| Native plants spontaneously growing on a multiple metals-mine tailings have been attracting a numerous attention in recent years,particularly owing to the diverse advantages e.g.the great adaptability to the extremely metal-polluted mine tailing soils.Especially,the native plant species with fast-growing,big biomass and bio-economy product,e.g.Populus spp.,Salix spp.,Morus alba,and Sophora japonica etc.show great potentials for the bio-economy product in the extremely metal-contaminated mine soils.In a previous field-survey,we found that two woody plant species i.e.Populus yunnanensis and Coriaria nepalensis,form a dominant plant community,and show the great adaptability to the abandoned heavy metal mine tailing soils without the obvious symptoms of chronic heavy metal poisoning,in Gejiu city,Yunnan province,southwestern China.While the distribution and function of microorganisms including arbuscular mycorrhizal fungus(AMF)and dark septate endophytes(DSE)in the roots of the two woody plants in the mining area have not been elucidated.In this paper,the colonization of AMF and DSE in the roots of P.yunnanensis and C.nepalensis,was assessed.The traditional technology of microbial isolation and culture was used to study the diversity of DSE.High-throughput sequencing technology was used to explore the diversity and community composition of endophytes colonizing the two woody plants.In the greenhouse,the representative DSE strains from P.yunnanensis were used to study the potential functions of DSE in the improved tolerance of P.yunnanensis to heavy metal stress.The following results and conclusions were achieved:(1)Under the compound light microscope,the diverse structures of AMF and DSE,including the abundant septate and melanized hyphae and microsclerotia of DSE,and diverse arbuscules and vesicles of AMF,were observed in the roots of P.yunnanensis and C.nepalensis,especially the dominant running hyphae.We noted that there was a higher colonization intensity of both AMF and DSE in P.yunnanensis with 66.7±1.6% and 73.5±3.0% than those in C.nepalensis with 54.7±0.8% and 37.4±0.8%,respectively(p < 0.05).In addition,a distinct trend in the colonization of AMF and DSE in the roots was observed,and there was a higher root colonization of DSE than that of AMF in P.yunnanensis,and conversely a lower value of DSE than AMF occurred in C.nepalensis.Our results suggest that both AMF and DSE were one of the integral of roots of both woody plant species,while there was a distinct characteristic of fungal colonization by AMF and DSE.(2)A total of 192 DSE strains were isolated from the roots of P.yunnanensis and C.nepalensis,of which 104(accounting for 54.17%)were from P.yunnanensis and 88(accounting for 45.83%)from C.nepalensis,respectively.Phylogenetic analyses of ITS1-5.8S-ITS2 genes showed that all 192 DSE strains belonged to 15 phylotypes,including the 9 shared phylotypes,and 4 P.yunnanensis– specific and 2 C.nepalensi – specific phylotypes.Among the 15 phylotypes,there were 8 and 4 dominant phylotypes with the proportions of over 5% for P.yunnanensis and C.nepalensi,respectively.Among them,4 dominant phylotypes i.e.Phialophora,Cladosprium,Cadophora and Paraphoma overlapped in the two woody plant species.As for P.yunnanensis,the remaining 4 dominant phylotypes were Cyphellophora,Herpotrichiellaceae,Sordariales and Pyrenochaeta.The results suggest that there was a different fungal assemblage colonizing the roots of two plants,and there was a higher diversity of cultivable DSE in P.yunnanensis than C.nepalensis in the abandoned Huangmaoshan mine tailings,in Gejiu city,Yunnan province,southwestern China.(3)Based on high throughput second-generation sequencing technology,the communities of root-associated endophytic fungi were comparatively evaluated.And found that there were diverse root-associated endophytic fungi colonizing the two plants,and the richness indeces and diversity indeces of the fungi colonizing the roots of P.yunnanensis were higher than those of C.nepalensis,which indicated that the fungi in the roots of P.yunnanensis had higher richness and diversity than C.nepalensis.The PCoA analyses of unweighted UniFrac distance algorithm showed that there was distinct assembly of fungi in the roots of both plant species,and the distribution of dominant fungi in the roots of the two dominant plants is also quite different.We noted that there was a total of 18 taxa with a proportion of more than 1% in C.nepalensis,which is higher the 12 taxa in P.yunnanensis.While among the dominant group of fungi with a proportion of more than 5%,there were 8 in the roots of P.yunnanensis,5 belong to Ascomycetes and 2 belong to Basidiomycetes,while only 3 in the C.nepalensis,all belonging to Ascomycetes.In addition,the results of Spearman correlation between 35 dominant fungal species in the roots of two dominant plants and their environmental factors revealed that,compared with the concentrations of heavy metals,soil nutrients showed more significant effects on the fungal assemblages colonizing the roots of two plant species,among which the most important environmental factors are total potassium,followed by total nitrogen,soluble nitrogen and soluble phosphorus.(4)Data from the experiments of inoculation with 4 representative DSE strains and the non-inoculated controls,showed that the inoculation with all 4 DSE strains promoted the growth of P.yunnanensis,especially the significantly increased biomass by the inoculation of both 3871 and 39112 strains,compared to CK groups,which indicated that DSE strains could alleviated the toxic metal stress caused by mine tailing soils,and enhanced the tolerance of heavy metals of P.yunnanensis.Meantime,we found that the inoculation of DSE reduced the concentrations of DTPA-extracted metal ions e.g.Zn and Cd in the rhizosphere soils,which maybe result from the bioaugmented chelation of free ions by the combined effects of plant roots and their associated DSE inoculants.Thus,it was reasonable that DSE inoculation restricted the uptake of metal ions by plant roots,and restricted the Zn and Cd accumulation in plant roots,twigs and leaves,which resulted in the alleviation of plants by metal stress and promoted the plant growth.We also noted that the effects of fungal inoculation were determinant on both the fungal strains and metal species,and among the 4 fungal strains used,the maximum effects of Phialophora sp.39112 were observed.Our results revealed that both AMF and DSE were an integral of two woody plant roots,which dominantly colonized the roots of plants in the abandoned tailing area of Huangmaoshan,Gejiu,Yunnan,Southwest China.In total,192 DSE strains were isolated and collected,and the member belonging to 15 fungal taxa including Phialophora,Cladosporium,Cadophora were identified by the phylogenetic analyses of ITS1-5.8S-ITS2 genes.Evidence from both culture-specific and culture-independent results showed that there was a distinct assemblage of fungi colonizing the roots of two woody plant species,and the soil nutritional parameters e.g.total K,total N,soluble N and soluble P,were the determinant factors,rather than the metal concentrations.The results from the greenhouse experiments revealed that the diverse DSE strains play an important functional role in the adaptability of P.yunnanensis in the extremely stressed mine tailing soils in Yunnan,China.While,the mechanisms need to be explored. |
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