Diversity And Functions Of Dark Septate Endophytes (DSE) Colonizing The Aquatic And Terrestrial Arundo Donax In Munai Pb/Zn Slag Heap, Yunnan, SW China

Unreclaimed mine tailings sites are a worldwide problem, with a high content of heavy metals, low level of macronutrients and acidity in abandoned substrates, which are the major constraints to restoration. More and more prevailing evidence reveals that inoculation with soil functional microbiota significantly enhances their host plants’ tolerance against heavy metal stress, and contributes to ecological rehabilitation on vegetation recruitment. Especially, it is true for those pioneer plant species in unreclaimed and stressful mine tailing sites. In the previous field surveys, we found that Giant reed (Arundo donax), one of fast-growing energy plants, adapts to survive on the heavily metal-polluted soils in Munai Pb/Zn slag heap, Yunnan, China. However, diversity and functions of dark septate endophytes (DSE) colonizing the roots of Giant reed have not yet been elucidated. In the current study, colonization, diversity and functions of DSE in the aquatic and terrestrial Arundo donax in Munai Pb/Zn slag heap, Yunnan, SW China were assessed, and made the key result points and main conclusions as the following:1. In total, physicochemical properties of rhizosphere soils and DSE colonization characteristics in roots of 35 terrestrial and 20 aquatic root samples of giant reed were evaluated using a modified acid fuchsin dyeing method in Munai Pb/Zn slag heap, Yunnan, China. It was found that terrestrial habitats and aquatic habitats were severely contaminated with excessive Pb, Zn and Cd contents, and Cd, Pb and Zn content were 26.78±7.83 mg/kg,9749.70±3469.44 mg/kg,1609.97±131.32 mg/kg, respectively. Total Cd, Pb and Zn contents in aquatic habitats also reached 23.22±4.01 mg/kg, 4019.40±447.89 mg/kg,1693.74±43.18 mg/kg, respectively. We found that both the aquatic and terrestrial Arundo donax roots were colonized by DSE, with colonization intensity of 4.37±1.76% and 14.08 ± 2.45%, respectively. Statistical analysis showed that DSE colonization intensity in terrestrial habitats was significantly higher than that of aquatic habitat (p= 0.003<0.05). Our results showed that DSE is an integral of roots of both quatic and terrestrial Arundo donax roots in Munai Pb/Zn slag heap, China.2. In total,120 DSE strains were isolated from the roots of both Giant reed ecotypes. Cadophora sp. was the most dominant with a proportion of 28.33%, then followed by Ophiosphaerella korrae (19.17%), Multiseptospora thailandica (8.33%), Pleosporales sp. (7.5%), Cladosporium tenuissimum (6.67%), and the other members were low. In total,120 DSEs were isolated from the roots of both terrestrial (108 strains) and aquatic Arundo donax (12 strains). The 12 aquatic DSE consisted of seven cluster groups, mainly including Lophiostoma sp. and Saccharicola sp. There were 30 cluster groups in 108 terrestrial DSE, dominantly by Cadophora sp. and Ophiosphaerella korrae. While, there were only three cluster groups in both two Giant reed ecotypes.3. Diversity of the root-associated endophytic fungi colonizing the aquatic and terrestrial Arundo donax ecotypes were assessed useing Illumina Miseq sequencing. And diverse endophytic fungi colonized the roots of both aquatic and terrestrial Arundo donax ecotypes. In total,772 OTUs were identified, including 628 aquatic OTUs and 481 terrrestrial OTUs. Our results showed that there were 337 shared OTUs in both two habitats, accounting for 43.7%, suggesting highly similar fungal community structures. The roots of Arundo donax dominantly colonized by the members of Ascomycota, such as Ascomycota sp., Xylaria sp. B101, Boeremia exgua var. populi, Paraphaeosphaeria pilleata, Harpophora oryzae, Mortierella sp., Stachybotrys sp., Exophiala sp., Sarocladium, Phoma calidophila. Statistical analysis showed that there was no significant difference between the assembles of roots associated fungi in both two Arundo donax ecotypes.4. In greenhouse, effects of DSE inoculation on the growth of Arundo donax under different Cd concentrations (0,5 and 50 mg/kg Cd2+). Comparing with the non-inoculated controls, all 6 DSE strains alleviated the deleterious effects of Cd stress and significantly promoted the giant reed growth (p<0.05), excluding the inhibited growth by P0824 inoculation under 5 mg/kg Cd2+stress. Interestingly, low concentrations of Cd stress (5 mg/kg Cd2+) promoted the growth of Arundo donax, regardless of DSE inoculation, then followed by non-Cd stress, and the minimum of biomass occurred under 50 mg/kg Cd stress. Statistical analysis showed that, in the absence of cadmium and high concentrations of Cd stress, there was no significant difference among 6 fungal inoculations, except significant promotion caused by P1424 colonization than the other 5 fungal strains, though all 6 fungal inoculations significantly higher than the corresponding non-inoculatedcontrols. But, at the low concentration of cadmium stress (5 mg/kg Cd2+), distinct promotion of all 6 fungal strains on the growth of Giant reed occurred, and maximum biomass promotion by both P1233 and P3134, was significantly higher than the other 4 fungal strains and non-inoculated controls, followed by P2127 and M1321, and by P1424. However, P0824 inoculation inhibited the growth oft Arundo donax compared to non-inoculated controls.In summary, diverse and abundant endophytic fungi dominantly colonized the roots of both aquatic and terrestrial Giant reed ecotypes in Munai Pb/Zn slag heap, Yunnan, China. Our results showed that ubiquitous DSE were integral to the functions of metal mine ecosystems, and more in-depth studies on the interaction between DSE and giant reed will ultimately lead to their potential applications in phytomanagement in mining area.