The response of dark septate endophytes (DSE) to heavy metals in pure culture

Abstract

Dark septate endophytes (DSE) occur widely in association with plants exposed to heavy metal stress. However, little is known about the response of DSE exposed to heavy metals. In this study, five DSE were isolated from the roots of Astragalus adsurgens Pall. seedlings growing on lead-zinc mine tailings in China. Based on morphological characteristics and DNA sequence analyses, the isolates were identified as Gaeumannomyces cylindrosporus, Paraphoma chrysanthemicola, Phialophora mustea, Exophiala salmonis, and Cladosporium cladosporioides. G. cylindrosporus was selected to explore responses to Pb stress. Scanning electron microscopic observations of G. cylindrosporus grown on solid medium revealed curling of hyphae and formation of hyphal coils in response to Pb. In contrast, in liquid medium, hyphae became thick and swollen with an increase in Pb (II) concentration. We interpret that these changes are related to the variation in cell wall components. We also demonstrated that fungal melanin content increased with the addition of Pb(II). Melanin, as an important component in the cell wall, is known to be an essential antioxidant responsible for decreasing heavy metal toxicity. We also measured the total soluble protein content and glutathione (GSH) concentrations in G. cylindrosporus and found that they initially increased and then decreased with the increase of Pb(II) concentrations. The antioxidant enzyme activities were also examined, and the results showed that superoxide dismutase (SOD) activity was significantly positively correlated with Pb(II) concentrations (r = 0.957, P<0.001). Collectively, our observations indicate that the intracellular antioxidant systems, especially fungal melanin, play an important role in abating the hazards of heavy metals.

Figure 1. Two-week-old colonies of the five isolates on PDA.

Two-week-old colonies of the five isolates on PDA. A, Gaeumannomyces cylindrosporus (B145); B, Paraphoma chrysanthemicola (B100); C, Phialophora mustea (BC42); D, Exophiala salmonis (BC5); E, Cladosporium cladosporioides (B142).

Figure 2. Neighbor-joining phylogenetic trees showing the placement of B145 and BC42 (A), B100(B), BC5(C), and B142(D) based on ITS1-5.8S-ITS2 sequences.

The Kimura two-parameter model was used for pairwise distance measurement. Numbers on branches were values generated from 1000 bootstrap replicates. Bootstrap values of 50% were shown above branch nodes. • denoted the isolates.

Figure 3. Typical structures of DSE fungi colonized in the roots of A. adsurgens seedlings.

A, Structure of microsclerotia formed by P. chrysanthemicola; B, Intercellular melanized septate hyphae formed by G. cylindrosporus.

Figure 4. The biomass of the five DSE fungi treated with different concentrations of Pb(II).

The biomass of the five DSE fungi grown on solid MMN medium amended with different concentrations of Pb(II), Zn (II), and Cu(II) for two weeks. Data are means ± SE (n = 4).

Figure 5. Colony morphology of G. cylindrosporus exposed to different concentrations of Pb(II).

Colonies of G. cylindrosporus on MMN medium with different concentrations of Pb(II) for 2 weeks. A, Colony of G. cylindrosporus with no Pb(II) stress; B, Colony of G. cylindrosporus on MMN medium supplemented with 0.2 mg/ml of Pb(II); C, Colony of G. cylindrosporus on MMN medium supplemented with 0.6 mg/ml of Pb(II); D, Colony morphology of G. cylindrosporus on MMN medium supplemented with 1.0 mg/ml of Pb(II).

Figure 6. Hyphal morphology of G. cylindrosporus cultured on solid medium.

Scanning electron micrographs of the hyphae from the colony edge of G. cylindrosporus. Twisting and looping of individual hyphae and formation of intertwined hyphal strands occurred when Pb(II) was added in the medium. However, there was no obvious relationship between the number of hyphal coils or the extent of hyphal twisting and the concentrations of Pb(II). A, Hyphae from the untreated control; B, Hyphae from a colony treated with 0.2 mg/ml Pb(II); C–F, Mycelial special morphology of G. cylindrosporus under Pb(II) stress.

Figure 7. Hyphal morphology of G. cylindrosporus cultured in liquid medium.

Scanning electron micrographs of the hyphae of G. cylindrosporus cultivated for a week in liquid medium supplemented with different concentrations of Pb(II). A–D, Mycelial morphology of G. cylindrosporus treated with 0, 0.1, 0.3, and 0.5 mg/ml Pb(II), respectively.

Figure 8. Melanin content in G. cylindrosporus under different Pb(II) concentrations stress.

The influence of Pb(II) on melanin content in G. cylindrosporus under liquid culture condition. DW represented dry weight. Different letters above bars indicate significant differences (P<0.05) assessed by Duncan's test. Data are means ± SE (n = 4).

Figure 9. The responses of antioxidant substances to the toxicity of Pb(II).

The contents of total soluble protein (A) and GSH (B) and the activities of SOD (C) and CAT (D) in the hyphae of G. cylindrosporus cultivated for a week in liquid MMN medium supplemented with a series of increasing Pb(II). FW represented fresh weight. Different letters above bars indicate significant differences (P<0.05) assessed by Duncan's test. Data are means ± SE (n = 4).