the coastal area (i.e., samples HS and HW), FUNGuild database analyses revealed a higher proportion of saprophytic fungi. The relative abundance of dung saprotroph, plant saprotroph, and wood saprotroph were 27.4%, 26.9%, and 27.1%, respectively, in the HS sample, indicating a disturbance from agricultural industry. It is noteworthy that plant pathogens accounted for 38.8% abundance of the fungal OTUs in sample TW, which cannot be ignored due to potential harmful effects on plant health.

Isolation of Endophytic Fungi from Plant Roots
A total of 29 endophytic fungal taxa were isolated from the roots of the plant distributed in the investigated sites. Among them, genera Alternaria, Fusarium, Monosporascus, and Podospora were the four most common genera that were detected in the roots of many plant species (Table S1). Alternaria was observed mostly in willow, while Fusarium, Monosporascus, and Podospora were mostly isolated in plant roots from site H. Besides the four genera, Aspergillus, Aureobasidium, Cladosporium, Clonostachys, Dactylonectria, Diaporthe, Mortierella, Penicillium, Sarocladium, Stemphylium, and Talaromyces were detected by both FUNGuild was used to examine the fungal community from an ecological perspective, which could explore fungal trophic type and metabolic function characteristics rather than taxonomic identity [28]. Based on FunGuild ecological guild assignments (Figure 1b), the highest relative abundances of AM fungi and endophytic fungi were observed in samples TS (21.4%) and TW (7.46%), respectively. A large proportion of the OTUs at site F belonged to ECM fungi, and the relative abundances of ECM fungi were 47.0% and 83.5% for samples FW and FS, respectively. According to the location of the sampling sites, AM fungi and endophytic fungi were more frequent at the sites close to the sea embankment (i.e., TS and TW, respectively), while ECM fungi were dominant at the site located in the protection forest along the highway (i.e., FS). The difference in relative abundances of AM and ECM fungi among the investigated sites was probably due to the different vegetation types. In general, AM fungi were prevalent in herbaceous plants, while ECM prefer to form symbiosis with temperate woody plant species [29]. Additionally, AM fungi generally establish symbiotic associations with plants in the early stages of growth, while ECM fungi dominate in mature plants [9,21,30]. The establishment of a dense woody plant (i.e., 5-year-old willow) cover at site F could explain the dominance of ECM. At the site close to a farm along the coastal area (i.e., samples HS and HW), FUNGuild database analyses revealed a higher proportion of saprophytic fungi. The relative abundance of dung saprotroph, plant saprotroph, and wood saprotroph were 27.4%, 26.9%, and 27.1%, respectively, in the HS sample, indicating a disturbance from agricultural industry. It is noteworthy that plant pathogens accounted for 38.8% abundance of the fungal OTUs in sample TW, which cannot be ignored due to potential harmful effects on plant health.

Isolation of Endophytic Fungi from Plant Roots
A total of 29 endophytic fungal taxa were isolated from the roots of the plant distributed in the investigated sites. Among them, genera Alternaria, Fusarium, Monosporascus, and Podospora were the four most common genera that were detected in the roots of many plant species (Table S1). Alternaria was observed mostly in willow, while Fusarium, Monosporascus, and Podospora were mostly isolated in plant roots from site H. Besides the four genera, Aspergillus, Aureobasidium, Cladosporium, Clonostachys, Dactylonectria, Diaporthe, Mortierella, Penicillium, Sarocladium, Stemphylium, and Talaromyces were detected by both root isolation and soil ITS sequencing. According to Hardoim et al. [31], the endophytic community can be classified into facultative endophytes, obligate endophytes, and passenger endophytes. The aforementioned genera belonged to the facultative endophytes that can live inside the host plant and the soil habitats. Strains from genera Aspergillus, Penicillium, and Talaromyces were distributed in the plant roots with low frequency of appearance. However, they were widely distributed with relatively low abundance in soil samples according to the ITS sequencing results. These three genera might belong to passenger endophytes, which randomly enter the plant. The fungi that were isolated from the plant roots but were not detected in the soil samples via ITS sequencing (Arthrobotrys, Botrytis, Macrophomina, Paecilomyces, Parengyodontium, Peniophora, Phaeomyces, Phialophora, Phoma, Pithomyces, and Rhizoctonia) could be classified into obligate endophytes.
Among the isolated fungi, genera Alternaria [32], Aspergillus [33], Cladosporium [34], Phoma [35], and Phialophora [36] contain strains classified as dark septate endophytes (DSE). Positive effects of DSE on the growth of host plants were reported, including the production of bioactive metabolites against pathogens [37], the synthesis of phytohormones, and the mineralization of organic N-containing compounds [38]. Additionally, strains from the genera Aspergillus, Aureobasidium, Cladosporium, Monosporascus, and Sarocladium have been reported as halophilic fungi [39]. Among them, genera Aspergillus, Aureobasidium, and Monosporascus were only isolated in plant roots from site H. Strains from the genera Mortierella, Penicillium, and Talaromyces have been recognized as plant growth promoting fungi [40,41]. The presence of the aforementioned fungal endophytes in the plant roots from the investigated sites indicates their important role in plant-fungus interactions. Nevertheless, previous literature showed that the majority of the genera listed (Table S1) include pathogenic fungal strains [42,43]. Among them, genus Fusarium is widely accepted as one of the major groups of soil-borne root pathogenic fungi [44]. In vitro tests need to be employed to evaluate the pathogenicity and confirm the function of the isolated fungi.

Spatial and Temporal Variation of Soil Fungal Communities
As the EC values and the average percentages of SN and SC contents in the soil samples differed significantly among the sampling sites and between the seasons, it was expected that these factors would be the key drivers for the distribution of fungi community. Indeed, the NMDS plot showed that the samples collected at different sites in two seasons were separated from each other, and the ANOSIM revealed a significant difference among them (R = 0.983, p = 0.001) (Figure 2). Seasonality is commonly regarded as an important parameter influencing plant-associated microbial communities [45]. In this study, the samples collected at different seasons were separated from each other. Besides providing essential nutrition (e.g., carbon and nitrogen source) for microbial growth, soil also affects the growth and distribution of microbe by its physicochemical properties (e.g., EC and pH) [10]. The samples collected at site F (distributed at the bottom right of the coordinate plane) were separated from those sampled at sites H and T. At site F, a dense tree canopy and relatively lower EC value and SN and SC contents were observed. The preferable environmental conditions could promote the dominance of ECM fungi, which explain the clustering pattern of the samples in the NMDS plot.
The random forest analysis was performed to identify the signature microbiota in response to the variation of the investigated sites and seasons, respectively. It is expected that random-forest analysis could be used to classify the fungi community as mirroring sampling sites and seasons, suggesting essential factors shaping soil fungi community. Figure 3 shows the result of the random forest classification preformed using generalevel composition data of the rhizosphere soil samples. The abundance of fungi genera with the 30 highest Gini scores in the random forest analysis is presented in the heatmap. Among these selected genera, four dominant genera (Tuber, Peziza, Tomentella, and Geopora) belonging to ECM fungi were the potential contributors to identify the sites at which rhizosphere soil samples were collected (Figure 3a). ECM fungi were abundant in the FS (Tuber) and HS (Tomentella) samples, while it was seldom observed at site T. The site T located in a young reclaimed region, in which the C% and N% contents of the soil samples were the lowest. Five genera (Pichia, Barnettozyma, Candida, Nigrospora, and Coprinopsis) with high abundance in winter contributed to distinguishing the winter samples from the summer samples (Figure 3b), among which three genera (Pichia, Barnettozyma, and Candida) were identified as yeast, and their dominance was observed in winter.
J. Fungi 2021, 7, x FOR PEER REVIEW 8 and HS (Tomentella) samples, while it was seldom observed at site T. The site T locate a young reclaimed region, in which the C% and N% contents of the soil samples were lowest. Five genera (Pichia, Barnettozyma, Candida, Nigrospora, and Coprinopsis) with h abundance in winter contributed to distinguishing the winter samples from the sum samples (Figure 3b), among which three genera (Pichia, Barnettozyma, and Candida) w identified as yeast, and their dominance was observed in winter. LEfSe analysis (LDA threshold 4.0) was conducted to identify and compare un fungal taxa in the soil samples with statistical difference among different groups (Fig  4a). For samples from site F, fungi that were differentially abundant include genera T and Peziza in the FS sample and genera Geopora and Tetracladium in the FW sample. At H, biomarkers mainly comprised of genera Podospora, Coprinellus and Tomentella in sample HS and genera Preussia, Glomus and Candida in the sample HW. At site T, ge Zopfiella was the biomarker in the TS sample, while the biomarkers in the TW sampl clude genera Nigrospora, Coniochaeta, Alternaria, Stemphylium, and Pichia.
A network interface was constructed to show the topological and taxonomic cha teristics of the fungal co-occurrence patterns in each region (Figure 4b-d). The dens of the co-occurrence network at sites F, H, and T were 0.189, 0.241, and 0.409, respectiv These results suggested that the fungal network at site T was more connected than th sites F and H. Nodes with high degree values and centrality metrics (betweenness or cl ness centrality) were recognized as keystone species in the co-occurrence network. keystone species at sites F, H, and T were identified, which could play critical roles in co-occurrence network. Genera Clitopilus and Aspergillus were identified as the keys genera at site F. At site H, keystone genera including Rhizophagus, Nigrospora, Pichia, Kazachstania were identified. Genera Papiliotrema, Phanerochaete, Entrophospora, and fiella were recognized as the keystone taxa at site T.
In the FS sample, the relative abundances of genera Tuber and Peziza were the high Genus Geopora was detected in most of the investigated sites, with the highest abunda in the sample FW. Genera Tuber, Peziza, and Geopora belong to order Pezizales. Many cies in order Pezizales were recognized as ECM fungi that grow in symbiosis with the p roots [46]. Both RF and LEfSe analyses indicated that genus Tuber had a preference fo distribution among the three sites. At site F, the higher soil SC and SN contents and lo LEfSe analysis (LDA threshold 4.0) was conducted to identify and compare unique fungal taxa in the soil samples with statistical difference among different groups (Figure 4a). For samples from site F, fungi that were differentially abundant include genera Tuber and Peziza in the FS sample and genera Geopora and Tetracladium in the FW sample. At site H, biomarkers mainly comprised of genera Podospora, Coprinellus and Tomentella in the sample HS and genera Preussia, Glomus and Candida in the sample HW. At site T, genus Zopfiella was the biomarker in the TS sample, while the biomarkers in the TW sample include genera Nigrospora, Coniochaeta, Alternaria, Stemphylium, and Pichia.
A network interface was constructed to show the topological and taxonomic characteristics of the fungal co-occurrence patterns in each region (Figure 4b-d). The densities of the co-occurrence network at sites F, H, and T were 0.189, 0.241, and 0.409, respectively. These results suggested that the fungal network at site T was more connected than that at sites F and H. Nodes with high degree values and centrality metrics (betweenness or closeness centrality) were recognized as keystone species in the co-occurrence network. The keystone species at sites F, H, and T were identified, which could play critical roles in the co-occurrence network. Genera Clitopilus and Aspergillus were identified as the keystone genera at site F. At site H, keystone genera including Rhizophagus, Nigrospora, Pichia, and Kazachstania were identified. Genera Papiliotrema, Phanerochaete, Entrophospora, and Zopfiella were recognized as the keystone taxa at site T.    In the FS sample, the relative abundances of genera Tuber and Peziza were the highest. Genus Geopora was detected in most of the investigated sites, with the highest abundance in the sample FW. Genera Tuber, Peziza, and Geopora belong to order Pezizales. Many species in order Pezizales were recognized as ECM fungi that grow in symbiosis with the plant roots [46]. Both RF and LEfSe analyses indicated that genus Tuber had a preference for its distribution among the three sites. At site F, the higher soil SC and SN contents and lower EC values compared with the other two sites could be responsible for the dominance of genus Tuber. As the keystone genera at site F, genus Clitopilus was only detected in sample FS, while genus Aspergillus was identified in all samples. Genus Clitopilus belongs to family Entolomataceae, in which many strains can form ectomycorrhizae with a wide range of tree species; being considered as a common saprotrophic genus, it was reported to increase plant growth via facilitated potassium uptake [47]. Genus Aspergillus was widely distributed in all the investigated sites. Liang et al. [48] reported a ribosomal protein from halophilic strain (Aspergillus glaucus) that could confer salt tolerance in heterologous organisms. It is noteworthy that genus Aspergillus was recognized as a culturable fungal endophyte, and it was only isolated from the plant roots at site H. Genus Tetracladium was mainly identified at site F, with a higher abundance in the sample FW than that in the sample FS. Sati and Pant [49] reported a Tetracladium strain isolated from healthy roots of Berberris vulgaris growing in a riparian area had the potential to solubilize phosphate. As an endophytic fungus, genus Tetracladium could be capable of promoting plant growth at site F.
In the sample HS, the highest abundance of genus Podospora was detected. Genus Coprinellus were dominant in the sample HS, while they were seldom observed in other samples. The abundance of genus Preussia in winter was higher than that in summer, and its highest abundance were observed in the sample HW. Genera Podospora, Coprinellus, and Preussia include coprophilous species inhabiting the dung of various herbivores [50][51][52]. The site H is close to a poultry farm, which might probably release poultry excrement to the surrounding environment. It could explain why the coprophilous species were dominant at site H. In the sample HS, genus Tomentella were dominant. It was commonly found as an ectomycorrhizal partner of many trees, such as willow, birch, or alder, with the tendency to dominate in unfavorable environmental conditions (e.g., salinity stress) [46]. The dominance of genera Glomus and Rhizophagus (belonging to AM fungi) was observed in HW and HS, respectively. Glomus species was reported to increase the number of fruits and yield in papaya plants [53], while genus Rhizophagus could stabilize soil aggregates, enhance plant nutrition, and improve plant growth [16]. AM fungi form a mutualistic association with the roots of 90% of the terrestrial plants, and the symbiosis between AM fungi and plant roots is a well-recognized beneficial interaction occurring in soil [54]. They were reported to be capable of improving rooting, enhancing plant nutrition, favoring nutrient renovation, and promoting tolerance to biotic and abiotic stress [53]. Unfortunately, the use of AM fungi as an inoculant on a large scale is not yet widely used because of several difficulties in obtaining a large amount of inoculum (e.g., low growth and high competition with native AM fungi) [16]. Genera Candida, Pichia, and Kazachstania were described as ascomycetous yeast species [55,56]. Yeasts are particularly suitable as biocontrol agents against several kinds of rot fungi because their activity does not usually rely on the production of toxic metabolites, but rather on their ability to compete with pathogens for space and nutrients [57]. Some species belonging to genus Candida were reported to stimulate rice seedling growth [58]. Their dominance at site H (especially in winter) could be beneficial for the plants to defend against pathogens and survive under the salinity stress. However, a Pichia strain (Pichia fermentans) with dual activity (an effective biocontrol agent and an aggressive pathogen) was reported [55]. To avoid unpredictable effects, it needs a thorough risk analysis before the application of yeast species as biocontrol agents. Many strains in the genus Nigrospora are characterized as plant endophytic microorganisms. This genus produces a broad range of bioactive secondary metabolites, which could be used to inhibit the growth of plant pathogenic fungi [59].
In the sample TS, the dominance of genus Entrophospora was observed. It was classified as an AM fungus [53], which could exchange nutrients gathered from soil for carbon compounds provided by plants. They are important microbial symbionts for plants, especially when soil total phosphorus (TP) and TN are limiting [60]. Although soil TP content was not measured in this study, the lower SC and SN contents at site T indicated a possible deficit in TP contents. The appearance of genus Entrophospora might favor plant growth at site T. Genera Nigrospora and Coniochaeta were observed in winter, especially in the sample TW. Genus Zopfiella was observed with the highest abundance in the sample TS. Some strains of the genera Nigrospora, Coniochaeta, and Zopfiella are characterized as plant endophytic micro-organisms, and they are capable of promoting the stress resistance of the host plant and producing antifungal compounds [61][62][63]. Genera Alternaria and Stemphylium include both plant-pathogenic and saprophytic species [64,65]. Their dominance in winter, especially in the sample TW, indicated possible damage or decay of plants after establishing pathogenic, saprotrophic, or endophytic relationships with the plant hosts. Additionally, genus Phanerochaete that was recognized as a saprotrophic fungus appeared in the sample TS. It is capable of degrading lignin and mineralizing a wide variety of priority aromatic pollutants [66].

The Relationship between Fungi Genera and Soil Physical-Chemical Parameters
The relationships between the fungi (top 10 dominant genera, biomarkers, and keystone taxa) and selected environmental factors were explored by CCA ( Figure 5). The two CCA axes explained a substantial proportion of the variation (88.8%) in the fungienvironment relationship. in the sample TS. It is capable of degrading lignin and mineralizing a wide variety of priority aromatic pollutants [66].

The Relationship between Fungi Genera and Soil Physical-Chemical Parameters
The relationships between the fungi (top 10 dominant genera, biomarkers, and keystone taxa) and selected environmental factors were explored by CCA ( Figure 5). The two CCA axes explained a substantial proportion of the variation (88.8%) in the fungi-environment relationship. The abundance of Tuber and Geopora (belonging to ECM fungi) and Peziza (containing both ECM and saprotrophic fungi) was positively related with SC content and negatively related with EC level. High soil salinity was generally recognized to have a negative impact on ECM associations [46]. Genera Tuber and Geopora were dominant at site F, which was characterized by high SC and SN contents and low soil EC value. ECM fungi are The abundance of Tuber and Geopora (belonging to ECM fungi) and Peziza (containing both ECM and saprotrophic fungi) was positively related with SC content and negatively related with EC level. High soil salinity was generally recognized to have a negative impact on ECM associations [46]. Genera Tuber and Geopora were dominant at site F, which was characterized by high SC and SN contents and low soil EC value. ECM fungi are capable of excreting oxidative and hydrolytic enzymes to break down soil organic matter and liberate unavailable nutrients [8,67]. Their predominance (e.g., Tuber and Geopora) at site F could be helpful to transfer nutrients from organic matter to their host tree. There are also some ECM fungi that are highly adapted to saline conditions [10], and their appearance (e.g., Tomentella) at site H (with relatively high values of soil EC) was observed. Additionally, the positive relationship between the abundance of Tetracladium and SC content was also observed. Some strain (Tetracladium setigerum) belonging to Tetracladium was reported as a phosphate solubilization fungi, which has the potential to solubilize various source of phosphates through the production of phytases and phosphatases enzymes and the organic acids [49]. The dominance of genus Tetracladium at site F could provide sufficient P for the good development of shelter forest.
Soil pH value has a positive influence on the distribution of the genera Zopfiella, Tomentella, and Coprinellus that were dominant in summer during the investigation. Zhao et al. [68] reported that genera Zopfiella was dominated in the soil with the highest pH values after disinfestation treatment using bean dregs. During the investigation on fungi in protected coastal Salix repens communities in the Netherlands, Geml et al. [60] reported that Tomentella showed a strong preference for alkaline soils. Liu et al. [69] indicated that Coprinellus is preferably dwelling alkaline environments. Additionally, several yeasts (Candida, Pichia, and Barnettozyma) were negatively related with pH values. Birkhofer et al. [70] investigated the soil yeast community composition and abundance in different land use types in Germany, and reported that yeasts were highly abundant in the region with a low soil pH and high soil moisture.
There was a positive relationship between the EC value and the abundance of genera Stemphylium and Glomus. Gonçalves et al. [13] isolated Stemphylium from the field-collected Salicornia, and found that the growth of Stemphylium sp. was unaffected by the presence of NaCl (200-800 mM) in the growth medium. Furthermore, the inoculation of Salicornia with the isolated Stemphylium strain positively influenced total biomass production and N concentration in roots in salinity conditions (150 mM NaCl). It is already known that AM fungi (e.g., Glomus) widely exist in salt-affected soils, and they were reported to play a vital role in mitigating the adverse effects of salinity on plants by the processes including the upregulation of the antioxidant system, the modulation of the biosynthesis of osmoprotectants, and the compartmentalization of excessive toxic ions into the vacuole [71][72][73].

Conclusions
The differences in soil physicochemical parameters (i.e., the EC values and contents of SN and SC), fungal diversity, and community structure were detected among the sampling sites (i.e., sites F, H and T) and between the seasons (summer and winter 2020). Based on FunGuild ecological guild assignments, the highest relative abundances of Ectomycorrhizal (ECM) fungi (83.5%), arbuscular mycorrhizal (AM) fungi (21.4%), and endophytic fungi (7.46%) were observed in the samples FS, TS, and TW, respectively. Genera Tuber and Geopora that belong to ECM fungi were dominant at site F, which was characterized by high SC and SN contents and low soil EC value. AM fungi, including genera Glomus, Rhizophagus, and Entrophospora, were dominant in the samples HW, HS, and TS, respectively. The positive relationship between the EC value and the abundance of genus Glomus indicated the ability of AM fungi to protect plants against the salt stress. According to LEfSe analysis and co-occurrence analysis, some biomarkers or keystone taxa belonging to endophytic fungi were recognized at sites F (Aspergillus and Tetracladium), H (Nigrospora), and T (Nigrospora, Coniochaeta and Zopfiella), which could contribute to the promotion of plant growth. However, most of them were not isolated from the plant roots in the investigated sites. A total of 15 endophytic fungi were detected by both root isolation and ITS sequencing of soil samples. It deserves further investigation to isolate more endophytic fungal strains from the plant roots and to evaluate their potential function in conferring stress tolerance to plants.