Isolation and Molecular Characterization of Heavy Metal-Resistant Azotobacter chroococcum from Agricultural Soil and Their Potential Application in Bioremediation

Pollution of soil with heavy metals, herbicides, antibiotics and other chemicals is known to have a negative effect on microbial activities. Therefore, the aim of this study was to isolate cultures of Azotobacter sp. from polluted and unpolluted soils and to study the effect of these pollutants on their growth. A total of 120 Azotobacter sp. were isolated from soils irrigated with wastewater (contaminated soils) and groundwater (uncontaminated soils). These isolates were screened for resistance to heavy metals, herbicide and antibiotics. Also, the soils from which the cultures were isolated were analyzed for the concentrations of Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺ and Mn²⁺ they contained. Contaminated soil showed high levels of heavy metals as compared to uncontaminated soil. The size of the Azotobacter population in contaminated soil was lower than that in uncontaminated soil. Of the Azotobacter isolates, 64 that were recovered from contaminated soil exhibited high resistance to heavy metals (Hg²⁺, Cd²⁺, Cu²⁺, Cr³⁺, Co²⁺, Ni²⁺, Zn²⁺ and Pb²⁺) and herbicide 2,4-D compared to 56 isolates from uncontaminated soil. Also, isolates from contaminated soil showed high resistance to chloramphenicol, nitrofurantoin and co-trimoxazole compared to those isolated from uncontaminated soil. The majority of Azotobacter isolates from contaminated soil showed multiple-resistance to different metal ions and antibiotics. All isolates failed to grow at pH less than 6. Salt concentration (5%) was found to be inhibitory to all isolates. The most potent isolates from contaminated soil that showed multiresistance to all substances tested were identified on the basis of morphological and biochemical characteristics, and 16S rRNA as A. chroococcum. These resistant isolates could be employed in contaminated soils and/or bioremediation.     

Metal resistance in Pseudomonas strains isolated from soil treated with industrial wastewater

Heavy metal concentrations in soil treated with industrial wastewater of Aligarh City (U.P.), India were determined. The analysis of test samples revealed high levels of Fe, Zn, Ni and Cu. A total of 45 Pseudomonas spp. were isolated from soil and were characterized on the basis of morphological, cultural and biochemical characteristics. MICs of Hg²⁺, Cd²⁺, Cu²⁺, Cr³⁺, and Zn²⁺ for each isolate were determined. Eighty percent of the strains isolated from soil harboured resistance to copper, whereas 73.3% of the isolates exhibited resistance to cadmium, 71.1% to chromium and zinc and 48.8% to mercury. A maximum MIC of 200 g/ml for mercury and 1600 g/ml for other metals was observed. Metal resistance was found to be plasmid mediated as evidenced by transformation studies. Further, the transmissible nature of chromium resistance was confirmed by conjugation. Agarose gel electrophoresis using the miniprep method for plasmid isolation revealed that these isolates harboured plasmids of molecular weights (45 & 47 kb) using EcoRI and HindIII digests of DNA and undigested DNA as standard markers.     

Tolerance and bioaccumulation of heavy metals in five populations of Cistus ladanifer L. subsp. ladanifer

The effects of heavy metals on the growth, mineral composition (P, K, Fe and Mn) and metal accumulation of five populations of Cistus ladanifer subsp. ladanifer from NE Portugal were investigated in hydroponic experiments. Plants were exposed to increasing concentrations (0–2000 M) of one of eight heavy metals: Cd, Co, Cr, Cu, Mn, Ni, Pb or Zn. Populations of C. ladanifer, whose origin was ultramafic soils (S and UB) or soils developed on basic rocks (B), showed a higher tolerance to the metals Cd, Co, Cr, and Mn, and a considerable degree of tolerance to Ni. In contrast, populations originating on acid-rock soils (M and SC) showed higher tolerance to the metals Cu and Zn. Populations showed different patterns of metal accumulation and distribution in the plant parts, suggesting different mechanisms of metal tolerance are used. The more Cd-, Co- and Mn-tolerant populations (S, UB, B and SC (Cd)) showed accumulation of these three metals in the shoots (shoot:root metal concentration ratios (S:R) > 1). Shoot concentrations of up to 309 g Cd g^–1, 2667 g Co g^–1 and 6214 g Mn g^–1 were found in these populations. The populations, UB and M, showed considerable tolerance to Ni and Zn, respectively. These populations accumulated up to 4164 g Ni g^–1 and 7695 g Zn g^–1 in their shoot tissues, and these metals were efficiently transported from the roots to aerial parts (S:R > 3 (Ni), S:R > 1 (Zn)). In contrast, the S and SC populations maintained higher growth rates in the presence of Ni and Zn, respectively, but showed exclusion mechanisms of metal tolerance: reduced Ni and Zn transport to shoots (S:R < 1). Cistus ladanifer was not able to efficiently transport Cr, Cu or Pb from its roots to its aerial parts (S:R ranged from 0–0.4). The more Cu-tolerant populations, M and SC, showed a greater restriction of Cu transport to the shoots than the ultramafic- or basic-rock populations. Significant changes in the plant mineral composition were found, however, concentrations were generally above mineral deficiency levels. Based on these preliminary results the possible usefulness of this plant for phytoremediation technologies is discussed. However, further investigations are necessary to evaluate its growth and metal accumulation under soil and field conditions.     

Phytoremediation potential of vetiver grass irrigated with wastewater for treatment of metal contaminated soil

A field experiment was conducted to understand the potential of vetiver grass (Vetiveria zizanioides) in heavy metal uptake from the soil and wastewater. Four main irrigation treatments including T₁ (treated industrial wastewater), T₂ (1:1 ratio of municipal:industrial wastewater), T₃ (treated municipal wastewater) and T₄ (fresh water) were applied. Moreover, the effect of arbuscular mycorrhizal fungus (AMF), Glomus mosseae, on plant growth and heavy metal concentration was evaluated. Three main criteria including bioconcentration factor (BCF), translocation factor (TF) and heavy metal uptake were applied to assess the potential of vetiver grass in accumulation and translocation of heavy metals to aerial parts. The highest concentration of heavy metals was found in plant and soil irrigated with T₁ treatment followed by T₂, T₃ and the lowest concentrations were found in T₄ treatment. Irrigation with treated municipal wastewater led to a significant increase in plant biomass and heavy metal uptake compared to other treatments. In T₁ treatment (industrial wastewater), vetiver grass caused a significant decrease in Zn, Fe, Cu, Cd and Pb concentrations in soil as compared to no-plant treatment (without planting vetiver grass). Therefore, vetiver grass, irrigated with treated industrial wastewater, is a promising method for the development of urban and industrial green space.     

Plasmid incidence in bacteria from agricultural and industrial soils

Heavy metal contents of agricultural and industrial soils were determined by atomic absorption spectrophotometry. The analysis of the samples collected from two different locations revealed significantly high levels of Fe, Zn, Cu, Cr and Ni. Certain microbiological parameters (total aerobic heterotrophs, asymbiotic N₂-fixers, total Actinomycetes and fungi) were also monitored from these soils. A total of 70 bacterial isolates from agricultural and industrial soils were examined for plasmid DNA content and resistance to the antibiotics amoxycillin, cloxacillin, chloramphenicol, doxycycline methicillin, nalidixic acid, and tetracycline. Minimum inhibitory concentrations (MICs) of Cu, Cr, Pb, Cd, Hg, Zn, and Ni for each isolate were also determined. Resistance was most frequent to methicillin (48.5%), cloxacillin (45.7%), and nalidixic acid (40%) for all isolates of bacteria. The highest MICs observed were 100 g/ml for mercury, 800 g/ml for Ni and 1600 g/ml for other metals. The incidences of metal resistance and MICs of metals for bacteria from industrial soil were significantly different to those of agricultural soil. On a percentage basis, 91.4% of the total bacterial isolates from industrial soil were found to harbour plasmids whereas 40% of the isolates from agricultural soil contained plasmids.     

A direct solid‐phase assay specific for heavy‐metal toxicity. II. Assessment of heavy‐metal immobilization in soils and bioavailability to plants

We have used the solid‐phase MetPLA TE, an enzyme assay that is specific for heavy‐metal toxicity, to investigate metal toxicity of soils that have been amended with urban wastewater sludges or contaminated with dry deposition from metal‐plating industries. We have shown that soil toxicity, using MetPLA TE, ranged from 21 to 72.5% inhibition of enzyme activity. Evin soil, which displayed the highest toxicity, also had the highest concentrations of Pb and Zn. Metal uptake studies with ryegrass grown on Evin soil, showed Zn, Cd, and Pb accumulation in the plant that exceeds the standard levels reported for grasses

Solid‐phase MetPLA TE was also used as a tool to study the reduction of heavy‐metal toxicity following soil amendments to immobilize metals in soil and thus reduce their toxicity. It was found that the addition of 1% hydrated manganese oxide significantly reduced dissolved metals in soil, their accumulation by ryegrass, and soil toxicity as shown by MetPLA TE.     

Toxic metals accumulation in <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> and <Emphasis Type="Italic">T. harzianum</Emphasis>

Heavy metal contamination represents an important environmental issue due to the toxic effects of metals on different organisms. Filamentous fungi play an important impact in the bioremediation of heavy metal-contaminated wastewater and soil. The purpose of this investigation was to observe fungal uptake behavior toward heavy metal. For this aim Trichoderma asperellum TS141 and T. harzianum TS103 at growth period were screened for their tolerance and uptake capability of cadmium (Cd), lead (Pb) and nickel (Ni) at different concentrations (0, 25, 50, 100, and 200 mg/L) in PDB media (potato dextrose broth as a complex medium). Results showed that both fungi were able to survive at the maximum concentration of 200 mg/L of the heavy metals, and remove them. T. asperellum had a better uptake capacity for Cd compared to Pb and Ni in the highest metal concentration in media. Maximum removal efficiency of Pb (68.4%) at 100 mg/L and Ni (78%) at 200 mg/L was performed by T. asperellum. For Cd, the highest removal efficiency (82.1%) was recorded by T. harzianum at 200 mg/L Cd in aqueous solution. The uptake of Cd was highly dependent on pH of solution than Pb and Ni so that the optimal pH of Cd uptake was 9 for T. asperellum and 4 for T. harzianum. Also, optimal temperature was 35°C for Cd and Pb uptake in both fungi, whereas for Ni uptake was 30 and 35°C in T. harzianum and T. asperellum, respectively. We propose that T. asperellum TS141 and T. harzianum TS103 can be used as a bioremediation agent for metal remediation from wastewater and heavy metal-contaminated soils.     

The use of indigenous plant species and calcium phosphate for the stabilization of highly metal-polluted sites in southern Poland

Highly metal-polluted (Pb, Cd, Zn) soil from a non-ferrous mine and smelter site in southern Poland, further referred to as Waryski soil, was used to test indigenous plant species for stabilization effectiveness of heavy metals in soils. Results of pilot investigations with commercially available cultivars of plant species showed that these cultivars could not grow on this highly polluted soil even with the application of soil amendments to stabilize the heavy metals. Based on these results, mesocosm and field experiments with an indigenous, metal-tolerant ecotype of Deschampsia cespitosa from the Waryñski site were carried out. The mesocosm experiment showed that applications of calcium phosphate (3.8% w/w) as a heavy metal-stabilizing amendment decreased Cd and Zn concentrations 2 and 3-fold respectively in leachates, whereas lead content was not significantly changed. This decrease in the concentration of heavy metals in leachates was correlated with a lower accumulation of Pb, Cd and Zn in the roots and shoots of D. cespitosa, ecotype Waryñski. In the field experiment, lower accumulations of Cd in roots and shoots and Zn in shoots in the amendment added plot were observed during the second year of investigations. In the first growing season, D. cespitosa plant cover in the amendment enriched mesocosms ranged from 95 to 100%, compared to 10% in mesocosms without calcium phosphate. In the second year of the experiment, in non-amendment enriched mesocosms D. cespitosa was substituted with Cardaminopsis arenosa(95% cover). C. arenosa is an undesirable species for phytostabilization, as it accumulates high amounts of zinc and cadmium in its shoots, even thought it provided better growth cover in not amended soils. However, in amended mesocosms, soil surface cover by D. cespitosa was still very high (90%). Similar results were obtained in field experiments. Addition of calcium phosphate to the soil also resulted in excellent D. cespitosa root system development when compared to soils without amendment. In amended mesocosms, high plant cover and root system development significantly decreased the volume of leachates and improved water retention. These results indicate that the use of D. cespitosa, ecotype Waryski in combination with calcium phosphate as a heavy metals immobilizing agent is sufficient to restore a dense vegetative cover to highly heavy metal-polluted soil.     

The accumulation and intracellular compartmentation of cadmium,lead, zinc and calcium in two earthworm species (Dendrobaena rubida and Lumbricus rubellus) living in highly contaminated soil

Summary The earthworm Lumbricus rubellus contained more Ca and Zn, and less Pb and Cd, than Dendrobaena rubida living in the same contaminated disused-mine soil. Differences in the kinetics of Ca turnover may account for some of the inter-specific differences in heavy metal burdens, although the calciferous glands do not seem to be directly involved in heavy metal excretion. A comparison of the present findings with published data indicated that the concentration of soil Ca and the bioavailability of heavy metals, both factors being allied to soil pH, are important exogenous determinants of heavy metal accumulation by different earthworm populations. Electron microprobe X-ray analysis of air-dried smears of chloragogenous tissue showed that the metals were fairly specifically compartmentalized into two distinct organelles in both worms: Ca, Pb and Zn were found (associated with P) in the chloragosomes; Cd was found (with S and probably in stoichiometric association) in a more electron-lucent vesicular component, designated the cadmosome, but which may be identical with the debris vesicles which are characteristic inclusions in conventionally-fixed chloragocytes. The in vivo incorporation of Pb by the chloragosomes of D. rubida was accompanied by the loss of Ca, Zn and P.     

Heavy-metal uptake and distribution in Silene vulgaris and Minuartia verna growing on mining-dump material containing lead and zinc

Plant and soil samples from a mining area in Carinthia (Austria) were investigated for their heavy metal content. In the soil surrounding roots of plants (Minuartia verna and Silene vulgaris) growing on the mining dumps, high concentrations of lead and zinc are to be expected. The two species (Minuartia and Silene) show very different heavy metal concentrations in their above- and belowground organs. From these observations it can be concluded that the divergent distribution of heavy metals within the plants is an important mechanism of tolerance to heavy metals.     

Bioremediation of Heavy Metals in Liquid Media Through Fungi Isolated from Contaminated Sources

Wastewater particularly from electroplating, paint, leather, metal and tanning industries contain enormous amount of heavy metals. Microorganisms including fungi have been reported to exclude heavy metals from wastewater through bioaccumulation and biosorption at low cost and in eco-friendly way. An attempt was, therefore, made to isolate fungi from sites contaminated with heavy metals for higher tolerance and removal of heavy metals from wastewater. Seventy-six fungal isolates tolerant to heavy metals like Pb, Cd, Cr and Ni were isolated from sewage, sludge and industrial effluents containing heavy metals. Four fungi (Phanerochaete chrysosporium, Aspegillus awamori, Aspergillus flavus, Trichoderma viride) also were included in this study. The majority of the fungal isolates were able to tolerate up to 400 ppm concentration of Pb, Cd, Cr and Ni. The most heavy metal tolerant fungi were studied for removal of heavy metals from liquid media at 50 ppm concentration. Results indicated removal of substantial amount of heavy metals by some of the fungi. With respect to Pb, Cd, Cr and Ni, maximum uptake of 59.67, 16.25, 0.55, and 0.55 mg/g was observed by fungi Pb3 (Aspergillus terreus), Trichoderma viride, Cr8 (Trichoderma longibrachiatum), and isolate Ni27 (A. niger) respectively. This indicated the potential of these fungi as biosorbent for removal of heavy metals from wastewater and industrial effluents containing higher concentration of heavy metals.     

The performance of vetivers (Chrysopogon zizaniodes and Chrysopogon nemoralis) on heavy metals phytoremediation: laboratory investigation

Abstract

Phytoremediation with vetiver was investigated in relation to heavy metal contaminated soil in Thailand. The work compared the performance of two species of vetiver named Songkhla 3 (Chrysopogon zizaniodes) and Prachuap Khiri Khan (Chrysopogon nemoralis) in absorbing lead, zinc, and cadmium in contaminated soils. Toxicity Characteristic Leaching Procedure (TCLP), and Allium tests were conducted to determine toxicity of treated soil. Ethylenediaminetetraacetic acid (EDTA) was also used to increase heavy metals concentration in solution in soil, which led to an increase in translocation and bioaccumulation factors. In general, results showed that concentration of heavy metals decreased in soil and increased in both the shoots and roots of vetivers during a 4-month treatment period. TCLP results indicated that the concentration of zinc and cadmium in contaminated soil was reduced over treatment time, and significantly increased after EDTA was applied. To confirm vetiver performance in phytoremediation, Allium testing showed that remained heavy metals in treated soils had no effect on nucleus aberration. Songkhla 3 and Prachuap Khiri Khan showed similar trends in their ability to remediate lead, zinc, and cadmium from contaminated soil. Both species could accumulate higher concentrations of heavy metals in their shoots and roots over time, and with EDTA application.     

From industrial sites to environmental applications with <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>

Cupriavidus metallidurans CH34 and related strains are adapted to metal contaminated environments. A strong resistance to environmental stressors and adaptation make it ideal strains for survival in decreasing biodiversity conditions and for bioaugmentation purposes in environmental applications. The soil bacterium C. metallidurans is able to grow chemolithoautotrophically on hydrogen and carbon dioxide allowing a strong resilience under conditions lacking organic matter. The biofilm growth on soil particles allows coping with starvation or bad conditions of pH, temperature and pollutants. Its genomic capacity of two megaplasmids encoding several heavy metal resistance operons allowed growth in heavy metal contaminated habitats. In addition its specific siderophores seem to play a role in heavy metal sequestration besides their role in the management of bioavailable iron. Efflux ATPases and RND systems pump the metal cations to the membrane surface where polysaccharides serve as heavy metal binding and nucleation sites for crystallisation of metal carbonates. These polysaccharides contribute also to flotation under specific conditions in a soil-heavy metals–bacteria suspension mixture. An inoculated moving bed sand filter was constructed to treat heavy metal contaminated water and to remove the metals in the form of biomass mixed with metal carbonates. A membrane based contactor allowed to use the bacteria as well in a versatile wastewater treatment system and to grow homogeneously formed heavy metal carbonates. Its behaviour toward heavy metal binding and flotation was combined in a biometal sludge reactor to extract and separate heavy metals from metal contaminated soils. Finally its metal-induced heavy metal resistance allowed constructing whole cell heavy metal biosensors which, after contact with contaminated soil, waste, solids, minerals and ashes, were induced in function of the bioavailable concentration (Cd, Zn, Cu, Cr, Co, Ni, Tl, Pb and Hg) in the solids and allowed to investigate the speciation of immobilization of those metals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of Cu,Pb and Zn on two Potamogeton species grown under field conditions

Two common macrophyte species, Potamogeton perfoliatus L. and Potamogeton pectinatus L. were grown for 12 weeks at shallow depths in sediments contaminated with 1250 or 2500 g Pb or Cu and/or Zn (gDW sediment)^-1. Control experiments were run at background levels of 4, 13, and 38 g Pb, Cu and Zn (gDW sediment)^-1, respectively. Effects of heavy metals on biomass production and metal uptake and distribution in plants are presented in relation to total amount and plant-available fraction of metals in the sediment.All three studied metals gave reduced biomass production, and the toxicity of the metals decreased in the order Zn>Cu>Pb. The root/shoot biomass ratio increased for P. pectinatus, but decreased for P. perfoliatus with metal treatment. The content of any single metal was higher in shoots than in roots of plants grown on sediments not contaminated with that specific metal, but addition of that metal increased the proportion in roots. The uptake by plants of any of the heavy metals increased with increased metal addition. The magnitude of the plant-available fraction of metals of untreated sediment was Zn>Cu>Pb, and increased in contaminated sediments. Addition of Cu decreased both the plant-available fraction and the total concentration of Zn in the sediment, while increased the uptake of Zn by the plants. The opposite was found for Cu when Zn was added. P. pectinatus accumulated about twice as much Cu as P. perfoliatus. On the other hand, the concentration of Pb was higher in P. perfoliatus than in P. pectinatus, and was negligible in P. pectinatus when cultivated in untreated sediments.     

Tolerance of pesticides and antibiotic resistance in bacteria isolated from wastewater-irrigated soil

A total of 64 bacterial isolates (40 Pseudomonas spp., 12 Azotobacter and 12 Rhizobium spp.) were characterized on the basis of morphological, cultural and biochemical characteristics. All the isolates were tested for their tolerance to the pesticides endosulfan, carbofuran, and malathion. 12.5% of the Pseudomonas isolates from soil tolerated concentrations of 1600 g malathion ml whereas 7.5% of isolates tolerated the same concentration of carbofuran. However, Pseudomonas isolates demonstrated a tolerance limit to endosulfan at a concentration of 800 g/ml. Asymbiotic N₂-fixers (Azotobacter) and symbiotic N₂-fixers (Rhizobium spp.) were also able to tolerate concentrations of pesticides up to 1600 g/ml. All the isolates were further tested for their antibiotic susceptibility against seven different antibiotics, nalidixic acid, cloxacillin, chloramphenicol, tetracycline, amoxycillin, methicillin, doxycycline. 100% of the Pseudomonas isolates were resistant to cloxacillin and 57.5% were resistant to methicillin. 7.5% of the isolates exhibited multiple resistance to five different antibiotics in three different combinations whereas 25% of the isolates showed multiple resistance to four different antibiotics in seven different combinations. Some of the resistant isolates were also screened for plasmid DNA and found to harbour a single plasmid.     

Heavy metal tolerance and accumulation of Cd, Cr and Ni by Cannabis sativa L.

Experiments in semi-natural conditions were undertaken to assess hemp metal tolerance and its ability to accumulate cadmium, nickel and chromium. Cannabis sativa was grown in two soils, S1 and S2, containing 27, 74, 126 and 82, 115, 139 g g^–1 of Cd, Ni and Cr, respectively. After two months from germination and at ripeness, no significant alteration in plant growth or morphology was detected. On the contrary, a high hemp reactivity to heavy metal stress with an increase in phytochelatin and DNA content was observed during development, suggesting the Cannabis sativa ability to avoid cell damage by activating different molecular mechanisms. Metals were preferentially accumulated in the roots and only partially translocated to the above-ground tissues. The mean shoot Cd content was 14 and 66 g g^–1 for S1 and S2 soil, respectively. Although not negligible concentrations they were about 100 times lower than those calculated for the hyperaccumulator Thlaspi caerulescens. Similarly Ni uptake was limited if compared with that of the Ni-hyperaccumulator Alyssum murale. Chromium uptake was negligible. As expected on the base of the metal concentration detected in ripe plants, no statistically significant variation in soil metal content was detected after one crop of hemp. Nevertheless, a consistent amount (g) of Cd and Ni is expected to be extracted by 1 ha biomass of hemp (about 10 t) per year and along the time a slow restoration of deeper soil portions can be obtained by its wide root system (at least 0,5 m deep). In addition, the possibilities of growing hemp easily in different climates and using its biomass in non-food industries can make heavy metal contaminated soils productive. This means economical advantage along with a better quality of soil.     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.     

In vitro susceptibility ofTrichophyton rubrum isolates to griseofulvin and tioconazole. Induction and isolation of a resistant mutant to both antimycotic drugs

The in vitro susceptibility of three clinicalTrichophyton rubrum isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0.5 to 1.0g/ml, respectively. One mutant (gril) obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC > 2000g/ml) and tioconazole (MIC=1.0g/ml). The clinical importance and the possibility of a multidrug resistance (MDR)-type mechanism being involved in this event is discussed.     

EDTA-assisted heavy-metal uptake by poplar and sunflower grown at a long-term sewage-sludge farm

Little information is available concerning the efficacy of chelates applied to biosolids (sewage-sludge)-treated soil for heavy-metal removal. The purpose of the experiment was to determine the availability to sunflower (Helianthus annuus L.) and hybrid poplar (Populus deltoides Marsh. × P. nigra L.) seedlings, of non-essential (Cd, Ni, Pb) and essential heavy metals (Cu, Fe, Mn, Zn) in field soil injected with biosolids since 1976 and treated with ethylenediamine-tetraacetic acid (EDTA) in 2001. Sunflower was grown at two densities, 20000 and 60000 plants/ha, and poplar at 10000 plants/ha. The tetrasodium salt of EDTA was applied at rates of 0, 0.5, 1, and 2 g EDTA salt per kg surface (25-cm depth) soil. The EDTA did not affect uptake by poplar of the three non-essential (Cd, Ni, Pb) and four essential (Cu, Fe, Mn, Zn) heavy metals. For sunflower, the 1.0 g/kg rate of chelate addition resulted in maximal removal of the three non-essential heavy metals (Cd, Ni, Pb). Uptake of the essential heavy metals by sunflower was little affected by the EDTA. At the 20000 plants/ha density, leaves of sunflower grown with 1.0 g EDTA Na₄2H₂O per kg soil accumulated more Cd, Ni, and Pb than leaves of sunflower grown without the EDTA salt. At this density, concentrations of Cd in leaves of sunflower without EDTA and with 1.0 g/kg EDTA salt were 2.2 and 6.5 g/g, respectively; for Ni, they were 6.7 and 19.2 g/g, respectively; and for Pb, they were 15.6 and 46.9 g/g, respectively. At the 60000 plants/ha density, stems of sunflower grown with 1.0 g EDTA Na₄2H₂O per kg soil accumulated more Cd, Ni, and Pb than stems of sunflower grown without the EDTA salt. At this density, concentrations of Cd in stems of sunflower without EDTA and with 1.0 g/kg EDTA salt were 0.6 and 4.6 g/g, respectively; for Ni, they were 1.7 and 17.6 g/g, respectively; and for Pb, they were 5.2 and 42.8 g/g, respectively. Removal of the non-essential heavy metals by sunflower was greater at the higher plant density (60000 plants/ha) compared to the lower one (20000 plants/ha).     

Response of ectomycorrhizalPinus banksiana andPicea glauca to heavy metals in soil

Pinus banksiana andPicea glauca inoculated or not with the ectomycorrhizal fungusSuillus luteus were grown in a sandy loam soil containing a range of Cd, Cu, Ni, Pb and Zn concentrations. Ectomycorrhizal colonization rates were significantly reduced on Pinus and Picea seedlings by the heavy metals, particularly Cd and Ni. Needle tissue metal concentrations were lower in ectomycorrhizal seedlings at low soil metal concentrations. However, at higher soil concentrations, heavy metal concentrations of needle tissue were similar in ectomycorrhizal and nonmycorrhizal plants. The growth of nonmycorrhizal seedlings exposed to heavy metals was reduced compared to those inoculated withSuillus luteus. Apparently ectomycorrhizal colonization can protect Pinus and Picea seedlings from heavy metal toxicity at low or intermediate soil concentrations of Cd, Cu, Ni, Pb and Zn.