Diversity and characterization of Cd-tolerant dark septate endophytes (DSEs) associated with the roots of Nepal alder (Alnus nepalensis) in a metal mine tailing of southwest China

Applied Soil Ecology 93 (2015) 11–18

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Diversity and characterization of Cd-tolerant dark septate endophytes (DSEs) associated with the roots of Nepal alder (Alnus nepalensis) in a metal mine tailing of southwest China Runbing Xu a,1, Tao Li a,1, Hongliang Cui a , Junling Wang a , Xin Yu a , Yanhua Ding a , Chaojun Wang a , Zhuliang Yang b , Zhiwei Zhao a, * a Laboratory of Conservation and Utilization for Bioresources, Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan University, Kunming 650091, China b Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 January 2015 Received in revised form 14 March 2015 Accepted 30 March 2015 Available online xxx

Nepal alder (Alnus nepalensis), being an ideal and fast-growing pioneer species to accelerate ecological restoration of the degraded habitats in eastern Himalayas, have received extensive attention. In the present study, colonization characteristics, diversity and cadmium tolerance of dark septate endophytes (DSEs) colonizing the roots of Nepal alders were assessed along environmental gradients in a Pb, Zn, and Cd mine tailing, southwestern China. All Nepal alder roots surveyed were conspicuously colonized by DSEs with 23.1–35.8% colonization intensity. In total, 72 culturable strains of melanized root-associated fungi were isolated from the 45 root samples. Most strains were closely related to well-known DSE fungi, such as fungi belonging to the genera Phialophora,Leptodontidium, Cladosporium, and Exophiala. DSEs from heavily metal-polluted sites showed a higher Cd2+ tolerance in vitro than those from the slightly polluted plots. These findings stressed that DSE was an integral component of the root system of Nepal alder growing in metal-contaminated soils, and it is more feasible to obtain heavy metal (HM) tolerant DSEs from mining areas than from no HM-polluted environments for their potential application in phytoremediation. We concluded with a discussion of further research for addressing the unresolved roles of DSEs in A. nepalensis restoration during the early stage of plant establishment in the freshly metal-exposed mining soils. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Dark septate endophyte (DSE) Diversity Cd tolerance Alnus nepalensis

1. Introduction Intensive anthropogenic activities, such as mining and industrial waste discharge, dramatically increase the amount of various heavy metals (HMs) in soil. Excessive toxic metal ions in soil cause direct and/or indirect effects on plants, affecting practically all physiological functions of the plants, and even posing a severe threat to plant survival (Sharma and Dietz, 2009; López-Climent et al., 2014). Some plants, however, such as excluders or accumulators, can tolerate high levels of heavy metals using a series of strategies that involve complex physiological traits, metabolic pathways, and molecular or gene networks (Deng et al., 2013; Milner et al., 2014). In addition, the active rhizospheric microorganisms, such as mycorrhizal fungi and dark septate

* Corresponding author. Tel.: +86 871 6503 4799; fax: +86 871 6503 4838. E-mail address: [email protected] (Z. Zhao). Runbing Xu and Tao Li contributed equally to this work.

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http://dx.doi.org/10.1016/j.apsoil.2015.03.013 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

endophytes (DSEs), are considered essential for the survival and development of plants in heavily metal-contaminated soils (Khosla et al., 2009; Ban et al., 2012; Babu et al., 2014a,b). This is especially true for the woody pioneer plants rather than the herbaceous pioneers because the former rely more on their symbiotic microorganisms during excess metal exposure (Colpaert, 2008; Colpaert et al., 2011). DSEs are a group of melanized fungi that frequently colonize in roots of different plants around the globe (Kauppinen et al., 2014; Massenssini et al., 2014; Muthuraja et al., 2014; Porras-Alfaro et al., 2014; Saravesi et al., 2014; Terhonen et al., 2014), especially plants growing in various stressed environments including the extreme HM-contaminated soil (Mandyam and Jumpponen, 2005; Likar and Regvar, 2013; Zhang et al., 2013; Chadha et al., 2014). More experimental evidence of the interaction between plants and their root-associated-fungi revealed that DSEs could stimulate plant growth under specific environmental conditions and enhance plant tolerance to a variety of environmental stresses (Mandyam et al., 2010; Mandyam et al., 2012). In metal-enriched soil, DSEs

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could ameliorate metal toxicity for their host plants by restricting the uptake of toxic metals (Li et al., 2011; Affholder et al., 2014), increasing chlorophyll, positively affecting transpiration rate (Likar and Regvar, 2013), and by improving the supply of essential elements (Mandyam and Jumpponen, 2014). Thus, the integrated development of the combination of plant roots and DSEs has received a wealth of research attention in recent years (Alberton et al., 2010; Newsham, 2011; Mukasa Mugerwa et al., 2013). More field surveys showed several Alnus species with optimal adaptation to abiotic environmental stresses, and these species may have potential application for large-scale afforestation in landslideaffected areas and rocky and eroded slopes in the eastern Himalayas (Sharma and Ambasht, 1984; Põlme et al., 2013). Even in extreme metal-contaminated soil, some alder species are able to survive and thrive and showed phytoremediation potential (Lee et al., 2009). For example, Rosselli et al. (2003) found Alnus incana to be the ideal excluder of heavy metals on a Cu- and Zn-contaminated site. Additionally, A. glutinosa was found to survive well by excluding HMs from their leaves after growing in an HM-contaminated dewatered dredged sediment and therefore, might be suitable for phytostabilization in polluted sites (Mertens et al., 2004). There is no information, however, about the relationships between Alnus species and their root-associated DSEs. In a previous field survey, we noticed that A. nepalensis, a fastgrowing pioneer tree, showed extreme metal tolerance and thrived well in the freshly exposed mine tailing soils that contained excessive Pb, Zn, and Cd. In the current study, the functional DSEs associated with the roots of A. nepalensis were surveyed in tailing piles containing metal concentration gradients in southwestern China, and we assessed DSE colonization characteristics, species diversity, and metal tolerance. 2. Material and methods 2.1. Sample site The area of sample is approximately 3 km
5 km and located in an abandoned mine area (99 290 –100 350 E, 22 010 –23160 N) in

Lancang County, southwestern China (Fig. 1), where is located in southwestern Yunnan below the Northern Tropic. It contains elements of both a tropical wet and dry climate and a humid subtropical climate. The monthly 24-h average temperature ranges from 13.0  C (in January) to 23.4  C (in June), while the annual mean is 19.4  C. Rainfall totals about 1580 mm annually, with nearly 70% of it occurring from June to September, when relative humidity averages above 85%. An A. nepalensis population has naturally developed and become dominant in the lead-zinc spoil heap, where lead–zinc mining has been carried out using Chinese traditional methods for approximately 600 years (Chen et al., 1997). It also grows together with several shrub, grass and forb species, such as Bidens pilosa, Cynodon dactylon, Ageratina adenophora, Arthraxon hispidus, Pseudocyclosorus esquirolii and Sphenomeris chinensis. Five sampling plots (P1, P2, P3, P4, P5) were established in the sampled area and were located at a distance of 0.2, 0.5, 1.7, 2.8 and 3.8 km from the metal smelt center, respectively. 2.2. Sample collection At each sampling plot (P1, P2, P3, P4 and P5), nine naturally regenerated A. nepalensis seedlings (approximately 2–3 years old) were selected in May 2010. The roots and rhizospheric soils (depth 10–25 cm, more than 500 g) of each seedling were collected in sterile polythene bags. The roots were divided into two portions: one was for the observation of DSE colonization and the other was used to isolate DSEs. 2.3. HM concentrations of the sampled soils The rhizospheric soil samples mentioned above were air-dried, ground to fine powder in a mortar, and passed through a 2 mm sieve. Samples from the same plot were mixed completely. Then, approximately 500 g of mixed soil was taken and divided into two portions by quartering. One portion was directly used for the determination of acid extractable soil Zn, Pb and Cd. Briefly, 5.0 g of soil was weighed and put into a 100 mL Erlenmeyer flask. Then,

Fig. 1. Geographic localization of the sample site. The solid black triangle in the map represents the location of the investigated site.

R. Xu et al. / Applied Soil Ecology 93 (2015) 11–18

50 mL of 0.1 M HCl was added, and the solution was shaken for 2 h at 20  C and filtered into a volumetric flask (Lu, 2000). Then, the extractable concentrations of Zn, Pb and Cd were determined by flame atomic absorption spectrometry (FAAS) using a Z2000 polarized Zeeman atomic absorption spectrophotometer (Hitachi, Japan) as described by Li et al. (2011). The other portion was further put through a 0.15 mm sieve for the analysis of soil total HMs. For each soil sample, 0.2 g of soil was digested in 5 mL of aqua regia (ultrapure mixture of concentrated HNO3/HCl, 1/3 (v/v)) in a teflon crucible and diluted with water to 50 mL, and then the total HM contents were analysed by FAAS (SEPA, 1997). 2.4. DSE colonization The roots (with tips) were collected from the 45 A. nepalensis seedlings separately, washed thoroughly under tap water, and then cleaned in 10% (w/v) KOH at 90  C in a water bath for 1.5 h. The cleaned roots were then stained with acid fuchsine (Berch and Kendrick, 1982). The stained roots were cut into approximately 1–2 cm fragments. More than 10 root fragments from each sample were chosen for examination under an optical microscope (OLYMPUS-BX51, Japan) at 400
magnification. The colonization status, including the DSE hyphae or microsclerotia, was observed. The root colonization intensity of DSE was estimated using the magnified intersection method by checking over 150 intersections (McGonigle et al., 1990; Massicotte et al., 2005; Olsrud et al., 2007). 2.5. DSE Isolation The fresh root samples were washed thoroughly under tap water, sterilized with 75% ethanol for 5 min and 10% NaClO for 5 min, then thoroughly washed three times in sterile distilled water, and finally air-dried under aseptic conditions (Arnold et al., 2000). These freshly surface-sterilized roots were aseptically sectioned into approximately 0.5 cm fragments. Each fragment was placed onto potato dextrose agar that contained both 50 mg L1 streptomycin and 50 mg L1 ampicillin in sterilized petri dishes. The root fragments were incubated in the dark at 28  C for two to three weeks (Silvani et al., 2008). During cultivation, root samples were checked every other day, and only new melanized hyphae emerging from root fragments were recorded and carefully transferred to the sterilized PDA slants in the dark at 28  C. Then, the DSE isolation rate ((number of isolates)/(total number of plants)) of each plot was finally calculated. 2.6. DSE Identification The melanized isolates were cultured on PDA medium for morphological identification. The sporulation characteristics of each isolate, especially the conidiogenous cells and conidia, were observed under a compound-light microscope (OLYMPUS-BX51, Japan) and recorded. All isolates were continuously monitored for sporulation at 28  C for 3 weeks. The isolates that failed to

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sporulate were recorded as “sterile” (Lacap et al., 2003). The sporulation isolates were identified based on their morphology, the manner of spore production, and spore characteristics with reference to the descriptions provided by Barnett and Hunter (1987) and Ellis (1988) and with their original species descriptions. For phylogenetic analysis, the total genomic DNA of each DSE strain was extracted and purified using the CTAB method (Sharrock et al., 2004). The primer set ITS1-F/ITS4 was used to amplify the ITS1-5.8S-ITS2 regions (White et al., 1990; Gardes and Bruns, 1993). The PCR reactions were carried out in a total volume of 50 mL with final concentrations of 10
PCR buffer (containing Mg2+), 2.5 mM dNTP, 5.0 U mL1 Taq DNA polymerase (TaKaRa, Japan), and 50–100 ng mL1 total DNA. The amplifications were performed on a Mastercycler (Eppendorf, Germany) under the following conditions: an initial pre-denaturing at 94  C for 4 min, 32 cycles at 94  C for 30 s, 58  C for 30 s, 72  C for 60 s, and a final extension at 72  C for 7 min. The amplification products were electrophoresed in 2% agarose gel and purified with a Gel Extraction Kit (UNIQ-10, Sangong, Shanghai, China). The purified products were ligated to the pMD18-T vector (TaKaRa, Japan) and transferred into competent Escherichia coli DH5a cells. Positive clones were identified based on blue-white screening, then sequenced on an ABI 3730 DNA Analyzer (PerkinElmer, America). Subsequently, the sequences were checked and analysed using the BLAST sequence similarity search tool provided by GenBank (Altschul et al., 1997). Then, sequences of high homology were retrieved and aligned by the MegAlign program of Lasergene Package (DNAStar Inc., Madison, WI, USA). The aligned sequences were then subjected to a neighbor-joining (NJ) analysis using PAUP*4.0b10 (Sworfford, 2003) with support values estimated using 1000 bootstrap. Modeltest 3.06 was used to determine the evolutionary model that best fit the dataset (Posada and Crandall, 1998). 2.7. Cadmium tolerance of DSE isolates To determine the Cd2+ tolerance, the range of MIC (minimum inhibition concentration) for all fungal isolates was evaluated in the Modified Melin–Norkrans (MMN) media supplemented with a gradient of Cd2+. Initially, stock solution of Cd2+ (CdCl22.5H2O) was filtered through a glass-fibre filter (0.22 mm) and diluted to yield the sterilized MMN liquid medium (a total volume of 1 L with 0.05 g CaCl2, 0.15 g MgSO4, 0.025 g NaCl, 1.2 mL FeCl3 (1%), 0.5 g K2HPO4, 0.1 mg VB1, 0.25 g (NH4)2HPO4, 10.0 g glucose, 3.0 g maltose, 3.0 g NaNO3, and pH 5.8) with a series of Cd2+ supplements (0, 100, 200, 300, 400 and 600 mg L1). Then, 5 mL of each solution was put into a sterilized tube (18
150 mm) and inoculated with two fungal disks (F0.6 cm) cut from the 14day-old PDA culture. The tubes were incubated at 28  C and 120 rpm agitation for 30 d for growth observation. Then, the fungal growth vigor was recorded, and the MIC range of each fungal isolate was determined. Five replicates of each fungal isolate were carried out.

Table 1 Heavy metal contents in the soil (mg kg1, mean  SE, n  6) from a Pb–Zn mine tailing, southwestern China. Plot

Zn Total

Acid extractable

Total

Acid extractable

Total

Acid extractable

P1 P2 P3 P4 P5

6285.3  1172.8a 4147.7  1108.3b 6351.4  1565.7a 415.8  206.4c 212.2  56.1d

1417.1  479.3a 900.7  105.6b 392.9  80.9c 17.9  2.6d 4.2  2.8d

36,845.5  10,895.1a 32,323.5  17,071.1a 15,189.7  20,029.1b 186.1  13.9c 40.5  36.3c

12,148.0  1707.0a 5544.4  1032.5b 2699.9  838.1c 13.1  1.5d 3.4  2.0d

81.8  43.0a 35.9  17.4b 25.7  14.9bc 12.0  6.3c 5.9  3.1d

32.4  19.0a 13.8  4.3b 6.8  1.3bc 2.6  1.2c 0.3  0.2d

Pb

Cd

Notes: different letters in the same group indicate significant differences at the p < 0.05 level according to a LSD test.

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Table 2 Relationships between soil acid extractable metals and total metals, DSE colonization (hyphae and microsclerotia), DSE isolation ratio and Cd tolerance of DSE in a Pb–Zn mine tailing, southwestern China.

Extractable Zn Extractable Pb Extractable Cd

Total metal

Hyphae

Microsclerotia

Isolation ratio

Cd tolerance

0.764 0.920* 0.997**

0.937* 0.958* 0.964**

0.037 0.098 0.078

0.498 0.379 0.375

0.852 0.904* 0.929*

2.8. Data analysis The statistical analysis was performed using SPSS software (version 17.0) after homoscedasticity of the variance was checked. The differences between means were considered statistically significant if the p value was less than 0.05. The HM ion concentration data were subjected to one-way ANOVA to test the differences among the five sampling plots. The relationships among the acid extractable soil metals and the total soil metals, hyphae, microsclerotia, isolation ratio and Cd tolerance of DSEs were determined by Pearson’s correlation analysis. The Shannon diversity index and the Simpson index of the fungal isolates were calculated from the sequence data using the open-source computer program Mothur v. 1.24.1 (Schloss et al., 2009). The identity was set to 97%. 3. Results

proportions of acid extractable HMs in the total soil HMs ranged from 2.0% to 22.5% for Zn, 8.5% to 17.2% for Pb and 4.8% to 39.3% for Cd. A significant positive correlation between total and acid extractable HMs was observed (Table 2). 3.2. Colonization of DSE Typical dark septate hyphae and microsclerotia were observed in all root samples surveyed (Fig. 2a–b). The colonization intensity of the DSE hyphae showed the lowest value (26.1%) in the samples from the heavily polluted plot (P1), a peak at 35.5% in the samples from the medium HM stressed soil (P3), and a slight decrease to 34.9% and 34.7% in the less polluted soils (P4 and P5). A significant difference did not exist between the plots (Fig. 3). However, all samples had a low colonization intensity of DSE microsclerotia, ranging from 1.86% to 2.94%. The colonization intensity was negatively correlated to the acid extractable HM concentrations in the soil (Table 2).

3.1. Soil HM concentrations 3.3. Isolation and identification of DSE The HM concentrations of the soil were shown in Table 1. The sampled soils contained high levels of total Zn, Pb and Cd, with maximal concentrations of 6351.4 mg kg1, 36,845.5 mg kg1 and 81.8 mg kg1, respectively. Compared with the slightly contaminated plots P4 and P5, P1, P2 and P3 were heavily polluted. The

In total, 72 DSE strains were isolated from the 45 root samples, including 41 strains isolated from the heavily HM-polluted plots (P1, P2 and P3) and 31 strains from the slightly HM-polluted plots (P4, P5) (Table 3). Moreover, the isolation ratios did not differ

Fig. 2. Morphological characteristics of DSE colonizing the roots of A. nepalensis and the dominant DSE fungi in a Pb–Zn mine tailing, southwestern China. (a) Abundant microsclerotia and hyphae of DSE colonized the roots of A. nepalensis in the heavily metal-contaminated plot 1. (b) A typical microsclerotium and its subtending septate hyphae in slightly metal-polluted soil (plot 5). (c) Brown conidia and lageniform-shaped phialides in small clusters of Cladosporium sp. B64. (d) Hyaline conidia in groups and flask- and bottle-shaped phialides of Phialophora sp. B61. (e) Long-ellipsoidal conidia with brown annellide and lageniform-shaped phialides of Exophiala sp. F21.

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were isolated from the slightly polluted area (P4 and P5) (Fig. 5). Among the 41 strains from P1, P2 and P3, most fungi (95%) showed a high tolerance with the minimum MIC value of 100 mg L1 Cd2+, whereas this value decreased to 52% (16/31) in the slightly polluted area. In total, 13 extreme tolerant fungal strains were collected and grew well in the medium that contained 600 mg L1 Cd2+, and approximately 70% (9/13) of them were isolated from the heavily polluted plots. 4. Discussion

Fig. 3. Colonization of DSE (H: hyphae; MS: microsclerotia) in A. nepalensis roots in a Pb–Zn mine tailing, southwestern China. Different letters in the same group indicate significant differences at the p < 0.05 level according to a LSD test. Error bars indicate standard deviation.

significantly in the HMs (total isolation ratio was 1.52:1) nor in the slightly polluted plots (total isolation ratio was 1.72:1). The 72 DSE strains were identified based on their morphological characteristics and molecular phylogenetic analysis (Figs. 2 and 4). Sporulation was observed in 16 isolates from the heavily polluted plots (P1, P2 and P3) and 5 from the slightly polluted plots (P4 and P5). According to their sporulation characteristics, these isolates could be identified as members of the Cladosporium, Phialophora and Exophiala genera (Fig. 2c–e). For phylogenetic analysis, a dataset containing 72 newly generated fungal sequences and 48 reference sequences retrieved from GenBank was constructed. Based on the results of the modeltest 3.7 analysis, the model of the hierarchical likelihood ratio test (hLRT) for the dataset was TrN with a gamma shape parameter (G) for intra-site variation (TrN + G). A rooted neighborjoining tree was created based on the above evolutionary model (Fig. 4). Phylogenetic analysis revealed that the 72 isolates were clustered into 8 clades, and approximately 39% of the fungi fell into clade 1, which was related to the well-known DSEs, such as Phialophora and Leptodontidium, with 99% bootstrap support. Clade 6 was the next most common (21%), and most of members of this clade also belonged to the common DSE genera Exophiala and Cladophialophora. Clade 8 included 7 strains that fell within Cladosporium, which had been previously reported as a DSE group (Yuan et al., 2011; Ban et al., 2012). There were 3 strains grouped into Clade 7, which were identified as Podospora curvicolla with 100% bootstrap support. The relative abundance of the other 4 clades was low and these fungi were closely related to the fungi of Pleosporales, Meliniomyces, Helotiales, and Berkleasmium. 3.4. Cadmium tolerance of the DSE strains Overall, fungi that were isolated from the heavily polluted plots (P1, P2 and P3) showed a higher Cd2+ tolerance than those that

Table 3 Isolation results of DSE strains from the roots of A. nepalensis from a Pb–Zn mine tailing, southwestern China. Plots

Root samples

Strain number

Isolation ratio

Frequency occurrence (%)

P1 P2 P3 P4 P5 Total

9 9 9 9 9 45

14 10 17 14 17 72

1.56: 1 1.11: 1 1.89: 1 1.56: 1 1.89: 1

19.44 13.89 23.61 19.44 23.61

Our results suggested that there was abundant DSE colonization in A. nepalensis growing in the mining spoil heap with a colonization intensity that ranged from 26.1% to 35.5%, which indicated that DSEs were an integral and common root component of A. nepalensis in HM-polluted soil. These results were consistent with other field data from various plants exposed to different metal stress around the globe (Pongrac et al., 2009; Regvar et al., 2010). For example, Gucwa-Przepióra et al. (2013) found that DSE fungi are one of the most frequent colonists in the roots of Deschampsia cespitosa growing in the immediate vicinity of a former Pb–Zn smelter in southern Poland. However, in the present study, a higher DSE colonization intensity in A. nepalensis was recorded compared with the other DSE colonization data (Ruotsalainen et al., 2007; Regvar et al., 2010; Zhang et al., 2013). In addition, we found that DSE colonization intensity was negatively correlated to the soil extractable HMs, which seems controversial based on previous reports. Several field surveys showed that with the increasing HM pollutants, the DSE hyphal colonization was not affected in Deschampsia flexuosa roots (Ruotsalainen et al., 2007) and even increased in the roots of Salix caprea L. (Regvar et al., 2010), or significantly showed more abundant mycorrhizal structures in the roots of Nicotiana rustica (Audet and Charest, 2006). Based on the excessive metals from the previous reports, it was reasonable that the significantly higher metal ions in our research sites, especially for Pb, might reduce the DSE colonization in the roots of A. nepalensis. For example, in a previous study, the maximum of Pb was only 1824 mg kg1, which was 20% less than in our investigation (Likar and Regvar, 2009). In the present study, we found diverse DSE groups colonizing the roots of A. nepalensis with a Shannon diversity index (H) of 2.59 at 97% similarity of ITS genes. This finding was consistent with the results of a study involving 14 shrub and forest tree species of the northern temperate zone (Ahlich and Sieber, 1996) in which a DSE group (Mycelium radices-atrovirens-complex) dominated the fungal assemblages of coniferous and ericaceous hosts. Additionally, the high diversity of DSEs with species belonging to the genera Phialophora, Phialocephala and Leptodontidium, was confirmed by temporal temperature gradient gel electrophoresis (TTGE) at the highly metal-enriched locations. In addition, some DSE genera (e. g. Phialophora, Exophiala and Phialocephala) had been widely reported in different ecosystems in the past decades (Addy et al., 2005; Newsham et al., 2009; Sonjak et al., 2009; Regvar et al., 2010; Terhonen et al., 2014). These pioneer or early stage fungal strains might be the basic materials to further explore their potential roles in the growth of plant species from one successional stage to another during the restoration of mining areas. In the present study, most DSEs showed great tolerance to the toxicity of excess Cd2+, and more than 50% of the tested isolates were Cd2+ tolerant with a concentration of 100 mg L1. Some isolates even grew well with 600 mg L1 Cd2+, which was consistent with the previous study by Li et al. (2012). Presumably, this may be due to the natural components of most DSE fungal walls, such as chitin and melanin, which can increase wall rigidity, reduce permeability and fortify DSE to withstand harsh environments (Gadd, 1993; Mandyam and Jumpponen, 2005; Zhan et al.,

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JN123359 Phialophora mustea JX145399 Phialophora mustea A201/B68/E62/F76/H62 95 A204/E61 B61 E82 RA 61 A143/E47 A202/B43/C112/E41/F75/H11/H12 Clade 1 JX145398 Phialophora mustea 38.89% A193/H23 Ascom ycota 57 A191 B51 75 GU479910 Leptodontidium orchidicola AF486133 Leptodontidium orchidicola 100 100 A141 HQ73163 5 Leptodontidium sp. 99 I21 85 B101/D51/E64 100 E31 DQ404351Cadophora melinii 98 93 HQ157927Meliniomyces vraolstadiae HQ157928Meliniomyces vraolstadiae C131 Clade 2 51 2.78% HM190124 Meliniomyces vraolstadiae 93 99 Meliniomy ces HM19012 6 Meliniomyces vari abilis 100 HM190128 Meliniomyces variabilis JQ272355 Meliniomyces sp. 52 C21 100 NR 103564 Phialocephala virens AF486132 Phialocephala virens Clade 3 59 2.78% C73/H21 61 DQ227258 Hyphodiscus hymeniophilus Helotiales KF313119 Paraphoma chrysanthemicola C81 B21/B23/B42/C53/C71/D47/E71 JN123358 Paraphoma chrysanthemicola KF251165 Paraphoma chrysanthemicola 87 D72 100 Clade 4 JX401946 Paraphoma sp. 19.44% B41 Pleosporales D44 92 HE608798 Alternaria sp. 100 JX164078 Alternaria sp. DQ023279 Alternaria alternata 79 I22/I81 99 76 JQ388267 Alternaria sp. 100 A142 GU934500 Alternaria sp. Clade 5 100 EU543255 Berkleasmium sp. 1.39% D78 Berkleasmium EU035406 Cladophialophora chaetospira 99 H22 EU035405 Cladophialophora chaetospira 100 EU035403 Cladophialophora chaetospira 55 EU035404 Cladophialophora chaetospira 100 I52 D61/F41/F62 F102 87 F21 Clade 6 94 20.83% I82 B71/B81 Ascomycota 100 HQ607852 Exophiala sp. 69 99 HQ839778 Exophiala pisciphila 100 100 DQ82673 9 Exophiala pisciphila 86 C111/I64 AB488490 Exophiala sp. 78 A42 C116 73 99 I23 100 AJ972801 Phaeococcomyces sp. AJ507323 Phaeococcomyces chersonesos GQ922547 Podospora curvicolla AY999122 Podospora curvicolla GQ922549 Podospora curvicolla Clade 7 72 GQ9225 4.17% 47 Podospora curvicolla 100 C145/E102 Podospora curvicolla 63 HQ631039 Podospora curvicolla I91 GU56622 2 Cl ado um cladosporioides 58 HQ631003 Cladospori sporium sp. A131/A146 90 B102 AJ300331 Cladosporium tenuissimum Clade 8 9.72% B67/F63/F101 99 Cladosporium EU570256 Cladosporium sphaerospermum B64 AF393709 Cladosporium lignicola 100 JQ768326 Cladosporium sphaerospermum AY471647 Melampsora epitea 61

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0.1 Fig. 4. Neighbor-joining phylogram of ITS genes of melanized fungal isolates from A. nepalensis from a Pb–Zn mine tailing, southwestern China, based on TrN + G substitution model, rooted tree by Melampsora epiteaas outgroup. Numbers above the nodes indicate bootstrap support in neighbor-joining analysis, 1000 times. Relative abundance (RA) here represents the number of melanized endophytic isolates in a given clade relative to the total number of DSE isolates.

2011). The results of the present study indicated that some native fungi that have been exposed to constant metal stress for a long period have a marked adaptive evolution and inherent tolerance to heavy metals, and the toxic metals were even used as micronutrients by these growth-stimulated DSEs. Therefore, it might be beneficial to inoculate these stress-adapted DSEs into plants for phytoremediation in heavy metal-contaminated soils (Dodd and

Thompson, 1994; Zhang et al., 2008; Affholder et al., 2014). Interestingly, in the present study, the tolerance of DSE fungi to Cd2 + had a significantly positive correlation with the soil extractable Pb and Cd, and the correlation coefficients were 0.904 and 0.929, respectively, (p<0.05). Additionally, DSEs of the heavily polluted plots (P1, P2 and P3) showed a higher Cd2+ tolerance than those from slightly polluted areas (P4 and P5). Colpaert and Van Assche

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mine tailing soils that contain excessive metal ions and low aggregate stability. In addition, one should keep in mind that the current analyses were only conducted on the culturable DSEs, and further work is needed to evaluate all community assembly of high metal-tolerant root-associated fungal communities using the combination of cultivation with metagenomic technology to directly amplify DNA in situ from environments without cultivation, which will contribute to a broader understanding of microbial ecology by linking microbial diversity with functional potential. Acknowledgments This research was financially supported by the National Natural Science Foundation of China (31160009, 41161083, 31460114 and 41461073), Key Project of Applied Basic Research of Yunnan (2013FA001), and Young Academic and Technical Leader Raising Foundation of Yunnan Province (2012HB006) . Fig. 5. Percentages of DSE with different Cd2+ tolerance colonizing the roots of A. nepalensis in 5 sampling plots from a Pb–Zn mine tailing, southwestern China. Cd2+ tolerance of DSE isolates was evaluated by the range of minimum inhibition concentration (MIC) with a gradient of Cd2+ that ranged from 100 to 600 mg/L Cd2+ supplements.

(1987) also reported that strains isolated from carpophores of Pinus and Betula spp. found on unpolluted soils were greatly inhibited by the metals, whereas most of the strains derived from polluted soils were strongly tolerant to HMs. Therefore, it was significant to screen metal-tolerant DSE fungi from the heavily contaminated sites rather than from the slightly or non-polluted ones. However, we must realize that the differences of DSE metaltolerance between in vitro and in vivo behavior can only result from the actual cellular metal concentrations and the inherent and genetic repertoire for DSE metal-adaptation (Fajardo et al., 2014), which highlights the importance of considering the interaction of the plant-fungi-soil matrix on a case by case basis. Numerous studies have indicated that fungal colonization may increase plant tolerance to heavy metals, especially arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) (Janoušková et al., 2005; Hildebrandt et al., 2007). The prevailing theory even suggested that these pioneer fungi may play a crucial role in the early stage of plant succession (Trowbridge and Jumpponen, 2004). Recently, experimental evidence from numerous in vivo researches confirmed that metal tolerant DSE fungi may significantly alleviate the toxicity of excess metal ions in host plants (Li et al., 2011; Likar, 2011; Likar and Regvar, 2013). Ban et al. (2014) also found that the root colonization by DSEs reduced the translocation of Zn and Pb from roots to shoots. Increasing data suggested that it presumably resulted from metal chelation of the abundant extraradical and intraradical hyphae in the mycorrhizosphere, by binding metals to hyphal cell walls, or by intracellular sequestration in fungal tissues or plant cells (Hall, 2002; Göhre and Paszkowski, 2006; Miransari, 2011). In a recent field survey of wild rosemary in metal contaminated soils, southeast France, Affholder et al. (2014) found a positive linear correlation between trace metal (loid) concentrations in soil and endophyte (dominant mycorrhizal fungi and DSE) root colonization rates, as well as the metal storage, thus they argued that these endomycorrhizal associations promoted to limit accumulation in aerial parts. Although the DSEs with great tolerance intensively colonized the roots of A. nepalensis, there is a need for more researches that will determine more precisely symbiont roles in these root-integral DSEs during the early stage of plant establishment in the freshly metal-exposed mining soils. Also, further studies are necessary to elucidate the mechanism and significance of the current findings for improving the metal tolerance of plants in freshly exposed

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