Effect of the high polycyclic aromatic hydrocarbon, benzo[a]pyrene, on the lipid content of Fusarium solani

The purpose of the present paper was to study the effect of the high polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, on the lipid [fatty acid (FA) and sterol] composition and content of the fungi Fusarium solani and F. oxysporum, respectively recognized as good and poor PAH degraders. The major FAs and the major sterol that characterized the tested Fusarium strains were C16:0, C18:1, C18:2, and ergosterol. Lipid profiles of F. solani remained unchanged with the addition of benzo[a]pyrene in the culture media at all concentrations and duration of treatment. However, in the presence of benzo[a]pyrene, significant decreases in FA content, which reached 18 % in young cultures and 28 % in mature colonies, were registered. Similarly, the sterol content of F. solani was reduced by 27 % in the presence of benzo[a]pyrene. In contrast, no modification in lipid profile and lipid content were observed with F. oxysporum, a strain recognized as a low benzo[a]pyrene degrader.     

Element distribution in mycorrhizal and nonmycorrhizal roots of the halophyte Aster tripolium determined by proton induced X-ray emission

Summary. The salt aster (Aster tripolium L.) colonized by the arbuscular mycorrhizal fungus Glomus intraradices Sy167 and noncolonized control plants were grown in a greenhouse for nine months with regular fertilization by Hoagland nutrient solution supplemented with 2% NaCl. Mycorrhizal roots showed a high degree of mycorrhizal colonization of 60–70% and formed approximately 25% more dry weight and much less aerenchyma than the nonmycorrhizal controls. Cryosectioning essentially preserved the root cell structures and apparently did not cause significant ion movements within the roots during cuttings. The experimental conditions, however, did not allow to discriminate between fungal and plant structures within the roots. Quantification of proton-induced X-ray emission (PIXE) data revealed that in control roots, Na⁺ was mainly concentrated in the outer epidermal and exodermal cells, whereas the Cl^– concentration was about the same in all cells of the roots. Cross sections of roots colonized by the mycorrhizal fungus did not show this Na1 gradient in the concentration from outside to inside but contained a much higher percentage of NaCl among the elements determined than the controls. PIXE images are also presented for the four other elements K, P, S, and Ca. Both in colonized and control roots, the concentration of potassium was high, probably for maintaining homoeostasis under salt stress. This is seemingly the first attempt to localize both Na⁺ and Cl^– in a plant tissue by a biophysical method and also demonstrates the usefulness of PIXE analysis for such kind of investigation.     

Polyploidy in tomato roots as affected by arbuscular mycorrhizal colonization

Nuclear changes in roots of tomato (Lycopersicon esculentum), a plant with a small genome, during the establishment of arbuscular mycorrhizal (AM) colonization were studied using light and electron microscopy, as well as flow and static cytometry. Nuclei of mycorrhizal root cortex cells were larger and had more decondensed chromatin than those of controls. Significant ploidy distribution differences were observed between nuclei of AM colonized and control roots, and a strong correlation between nuclear polyploidization and AM colonization was found. Polyploidization and decondensation are usually associated with high metabolic activity. The metabolic activity of mycorrhizal root cells, evaluated in this work as respiratory activity by using a cytochemical assay for succinate dehydrogenase combined with image analysis, increased in comparison to controls. The meaning of polyploidization is discussed in relation to the structural and metabolic modifications induced by mycorrhization.     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Localization of the yellow pigment formed in roots of gramineous plants colonized by arbuscular fungi

Summary Many plants form yellow coloured roots when colonized by arbuscular mycorrhizal (AM) fungi. In maize, a yellow pigment is first visible as small droplets in parenchyma cells of roots in the vicinity of arbuscules, 3–4 weeks after mycorrhizal colonization. During the course of the development of the plants, the yellow pigment spreads all over the cells of the cortex (with the exception of the exodermis) and of the endodermis, whereas the other stelar elements remain uncoloured. Other gramineous plants (wheat, barley, millet) show the same pattern of pigment formation. In contrast, the deposition of this pigment is not detected in roots ofTagetes, garden bean, onion, or leek. Weak yellow fluorescence is also seen in the fungal structures, particularly in the arbuscules of the investigated probes. This is, however, clearly different from the intense yellow colour of the pigment formed in root cells of grasses. The yellow pigment is even detected in such cells which are never colonized by fungal structures (e.g., endodermal cells). A major constituent of the yellow pigment of AM-colonized root cells has been identified as a carotenoid with 14 carbon atoms and two carboxylic groups and termed mycorradicin. This carotenoid is likely deposited in the vacuoles of root cells as a result of the colonization specifically by arbuscular fungi.     

Effects of NaCl and Mycorrhizal Fungi on Antioxidative Enzymes in Soybean

The effects of different concentrations of NaCl on the activities of antioxidative enzymes in the shoots and roots of soybean (Glycine max [L.] Merr cv. Pershing) inoculated or not with an arbuscular mycorrhizal fungus, Glomus etunicatum Becker & Gerdemann, were studied. Furthermore, the effect of salt acclimated mycorrhizal fungi on the antioxidative enzymes in soybean plants grown under salt stress (100 mM NaCl) was investigated. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased in the shoots of both mycorrhizal (M) and nonmycorrhizal (NM) plants grown under NaCl salinity. Salinity increased SOD activity in the roots of M and NM plants, but had no effect on CAT and polyphenol oxidase activities in the roots. M plants had greater SOD, POD and ascorbate peroxidase activity under salinity. Under salt stress, soybean plants inoculated with salt pre-treated mycorrhizal fungi showed increased SOD and POD activity in shoots, relative to those inoculated with the non pre-treated fungi.     

Effects of AM colonization on “wild tobacco” plants grown in zinc-contaminated soil

This greenhouse study aimed to determine the effect of colonization by the arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) on the “wild” tobacco (Nicotiana rustica L. var. Azteca), under soil–zinc (Zn) conditions. Plants of N. rustica were grown in AM or non-AM inoculated substrate and subjected to four soil–[Zn] concentrations (0, 50, 100, and 250 mg Zn kg⁻¹ dry soil). The AM root colonization increased markedly from 14 to 81% with the increasing soil–[Zn] and the mycorrhizal structures were significantly more abundant at the highest soil–[Zn], suggesting that Zn may be involved directly or indirectly in AM root colonization. In addition, total Zn content or Zn concentrations in shoots and roots were shown to increase as soil–[Zn] increased in both AM and non-AM plants. As for the growth parameters studied, there were no significant differences between treatments despite the increase in Zn content or concentration. The AM roots subjected to the highest soil–[Zn] had a significant reduction by about 50% of total Zn content and Zn concentration compared to non-AM roots. Still, the relative extracted Zn percentage decreased dramatically as soil–[Zn] increased. Soil pH was significantly lower in non-AM than AM treatments at the highest soil–[Zn]. In summary, AM plants (particularly roots) showed lower Zn content and concentration than non-AM plants. In this regard, the AM fungi have a protective role for the host plant, thus playing an important role in soil-contaminant immobilization processes; and, therefore, are of value in phytoremediation, especially when heavy metals approach toxic levels in the soil.     

Effectiveness of three Glomus species in protecting pepper (Capsicum annuum L.) against verticillium wilt

The objective of this work was to study the influence of three Glomus species—Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, Glomus intraradices (Schenck and Smith) and Glomus deserticola (Trappe, Bloss, and Menge)—on the development of Verticillium-induced wilt in Capsicum annuum cv. Piquillo. Results showed that the effectiveness of arbuscular mycorrhizal fungi (AMF) as biocontrol agents varied among different Glomus species. In pepper colonized by G. intraradices the severity of the disease was even higher than that observed in non-mycorrhizal plants in terms of plant growth and pepper yield. On the other hand, the high effectiveness exhibited by G. mosseae in improving plant growth and the early beginning of the reproductive stage in these plants was not associated with great plant protection and high pepper yield in diseased plants. Only plants associated with G. deserticola had greater yield than non-mycorrhizal ones despite the lower P fertilization applied to the mycorrhizal treatment and this fact was observed in both healthy and diseased plants. It is suggested that the higher specific phosphorus uptake in Verticillium-inoculated plants associated with G. deserticola could contribute to diminish the deleterious effect of pathogen on yield. On the other hand, the possible influence of endogenous phenolics in roots on the tolerance or resistance of pepper against wilt induced by Verticillium dahliae remains unclear.     

Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14

A fungal strain BAP14 isolated from marine sediments of coast in Xiamen city, was found to have the ability to degrade benzo[a]pyrene (BaP), and identified as Aspergillus sp. based on 18S rRNA gene sequence. Aspergillus sp. BAP14 was able to remove about 30 and 60% of BaP with initial concentration of 10 mg l⁻¹ in 3 and 12 days of incubation, respectively. Addition of saccharides and low molecular weight polycyclic aromatic hydrocarbons appeared to have effect on the degradation ability, in particularly the addition of lactose and naphthalene. Furthermore, we demonstrated that lipidic particles could be observed in the presence of benzo[a]pyrene based on the morphologic performance of Aspergillus sp. BAP14 through scanning electronic microscopy (SEM) and atomic force microscopy (AFM), respectively.     

Arbuscular mycorrhiza alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae)

The effect of root colonization by Glomus mosseae on the qualitative and quantitative pattern of essential oils (EO) was determined in three oregano genotypes (Origanum sp.). To exclude a simple P-mediated effect through mycorrhization the effect of P application to plants on the EO accumulation was also tested. In two genotypes the leaf biomass was increased through mycorrhization. Root colonization by the arbuscular mycorrhizal fungus (AMF) did not have any significant effect on the EO composition in oregano; however, in two genotypes the EO concentration significantly increased. As EO levels in P-treated plants were not enhanced, we conclude that the EO increase observed in mycorrhizal oregano plants is not due to an improved P status in mycorrhizal plants, but depends directly on the AMF–oregano plant association.     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).     

Extraradical mycelium network of arbuscular mycorrhizal fungi allows fast colonization of seedlings under in vitro conditions

Actively growing extraradical hyphae extending from mycorrhizal plants are an important source of inoculum in soils which has seldom been considered in vitro to inoculate young plantlets. Seedlings of Medicago truncatula were grown in vitro in the extraradical mycelium network extending from mycorrhizal plants. After 3, 6, 9, 12, and 15 days of contact with the mycelium, half of the seedlings were harvested and analyzed for root colonization. The other half was carefully transplanted in vitro on a suitable growth medium and mycelium growth and spore production were evaluated for 4 weeks. Seedlings were readily colonized after 3 days of contact with the mycelium. Starting from 6 days of contact, the newly colonized seedlings were able to reproduce the fungal life cycle, with the production of thousands of spores within 4 weeks. The fast mycorrhization process developed here opens the door to a broad range of in vitro studies for which either homogenous highly colonized seedlings or mass-produced in vitro inoculum is necessary. Liesbeth Voets and Ivan Enrique de la Providencia contributed equally to this work. MUCL is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

Effects of anthracene on development of an arbuscular mycorrhizal fungus and contribution of the symbiotic association to pollutant dissipation

The influence of anthracene, a low molecular weight polycyclic aromatic hydrocarbon (PAH), on chicory root colonization by Glomus intraradices and the effect of the root colonization on PAH degradation were investigated in vitro. The fungus presented a reduced development of extraradical mycelium and a decrease in sporulation, root colonization, and spore germination when exposed to anthracene. Mycorrhization improved the growth of the roots in the medium supplemented containing 140 mg l⁻¹ anthracene, suggesting a positive contribution of G. intraradices to the PAH tolerance of roots. Anthracene disappearance from the culture medium was quantified; results suggested that nonmycorrhizal chicory roots growing in vitro were able to contribute to anthracene dissipation, and in addition, that mycorrhization significantly enhanced anthracene dissipation. These monoxenic experiments demonstrated a positive contribution of the symbiotic association to anthracene dissipation in the absence of other microorganisms. In addition to anthracene dissipation, intracellular accumulation of anthracene was detected in lipid bodies of plant cells and fungal hyphae, indicating intracellular storage capacity of the pollutant by the roots and the mycorrhizal fungus.     

Simple luminescence method for estimation of benzo[a]pyrene in a complex mixture of polycyclic aromatic hydrocarbons without a pre‐separation procedure

Benzo[a]pyrene causes cancer at cellular level and is widely present in the environment. Conventional spectroscopic methods for analysis of this compound need a pre‐separation procedure due to severe spectral overlap from other polycyclic aromatic hydrocarbons. We report a simple method that avoids spectral overlap of benzo[a]pyrene from other impurities or polycyclic aromatic hydrocarbons (PAHs), thus it can easily identify benzo[a]pyrene in a complex PAH mixture. The method could easily identify benzo[a]pyrene in an 18‐component PAH mixture. Calibration plots in methanol solution and in micellar media show a good linearity (R > 0.9997) in the benzo[a]pyrene concentration range generally found in the environment. The method gives a detection limit of 1.52 × 10^?9 mol/L in CTAB micellar medium and 2.55 × 10^?9 mol/L in methanol solution. The proposed method is selective, sensitive and fast. The fluorescence response of benzo[a]pyrene is found to be a potential candidate to sense the critical micellar concentration (CMC) of CTAB micelles. Copyright © 2003 John Wiley & Sons, Ltd.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Contribution of an arbuscular mycorrhizal fungus to the uptake of cadmium and nickel in bean and maize plants

Two experiments were carried out in pots with three compartments, a central one for root and hyphal growth and two outer ones which were accessible only for hyphae of the arbuscular mycorrhizal fungus, Glomus mosseae ([Nicol. and Gerd.] Gerdemann and Trappe). In the first experiment, mycorrhizal and nonmycorrhizal bean (Phaseolus vulgaris L.) plants were grown in two soils with high geogenic cadmium (Cd) or nickel (Ni) contents. In the second experiment, mycorrhizal and nonmycorrhizal maize (Zea mays L.) or bean plants were grown in a non-contaminated soil in the central compartment, and either the Cd- or Ni-rich soil in the outer compartments. In additional pots, mycorrhizal plants were grown without hyphal access to the outer compartments. Root and shoot dry weight was not influenced by mycorrhizal inoculation, but plant uptake of metals was significantly different between mycorrhizal and nonmycorrhizal plants. In the first experiment, the contribution of mycorrhizal fungi to plant uptake accounted for up to 37% of the total Cd uptake by bean plants, for up to 33% of the total copper (Cu) uptake and up to 44% of the total zinc (Zn) uptake. In contrast, Ni uptake in shoots and roots was not increased by mycorrhizal inoculation. In the second experiment, up to 24% of the total Cd uptake and also up to 24% of the total Cu uptake by bean could be attributed to mycorrhizal colonisation and delivery by hyphae from the outer compartments. In maize, the mycorrhizal colonisation and delivery by hyphae accounted for up to 41% of the total Cd uptake and 19% of the total Cu uptake. Again, mycorrhizal colonisation did not contribute to Ni uptake by bean or maize. The results demonstrate that the arbuscular mycorrhizal fungus contributed substantially not only to Cu and Zn uptake, but also to uptake of Cd (but not Ni) by plants from soils rich in these metal cations. Deceased 21 September 1996 Deceased 21 September 1996     

Field response of wheat to arbuscular mycorrhizal fungi and drought stress

Mycorrhizal plants often have greater tolerance to drought than nonmycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on growth, grain yield and mineral acquisition of two winter wheat (Triticum aestivum L.) cultivars grown in the field under well-watered and water-stressed conditions. Wheat seeds were planted in furrows after treatment with or without the AM fungi Glomus mosseae or G. etunicatum. Roots were sampled at four growth stages (leaf, tillering, heading and grain-filling) to quantify AM fungi. There was negligible AM fungi colonization during winter months following seeding (leaf sampling in February), when soil temperature was low. During the spring, AM fungi colonization increased gradually. Mycorrhizal colonization was higher in well-watered plants colonized with AM fungi isolates than water-stressed plants. Plants inoculated with G. etunicatum generally had higher colonization than plants colonized with G. mosseae under both soil moisture conditions. Biomass and grain yields were higher in mycorrhizal than nonmycorrhizal plots irrespective of soil moisture, and G. etunicatum inoculated plants generally had higher biomass and grain yields than those colonized by G. mosseae under either soil moisture condition. The mycorrhizal plants had higher shoot P and Fe concentrations than nonmycorrhizal plants at all samplings regardless of soil moisture conditions. The improved growth, yield and nutrient uptake in wheat plants reported here demonstrate the potential of mycorrhizal inoculation to reduce the effects of drought stress on wheat grown under field conditions in semiarid areas of the world.     

Growth and arsenic uptake by Chinese brake fern inoculated with an arbuscular mycorrhizal fungus

A split-root experiment investigated the effects of inoculation with the arbuscular mycorrhizal fungus Glomus mosseae and arsenic (As) addition on As uptake by Pteris vittata L. Either part or all of the root system was inoculated with G. mosseae or exposed to As addition (50 ml 1000 μmol L⁻¹ As 1 week before harvest). Mycorrhizal colonization substantially increased frond and root dry weight and P and As contents irrespective of As addition. Frond As contents in mycorrhizal plants were highest when the whole root system was exposed to As. Frond As concentrations and contents were higher when inoculation and As addition were in the same parts of the root system than when spatially separate. There were positive effects of arbuscular mycorrhiza inoculation on plant growth and As uptake, and inoculation of part of the roots seemed to be as effective as inoculation of the whole root system.