Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination

Abundance and diversity of arbuscular mycorrhizal fungi (AMF) associated with dominant plant species were studied along a transect from highly lead (Pb) and zinc (Zn) polluted to non-polluted soil at the Anguran open pit mine in Iran. Using an established primer set for AMF in the internal transcribed spacer (ITS) region of rDNA, nine different AMF sequence types were distinguished after phylogenetic analyses, showing remarkable differences in their distribution patterns along the transect. With decreasing Pb and Zn concentration, the number of AMF sequence types increased, however one sequence type was only found in the highly contaminated area. Multivariate statistical analysis revealed that further factors than HM soil concentration affect the AMF community at contaminated sites. Specifically, the soils’ calcium carbonate equivalent and available P proved to be of importance, which illustrates that field studies on AMF distribution should also consider important environmental factors and their possible interactions.     

The use of olive-mill waste compost to promote the plant vegetation cover in a trace-element-contaminated soil

The applicability of a mature compost as a soil amendment to promote the growth of native species for the phytorestoration of a mine-affected soil from a semi-arid area (SE Spain), contaminated with trace elements (As, Cd, Cu, Mn, Pb and Zn), was evaluated in a 2-year field experiment. The effects of an inorganic fertiliser were also determined for comparison. Bituminaria bituminosa was the selected native plant since it is a leguminous species adapted to the particular local pedoclimatic conditions. Compost addition increased total organic-C concentrations in soil with respect to the control and fertiliser treatments, maintained elevated available P concentrations throughout the duration of the experiment and stimulated soil microbial biomass, while trace elements extractability in the soil was rather low due to the calcareous nature of the soil and almost unaltered in the different treatments. Tissue concentrations of P and K in B. bituminosa increased after the addition of compost, associated with growth stimulation. Leaf Cu concentration was also increased by the amendments, although overall the trace elements concentrations can be considered non-toxic. In addition, the spontaneous colonisation of the plots by a total of 29 species of 15 different families at the end of the experiment produced a greater vegetation cover, especially in plots amended with compost. Therefore, the use of compost as a soil amendment appears to be useful for the promotion of a vegetation cover and the phytostabilisation of moderately contaminated soils under semi-arid conditions.     

Diversity of arbuscular mycorrhizal fungi in grassland spontaneously developed on area polluted by a fertilizer plant

Mycorrhizal colonization and diversity of arbuscular mycorrhizal fungi (AMF) were analyzed in a calcareous grassland with residual phosphate contamination 10 years after the closure of a pollutant fertilizer plant in Thuringia (Germany). AMF were detected in 21 of 22 plant species analyzed. Mean mycorrhization levels reached up to 74.5% root length colonized. AMF diversity was analyzed based on 104 sequences of the internal transcribed spacer (ITS) of the ribosomal DNA. Phylogenetic analyses revealed a total of 6 species all belonging to the genus Glomus. There was no overlap between species detected as active mycorrhizas on roots (2 taxa) or as spores (4 taxa). Compared to the regional context, the diversity of AMF at our field site was reduced, which may reflect a residual disturbance effect. However, none of the detected species was exclusive to the polluted site as they are commonly found in the region.     

重金属污染土壤接种丛枝菌根真菌对蚕豆毒性的影响

采用盆栽实验的方法,研究了重金属(包括Cu、Zn、Pb和Cd)复合污染和接种丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)Glomus mosseae对蚕豆(Vicia faba)生长及DNA损伤的影响.结果表明,虽然接种菌根真菌对蚕豆生物量的影响并不显著,但是却显著影响植物对重金属的吸收,接种菌根真菌对蚕豆吸收4种重金属元素的作用有差异.采用单细胞凝胶电泳(single cell gel electrophoresis,SCGE)法研究接种菌根真菌对蚕豆叶片的DNA损伤的影响,与重金属吸收的结果相吻合.结果表明,接种处理可显著增加蚕豆叶片的DNA损伤程度,这与接种处理可提高植物的重金属吸收相一致.     

EDDS and EDTA-enhanced zinc accumulation by Solanum nigrum inoculated with arbuscular mycorrhizal fungi grown in contaminated soil

The effect of two different chelating agents [EDTA and EDDS (S,S-ethylenediaminedissucinic acid)] on Zn tissue accumulation in Solanum nigrum L. grown in a naturally contaminated soil was assessed. Under those conditions, the response of the plant to the inoculation with two different isolates of arbuscular mycorrhizal fungi (AMF)--Glomus claroideum and Glomus intraradices--was also studied. Plants grown in the local contaminated soil (Zn levels of 433mg kg(-1)) accumulated up to 1191mg kg(-1) of Zn in the roots, 3747mg kg(-1) in the stems and 3409mg kg(-1) in the leaves. S. nigrum plants grown in the same soil spiked with extra Zn (Zn levels of 964mg kg(-1)) accumulated up to 4735, 8267 and 7948mg Zn kg(-1) in the leaves, stems and roots, respectively. The addition of EDTA promoted an increase in the concentration of Zn accumulated by S. nigrum of up to 231% in the leaves, 93% in the stems and 81% in the roots, while EDDS application enhanced the accumulation in leaves, stems and roots up to 140, 124 and 104%, respectively. In the stems, the presence of Zn was predominantly detected in the cortex collenchyma cells, the starch sheath and the internal phloem and xylem parenchyma, and the addition of chelating agents did not seem to have an effect on the localisation of accumulation sites. The devise of a chelate-enhanced phytoextraction strategy, using chelating agents and AMF, is discussed.     

Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil

A heavy metal-resistant bacterial strain was isolated from heavy metal-contaminated soils and identified as Burkholderia sp. J62 based on the 16S rDNA gene sequence analysis. The heavy metal- and antibiotic resistance, heavy metal solubilization of the isolate were investigated. The isolate was also evaluated for promoting plant growth and Pb and Cd uptakes of the plants from heavy metal-contaminated soils in pot experiments. The isolate was found to exhibit different multiple heavy metal and antibiotic resistance characteristics. Atomic absorption spectrometer analysis showed increased bacterial solubilization of lead and cadmium in solution culture and in soils. The isolate produced indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. The isolate also solubilized inorganic phosphate. Inoculation with the isolate was found to significantly (p < 0.05) increase the biomass of maize and tomato plants. Increase in tissue Pb and Cd contents varied from 38% to 192% and from 5% to 191% in inoculated plants growing in heavy metal-contaminated soils compared to the uninoculated control, respectively. These results show that heavy metal-solubilizing and plant growth promoting bacteria are important for plant growth and heavy metal uptake which may provide a new microbial enhanced-phytoremediation of metal-polluted soils.     

Solanum nigrum grown in contaminated soil: effect of arbuscular mycorrhizal fungi on zinc accumulation and histolocalisation

Zn tissue accumulation in Solanum nigrum grown in a non-contaminated and a naturally contaminated Zn matrix and the effect of inoculation with different arbuscular mycorrhizal fungi (AMF) on metal uptake were assessed. S. nigrum grown in the contaminated soil always presented higher Zn accumulation in the tissues, accumulating up to 1622 mg Zn kg(-1). The presence of both Glomus claroideum and Glomus intraradices enhanced the uptake and accumulation of Zn by S. nigrum (up to 83 and 49% higher Zn accumulation, respectively). The main deposits of the metal were found in the intercellular spaces and in the cell walls of the root tissues, as revealed by autometallography, with the inoculation with different AMF species causing no differences in the location of Zn accumulation. These findings indicate that S. nigrum inoculated with selected heavy metal tolerant AMF presents extracting and accumulating capacities, constituting a potentially suitable remediation method for Zn polluted soils.     

Two bacterial strains isolated from a Zn-polluted soil enhance plant growth and mycorrhizal efficiency under Zn-toxicity

In this study we investigated the interactions among plant, rhizosphere microorganisms and Zn pollution. We tested the influence of two bacterial strains isolated from a Zn-polluted soil on plant growth and on the symbiotic efficiency of native arbuscular mycorrhizal fungi (AMF) under Zn toxicity. The two bacterial strains exhibited Zn tolerance when cultivated under increasing Zn levels in the medium. However, strain B-I showed a higher Zn tolerance than strain B-II at the two highest Zn levels in the medium (75 and 100 mg l(-1) Zn). Molecular identification placed the strain B-I within the genus Brevibacillus. Our results showed that bacterial strain B-I consistently enhanced plant growth, N and P accumulation, as well as nodule number and mycorrhizal infection which demonstrated its plant-growth promoting (PGP) activity. This strain B-I has been shown to produce IAA (3.95 microg ml) and to accumulate 5.6% of Zn from the growing medium. The enhanced growth and nutrition of plants dually inoculated with the AMF and bacterium B-I was observed at three Zn levels assayed. This effect can be related to the stimulation of symbiotic structures (nodules and AMF colonization) and a decreased Zn concentration in plant tissues. The amount of Zn acquired per root weight unit was reduced by each one of these bacterial strains or AMF and particularly by the mixed bacterium-AMF inocula. These mechanisms explain the alleviation of Zn toxicity by selected microorganisms and indicate that metal-adapted bacteria and AMF play a key role enhancing plant growth under soil Zn contamination.     

Combined effect of diuron and simazine on photosystem II photochemistry in a sandy soil and soil amended with solid olive-mill waste

Diuron (3-(3,4-dichlorophenyl)- = 1,1-dimethylurea) and simazine (6-chloro-N(2), N(4)-diethyl-1,3,5-triazine-2,4-diamine) are soil-applied herbicides used in olive crops. The objective of this study is to investigate the effect of these herbicides on Photosystem II photochemistry of Olea europaea L., and whether the amendment of soil with an organic waste (OW) from olive oil production industry modifies this effect. For this purpose, herbicide soil adsorption studies, with unamended and OW-amended soil, and chlorophyll fluorescence measurements in adult olive leaves, after one, two and three weeks of soil herbicide treatment and/or OW amendment, were performed. Soil application of these herbicides reduced the efficiency of Photosystem II photochemistry of olive trees due to chronic photoinhibition, and this effect is counterbalanced by the addition of OW to the soil. OW reduces herbicide uptake by the plant due to an increase in herbicide adsorption.     

Combined effect of diuron and simazine on photosystem II photochemistry in a sandy soil and soil amended with solid olive-mill waste

Diuron (3-(3,4-dichlorophenyl)- = 1,1-dimethylurea) and simazine (6-chloro-N ², N ⁴-diethyl-1,3,5-triazine-2,4-diamine) are soil-applied herbicides used in olive crops. The objective of this study is to investigate the effect of these herbicides on Photosystem II photochemistry of Olea europaea L., and whether the amendment of soil with an organic waste (OW) from olive oil production industry modifies this effect. For this purpose, herbicide soil adsorption studies, with unamended and OW-amended soil, and chlorophyll fluorescence measurements in adult olive leaves, after one, two and three weeks of soil herbicide treatment and/or OW amendment, were performed. Soil application of these herbicides reduced the efficiency of Photosystem II photochemistry of olive trees due to chronic photoinhibition, and this effect is counterbalanced by the addition of OW to the soil. OW reduces herbicide uptake by the plant due to an increase in herbicide adsorption.     

Effects of arbuscular mycorrhizal fungi on the root uptake and translocation of radiocaesium

Because mycorrhizal fungi are intimately associated with plant roots, their importance in radionuclide (RN) recycling and subsequent dispersion into the biosphere has received an increasing interest. Recently, the capacity of arbuscular mycorrhizal fungi to take up and translocate radiocaesium to their host was demonstrated. However, the relative contribution of these processes in comparison to the ones of roots remains unknown. Here, the respective contributions of the hyphae of a Glomus species and the transformed carrot (Daucus carota L.) roots on radiocaesium uptake and translocation were compared and quantified. We observed that radiocaesium uptake by hyphae was significantly lower as compared to that of the roots, while the opposite was noted for radiocaesium translocation/uptake ratio. We also observed that the intraradical fungal structures might induce a local accumulation of radiocaesium and concurrently reduce its translocation within mycorrhizal roots. We believe that intraradical fungal structures might induce the down-regulation of radiocaesium channels involved in the transport processes of radiocaesium towards the xylem.     

Cu污染土壤接种丛枝菌根真菌对旱稻生长的影响

采用盆栽实验的方法,研究了不同Cu处理水平(0、100和200 mg/kg)下,接种丛枝菌根真菌(arbuscular my-corrhizal fungi,AMF)Glomus mosseae对旱稻(Oryzal Sativa L.)生长的影响。结果表明,未添加Cu处理下,旱稻菌根侵染率可达69%,随着土壤中Cu添加量增加,旱稻菌根侵染率显著下降(P<0.05)。与未接种处理相比,接种处理显著提高100mg/kg Cu处理下根系生物量以及200 mg/kg Cu处理下地上部分生物量(P<0.05);接种处理显著降低了100 mg/kg Cu处理下旱稻地下部Cu含量,却显著增加了200 mg/kg Cu处理下旱稻地上部分以及地下部分Cu含量(P<0.05)。     

In situ phytoremediation of arsenic- and metal-polluted pyrite waste with field crops: effects of soil management

Sunflower, alfalfa, fodder radish and Italian ryegrass were cultivated in severely As-Cd-Co-Cu-Pb-Zn-contaminated pyrite waste discharged in the past and capped with 0.15 m of unpolluted soil at Torviscosa (Italy). Plant growth and trace element uptake were compared under ploughing and subsoiling tillages (0.3 m depth), the former yielding higher contamination (∼30%) in top soil.Tillage choice was not critical for phytoextraction, but subsoiling enhanced above-ground productivity, whereas ploughing increased trace element concentrations in plants. Fodder radish and sunflower had the greatest aerial biomass, and fodder radish the best trace element uptake, perhaps due to its lower root sensitivity to pollution. Above-ground removals were generally poor (maximum of 33 mg m⁻² of various trace elements), with Zn (62%) and Cu (18%) as main harvested contaminants. The most significant finding was of fine roots proliferation in shallow layers that represented a huge sink for trace element phytostabilisation.It is concluded that phytoextraction is generally far from being an efficient management option in pyrite waste. Sustainable remediation requires significant improvements of the vegetation cover to stabilise the site mechanically and chemically, and provide precise quantification of root turnover.     

Dynamics of arbuscular mycorrhizal symbiosis in heavy metal phytoremediation: meta-analytical and conceptual perspectives

To estimate dynamics of arbuscular mycorrhizal (AM) symbiosis in heavy metal (HM) phytoremediation, we conducted a literature survey and correlated HM uptake and relative plant growth parameters from published data. After estimating AM feedback responses for these parameters at low and high soil-HM concentration intervals, we determined that the roles of AM symbiosis are characterized by (1) an increased HM phytoextraction via mycorrhizospheric 'Enhanced Uptake' at low soil-HM concentrations, and (2) a reduced HM bioavailability via AM fungal 'Metal-Binding' processes at high soil-HM levels, hence resulting in increased plant biomass and enhanced plant tolerance through HM stress-avoidance. We present two conceptual models which illustrate the important compromise between plant growth, plant HM uptake and HM tolerance, and further emphasize the importance of AM symbiosis in buffering the soil environment for plants under such stress conditions.     

The role of arbuscular mycorrhizal fungi in the transfer of nutrients between white clover and perennial ryegrass

A glasshouse experiment was conducted in which 15N was used as a tracer applied as (15NH4)2SO4 to donor plants of white clover and perennial ryegrass. Nitrogen transfer via hyphae of arbuscular mycorrhizal fungi (AMF) or by other routes was studied by separating the root systems of the two plant species, as donors and receivers, when growing in the same pot, with selective mesh barriers of varying pore sizes in the presence and absence of AMF. Inoculation with AMF increased DM production and nitrogen (N) yield of clover plants. Transfer of 15N occurred between white clover and grass plants but was independent of AMF. Pore size of the mesh barriers controlled the degree of 15N enrichment in the grass, suggesting that transfer was mediated by mass flow and/or diffusion. Additional experiments showed that grass roots could pass through pores of 60-microm diameter, and hyphal links could not be detected by autoradiography, thus supporting the conclusions of the tracer experiment.     

The arbuscular mycorrhizal fungus Glomus mosseae can enhance arsenic tolerance in Medicago truncatula by increasing plant phosphorus status and restricting arsenate uptake

A pot experiment examined the biomass and As uptake of Medicago truncatula colonized by the arbuscular mycorrhizal (AM) fungus Glomus mosseae in low-P soil experimentally contaminated with different levels of arsenate. The biomass of G. mosseae external mycelium was unaffected by the highest addition level of As studied (200 mg kg⁻¹) but shoot and root biomass declined in both mycorrhizal and non-mycorrhizal plants, indicating that the AM fungus was more tolerant than M. truncatula to arsenate. Mycorrhizal inoculation increased shoot and root dry weights by enhancing host plant P nutrition and lowering shoot and root As concentrations compared with uninoculated plants. The AM fungus may have been highly tolerant to As and conferred enhanced tolerance to arsenate on the host plant by enhancing P nutrition and restricting root As uptake.     

DDT uptake by arbuscular mycorrhizal alfalfa and depletion in soil as influenced by soil application of a non-ionic surfactant

A greenhouse pot experiment was conducted to investigate the colonization of alfalfa roots by the arbuscular mycorrhizal (AM) fungus Glomus etunicatum and application of the non-ionic surfactant Triton X-100 on DDT uptake by alfalfa and depletion in soil. Mycorrhizal colonization led to an increase in the accumulation of DDT in roots but a decrease in shoots. The combination of AM inoculation and Triton X-100 application enhanced DDT uptake by both the roots and shoots. Application of Triton X-100 gave much lower residual concentrations of DDT in the bulk soil than in the rhizosphere soil or in the bulk soil without Triton X-100. AM colonization significantly increased bacterial and fungal counts and dehydrogenase activity in the rhizosphere soil. The combined AM inoculation of plants and soil application of surfactant may have potential as a biotechnological approach for the decontamination of soil polluted with DDT.     

堆肥污泥施用于杨树后土壤理化性质及土壤污染风险

污泥可作为一种土壤改良剂应用于树木,特别是杨树。采用田间实验,探讨堆肥污泥施用于杨树后的土壤理化性质和土壤污染风险,以期为污泥的合理利用提供科学依据。通过2年的田间实验研究了不同堆肥污泥用量对土壤营养成分和重金属污染的影响,实验设置4个处理：对照、低量、中量和高量。结果表明,随着污泥用量的增加,土壤营养成分和重金属的质量分数均有所提高,而且堆肥污泥可显著降低土壤pH,高量污泥可使土壤碱性降为中性。污泥累计施用后,除了低量对K、Na、Cd 和Pb质量分数影响不显著外,污泥处理均对土壤营养成分和重金属的质量分数影响显著;中量和高量污泥处理使土壤有机质增加1.2和1.6倍,有效氮增加1.2和1.9倍,N、P及K质量分数分别增加2.7和2.9倍、2.8和4.3倍及0.6和0.7倍。土壤中Cu、Zn、Ni、Cr、Hg和Pb质量分数均没有超出我国农用地土壤污染风险筛选值和管制值范围(GB 15618-2018);Cd质量分数超过了土壤污染风险筛选值,但也远低于土壤污染风险管制值。本研究可为将堆肥污泥作为土壤改良剂应用于杨树提供参考。     

The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements

Naturally occurring soil organic compounds stabilize potentially toxic elements (PTEs) such as Cu, Cd, Pb, and Mn. The hypothesis of this work was that an insoluble glycoprotein, glomalin, produced in copious amounts on hyphae of arbuscular mycorrhizal fungi (AMF) sequesters PTEs. Glomalin can be extracted from laboratory cultures of AMF and from soils. Three different experiments were conducted. Experiment 1 showed that glomalin extracted from two polluted soils contained 1.6-4.3 mg Cu, 0.02-0.08 mg Cd, and 0.62-1.12 mg Pb/g glomalin. Experiment 2 showed that glomalin from hyphae of an isolate of Gigaspora rosea sequestered up to 28 mg Cu/g in vitro. Experiment 3 tested in vivo differences in Cu sequestration by Cu-tolerant and non-tolerant isolates of Glomus mosseae colonizing sorghum. Plants were fed with nutrient solution containing 0.5, 10 or 20 microM of Cu. Although no differences between isolates were detected, mean values for the 20 microM Cu level were 1.6, 0.4, and 0.3 mg Cu/g for glomalin extracted from hyphae, from sand after removal of hyphae and from hyphae attached to roots, respectively. Glomalin should be considered for biostabilization leading to remediation of polluted soils.     

Changes in soil solution Zn and pH and uptake of Zn by arbuscular mycorrhizal red clover in Zn-contaminated soil

Red clover plants inoculated with Glomus mosseae were grown in a sterile pasture soil containing 50 mg Zn kg(-1) in 'Plexiglas' (acrylic) containers with nylon net partitions (30 microm mesh) designed to separate the soil into a central root zone and two outer zones for hyphal growth with no root penetration. Two porous plastic soil moisture samplers were installed in each pot, one in the root compartment and the other in one of the hyphal compartments. The soil in the outer compartments was amended with one of the four application rates of Zn (as ZnSO4) ranging from 0 to 1000 mg kg(-1). Non-mycorrhizal controls were included, and there were five replicates of each treatment in a randomised block in a glasshouse. Uninoculated plants received supplementary P to avoid yield limitation due to low soil P status. Plants grew in the central compartment for nine weeks. Soil moisture samples were collected 4, 24 and 62 days after sowing to monitor changes in the Zn concentration and pH of the soil solution. At harvest, the mean mycorrhizal infection rate of inoculated plants ranged from 29% to 34% of total root length and was little affected by Zn application. Root and shoot yields were not affected by mycorrhizal infection. Plant Zn concentration and uptake were lower in mycorrhizal plants than non-mycorrhizal controls, and this effect was more pronounced with increasing Zn application rate to the soil. Soil solution Zn concentrations were lower and pH values were higher in mycorrhizal treatments than non-mycorrhizal controls and the mycorrhiza effect was more pronounced at higher Zn application rates. The protective effect of mycorrhiza against plant Zn uptake may have been associated with changes in Zn solubility mediated by changes in the soil solution pH, or by immobilisation of Zn in the extraradical mycelium.