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Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) Exophiala pisciphila
Science of the Total Environment 409 (2011) 1069–1074
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) Exophiala pisciphila T. Li a,1, M.J. Liu a,1, X.T. Zhang a, H.B. Zhang a,b, T. Sha a, Z.W. Zhao a,⁎ a b
Key Laboratory of Conservation and Utilization for Bioresources, Yunnan University, Kunming, 650091 Yunnan, China Department of Biology, Yunnan University, Kunming, 650091 Yunnan, China
a r t i c l e
i n f o
Article history: Received 20 August 2010 Received in revised form 2 December 2010 Accepted 2 December 2010 Available online 30 December 2010 Keywords: Dark septate endophyte (DSE) Exophiala pisciphila H93 Heavy metals Tolerance Zea mays L
a b s t r a c t Dark septate endophytes (DSE) are ubiquitous and abundant in stressful environments including heavy metal (HM) stress. However, our knowledge about the roles of DSE in improving HM tolerance of their host plants is poor. In this study, maize (Zea mays L.) was inoculated with a HM tolerant DSE strain Exophiala pisciphila H93 in lead (Pb), zinc (Zn), and cadmium (Cd) contaminated soils. E. pisciphila H93 successfully colonized and formed typical DSE structures in the inoculated maize roots. Colonization of E. pisciphila H93 alleviated the deleterious effects of excessive HM supplements and promoted the growth of maize (roots and shoots) under HM stress conditions, though it significantly decreased the biomass of inoculated maize under no HM stress. Further analysis showed that the colonization of E. pisciphila H93 improved the tolerance of maize to HM by restricting the translocation of HM ions from roots to shoots. This study demonstrated that under higher HM stress, such a mutual symbiosis between E. pisciphila and its host (maize) may be an efficient strategy to survive in the stressful environments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction All plants in natural ecosystems appear to be symbiotic with fungal endophytes (Rodriguez et al., 2009). Among this highly diverse group of endophytic fungi, dark septate endophytes (DSE) have received much attention in the recent years (Peterson et al., 2004). DSE comprise a heterogeneous group of root-associated endophytic fungi which are characterized by melanized intercellular and intracellular running hyphae and so-called microsclerotium (aggregation of dark, thick-walled, closely packed inflated cells) within epidermis and cortex of plant roots (Jumpponen and Trappe, 1998; Mandyam and Jumpponen, 2005; Silvani et al., 2008). Increasing evidence suggests that DSE may benefit their host plants by facilitating the uptake of plant mineral nutrients including P, N and water (Haselwandter and Read, 1982; Mullen et al., 1998; Newsham, 1999; Upson et al., 2009), suppressing the infection of plant pathogens (Narisawa et al., 2000; 2004; Barrow, 2003) and alleviating the harmful effects of severely stressful environments (Treu et al., 1996). Subsequently, experiments conducted by Usuki and Narisawa (2007) further confirm that DSE (Heteroconium chaetospira) can form a mutualistic symbiosis with Chinese cabbage (Brassica campestris) by nutrient exchange (the associated fungi providing organic nitrogen in exchange for their host plant carbon), though some controversial and speculative aspects of
⁎ Corresponding author. Tel.: +86 871 503 4799; fax: +86 871 503 4838. E-mail address: [email protected] (Z.W. Zhao). 1 Contributed equally to this work. 0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2010.12.012
deleterious, neutral DSE-plant associations are still debated (Mandyam and Jumpponen, 2005; Peterson et al., 2008; Smith and Read, 2008). Increasing field studies reveal that DSE are ubiquitous root-associated fungi, and especially common in stressful environments such as cool, nutritionally poor, alpine or subalpine ecosystems, high saline environments and polar regions (Read and Haselwandter, 1981; Hambleton and Currah, 1997; Mandyam and Jumpponen, 2005; Newsham et al., 2009; Sonjak et al., 2009). Even in severely stressful heavy metal (HM) polluted soils (5705 mg kg−1 Pb, 58007 mg kg−1 Zn and 77 mg kg−1 Cd) of the Ancient Lead and Zinc Smelting Site of Huize, Yunnan Province, southwest China, where non-ferrous metals Pb and Zn have been smelted by ancient smelting methods for hundreds of years (Xia et al., 1980), most plants of natural revegetation are colonized by DSE (Liang et al., 2007). Recent studies show that DSE are one of the most common root-associated fungi in metal polluted soils (Likar and Regvar, 2009; Regvar et al., 2010), and these DSE have an inherent tolerance (Zhang et al., 2008) and a low sensitivity to HM (Gibson and Mitchell, 2006). While many strides have been made in understanding the ecological significance of mycorrhizal fungi, our knowledge about the functions of DSE in HM polluted environments is poor (Likar and Regvar, 2009). To understand the possible functions of this poorly understood group of root-associated fungi in HM polluted soils, on the basis of known information we hypothesized that, DSE as well as mycorrhizal fungi, could improve tolerance of host plants to heavy metals beyond nutrient acquisition and resultant positive host growth responses. To test this hypothesis, one of the most dominant and highly metal resistant DSE strains, Exophiala pisciphila H93, isolated from the roots of Arundinella bengalensis naturally growing in Ancient Lead and Zinc
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Smelting Site of Huize, China, was used for inoculum. Greenhouse pot experiments were conducted under different concentrations of HM stress (Pb, Zn, Cd) to assess the effects of inoculation with E. pisciphila H93 on (1) maize biomass, (2) the absorption, translocation and accumulation of heavy metals in shoots and roots of maize and (3) the tolerance of host plants to HM. Possible mechanisms of DSE enhancing the tolerance of their host plants to HM are discussed.
Table 1 Heavy metal (HM) concentrations of cultural substrata in the four test groups. Groups
I II III IV
HM concentrations (mg kg−1 DW) Pb(NO3)2
ZnSO4·7H2O
CdSO4·8H2O
0 1000 2000 3000
0 500 1000 1500
0 50 100 150
2. Materials and methods 2.1. Preparation of fungal colonized and non-colonized maize seedlings This experiment was performed to get uniform fungal (E. pisciphila H93) colonized and non-colonized maize (Zea mays L.) seedlings to be used for the subsequent pot experiments (2.2). Maize seeds were surface-sterilized by dipping in 75% ethanol for 5 min and then in 10% sodium hypochlorite for 10 min under agitation. Sterilized seeds were thoroughly rinsed with sterile water and then aseptically planted onto the water agar medium (agar 8 g L−1) contained in Petri dishes (90 mm) for germinating at 25 °C, and 6 seeds were used per Petri dish. After 3 days of incubation, the germinating seeds were transplanted into sterile glass bottles (Φ60 × 200 mm) containing culture substrata for fungal inoculation. Each glass bottle was filled with about 130 g culture substrata that 100 g river sand mixed with 30 mL improved MS liquid medium (containing in mg L−1: NH4NO3 1650, KNO3 1900, CaCl2·2H2O 440, MgSO4·7H2O 370, KH2PO4 170, KI 0.83, H3BO3 6.2, MnSO4·4H2O 22.3, ZnSO4·7H2O 8.6, Na2MoO4·2H2O 0.25, CuSO4·5H2O 0.025, CoCl2·6H2O 0.025, FeSO4·7H2O 27.8, Na2-EDTA·2H2O 37.3, linositol 100, VB3 0.5, VB6 0.5, VB1 0.5, Glycine 2, supplemented with 20 g L−1 sucrose). Before using, river sand was sieved through a 0.2 cm sieve and washed by tap water to remove the soil silt and nutrients, and placed in the 80 °C oven and dried to constant weight (this usually required 5–12 h) and then autoclaved for 2 h at 121 °C (three times with 2-day intervals). The germinating seeds were planted into the culture substrata contained in the bottles (2 seeds for each bottle) and each seed was inoculated with a fungal disk (Φ 0.5 cm) cut from a 14-day-old PDA culture (potato 200 g, dextrose 20 g, agar 18 g, and water 1000 mL), by attaching the fungal disk to the root of maize. For a control treatment, other germinating seeds were also inoculated with a disk (Φ 0.5 cm) cut from PDA plate without fungus and planted into the bottle as above. All the inoculation processes were operated in an SW-CJ-1DF clean bench (Airtech, China). The glass bottles were covered with sterile AeraSeal films (150 × 150 mm) (Mycomebio (Beijing) Bio-medical Science Technology Center, China) and cultivated under a day temperature of 25 °C, a night temperature of 18 °C with a photoperiod of 12 h for 4 weeks, and watered with 5 ml deionized–distilled water every 5 days per bottle. After 4 weeks’ cultivation, the maize seedlings were ready for the subsequent pot experiments (2.2).
shown in Table 1, respectively. For example, the 1000 mg kg−1 of Pb (II) substrata means 1 kg dried sand contained 1000 mg Pb (II). Therefore, to make the 1000 mg kg−1 of Pb (II) substrata, 1 kg of the dried sand was mixed with 150 ml solution that contained 1000 mg Pb (II). Similarly, to make the 500 mg kg−1 of Zn (II) substrata, 1 kg of the dried sand was mixed with 150 ml solution that contained 500 mg Zn (II). To make the non-polluted substrata (control), 1 kg of the dried sand was mixed with 150 ml of deionized-distilled water. The concentrations of HCl-extractable Pb (II), Zn (II), Cd (II) from the sand were trivial, being 4.3, 4.5 and 0.006 mg kg−1respectively, and thus were omitted in the subsequent analysis. Plastic pots (20 cm diameter × 16 cm height) each filled with 2 kg different pot culture substrata were used for plant growing. Before the transplantation of the above 4-week-old maize seedlings from the bottles to pots, the colonization intensity of E. pisciphila H93 in maize roots was evaluated. Five 0.5 cm root fragments were randomly collected from each maize seedling and cleaned in 5 mL 10% (w/v) KOH contained in a test tube for 2 h at 90 °C in a water bath and then stained with 0.5% acid fuchsin (Berch and Kendrick, 1982). Colonization intensity of E. pisciphila H93 was determined by using the magnified intersection method under a compound-light microscope (Olympus-BX51) at 200× magnification (McGonigle et al., 1990). Fungal colonization intensity, i.e. the percentage of the presence of E. pisciphila H93 in all intersections observed (more than 150 intersections were used for each sample) was determined for each sample. The fungal colonized (only those seedlings each had 93–95% root colonization intensity from inoculation of E. pisciphila H93 were used) and non-fungal colonized (without inoculation) maize seedlings were transplanted into pots filled with different pot culture substrata as described in Table 1. Three plants (one plant per pot) were used for each treatment (each group × each metal species), and thus a total of 60 pots were used (3 HM × 3 groups × 2 treatments × 3 replicates + 1 nonHM × 1 group × 2 treatments × 3 replicates). Then maize seedlings were grown in a glasshouse with a day temperature varying between 16 and 28 °C, a night temperature varying between 8 and 16 °C. Seedlings were regularly watered with deionized–distilled water and each pot received 100 mL sterile Hoagland′s nutrient weekly. 2.3. Harvest and analysis of plant HM
2.2. Greenhouse pot cultivation Pot culture experiments in a greenhouse were established to determine the effects of E. pisciphila H93 on maize (Zea mays L.) growing under different kinds and levels of HM (Pb, Zn, Cd) stress. River sand containing different kinds and concentrations of HM (Pb, Zn, Cd) were applied as pot culture substrata. According to the environmental quality standard for soils of China (GB15618-1995) and the contents of HCl-extractable Pb, Zn and Cd in the field soils where the test fungus was isolated (Liang et al., 2009), three HM polluted levels of each HM species (Pb, Zn and Cd) and one nonpolluted level (control) were conducted and thus formed four test treatments in total (Table 1). River sand was cleaned and dried as above before using. To make the four groups of pot culture substrata, 1 kg of the dried river sand was mixed with 150 ml different concentrations of HM salt solutions to achieve the concentrations
After 3 months of growth, the leaves of maize at the time of male flowering were turning brownish yellow and all maize was harvested. Fungal colonization intensity was evaluated at this stage for each plant as described above. Shoots (leaves+ stems) and roots were collected, respectively, and roots were thoroughly washed by tap water to remove the soil silt. The shoots and roots then were placed in the 80 °C oven and dried to constant weight and weighed. All dried root and shoot samples were ground separately by a mini-vegetation disintegrator (FZ102, Tianjing City Test Instrument Co. Ltd, China), and 0.5 g shoot and 0.5 g root powder of each sample were then subsampled by the usual method of coning and quartering. These representative subsamples were digested by HNO3 + HClO4 respectively and the concentrations of HM ions were determined by the flame atomic absorption spectrometry (FAAS) using a Z2000 polarized Zeeman atomic absorption spectrophotometer (Hitachi, Japan). Quantification was carried out with a
T. Li et al. / Science of the Total Environment 409 (2011) 1069–1074
calibration curve using a graded series of diluted lead (GSW08619) (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg ml−1), zinc (GSW08620) and cadmium (GSW08612) (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg ml− 1) (National Research Center for Certified Reference Materials, China) given in parentheses. A standard reference material of poplar leaves (GBW 07604, National Research Center for Certified Reference Materials, China) was carried through the digestion and analyzed as a part of the quality control protocol (accuracies within 100 ± 20%). The hollow cathode lamp was operated at 4 mA for Cd and 5 mA for Pb and Zn, and the analytical wavelengths were set at 217.0, 213.9, 228.8 nm for detection of Pb (II), Zn (II) and Cd (II), respectively (AOAC, 1984). 2.4. Statistical analysis The differences of plant biomass and HM concentrations in roots and shoots between inoculated and non-inoculated plants under deferent substrata were determined using two-way (HM concentration in substrata is one factor, inoculation or not is the other factor) analysis of variance (ANOVA) followed by Duncan's multiple-range test for multiple comparisons. Pearson's correlation coefficient (r) was used to determine the relationships between soils, plant HM concentrations and colonization intensity. Rejection level was set at α b 0.05 in all analyses. All analyses were made using SPSS software (version 16.0). Unless stated otherwise all values reported are means ± SD. 3. Results No DSE structures were observed in the roots of non-inoculated maize in pot experiments after harvesting. In inoculated treatments, typical structures of DSE-plant associations were observed under all test concentrations of the three HM and abundant intra-radical and extraradical hyphae and microsclerotia colonized the roots of maize similar to
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their colonization in nature (Fig. 1). The lowest colonization intensity of E. pisciphila H93 occurred in non-HM stressed soils (17.58 ± 10.30%), and this value increased with increasing HM concentrations and peaked at 78.90 ± 6.11% in the roots of maize grown in the substrata supplemented with 1500 mg kg−1 ZnSO4·7H2O (Table 2). Colonization intensity was significantly positively correlated to the HM concentrations of cultural substrata (Pb, r = 0.999, p b 0.01; Zn, r = 0.964 p b 0.05; Cd, r = 0.955 p b 0.05). Biomass of maize was significantly decreased with the increasing HM supplements both in inoculated and non-inoculated treatments (Fig. 2). Inoculation of E. pisciphila H93 significantly decreased maize biomass (roots and shoots) compared with the non-inoculated maize under no HM stress. However, under higher HM stress conditions, inoculation of E. pisciphila H93 alleviated the negative effects of increasing toxicity of heavy metals, which was shown by the significantly higher maize biomass (roots and shoots) in fungal inoculated maize than those of non-inoculated maize. The root and shoot HM concentrations of inoculated and noninoculated maize growing in all test substrata are summarized in Table 3. With increasing HM concentrations, HM were also absorbed and accumulated increasingly by roots and shoots of both inoculated and non-inoculated treatments. However, the majority of HM contents were accumulated in maize roots both in inoculated and non-inoculated treatments, and thus ratio of shoot to root HM concentrations (RS/R) remained far below 1.0 in almost all treatments except the essential micronutrient Zn under the trace level (Table 3). More importantly, significantly lower shoot HM as well as significantly higher root HM of inoculated treatments were accumulated than those of non-inoculated maize, though the significant effects of inoculation with E. pisciphila H93 on plant growth varied greatly due to the different HM and their concentrations (Table 3). Root Zn and Cd concentrations were significantly correlated to the colonization intensity of E. pisciphila H93 (Zn,
Fig. 1. Morphology of Exophiala pisciphila H93 colonized in host plant roots. (a) Abundant intra-radical and extra-radical hyphae of H93 colonized maize seedling roots before transplant to pot experiments. (b) Intracellular running hyphae of H93 in maize roots grown in the pot supplemented with 100 mg kg−1 CdSO4·8H2O. (c) Hyphae and melanized microsclerotia of H93 colonized the roots of Arundinella bengalensis in field at Ancient Lead and Zinc Smelting Site of Huize, Yunnan Province, southwest China. (d) Melanized microsclerotia of H93 in maize roots grown in the pot supplemented with 1500 mg kg−1 ZnSO4·7H2O. Bars = 50 μm (a) and 10 μm (b–d).
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Table 2 Colonization intensity of E. pisciphila H93 in the inoculated treatments of four test groups (no DSE structures were observed in the roots of non-inoculated maize, and thus not provided here) (mean ± SD, n = 3). Groups
I II III IV
Colonization intensity of H93 (%) Pb
Zn
Cd
17.58 ± 17.84 33.50 ± 19.45 54.05 ± 32.96 69.61 ± 16.63
17.58 ± 17.84 23.49 ± 11.97 63.11 ± 27.15 78.90 ± 10.58
17.58 ± 17.84 20.56 ± 17.34 42.89 ± 47.39 70.00 ± 12.73
r=0.978 pb 0.05; Cd, r=0.994 pb 0.01), except Pb (r=0.902 pN 0.05). The shoot Pb concentrations were significantly correlated with the colonization intensity of E. pisciphila H93 (r=0.986 pb 0.05), but Zn (r=0.877 pN 0.05) and Cd (r=0.946 pN 0.05). 4. Discussion Colonization intensity of maize roots by E. pisciphila H93 obviously increased with increasing HM toxicity, which indicated that E. pisciphila H93 isolated from metal polluted soil had a high inherent tolerance or a low sensitivity to HM (Gibson and Mitchell, 2006; Zhang et al., 2008). Field surveys showed that 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) with increasing pollutants. Our findings agree with Audet and Charest's
Fig. 2. Shoot and root biomass (DW, g) of inoculated (H93) and non-inoculated maize in the four test groups (group I for control without heavy metal supplements, II, III and IV for groups with different concentrations of Pb, Zn and Cd as described in Table 1). Different letters refer to significant differences at the 0.05 level according to Duncan's multiple-range test.
(2006) observation that colonization of mycorrhizal fungus (Glomus intraradices) on the roots of “wild” tobacco (Nicotiana rustica) markedly increased with increasing soil Zn and the mycorrhizal structures were significantly more abundant at the higher soil Zn (250 mg kg−1). Janoušková and Vosátka (2005) also reported that the colonization of carrot roots by G. intraradices increased when the more Cd was applied in the medium. Presumably, the increasing colonization of E. pisciphila H93 might be a mutual response of endophyte–plant associations to the increasing HM toxicity, which improved the fitness of both plant and fungal symbionts, and thus it may be an efficient strategy to survive in stressfully HM contaminants for both DSE and their hosts. It is now well recognized that the change in plant growth resulting from their associated mycorrhizal symbionts varies widely, which can span a wide range of species interactions from mutualism to parasitism under different environmental conditions (Johnson et al., 1997; Smith et al., 2009). Responses of host plants to DSE colonization like mycorrhizal associations varied greatly with fungus and host plant specificity and cultural conditions, and even intra-individual variation in host plant response to E. pisciphila H93 occurred in this study. Consequently, beneficial, amensal, neutral or parasitic associations have been often debated (Wilcox and Wang, 1987; Fernando and Currah, 1996). Clearly, because of the complexity of the DSE–soil–plant system, more data from other repeated experiments within different DSE-plant combination are needed to confirm DSE–plant association. Here we suppose that the potential trade-offs in carbon economy and nutrients between host plants and their associated fungi may influence their association relationship. In this study, low nutrient sand substrata greatly restricted DSE potential nutrient benefits to its host in exchange for their carbon, and the root-associated fungi can even be considered to be parasitic on plants when the net cost of the symbiosis exceeds the net benefits. Therefore, photosynthesis products of maize repartitioning to E. pisciphila H93 resulted in the significantly lower maize biomass under no HM stress. However, under HM stress, the higher biomass of inoculated maize than non-inoculated maize suggested that their association relationship has changed from parasitism to mutualism (Fig. 2). Because of the complexity of fungi–plant associations, an understanding of the several parameters affecting endophyte functioning, such as the morphology and physiology of both symbionts, and biotic and abiotic factors at the rhizosphere, community and ecosystem levels, is required to construct predictive models of fungal functioning (Johnson et al., 1997). Like other root-associated fungi including arbuscular and ectomycorrhizal fungi (Turnau et al., 1996; Liang et al., 2009), inoculation of E. pisciphila H93 influenced the HM uptake, translocation and accumulation in its host plants (Table 3). Lower RS/R of inoculated maize than those of non-inoculated maize showed that E. pisciphila H93 colonization restricted the translocation of HM from roots to shoots, though more HM ions were absorbed and accumulated in the roots of inoculated maize. Different from the higher root accumulation of DSE inoculation, mycorrhizal inoculation (Glomus sp and G. mosseae) significantly reduced the uptake and accumulation of HM in host plant roots (Liang et al., 2009). By using micro-protoninduced X-ray emission (PIXE), a clear biofiltering pattern in the ectomycorrhiza between Suillus luteus and Pinus sylvestris was studied, and strong accumulation of Ca, Fe, Zn and Pb within the fungal mantle and in the rhizomorph was observed (Turnau et al., 2001). Galli et al. (1994) summarized that the retention of HM by fungal mycelia may involve adsorption to cell walls, and even E. pisciphila H93 accumulated and adsorbed over 20% lead and 5% cadmium of their biomass (DW) (Zhang et al., 2008), thereby minimizing metal translocation to the shoots (Galli et al., 1994). Furthermore, more complex molecular mechanisms may be involved in the improved tolerance against metal toxicity of endophytes to their host plants. Experiments conducted by Cicatelli et al. (2010) showed that inoculation with mycorrhizal fungi (G. mosseae or G. intraradices) restored normal growth of a white poplar clone grown on HM-contaminated soil and enhanced the stress tolerance of
11.31** 549.15*** 62.38*** 5.75* 91.62*** 2.78NS 0.36NS 89.32*** 0.33NS 15.21** 1544.05*** 1.04NS 6.06* 721.97*** 4.53* 3.48NS 189.96*** 1.83NS 7.72** 80.04*** 1.21NS 36.29*** 72.94*** 10.67***
Values followed by different letters within a column refer to significant difference at the 0.05 level according to Duncan's multiple-range test. ***, pb 0.001; **, pb 0.01; *, pb 0.05; NS: not significant.
IV
III
45.24*** 183.08*** 37.69***
4.55 ± 0.80d 4.27 ± 1.51d 56.80 ± 6.27d 49.84 ± 6.67d 177.88 ± 58.20c 173.01 ± 49.14c 757.63 ± 76.59a 651.85 ± 94.24b II
F-value and level of significance H93 HM H93 × HM
0.33 ± 0.02b 0.50 ± 0.05a 0.22 ± 0.00c 0.20 ± 0.02c 0.11 ± 0.02d 0.08 ± 0.00d 0.20 ± 0.00c 0.09 ± 0.02d
RS/R Shoot
0.01 ± 0.00d 0.01 ± 0.00d 5.43 ± 0.73c 5.10 ± 0.95cd 8.03 ± 1.78c 6.56 ± 2.15c 20.32 ± 7.27a 14.76 ± 4.76b 0.03 ± 0.02d 0.02 ± 0.02d 24.8 ± 5.91d 25.17 ± 4.64d 75.55 ± 17.42c 87.43 ± 12.35c 103.53 ± 40.15bc 156.25 ± 33.77a
Root Shoot
4.33 ± 0.55e 4.26 ± 0.26e 463.25 ± 61.00d 483.74 ± 30.97d 1236.73 ± 582.85c 1594.75 ± 207.39bc 1813.25 ± 340.20a 1889.00 ± 200.01a 0.51 ± 0.17a 0.43 ± 0.16a 0.16 ± 0.03b 0.09 ± 0.00b 0.14 ± 0.02b 0.06 ± 0.00b 0.12 ± 0.02b 0.03 ± 0.02b 10.33 ± 1.94e 9.28 ± 1.51e 28.79 ± 3.79de 23.45 ± 5.44de 161.12 ± 72.93ab 69.74 ± 32.27cd 207.78 ± 31.96a 106.19 ± 49.24bc
Shoot Root
C H93 C H93 C H93 C H93 I
20.32 ± 1.37e 21.62 ± 2.06e 183.30 ± 21.67d 255.23 ± 32.73d 1136.22 ± 375.17c 1238.75 ± 297.09c 1691.25 ± 220.15bc 4147.50 ± 1048.41a
RS/R
Root
RS/R
1.05 ± 0.08a 1.00 ± 0.07a 0.12 ± 0.02c 0.10 ± 0.00c 0.14 ± 0.03c 0.11 ± 0.02c 0.42 ± 0.05b 0.35 ± 0.02b
Cd (mg kg−1 plant DW) Zn (mg kg− 1 plant DW) Pb (mg kg−1 plant DW) Treatments Groups
Table 3 Root and shoot heavy metal (HM) concentrations (mg kg− 1 plant DW) and ratio of shoot/root HM concentrations (RS/R) of maize with inoculated (H93) and non-inoculated (C) treatments of all four test groups (mean ± SD, n = 3).
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host plants to accumulate higher Cu and Zn in plant organs, especially roots. Molecular mechanism analysis showed that the improved tolerance of poplar against HM was due to an overall induction of the poplar transcriptional upregulation of several stress-related genes and the protective role of polyamines. Moreover, the differential transcription expression patterns induced by mycorrhizal fungi occurred between leaves and root, and even among different organ cells of roots of mycorrhizal (Gl. intraradices) and non-mycorrhizal tomato (Ouziad et al., 2005). To the best of our knowledge, this is the first report that DSE (E. pisciphila H93) alleviated the deleterious effects of excessive HM ions and promoted the growth of host plants (roots and shoots), and improved the tolerance of maize by restricting the translocation of toxic HM from the roots to shoots under HM stressed soils. This study demonstrated that the facultative associations between E. pisciphila and its host (maize) had transformed from parasitism in the low nutrient substrata without HM pollutants to mutualism when net benefits exceed net cost under higher HM stress. Such a mutual symbiosis within these species may be an efficient strategy to survive in the stressful environments. Acknowledgements We are grateful to Associate Editor Dr. J. Bennett and three anonymous referees for their constructive comments to improve our manuscript. Many thanks to our colleagues Zhang Jie, Kang Yu, and Yan Jun for carrying out partial experiments. We would like to also express our appreciation to Mr. Hu Bin and Cheng Lizhong (Biology Department of Yunnan University) for FAAS analysis and to Dr. Jing Yuebo (English editor of Journal of West China Forestry Science) and Dr. Xu Jin (Massey University, New Zealand) for their help to improve English writing. This research was financially supported by the National Natural Science Foundation of China (30770052, 40763003) and Yunnan Province (2007C007M). References AOAC. Official methods of analysis of the Association of Official Analytical Chemists14th ed. ; 1984. p. 344. Washington, DC. Audet P, Charest C. Effects of AM colonization on “wild tobacco” plants grown in zinccontaminated soil. Mycorrhiza 2006;16:277–83. Barrow JR. Atypical morphology of dark septate fungal root endophytes of Bouteloua in arid southwestern USA rangelands. Mycorrhiza 2003;13:239–47. Berch SM, Kendrick B. Vesicular-arbuscular mycorrhizae of southern Ontario ferns and fern-allies. Mycologia 1982;74:769–76. Cicatelli A, Lingua G, Todeschini V, Biondi S, Torrigiani P, Castiglione S. Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression. Ann Bot 2010;106: 791–802. Fernando AA, Currah RS. A comparative study of the effects of the root endophytes Leptodontidium orchidicola and Phialocephala fortinii (Fungi Imperfecti) on the growth of some subalpine plants in culture. Can J Bot 1996;74:1071–8. Galli U, Schüepp H, Brunold C. Heavy metal binding by mycorrhizal fungi. Physiol Plant 1994;92:364–8. Gibson BR, Mitchell DT. Sensitivity of ericoid mycorrhizal fungi and mycorrhizal Calluna vulgaris to copper mine spoil from Avoca, county Wicklow. Biol Environ Proc Roy Ir Acad 2006;106B:9-18. Hambleton S, Currah RS. Fungal endophytes from the roots of alpine and boreal Ericaceae. Can J Bot 1997;75:1570–81. Haselwandter K, Read DJ. The significance of root-fungus association in two Carex species of high-alpine plant communities. Oecologia 1982;53:352–4. Janoušková M, Vosátka M. Response to cadmium of Daucus carota hairy roots dual cultures with Glomus intraradices or Gigaspora margarita. Mycorrhiza 2005;15: 217–24. Johnson NC, Graham JH, Smith FA. Functioning of mycorrhizal associations along the mutualism–parasitism continuum. New Phytol 1997;135:575–85. Jumpponen A, Trappe JM. Dark septate endophytes: a review of facultative biotrophic root colonizing fungi. New Phytol 1998;140:295–310. Liang CC, Li T, Xiao YP, Liu MJ, Zhang HB, Zhao ZW. Effects of inoculation with arbuscular mycorrhizal fungi on maize grown in multimetal contaminated soils. Int J Phytorem 2009;11:692–703. Liang CC, Xiao YP, Zhao ZW. Arbuscular mycorrhiza and dark septate endophytes in an abandoned lead-zinc mine in Huize, Yunnan, China. Chin J Appl Environ Biol 2007;13:811–7.
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Likar M, Regvar M. Application of temporal temperature gradient gel electrophoresis for characterisation of fungal endophyte communities of Salix caprea L. in a heavy metal polluted soil. Sci Total Environ 2009;407:6179–87. Mandyam K, Jumpponen A. Seeking the elusive function of the root-colonising dark septate endophytic fungi. Stud Mycol 2005;53:173–89. McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 1990;115:495–501. Mullen RB, Schmidt SK, Jaeger CH. Nitrogen uptake during snowmelt by the snow buttercup, Ranunculus adoneus. Arct Alp Res 1998;30:121–5. Narisawa K, Ohki KT, Hashiba T. Suppression of clubroot and Verticillium yellows in Chinese cabbage in the field by the root endophytic fungus, Heteroconium chaetospira. Plant Pathol 2000;49:141–6. Narisawa K, Usuki F, Hashiba T. Control of Verticillium yellows in Chinese cabbage by the dark septate endophytic fungus LtVB3. Phytopathology 2004;94:412–8. Newsham KK, Upson R, Read DJ. Mycorrhizas and dark septate root endophytes in polar regions. Fung Ecol 2009;2:10–20. Newsham KK. Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua. New Phytol 1999;144:517–24. Ouziad F, Hildebrandt U, Schmelzer E, Bothe H. Differential gene expressions in arbuscular mycorrhizal colonized tomato grown under heavy metal stress. J Plant Physiol 2005;162:634–49. Peterson RL, Massicotte HB, Melville LH. Mycorrhizas: anatomy and cell biology. Ottawa: NRC Research Press; 2004. Peterson RL, Wagg C, Pautler M. Associations between microfungal endophytes and roots: do structural features indicate function? Botany 2008;86:445–56. Read DJ, Haselwandter K. Observations on the mycorrhizal status of some alpine plant communities. New Phytol 1981;88:341–52. Regvar M, Likar M, Piltaver A, Kugonič N, Smith JE. Fungal community structure under goat willows (Salix caprea L.) growing at metal polluted site: the potential of screening in a model phytostabilisation study. Plant Soil 2010;330:345–56. Rodriguez RJ, White Jr JF, Arnold AE, Redman RS. Fungal endophytes: diversity and functional roles. 2009; 182: 314-30. Ruotsalainen AL, Markkola A, Kozlov MV. Root fungal colonisation in Deschampsia flexuosa: effects of pollution and neighbouring trees. Environ Pollut 2007;147:723–8.
Silvani VA, Fracchia S, Fernández L, Pérgola M, Godeas A. A simple method to obtain endophytic microorganisms from field-collected roots. Soil Biol Biochem 2008;40: 1259–63. Smith FA, Grace EJ, Smith SE. More than a carbon economy: nutrient trade and ecological sustainability in facultative arbuscular mycorrhizal symbioses. New Phytol 2009;182:347–58. Smith SE, Read DJ. Mycorrhizal symbiosis. 3rd ed. San Diego: Academic Press; 2008. Sonjak S, Udovič M, Wraber T, Likar M, Regvar M. Diversity of halophytes and identification of arbuscular mycorrhizal fungi colonising their roots in an abandoned and sustained part of Sečovlje salterns. Soil Biol Biochem 2009;41: 1847–56. Treu R, Laursen GA, Stephenson SL, Landolt JC, Densmore R. Mycorrhizae from Denali National Park and Preserve, Alaska. Mycorrhiza 1996;6:21–9. Turnau K, Miszalski Z, Trouvelot A, Bonfante P, Gianinazzi S. Oxalis acetosella as a monitoring plant on highly polluted soils. In: Azcon-Aguilar C, Barea JM, editors. Mycorrhizas in integrated systems, from genes to plant development. Proceedings of the Fourth European Symposium on Mycorrhizas, COST Edition. Brussels, Luxemburg: European Commission; 1996. p. 483–6. Turnau K, Przybyłowicz WJ, Mesjasz-Przybyłowicz J. Heavy metal distribution in Suillus luteus mycorrhizas - as revealed by micro-PIXE analysis. Nucl Instrum Methods Phys Res B 2001;181:649–58. Upson R, Read DJ, Newsham KK. Nitrogen form influences the response of Deschampsia antarctica to dark septate root endophytes. Mycorrhiza 2009;201:1-11. Usuki F, Narisawa K. A mutualistic symbiosis between a dark septate endophytic fungus, Heteroconium chaetospira, and a nonmycorrhizal plant, Chinese cabbage. Mycologia 2007;99:175–84. Wilcox HE, Wang CJK. Ectomycorrhizal and ectendomycorrhizal associations of Phialophora finlandia with Pinus resinosa, Picea rubens and Betula alleghaensis. Can J For Res 1987;17:976–90. Xia XR, Li ZJ, Wang GY. Development history of the Mining Industry in Ancient China. Beijing: Geology Press; 1980. Zhang YJ, Zhang Y, Liu MJ, Shi XD, Zhao ZW. Dark septate endophyte (DSE) fungi isolated from metal polluted soils: their taxonomic position, tolerance, and accumulation of heavy metals in vitro. J Microbiol 2008;46:624–32.
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) Exophiala pisciphila T. Li a,1, M.J. Liu a,1, X.T. Zhang a, H.B. Zhang a,b, T. Sha a, Z.W. Zhao a,⁎ a b
Key Laboratory of Conservation and Utilization for Bioresources, Yunnan University, Kunming, 650091 Yunnan, China Department of Biology, Yunnan University, Kunming, 650091 Yunnan, China
a r t i c l e
i n f o
Article history: Received 20 August 2010 Received in revised form 2 December 2010 Accepted 2 December 2010 Available online 30 December 2010 Keywords: Dark septate endophyte (DSE) Exophiala pisciphila H93 Heavy metals Tolerance Zea mays L
a b s t r a c t Dark septate endophytes (DSE) are ubiquitous and abundant in stressful environments including heavy metal (HM) stress. However, our knowledge about the roles of DSE in improving HM tolerance of their host plants is poor. In this study, maize (Zea mays L.) was inoculated with a HM tolerant DSE strain Exophiala pisciphila H93 in lead (Pb), zinc (Zn), and cadmium (Cd) contaminated soils. E. pisciphila H93 successfully colonized and formed typical DSE structures in the inoculated maize roots. Colonization of E. pisciphila H93 alleviated the deleterious effects of excessive HM supplements and promoted the growth of maize (roots and shoots) under HM stress conditions, though it significantly decreased the biomass of inoculated maize under no HM stress. Further analysis showed that the colonization of E. pisciphila H93 improved the tolerance of maize to HM by restricting the translocation of HM ions from roots to shoots. This study demonstrated that under higher HM stress, such a mutual symbiosis between E. pisciphila and its host (maize) may be an efficient strategy to survive in the stressful environments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction All plants in natural ecosystems appear to be symbiotic with fungal endophytes (Rodriguez et al., 2009). Among this highly diverse group of endophytic fungi, dark septate endophytes (DSE) have received much attention in the recent years (Peterson et al., 2004). DSE comprise a heterogeneous group of root-associated endophytic fungi which are characterized by melanized intercellular and intracellular running hyphae and so-called microsclerotium (aggregation of dark, thick-walled, closely packed inflated cells) within epidermis and cortex of plant roots (Jumpponen and Trappe, 1998; Mandyam and Jumpponen, 2005; Silvani et al., 2008). Increasing evidence suggests that DSE may benefit their host plants by facilitating the uptake of plant mineral nutrients including P, N and water (Haselwandter and Read, 1982; Mullen et al., 1998; Newsham, 1999; Upson et al., 2009), suppressing the infection of plant pathogens (Narisawa et al., 2000; 2004; Barrow, 2003) and alleviating the harmful effects of severely stressful environments (Treu et al., 1996). Subsequently, experiments conducted by Usuki and Narisawa (2007) further confirm that DSE (Heteroconium chaetospira) can form a mutualistic symbiosis with Chinese cabbage (Brassica campestris) by nutrient exchange (the associated fungi providing organic nitrogen in exchange for their host plant carbon), though some controversial and speculative aspects of
⁎ Corresponding author. Tel.: +86 871 503 4799; fax: +86 871 503 4838. E-mail address: [email protected] (Z.W. Zhao). 1 Contributed equally to this work. 0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2010.12.012
deleterious, neutral DSE-plant associations are still debated (Mandyam and Jumpponen, 2005; Peterson et al., 2008; Smith and Read, 2008). Increasing field studies reveal that DSE are ubiquitous root-associated fungi, and especially common in stressful environments such as cool, nutritionally poor, alpine or subalpine ecosystems, high saline environments and polar regions (Read and Haselwandter, 1981; Hambleton and Currah, 1997; Mandyam and Jumpponen, 2005; Newsham et al., 2009; Sonjak et al., 2009). Even in severely stressful heavy metal (HM) polluted soils (5705 mg kg−1 Pb, 58007 mg kg−1 Zn and 77 mg kg−1 Cd) of the Ancient Lead and Zinc Smelting Site of Huize, Yunnan Province, southwest China, where non-ferrous metals Pb and Zn have been smelted by ancient smelting methods for hundreds of years (Xia et al., 1980), most plants of natural revegetation are colonized by DSE (Liang et al., 2007). Recent studies show that DSE are one of the most common root-associated fungi in metal polluted soils (Likar and Regvar, 2009; Regvar et al., 2010), and these DSE have an inherent tolerance (Zhang et al., 2008) and a low sensitivity to HM (Gibson and Mitchell, 2006). While many strides have been made in understanding the ecological significance of mycorrhizal fungi, our knowledge about the functions of DSE in HM polluted environments is poor (Likar and Regvar, 2009). To understand the possible functions of this poorly understood group of root-associated fungi in HM polluted soils, on the basis of known information we hypothesized that, DSE as well as mycorrhizal fungi, could improve tolerance of host plants to heavy metals beyond nutrient acquisition and resultant positive host growth responses. To test this hypothesis, one of the most dominant and highly metal resistant DSE strains, Exophiala pisciphila H93, isolated from the roots of Arundinella bengalensis naturally growing in Ancient Lead and Zinc
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Smelting Site of Huize, China, was used for inoculum. Greenhouse pot experiments were conducted under different concentrations of HM stress (Pb, Zn, Cd) to assess the effects of inoculation with E. pisciphila H93 on (1) maize biomass, (2) the absorption, translocation and accumulation of heavy metals in shoots and roots of maize and (3) the tolerance of host plants to HM. Possible mechanisms of DSE enhancing the tolerance of their host plants to HM are discussed.
Table 1 Heavy metal (HM) concentrations of cultural substrata in the four test groups. Groups
I II III IV
HM concentrations (mg kg−1 DW) Pb(NO3)2
ZnSO4·7H2O
CdSO4·8H2O
0 1000 2000 3000
0 500 1000 1500
0 50 100 150
2. Materials and methods 2.1. Preparation of fungal colonized and non-colonized maize seedlings This experiment was performed to get uniform fungal (E. pisciphila H93) colonized and non-colonized maize (Zea mays L.) seedlings to be used for the subsequent pot experiments (2.2). Maize seeds were surface-sterilized by dipping in 75% ethanol for 5 min and then in 10% sodium hypochlorite for 10 min under agitation. Sterilized seeds were thoroughly rinsed with sterile water and then aseptically planted onto the water agar medium (agar 8 g L−1) contained in Petri dishes (90 mm) for germinating at 25 °C, and 6 seeds were used per Petri dish. After 3 days of incubation, the germinating seeds were transplanted into sterile glass bottles (Φ60 × 200 mm) containing culture substrata for fungal inoculation. Each glass bottle was filled with about 130 g culture substrata that 100 g river sand mixed with 30 mL improved MS liquid medium (containing in mg L−1: NH4NO3 1650, KNO3 1900, CaCl2·2H2O 440, MgSO4·7H2O 370, KH2PO4 170, KI 0.83, H3BO3 6.2, MnSO4·4H2O 22.3, ZnSO4·7H2O 8.6, Na2MoO4·2H2O 0.25, CuSO4·5H2O 0.025, CoCl2·6H2O 0.025, FeSO4·7H2O 27.8, Na2-EDTA·2H2O 37.3, linositol 100, VB3 0.5, VB6 0.5, VB1 0.5, Glycine 2, supplemented with 20 g L−1 sucrose). Before using, river sand was sieved through a 0.2 cm sieve and washed by tap water to remove the soil silt and nutrients, and placed in the 80 °C oven and dried to constant weight (this usually required 5–12 h) and then autoclaved for 2 h at 121 °C (three times with 2-day intervals). The germinating seeds were planted into the culture substrata contained in the bottles (2 seeds for each bottle) and each seed was inoculated with a fungal disk (Φ 0.5 cm) cut from a 14-day-old PDA culture (potato 200 g, dextrose 20 g, agar 18 g, and water 1000 mL), by attaching the fungal disk to the root of maize. For a control treatment, other germinating seeds were also inoculated with a disk (Φ 0.5 cm) cut from PDA plate without fungus and planted into the bottle as above. All the inoculation processes were operated in an SW-CJ-1DF clean bench (Airtech, China). The glass bottles were covered with sterile AeraSeal films (150 × 150 mm) (Mycomebio (Beijing) Bio-medical Science Technology Center, China) and cultivated under a day temperature of 25 °C, a night temperature of 18 °C with a photoperiod of 12 h for 4 weeks, and watered with 5 ml deionized–distilled water every 5 days per bottle. After 4 weeks’ cultivation, the maize seedlings were ready for the subsequent pot experiments (2.2).
shown in Table 1, respectively. For example, the 1000 mg kg−1 of Pb (II) substrata means 1 kg dried sand contained 1000 mg Pb (II). Therefore, to make the 1000 mg kg−1 of Pb (II) substrata, 1 kg of the dried sand was mixed with 150 ml solution that contained 1000 mg Pb (II). Similarly, to make the 500 mg kg−1 of Zn (II) substrata, 1 kg of the dried sand was mixed with 150 ml solution that contained 500 mg Zn (II). To make the non-polluted substrata (control), 1 kg of the dried sand was mixed with 150 ml of deionized-distilled water. The concentrations of HCl-extractable Pb (II), Zn (II), Cd (II) from the sand were trivial, being 4.3, 4.5 and 0.006 mg kg−1respectively, and thus were omitted in the subsequent analysis. Plastic pots (20 cm diameter × 16 cm height) each filled with 2 kg different pot culture substrata were used for plant growing. Before the transplantation of the above 4-week-old maize seedlings from the bottles to pots, the colonization intensity of E. pisciphila H93 in maize roots was evaluated. Five 0.5 cm root fragments were randomly collected from each maize seedling and cleaned in 5 mL 10% (w/v) KOH contained in a test tube for 2 h at 90 °C in a water bath and then stained with 0.5% acid fuchsin (Berch and Kendrick, 1982). Colonization intensity of E. pisciphila H93 was determined by using the magnified intersection method under a compound-light microscope (Olympus-BX51) at 200× magnification (McGonigle et al., 1990). Fungal colonization intensity, i.e. the percentage of the presence of E. pisciphila H93 in all intersections observed (more than 150 intersections were used for each sample) was determined for each sample. The fungal colonized (only those seedlings each had 93–95% root colonization intensity from inoculation of E. pisciphila H93 were used) and non-fungal colonized (without inoculation) maize seedlings were transplanted into pots filled with different pot culture substrata as described in Table 1. Three plants (one plant per pot) were used for each treatment (each group × each metal species), and thus a total of 60 pots were used (3 HM × 3 groups × 2 treatments × 3 replicates + 1 nonHM × 1 group × 2 treatments × 3 replicates). Then maize seedlings were grown in a glasshouse with a day temperature varying between 16 and 28 °C, a night temperature varying between 8 and 16 °C. Seedlings were regularly watered with deionized–distilled water and each pot received 100 mL sterile Hoagland′s nutrient weekly. 2.3. Harvest and analysis of plant HM
2.2. Greenhouse pot cultivation Pot culture experiments in a greenhouse were established to determine the effects of E. pisciphila H93 on maize (Zea mays L.) growing under different kinds and levels of HM (Pb, Zn, Cd) stress. River sand containing different kinds and concentrations of HM (Pb, Zn, Cd) were applied as pot culture substrata. According to the environmental quality standard for soils of China (GB15618-1995) and the contents of HCl-extractable Pb, Zn and Cd in the field soils where the test fungus was isolated (Liang et al., 2009), three HM polluted levels of each HM species (Pb, Zn and Cd) and one nonpolluted level (control) were conducted and thus formed four test treatments in total (Table 1). River sand was cleaned and dried as above before using. To make the four groups of pot culture substrata, 1 kg of the dried river sand was mixed with 150 ml different concentrations of HM salt solutions to achieve the concentrations
After 3 months of growth, the leaves of maize at the time of male flowering were turning brownish yellow and all maize was harvested. Fungal colonization intensity was evaluated at this stage for each plant as described above. Shoots (leaves+ stems) and roots were collected, respectively, and roots were thoroughly washed by tap water to remove the soil silt. The shoots and roots then were placed in the 80 °C oven and dried to constant weight and weighed. All dried root and shoot samples were ground separately by a mini-vegetation disintegrator (FZ102, Tianjing City Test Instrument Co. Ltd, China), and 0.5 g shoot and 0.5 g root powder of each sample were then subsampled by the usual method of coning and quartering. These representative subsamples were digested by HNO3 + HClO4 respectively and the concentrations of HM ions were determined by the flame atomic absorption spectrometry (FAAS) using a Z2000 polarized Zeeman atomic absorption spectrophotometer (Hitachi, Japan). Quantification was carried out with a
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calibration curve using a graded series of diluted lead (GSW08619) (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg ml−1), zinc (GSW08620) and cadmium (GSW08612) (0, 0.1, 0.2, 0.4, 0.8, 1.2 μg ml− 1) (National Research Center for Certified Reference Materials, China) given in parentheses. A standard reference material of poplar leaves (GBW 07604, National Research Center for Certified Reference Materials, China) was carried through the digestion and analyzed as a part of the quality control protocol (accuracies within 100 ± 20%). The hollow cathode lamp was operated at 4 mA for Cd and 5 mA for Pb and Zn, and the analytical wavelengths were set at 217.0, 213.9, 228.8 nm for detection of Pb (II), Zn (II) and Cd (II), respectively (AOAC, 1984). 2.4. Statistical analysis The differences of plant biomass and HM concentrations in roots and shoots between inoculated and non-inoculated plants under deferent substrata were determined using two-way (HM concentration in substrata is one factor, inoculation or not is the other factor) analysis of variance (ANOVA) followed by Duncan's multiple-range test for multiple comparisons. Pearson's correlation coefficient (r) was used to determine the relationships between soils, plant HM concentrations and colonization intensity. Rejection level was set at α b 0.05 in all analyses. All analyses were made using SPSS software (version 16.0). Unless stated otherwise all values reported are means ± SD. 3. Results No DSE structures were observed in the roots of non-inoculated maize in pot experiments after harvesting. In inoculated treatments, typical structures of DSE-plant associations were observed under all test concentrations of the three HM and abundant intra-radical and extraradical hyphae and microsclerotia colonized the roots of maize similar to
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their colonization in nature (Fig. 1). The lowest colonization intensity of E. pisciphila H93 occurred in non-HM stressed soils (17.58 ± 10.30%), and this value increased with increasing HM concentrations and peaked at 78.90 ± 6.11% in the roots of maize grown in the substrata supplemented with 1500 mg kg−1 ZnSO4·7H2O (Table 2). Colonization intensity was significantly positively correlated to the HM concentrations of cultural substrata (Pb, r = 0.999, p b 0.01; Zn, r = 0.964 p b 0.05; Cd, r = 0.955 p b 0.05). Biomass of maize was significantly decreased with the increasing HM supplements both in inoculated and non-inoculated treatments (Fig. 2). Inoculation of E. pisciphila H93 significantly decreased maize biomass (roots and shoots) compared with the non-inoculated maize under no HM stress. However, under higher HM stress conditions, inoculation of E. pisciphila H93 alleviated the negative effects of increasing toxicity of heavy metals, which was shown by the significantly higher maize biomass (roots and shoots) in fungal inoculated maize than those of non-inoculated maize. The root and shoot HM concentrations of inoculated and noninoculated maize growing in all test substrata are summarized in Table 3. With increasing HM concentrations, HM were also absorbed and accumulated increasingly by roots and shoots of both inoculated and non-inoculated treatments. However, the majority of HM contents were accumulated in maize roots both in inoculated and non-inoculated treatments, and thus ratio of shoot to root HM concentrations (RS/R) remained far below 1.0 in almost all treatments except the essential micronutrient Zn under the trace level (Table 3). More importantly, significantly lower shoot HM as well as significantly higher root HM of inoculated treatments were accumulated than those of non-inoculated maize, though the significant effects of inoculation with E. pisciphila H93 on plant growth varied greatly due to the different HM and their concentrations (Table 3). Root Zn and Cd concentrations were significantly correlated to the colonization intensity of E. pisciphila H93 (Zn,
Fig. 1. Morphology of Exophiala pisciphila H93 colonized in host plant roots. (a) Abundant intra-radical and extra-radical hyphae of H93 colonized maize seedling roots before transplant to pot experiments. (b) Intracellular running hyphae of H93 in maize roots grown in the pot supplemented with 100 mg kg−1 CdSO4·8H2O. (c) Hyphae and melanized microsclerotia of H93 colonized the roots of Arundinella bengalensis in field at Ancient Lead and Zinc Smelting Site of Huize, Yunnan Province, southwest China. (d) Melanized microsclerotia of H93 in maize roots grown in the pot supplemented with 1500 mg kg−1 ZnSO4·7H2O. Bars = 50 μm (a) and 10 μm (b–d).
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Table 2 Colonization intensity of E. pisciphila H93 in the inoculated treatments of four test groups (no DSE structures were observed in the roots of non-inoculated maize, and thus not provided here) (mean ± SD, n = 3). Groups
I II III IV
Colonization intensity of H93 (%) Pb
Zn
Cd
17.58 ± 17.84 33.50 ± 19.45 54.05 ± 32.96 69.61 ± 16.63
17.58 ± 17.84 23.49 ± 11.97 63.11 ± 27.15 78.90 ± 10.58
17.58 ± 17.84 20.56 ± 17.34 42.89 ± 47.39 70.00 ± 12.73
r=0.978 pb 0.05; Cd, r=0.994 pb 0.01), except Pb (r=0.902 pN 0.05). The shoot Pb concentrations were significantly correlated with the colonization intensity of E. pisciphila H93 (r=0.986 pb 0.05), but Zn (r=0.877 pN 0.05) and Cd (r=0.946 pN 0.05). 4. Discussion Colonization intensity of maize roots by E. pisciphila H93 obviously increased with increasing HM toxicity, which indicated that E. pisciphila H93 isolated from metal polluted soil had a high inherent tolerance or a low sensitivity to HM (Gibson and Mitchell, 2006; Zhang et al., 2008). Field surveys showed that 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) with increasing pollutants. Our findings agree with Audet and Charest's
Fig. 2. Shoot and root biomass (DW, g) of inoculated (H93) and non-inoculated maize in the four test groups (group I for control without heavy metal supplements, II, III and IV for groups with different concentrations of Pb, Zn and Cd as described in Table 1). Different letters refer to significant differences at the 0.05 level according to Duncan's multiple-range test.
(2006) observation that colonization of mycorrhizal fungus (Glomus intraradices) on the roots of “wild” tobacco (Nicotiana rustica) markedly increased with increasing soil Zn and the mycorrhizal structures were significantly more abundant at the higher soil Zn (250 mg kg−1). Janoušková and Vosátka (2005) also reported that the colonization of carrot roots by G. intraradices increased when the more Cd was applied in the medium. Presumably, the increasing colonization of E. pisciphila H93 might be a mutual response of endophyte–plant associations to the increasing HM toxicity, which improved the fitness of both plant and fungal symbionts, and thus it may be an efficient strategy to survive in stressfully HM contaminants for both DSE and their hosts. It is now well recognized that the change in plant growth resulting from their associated mycorrhizal symbionts varies widely, which can span a wide range of species interactions from mutualism to parasitism under different environmental conditions (Johnson et al., 1997; Smith et al., 2009). Responses of host plants to DSE colonization like mycorrhizal associations varied greatly with fungus and host plant specificity and cultural conditions, and even intra-individual variation in host plant response to E. pisciphila H93 occurred in this study. Consequently, beneficial, amensal, neutral or parasitic associations have been often debated (Wilcox and Wang, 1987; Fernando and Currah, 1996). Clearly, because of the complexity of the DSE–soil–plant system, more data from other repeated experiments within different DSE-plant combination are needed to confirm DSE–plant association. Here we suppose that the potential trade-offs in carbon economy and nutrients between host plants and their associated fungi may influence their association relationship. In this study, low nutrient sand substrata greatly restricted DSE potential nutrient benefits to its host in exchange for their carbon, and the root-associated fungi can even be considered to be parasitic on plants when the net cost of the symbiosis exceeds the net benefits. Therefore, photosynthesis products of maize repartitioning to E. pisciphila H93 resulted in the significantly lower maize biomass under no HM stress. However, under HM stress, the higher biomass of inoculated maize than non-inoculated maize suggested that their association relationship has changed from parasitism to mutualism (Fig. 2). Because of the complexity of fungi–plant associations, an understanding of the several parameters affecting endophyte functioning, such as the morphology and physiology of both symbionts, and biotic and abiotic factors at the rhizosphere, community and ecosystem levels, is required to construct predictive models of fungal functioning (Johnson et al., 1997). Like other root-associated fungi including arbuscular and ectomycorrhizal fungi (Turnau et al., 1996; Liang et al., 2009), inoculation of E. pisciphila H93 influenced the HM uptake, translocation and accumulation in its host plants (Table 3). Lower RS/R of inoculated maize than those of non-inoculated maize showed that E. pisciphila H93 colonization restricted the translocation of HM from roots to shoots, though more HM ions were absorbed and accumulated in the roots of inoculated maize. Different from the higher root accumulation of DSE inoculation, mycorrhizal inoculation (Glomus sp and G. mosseae) significantly reduced the uptake and accumulation of HM in host plant roots (Liang et al., 2009). By using micro-protoninduced X-ray emission (PIXE), a clear biofiltering pattern in the ectomycorrhiza between Suillus luteus and Pinus sylvestris was studied, and strong accumulation of Ca, Fe, Zn and Pb within the fungal mantle and in the rhizomorph was observed (Turnau et al., 2001). Galli et al. (1994) summarized that the retention of HM by fungal mycelia may involve adsorption to cell walls, and even E. pisciphila H93 accumulated and adsorbed over 20% lead and 5% cadmium of their biomass (DW) (Zhang et al., 2008), thereby minimizing metal translocation to the shoots (Galli et al., 1994). Furthermore, more complex molecular mechanisms may be involved in the improved tolerance against metal toxicity of endophytes to their host plants. Experiments conducted by Cicatelli et al. (2010) showed that inoculation with mycorrhizal fungi (G. mosseae or G. intraradices) restored normal growth of a white poplar clone grown on HM-contaminated soil and enhanced the stress tolerance of
11.31** 549.15*** 62.38*** 5.75* 91.62*** 2.78NS 0.36NS 89.32*** 0.33NS 15.21** 1544.05*** 1.04NS 6.06* 721.97*** 4.53* 3.48NS 189.96*** 1.83NS 7.72** 80.04*** 1.21NS 36.29*** 72.94*** 10.67***
Values followed by different letters within a column refer to significant difference at the 0.05 level according to Duncan's multiple-range test. ***, pb 0.001; **, pb 0.01; *, pb 0.05; NS: not significant.
IV
III
45.24*** 183.08*** 37.69***
4.55 ± 0.80d 4.27 ± 1.51d 56.80 ± 6.27d 49.84 ± 6.67d 177.88 ± 58.20c 173.01 ± 49.14c 757.63 ± 76.59a 651.85 ± 94.24b II
F-value and level of significance H93 HM H93 × HM
0.33 ± 0.02b 0.50 ± 0.05a 0.22 ± 0.00c 0.20 ± 0.02c 0.11 ± 0.02d 0.08 ± 0.00d 0.20 ± 0.00c 0.09 ± 0.02d
RS/R Shoot
0.01 ± 0.00d 0.01 ± 0.00d 5.43 ± 0.73c 5.10 ± 0.95cd 8.03 ± 1.78c 6.56 ± 2.15c 20.32 ± 7.27a 14.76 ± 4.76b 0.03 ± 0.02d 0.02 ± 0.02d 24.8 ± 5.91d 25.17 ± 4.64d 75.55 ± 17.42c 87.43 ± 12.35c 103.53 ± 40.15bc 156.25 ± 33.77a
Root Shoot
4.33 ± 0.55e 4.26 ± 0.26e 463.25 ± 61.00d 483.74 ± 30.97d 1236.73 ± 582.85c 1594.75 ± 207.39bc 1813.25 ± 340.20a 1889.00 ± 200.01a 0.51 ± 0.17a 0.43 ± 0.16a 0.16 ± 0.03b 0.09 ± 0.00b 0.14 ± 0.02b 0.06 ± 0.00b 0.12 ± 0.02b 0.03 ± 0.02b 10.33 ± 1.94e 9.28 ± 1.51e 28.79 ± 3.79de 23.45 ± 5.44de 161.12 ± 72.93ab 69.74 ± 32.27cd 207.78 ± 31.96a 106.19 ± 49.24bc
Shoot Root
C H93 C H93 C H93 C H93 I
20.32 ± 1.37e 21.62 ± 2.06e 183.30 ± 21.67d 255.23 ± 32.73d 1136.22 ± 375.17c 1238.75 ± 297.09c 1691.25 ± 220.15bc 4147.50 ± 1048.41a
RS/R
Root
RS/R
1.05 ± 0.08a 1.00 ± 0.07a 0.12 ± 0.02c 0.10 ± 0.00c 0.14 ± 0.03c 0.11 ± 0.02c 0.42 ± 0.05b 0.35 ± 0.02b
Cd (mg kg−1 plant DW) Zn (mg kg− 1 plant DW) Pb (mg kg−1 plant DW) Treatments Groups
Table 3 Root and shoot heavy metal (HM) concentrations (mg kg− 1 plant DW) and ratio of shoot/root HM concentrations (RS/R) of maize with inoculated (H93) and non-inoculated (C) treatments of all four test groups (mean ± SD, n = 3).
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host plants to accumulate higher Cu and Zn in plant organs, especially roots. Molecular mechanism analysis showed that the improved tolerance of poplar against HM was due to an overall induction of the poplar transcriptional upregulation of several stress-related genes and the protective role of polyamines. Moreover, the differential transcription expression patterns induced by mycorrhizal fungi occurred between leaves and root, and even among different organ cells of roots of mycorrhizal (Gl. intraradices) and non-mycorrhizal tomato (Ouziad et al., 2005). To the best of our knowledge, this is the first report that DSE (E. pisciphila H93) alleviated the deleterious effects of excessive HM ions and promoted the growth of host plants (roots and shoots), and improved the tolerance of maize by restricting the translocation of toxic HM from the roots to shoots under HM stressed soils. This study demonstrated that the facultative associations between E. pisciphila and its host (maize) had transformed from parasitism in the low nutrient substrata without HM pollutants to mutualism when net benefits exceed net cost under higher HM stress. Such a mutual symbiosis within these species may be an efficient strategy to survive in the stressful environments. Acknowledgements We are grateful to Associate Editor Dr. J. Bennett and three anonymous referees for their constructive comments to improve our manuscript. Many thanks to our colleagues Zhang Jie, Kang Yu, and Yan Jun for carrying out partial experiments. We would like to also express our appreciation to Mr. Hu Bin and Cheng Lizhong (Biology Department of Yunnan University) for FAAS analysis and to Dr. Jing Yuebo (English editor of Journal of West China Forestry Science) and Dr. Xu Jin (Massey University, New Zealand) for their help to improve English writing. This research was financially supported by the National Natural Science Foundation of China (30770052, 40763003) and Yunnan Province (2007C007M). References AOAC. Official methods of analysis of the Association of Official Analytical Chemists14th ed. ; 1984. p. 344. Washington, DC. Audet P, Charest C. Effects of AM colonization on “wild tobacco” plants grown in zinccontaminated soil. Mycorrhiza 2006;16:277–83. Barrow JR. Atypical morphology of dark septate fungal root endophytes of Bouteloua in arid southwestern USA rangelands. Mycorrhiza 2003;13:239–47. Berch SM, Kendrick B. Vesicular-arbuscular mycorrhizae of southern Ontario ferns and fern-allies. Mycologia 1982;74:769–76. Cicatelli A, Lingua G, Todeschini V, Biondi S, Torrigiani P, Castiglione S. Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression. Ann Bot 2010;106: 791–802. Fernando AA, Currah RS. A comparative study of the effects of the root endophytes Leptodontidium orchidicola and Phialocephala fortinii (Fungi Imperfecti) on the growth of some subalpine plants in culture. Can J Bot 1996;74:1071–8. Galli U, Schüepp H, Brunold C. Heavy metal binding by mycorrhizal fungi. Physiol Plant 1994;92:364–8. Gibson BR, Mitchell DT. Sensitivity of ericoid mycorrhizal fungi and mycorrhizal Calluna vulgaris to copper mine spoil from Avoca, county Wicklow. Biol Environ Proc Roy Ir Acad 2006;106B:9-18. Hambleton S, Currah RS. Fungal endophytes from the roots of alpine and boreal Ericaceae. Can J Bot 1997;75:1570–81. Haselwandter K, Read DJ. The significance of root-fungus association in two Carex species of high-alpine plant communities. Oecologia 1982;53:352–4. Janoušková M, Vosátka M. Response to cadmium of Daucus carota hairy roots dual cultures with Glomus intraradices or Gigaspora margarita. Mycorrhiza 2005;15: 217–24. Johnson NC, Graham JH, Smith FA. Functioning of mycorrhizal associations along the mutualism–parasitism continuum. New Phytol 1997;135:575–85. Jumpponen A, Trappe JM. Dark septate endophytes: a review of facultative biotrophic root colonizing fungi. New Phytol 1998;140:295–310. Liang CC, Li T, Xiao YP, Liu MJ, Zhang HB, Zhao ZW. Effects of inoculation with arbuscular mycorrhizal fungi on maize grown in multimetal contaminated soils. Int J Phytorem 2009;11:692–703. Liang CC, Xiao YP, Zhao ZW. Arbuscular mycorrhiza and dark septate endophytes in an abandoned lead-zinc mine in Huize, Yunnan, China. Chin J Appl Environ Biol 2007;13:811–7.
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