Mechanisms Of Dark Septate Endophyte Isolated From Pb-zn Mine Improving Plant Lead Tolerance

In this study, we investigated the distributions of arbuscular mycorrhizal fungi （AMF）and dark septate endophytes （DSE） in different degree Pb-Zn polluted areas at QiandongshanPb-Zn mine, located in Feng county, Shaanxi province, and isolated several DSE strains fromthe roots of Astragalus adsurgens according to the DSE colonization. Based on morphologicalcharacteristics and molecular identification, the resynthesis experiment and the screening andselection of heavy metal-resistant DSE fungi, we finally chose the most resistantGaeumannomyces cylindrosporus as the object for further study. We studied the responses ofG. cylindrosporus to Pb stress, the effects of pH, temperature, Pb concentration and culturetime on the biosorption characteristics of Pb. In addition, we researched the effects ofinoculation with G. cylindrosporus on the growth characteristics, photosynthetic properties,and the uptake and translocation of Pb in maize seedings under Pb stress. The main results asfollows:1. Colonization characteristics of AMF and DSE in Pb-Zn polluted areasThe resources and distribution of AMF and DSE in four sampling areas in Pb-Zn mine,Feng county, Shaanxi province, were investigated.55root samples of36different plantsspecies in20families were analyzed, and the results showed that49samples （about89.1%ofthe total） were typically colonized by AMF, and Arum-type was more dominant than othertypes. No correlations were observed between the spore denstity and the AMF colonizationrate （P>0.05）.46root samples （about83.6%of the total） were found colonized by DSE.40samples were simultaneously colonized by AMF and DSE and accounted for72.7%of thetotal samples. DSE colonization rate was significantly positively correlated with the total Zncontent of soil （P<0.05）. These results indicated that AMF and DSE commonly occur inQiandongshan Pb-Zn mine, especially DSE has excellent heavy metal tolerance andadaptability.2. The relationships of AMF and DSE with soil chemistry propertiesWe explored the relationships of root colonization by AMF and DSE and GRSP contentin rhizosphere soils of six dominant plant species, including A. adsurgens, Artemisiabrachyloba, Potentilla anserina, Phragmites australis, Macleaya cordata, andGueldenstaedtia stenophylla, grown on Qiandongshan Pb-Zn mine tailings with soilchemistry properties, such as Pb and Zn content in soil, pH, available N, available P, available K, and organic matter. The results showed that AMF colonization was negatively correlatedwith organic matter content and the translocation of Zn in plant, and had extreme significantnegative correlation with available P content. DSE colonization was negatively correlatedwith the translocation of Pb in plant. Redundancy analysis revealed a significant effect of Pbcontent in soil on AMF community structure in soil samples, and canonical correspondenceanalysis of DGGE profiles showed that soil organic matter had significant relationship withAMF community structure in the roots.3. Identification and screening of heavy metal-resistant DSE fungiFive DSE isolates have been obtained from the roots of A. adsurgens, and all of themproduced melanized septate hyphae and microsclerotia in host plant roots after resynthesis.Based on morphological characteristics and DNA sequence analyses, the isolates wereidentified as G. cylindrosporus, Paraphoma chrysanthemicola, Phialophora mustea,Exophiala salmonis, and Cladosporium cladosporioides, respectively. The minimuminhibitory concentration （MIC） and the50%effective concentration （EC50） indicated that G.cylindrosporus was specifically resistant to Pb. Meanwhile, we found that the best culturingconditions for G. cylindrosporus were25°C,120rpm, and pH5.0, and the best carbon andnitrogen source were mannitol and peptone, respectively.4. The responses of G. cylindrosporus to Pb stressThere were remarkable changes in colony morphology in the presence of different Pbconcentrations. In the concentration range of0¹.0mg/mL of Pb²⁺, the colony color turnedblack firstly and then yellow with the increasing Pb concentration. There was twisting andlooping of individual hyphae and formation of intertwined hyphal strands under Pb stress,however, there was no obvious relationship between the number of hyphal coils or the extentof hyphal twisting and the concentrations of Pb. Under liquid culture condition, hyphae curledand twisted when Pb concentration was low, and obviously became thick and swollen hyphaewith the increase of Pb concentration.In the concentration range of0⁰.3mg/mL of Pb²⁺, melanin content of the hyphaegradually increased with the increase of Pb concentration. Melanin content declined when theconcentration of Pb²⁺was up to0.4and0.5mg/mL, however, still was4.1and3.5times thatof the control, respectively. The contents of soluble protein and GSH and the activities ofSOD and CAT in G. cylindrosporus were significantly higher than controls under low Pbconcentration, but higher Pb concentrations decreased these parameters. SOD activity wassignificantly positively correlated with Pb concentrations, thus, SOD may play an importantrole in abating the hazards of heavy metals.5. Adsorption characteristics of G. cylindrosporus for Pb We found G. cylindrosporus had strong ability to absorb Pb ions. When the Pbconcentrations increased from50to500mg/L, the Pb content of the hyphae absorbedincreased gradually, and the absorption content and rate of dead hyphae were higher than livehyphae. Under the condition of30°C and pH5.0, the live and dead hyphae reached themaximum absorption capacity. In a range of agitation speeds from80to140rpm, theabsorption capacity of Pb in dead hyphae gradually increased with the increase of agitationspeed, and the live hyphae reached the maximum absorption capacity at120rpm. Fouriertransform infrared spectroscopy showed that the stretch vibration absorption peaks of C-O,C=O and N-H and the bending vibration absorption peak of N-H were broadened, whichindicated that the sugar and protein content significantly changed after the adsorption of Pb.6. The effects of G. cylindrosporus inoculation on plant growth and the uptake andtranslocation of Pb in maizeG. cylindrosporus inoculation significantly enhanced the seedling height, basal diameter,biomass and the root activity of maize seedlings, the enhancements were more obvious underhigh Pb stress. Relative water content of leaves was increased by19.3%while watersaturation deficient was declined by8.5%after inoculated with G. cylindrosporus under Pb²⁺concentration of1000mg/kg, indicating that the water use efficiency of maize leaves hadbeen significantly improved. When the concentration of Pb²⁺was1000mg/kg, the content ofchlorophyll a, chlorophyll b and total chlorophyll under inoculation treatment were increasedby66.9%,36.8%and59.5%, respectively. Under the Pb²⁺concentration of500and1000mg/kg, Pn under inoculation treatment were2.14and4.11times that of the control, Tr wereimproved by45.3%and39.0%, and Ci under inoculation treatment were45.3%and39.0%ofthe control, respectively; Fv/Fmof leaves after inoculation was11.3times that of the control atthe concentration of1000mg/kg.Pb content of maize seedlings inoculated with G. cylindrosporus were1.17and1.36times that of the control under the Pb²⁺concentration of500and1000mg/kg, respectively.More Pb content were accumulated in the roots under inoculation treatment, indicating that G.cylindrosporus inoculation decreased Pb translocation from roots to shoots, and decreased Pbcontent of shoots, thus it alleviated the Pb toxicity to maize. In pot experiment, the maize rootcolonization by G. cylindrosporus increased gradually with the increasing Pb stress, whichwas consistent with the field investigation results.