Study On The Tolerance Mechanisms Of Exophiala Pisciphila ACCC32496for Cadmium

Cadmium (Cd) pollution caused by mining of Lead (Pb) and Zinc (Zn) in Pb-Zn mine area is one of the most serious environmental problems in Yunnan. The roots of plants naturally growing in Pb-Zn mine areas are commonly colonized by dark septate endophytes (DSE) suggestes that DSE may play an important role in adaptation to heavy metal stress of these plants. Based on these facts, to relise the Cd tolerance and mechanisms of DSE from Pb-Zn mine area may help us to elucidate biological functions of DSE in the special ecosystem and to explain the relationship between DSE and heavy metal tolerance of their host plants in Pb-Zn mine area. Meanwhile, this research work is also theoretically and practically significance for making use of DSE-plants association to improve the heavy metal tolerance of plants to restore vegetation in Cd polluted environment.Exophiala pisciphila ACCC32496, a fungus of DSE isolated from the roots of Arundinella bengalensis (Spreng.) Druce which is wild plant grown on Pb-Zn mine area with serious heavy metals pollution in Huze County, Yunnan Province was selected in this thesis. The extracellular and intracellular tolerance mechanisms to Cd of Exophiala pisciphila ACCC32496are analyzed by studies on Cd accumulation of mycelium, Cd chemical forms and subcellular distribution, Cd adsorption capacity and characteristics of cell wall, chemical groups for Cd adsorption, physical and chemical characteristics of melanin from mycelium, secretion of low molecular weight organic acid, mineral nutrition, antioxidant system and so on which are responses to Cd stress with concentration of0～400mg/L.The main results are as follows:(1) A relative high Cd tolerance and accumulation ability of E. pisciphila ACCC32496was founded. The50%effective inhibiting concentration (EC50) of Cd to E. pisciphila ACCC32496was calculated by linear interpolation and the result was332.2and111.2mg/L by solid and liquid culture respectively.89.02mg Cd per g dry mycelium was accumulated by E. pisciphila ACCC32496when the concentration of Cd was400mg/L in liquid medium.(2) The mycelium of E. pisciphila ACCC32496were analyzed by SEM-EDS, TEM and TEM-EDS, and the subcellular fractions, ie cell wall, organelles and soluble fraction, were separated by differential centrifugation. The results showed that Cd was mainly distributed in the cell wall of E. pisciphila ACCC32496. Cd associated with different chemical forms was successively extracted by designated solutions in the following order,80%ethanol, deionized water,1M NaCl,2%acetic acid and0.6M HCl. Acetic acid extractable (FHAc) and sodium chloride extractable (FNaCl) was the main chemical form of Cd.(2) About63.0%～83.5%of the total Cd in the mycelium of E. pisciphila ACCC32496could be desorbed by0.1M HCl or0.1M Na2EDTA,8.3%-43.8%by0.1M CaCl2, and0.9%-5.6%by0.1M NaOH.(3) Cd adsorption process of E. pisciphila ACCC32496mycelium can be well described by Langmuir adsorption isotherm model. Kinetics data for the adsorption of Cd from aqueous solutions by E. pisciphila ACCC32496mycelium were in good agreement with the pseudo-second rate equation within an initial concentration range of25-400mg/L, pH value of5.0, an agitation speed range of120rpm and an experimental temperature of30℃. The chemical groups of E. pisciphila ACCC32496mycelium involving in adsorption of Cd involved amino, amido, carboxyl, hydroxyl, and phosphoryl and so on.(4) The melanin isolated from E. pisciphila ACCC32496mycelium is typical of DHN melanin. The melanin synthesis of E. pisciphila ACCC32496mycelium could be specifically inhibited by tricyclazole. There was no significant effect of tricyclazole on the mycelial growth and spore production of E. pisciphila ACCC32496when the concentration of tricyclazole was not higher than10μg/ml. The melanin content of E. pisciphila ACCC32496mycelium was increased significantly when the range of Cd concentration in liquid medium was50-350mg/L. Biomass of the mycelium in which the melanin synthesis was inhibited by tricyclazole was significantly smaller than normal mycelium under50-200mg/L Cd stress, which indicated the melanin had a role in the Cd tolerance of E. pisciphila ACCC32496. (5) The content of N, K and Fe was decreased significantly, that of P, Mg and Ca was increased significantly in the mycelium of E. pisciphila ACCC32496under Cd stress. The content of S was increased significantly when the concentration of Cd in liquid medium was100mg/L. The production of organic acids by E. pisciphila ACCC32496mycelium was promoted by Cd stress, and the pH value in liquid medium was decreased. Oxalic acid was the main organic acid produced by E. pisciphila ACCC32496mycelium. The content of oxalic acid was increased significantly with the increase of Cd concentration in liquid medium.(6) The rate of superoxide anion formation, content of MDA and H2O2was increased significantly in the mycelium of E. pisciphila ACCC32496under Cd stress. The activity of SOD and CAT, content of GSH and-SH compounds, and the T-AOC was increased with the increase of Cd concentration within the certain range.These results suggested that E. pisciphila ACCC32496, a DSE strain isolated from Pb-Zn mine area with serious heavy metal pollution, had a strong tolerance and accumulation ability for Cd. The tolerance mechanisms to Cd of E. pisciphila ACCC32496included:①Extracellular mechanisms included increased secretion of extracellular low molecular weight organic acids, especially oxalic acid; the adsorption of Cd by cell wall; and increased synthesis of the special components-melanin in the mycelium.②Intracellular mechanisms included enhanced activity of antioxidant enzymes such as SOD and CAT in the mycelium; increased synthesis of GSH and other thiol compounds; and increased contents of mineral elements (P, S, Mg and Ca) in mycelium. So the tolerance of E. pisciphila ACCC32496was enhanced.