Characteristics Of Nitrogen And Potassium Nutrition Of Ectomycorrhizal Fungi And Their Response To Mercury Stress

Nitrogen and potassium, both of which play critical roles in origin, development, succession and decline of forests, are most needed essential elements by trees. In forest ecosystems, the exchangeable N in the soil solution is very low relative to total soil N and most of the soil nitrogen is in organic form. Large areas of red, yellow and limestone soil that distribute in the south of China are leaching strongly, thus the readily available potassium in soil is also very low. Since fertilization is difficult in forest, nitrogen and potassium nutrition of plants are mainly supplied by soil. On the other hand, mercury pollution becomes a global problem due to anthropic mercury release, which is more than 2909 tons per year with the largest part contributed by China. In areas with severe mercury pollution, absorption of nutrients including nitrogen and potassium by plants is restrained, development and renewal of trees are disturbed, and ultimately lead to forest degradation and decline. Ectomycorrhizal fungi are symbiotically associated with the roots of many woody plants and can dramatically enhance plant growth. Ectomycorrhiza-forming fungi alter the nutrient-acquisition capabilities of vascular plants, and may play an important role in tolerance of plant to drought, disease and heavy metal. Therefor, studying nitrogen and potassium nutrition of ectomycorrhizal fungi and their response to mercury stress is important to natural forest protection, afforestation and tree planting on mercury contaminated soil.14 ectomycorrhizal fungal strains involved in this study, including Pisolithus tinctorius (Pers.) Coker & Couch (Strain Pt 715, isolated from soils under eucalyptus, Xichang, Sichuan province), Lactarius deliciosus (L.:Fr.) Gray (strain Ld-1, Ld-2 & Ld-3, isolated from soils under masson pine, Jinyun mountain, Chongqin), Scleroderma polyrhizum Pers. (strain Sp-01, isolated from soils under masson pine, Jinfo mountain, Chongqin), Scleroderma polyrhizum Pers. (strain Sp-01, isolated from soils under masson pine, Jinfo mountain, Chongqin), Boletus spp. (strain Bo-02, isolated from soils under masson pine, Jinfo mountain, Chongqin), Suillus luteus(L.:Fr.) Gray (strain Sl 07 & Sl 14, isolated from soils under masson pine, Jinfo mountain, Chongqin), Gyroporus cyanescens(Bull.:Fr.) Quel.(strain Gc 99, isolated from Daqing mountain, Inner Mongolia), Cenococcum geophilum Fr. (strain Cg 04, isolated from Daqing mountain, Inner Mongolia), Suillus grevillei (Kl.)Sing. (strain Sg 03, isolated from Daqing mountain, Inner Mongolia), Lycoperdon perlatum Pers (strain Lp 01, isolated from Daqing mountain, Inner Mongolia), Suillus bovinus (L.:Fr.)Kuntze (strain Sb 05, isolated from Daqing mountain, Inner Mongolia), Cenococcum geophilum Fr. (Strain Cg SIV, from Spain). Ectomycorrhizal fungi were cultured in Pachlewski liquid media with different nitrogen resources, different potassium concentrations or different mercury concentrations. Nitrogen utilization to different nitrogen resources (NH₄⁺-N, NO₃^--N, urea and protein)by ectomycorrhizal fungi, growth rate, absorption of nitrogen, potassium and phosphorus, organic acids secretion and activities of key enzymes in oxalate metabolism pathways of ectomycorrhizal fungi under different potassium concentrations, as well as effects of mercury on growth rate, absorption of nitrogen, phosphorus and potassium, organic acids secretion and activities of nitrogen utilizing enzymes of ectomycorrhizal fungi, were studied. The result showed:Growth rate of most ectomycorrhizal fungi strains was promoted by ammonium as nitrogen resource. Protein was in the next place, whereas urea and nitrate performed poorly in promoting growth of ectomycorrhizal fungi. It is implied that the tested strains might not only benefit to nitrogen utilization diversity of host plants, but also favored different nitrogen sources. It suggested that the adaptation between soil and ectomycorrhizal fungal strains should be considered during afforestation. All of the four tested strains expressed protease, chitinase, urease and nitrate reductase, and there were significant differences among them. The existence and activity of nitrogen utilizing enzymes in the mycelia are the physiological basis of utilizing of different nitrogen resources by ectomycorrhizal fungi.Potassium concentration in the media influenced the growth rate of ectomycorrhizal fungi significantly. Growth was restrained without potassium, while most strains growed optimized in the concentration of middle-potassium. Sp 01 and Pt 715 got the fastest growth rate in low-potassium media, which suggested that Sp 01 and Pt 715 might both adapt to soils short of potassium. Effects of potassium on the nitrogen concentration of ectomycorrhizal fungi differed between strains. Higher concentration of potassium restrained phosphorus absorption by ectomycorrhizal fungi to some extents except SI 14. potassium concenteation in the media determined the potassium content in ectomycorrhizal fungal hyphae, as the increase of potassium level promoted potassium absorption by ectomycorrhizal fungi. K⁺ affected secretion of oxalate, acetic acid and H⁺ significantly. The secretion of oxalate and acetic acid reached the highest in no-K⁺ or low-K⁺ media, while was the lowest in middle or high-K⁺ media. Most strains secreted H⁺ at the utmost in no-K⁺ or low-K⁺ media, but Bo 02 and Lp 01, Pt 715 and Sb 05 secreted H⁺ at the utmost in middle or high-K⁺ media, respectively. Oxalate, acetic acid and proton secreted by ectomycorrhizal fungi may benefit to mineral weathering and the release of K trapped between clay layers.Activities of malate dehydrogenase, glyoxylate dehydrogenase and oxaloacetate hydrase in ectomycorrhizal fungi increased when lack of K, which showed some similarities with oxalate secretion. It suggested that low concentration of potassium may promote the expression of key enzymes in oxlate metabolism pathways. Furthermore, Activities of malate dehydrogenase, glyoxylate dehydrogenase and oxaloacetate hydrase in different strains differed siglificantly, which may imply the relative importance of these oxalate metabolism enzymes is different in different strains.Growth of fungal hyphae was restrained by mercury, whereas Cg SIV showed tolerance to Hg in some extent, with the least decrease on biomass. Although the biomass of Pt 715 and Ld-3 decreased by mercury treatment, their biomass are still the highest among tested strains.Effects of Hg on activities of nitrogen utilizing enzymes in ectomycorrhizal fungi varied with strains, enzyme categories and Hg²⁺ concentration. Since the inherited characteristics differ among strains, enzyme synthesis may vary under condition of the mercury existence, and express allozymes with different sensitivity to Hg. Moreover, low to medium Hg concentration promoted or did not affect the activities of nitrogen utilizing enzymes, which suggested that nitrogen utilization by ectomycorrhizal fungi might not be restraint or even be promoted. Activities of protease, urease, nitrate reductase and chitinase were the highest in Pt15 and Ld-3 with or without Hg treatment, which showed relatively high capacity of nitrogen utilization. Inoculating Pt715 and Ld-3 may improve nitrogen nutrition of eucalyptuses and pines planting in mercury contaminated soils.Under mercury stress, all of the tested strains secreted oxalate, while Sp 01, Sl 14 and Cg 04 secreted tartaric acid, and Sp 01, Cg 04 and Pt 715 secreted succinic acid. The efflux rate of oxalate, tartaric acid and succinic acid all showed the order of high Hg> middle Hg> low Hg> no Hg. Oxalate, tartaric acid and succinic acid are chelators of heavy metals, thus may reduce mercury concentration in the hyphae and the environment.