Fungal diversity of rhizosphere
ECM fungi is a highly mutual symbiotic combination between mycorrhizal fungi and host plants . This unique symbiotic relationship can help plants survive in adverse conditions, especially in the soil contaminated with heavy metals [9, 32]. In our previous study also found the ECM fungi enhanced the survival and growth of masson’s pine in Pb/Zn tailing area . In this current study, we used the Illumina MiSeq technique to investigate diversity and structure of rhizospheric fungal community at different inoculation treatments of masson’s pine in mining area contaminated with Pb and Zn. The results indicated that the fungal diversity and richness in the rhizospheric soil of masson’s pine were significantly higher than bulk soil. However, there was little difference between the soil with/without ECM fungi inoculation (Table 2), which illustrated that the difference of plant rhizospheric fungal diversity was mainly determined by “rhizosphere effect” of plant [26, 34]. Plants exchange the nutrients and water frequently with the surrounding environment of the root system during the growth process, which caused the micro-environments in the rhizosphere different from that in the non-rhizospheric soil. Those differences could result in the great discrepancy of the richness and diversity in the rhizospheric soil of the fungal groups.
Previous studies showed that the fungal ergosterol content was much more higher in potato rhizospheric soil than soil without plants in the stage of growing season . Besides, Thion et al.  found that the diversity of those fungi in the rhizospheric soil planted with alfalfa was significantly higher than that of non-rhizospheric soil of in situ experiment. Obviously, there are many factors that determine the relative abundance and diversity of the soil fungal communities, among them, the plants’ root seems to changed soil microenvironment of physicochemical property, such as soil organic matter, water content, and soil enzymes , which greatly impact the growth and the number of fungi species indirectly . Those results strongly support that the importance of plants and their “rhizosphere effects” was the main reason leading to the discrepancy of the diversity and structure of soil microorganism. Some other studies also showed that the ECM fungi inoculation would decrease the diversity of fungal community. According to Li et al. , the bacterial and fungal diversity reduced when Pinus armandii inoculated with Tuberindicum during early symbiotic stage as compared with non-ectomycorrhizal fungi soil. Compared with bulk soil, the great reduction of microbial diversity in the rhizosphere, was mainly attributed to large discrepancies between plant host species , or related to the surrounding sampling soil properties of the rhizosphere .
All together, our results revealed that even in a highly polluted mining area (Pb:131.25 mg/kg; Zn:262.67mg/kg), plants’ rhizosphere had a protective effect on the growth of microbial abundance and diversity host in the plant when compared to the soil without plants, which maybe related to the reducing of the toxicity of heavy metal on plants’ rhizosphere.
Indicators of the representative fungal genus
The difference in fungi community structure reflects the mycorrhiza and its metabolites, such as enzymes, extracellular polysaccharides, amino acids and growth hormone that have greatly effects on the surrounding environment. The differences in dominant populations may indicate that some mycorrhizal fungi can adapt to the specific environmental stress, especially in the rhizospheric soil polluted by heavy metals [8, 9]. Although there was little difference of rhizospheric fungal community diversity between WE and NE treatments, the community composition and dominant species of rhizospheric fungi were obviously different in the soil when inoculated with Suillus fungi (Fig.3, 4). Previous studies in forest ecosystem found that the phylum of Ascomycota and Basidiomycota were the dominant fungal communities either in rhizospheric soil or root samples of Pinus armandii with Tuber indicum inoculation . By 454 pyrosequencing, Liu et al. also found that the rhizosphere of Xinjiang jujube were mainly colonized by Ascomycotaand and Basidiomycota, whereas Chytridiomycota and Glomeromycota were probably restrained. However, in our research, besides Basidiomycota, there also a more abundant of Chytridiomycota was existed in the soil of ECM fungi inoculation compared with bulk soil, while the relative abundance of Ascomycota was low, which suggested that Basidiomycota and Chytridiomycota might closely related to the presence of ECM fungi (Suillus luteus) synthesis (Fig.5, 6). It also should be noted that fungal structure and composition were highly related with soils environment, and plant-fungus symbiont was undoubtedly shifted with soil pH, moisture, and soil nutrients, which indirectly determines fungal community composition .
In addition, our results showed that the relative abundance of Suillus, Paraglomus, Agaricus, Tulasnella and Melanotaenium were the functionality of abundant fungal genus in the WE (Fig.5). Some of these fungal genera are the important mycobiont with plants and enforce adaptability of plant for grow in stress conditions, such as metal-contaminated soils. Previous studies showed that Pinus massoniana seedlings were significantly resistant to heavy metals than non-mycorrhizal plants when inoculated with S. luteus . Besides Suillus luteus, some mycorrhizal fungi (Suillus bovinus) inoculated plants (e.g. Pinaceae and Fagaceae) have been reported to develop tolerance to heavy metals such as Zn and Cd [12, 14], and improve the survival of plants on heavy metal-contaminated soils . Others, such as Paraglomus, which was a common arbuscular mycorrhizal (AM) fungi and geographically widespread through locally sporadic in tilled agricultural soils , can increase growth performance of the peach seedlings and elevate the absorption of essential elements (K, Mg, Fe and Zn) for plants’ growth and photosynthetic, and fix the heavy metal such as Cu2+ and Mn2+ to the root system under the potted conditions ; Agaricus species can also increase the inorganic nutrients uptake in the soil, thus accelerate the adaptation process of plant in the stressed environment . These fungal mycobionts adhering to the roots of plants can form a huge network system underground (hartig network)  and release related antioxidant enzymes to acquire nutrients from organic compounds, such as chitin, melanin, and cellulose [43, 44]. The unique species adapted to these special environments would help plants survive against the environmental stress.
Relationships between fungal community structure and environmental variation
Soil processes may be primarily affected by interaction among surrounding environmental factors, structure of soil microbia communities and their reciprocal relationship . Alternatively, these mutural interrelationship have significant impact on the processes of ecosystem through feedback mechanism. Fungal communities act as a “bridge” between the plants, the soil environmental condition and their relationship , which play a vital role in driving nutrient and carbon cycling processes .
Numerous studies have indicated that soil physicochemical properties, such as pH, soil C:N, and soil water content could shape fungal communities composition and structure in the plant’ s rhizosphere , especially, in those heavy metal soils . Our result showed that the environmental variables of soil water content, C/N, and bulk density were important factor determining the composition and diversity of soil fungi (Fig.7). Previous studies also indicated that fungal symbionts formed an important interface between the soil and the plant’s rhizosphere that greatly affected soil physicochemical properties and microbial community structure in the rhizospheric soil , which was consistent with our results. As such, the existence of ECM fungi interact with the rhizospheric environment of host plant could thus in return affect the abundance and ECM could influence fungal community via altering physicochemical properties in the heavy metal polluted mining tails.
Soil extracellular enzyme is a kind of bioactive substance with catalytic ability, which decomposed and released by microorganisms and plants. Soil fungi play an important part in enzyme secretion that related with nutrient absorption and mobilization, such as nitrogen and/or phosphorus [19, 49]. Among these fungal communities, groups of ECM fungi are usually form symbiotic relationship with the roots of the plant , especially, on the condition of nutrient limitation, ECM fungi could receive carbohydrates from their plant host to power nutrient transformation  and soil enzyme expression . Here, in our research, by combining 18S rDNA sequencing with soil enzyme analyses, we demonstrated relationships between fungal community structures and soil enzyme activities with ECM fungi inoculated in the masson’s pine (Fig. 7). Obviously, ECM fungi had a major influence on soil enzyme activities during processes of restoration (Table 1). According to Burke et al. , ECM fungi were positively associated with the soil phosphate content and some soil enzymes of phosphatase (organic P degradation) and N-acetyl-β-glucosaminidase (NAG; organic N and chitin degradation) in field experiment of northern hardwood forest. Other researches also showed that some certain ECM fungi, such as the genera Cortinarius and Russula, were significantly associated with the activities of enzymes of lignin-degrading Mn-peroxidase  and plant cell wall-degrading in deeper layer of humus  in mineralizing nitrogen and phosphorus for the mutualistic symbiotic system , which was consistence with our research.
It is noteworthy that the discrepancy of diversity in plant’s rhizosphere fungal community might be determined by “rhizospheric effect” of plant, while the dominated keystone fungal species chosen by Suillus-Pinus symbionts showed a more effective way in establishing of soil microenvironment system and promoting the growth of plants in heavy metal polluted mining tails.