The sterol biosynthesis inhibitor molecule fenhexamid impacts the vegetative compatibility of Glomus clarum

The vegetative compatibility of the arbuscular mycorrhizal fungus (AMF) Glomus clarum MUCL 46238 was evaluated after continuous exposure to fenhexamid, a sterol biosynthesis inhibitor (SBI). Three lineages of this AMF were cultured in vitro for five generations in association with Ri T-DNA transformed carrot roots in the presence of 0, 5 or 10 mg l⁻¹ of fenhexamid. Whatever the AMF generation, fenhexamid at 5 and 10 mg l⁻¹ had no significant impact on the number of spores produced. However, vegetative compatibility tests (VCT) conducted with spores from the three lineages, in the presence of 10 mg l⁻¹ of fenhexamid, impacted the anastomosis process. At this concentration, the morphology of the germ tubes was modified. In addition, nitrotetrazolium–trypan blue staining revealed that 10 mg l^−l of fenhexamid significantly reduced the probability of fusion between the germ tubes regardless of the culture conditions (i.e. absence or presence of fenhexamid) preceding the VCT. Our results demonstrated that spore production was not affected by fenhexamid, while anastomosis between germ tubes was decreased. This suggested that high concentrations, accumulation or repeated application of this SBI fungicide may impact the community structure of AMF in soil.     

Fenpropimorph slows down the sterol pathway and the development of the arbuscular mycorrhizal fungus Glomus intraradices

The direct impact of fenpropimorph on the sterol biosynthesis pathway of Glomus intraradices when extraradical mycelia alone are in contact with the fungicide was investigated using monoxenic cultures. Bi-compartmental Petri plates allowed culture of mycorrhizal chicory roots in a compartment without fenpropimorph and exposure of extraradical hyphae to the presence of increasing concentrations of fenpropimorph (0, 0.02, 0.2, 2, 20 mg l⁻¹). In the fungal compartment, sporulation, hyphal growth, and fungal biomass were already reduced at the lowest fungicide concentration. A decrease in total sterols, in addition to an increase in the amount of squalene and no accumulation of abnormal sterols, suggests that the sterol pathway is severely slowed down or that squalene epoxidase was inhibited by fenpropimorph in G. intraradices. In the root compartment, neither extraradical and intraradical development of the arbuscular mycorrhizal (AM) fungus nor root growth was affected when they were not in direct contact with the fungicide; only hyphal length was significantly affected at 2 mg l⁻¹ of fenpropimorph. Our results clearly demonstrate a direct impact of fenpropimorph on the AM fungus by a perturbation of its sterol metabolism.     

Co-occurrence of Δ5- and Δ7 -sterols in the fungus Dictyuchus monosporus

The desmethyl sterol composition of the oomycete Dictyuchus monosporus is unusual in that it is a mixture of 56.9 % Δ⁵-sterols and 42.6 % Δ⁷-sterols. The Δ⁵-sterols are cholesterol, 24 methylenecholesterol and fucosterol; the Δ⁷-sterols are cholest-7-enol, ergosta-7,24(28)-dienol and stigmasta-7,E-24(28)-dienol. Stigmasta-7,E-24(28)-dienol, is identified for the first time from natural sources. In addition, traces of lanosterol are present.     

An in vitro propagation protocol of two submerged macrophytes for lake revegetation in east China

Thirty-six programs have been set up to revegetate the degraded lake wetlands in east China since 2002. Most projects however faced deficiency of submerged macrophyte propagules. To solve the problem, alternative seedling sources must be found besides traditional field collection. This paper deals with an in vitro propagation protocol for two popularly used submerged macrophytes, Myriophyllum spicatum L. and Potamogeton crispus L. Full strength Murashige and Skoog-based liquid media (MS) plus 3% sucrose in addition to 0–2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0–1.0 mg l⁻¹ indoleacetic acid (IAA) were tried for shoot regeneration. Meanwhile, full, half or quarter strength MS in addition to 0, 0.1 or 0.2 mg l⁻¹ naphthaleneacetic acid (NAA) were tested for root induction, respectively. Results indicated that both species had the ability of regeneration from stem fragments in MS without further regulators. However, the addition of 2.0 mg l⁻¹ BA with 0.2 or 1.0 mg l⁻¹ IAA in MS drastically stimulated the regeneration efficiency of M. spicatum, while the addition of 2.0 mg l⁻¹ BA with 0.2 or 0.5 mg l⁻¹ IAA in MS significantly stimulated that of P. crispus. For root induction, full strength MS in combination with 0.1or 0.2 mg l⁻¹ NAA was preferred by M. spicatum, and the same MS without or with 0.1 mg l⁻¹ NAA was preferred by P. crispus. Seedlings of each species produced from tissue culture room had a 100% survival rate on clay, sandy loam or their mixture (1:1) in an artificial pond, and phenotypic plasticity was exhibited when the nutrient levels varied among the three types of sediments. This acclimation of seedlings helped develop the shoot and root systems, which ensured seedling quality and facilitated the transplantation. Our study has established an effective protocol to produce high quality seedlings for lake revegetation programs at a larger scale. Since the two species we tested represent different regeneration performances in nature but shared similar in vitro propagation conditions, this study has indicated a potentially wide use of the common media for preparing seedlings of other submerged macrophytes.     

Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization

Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1¹ In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host.     

Arbuscular mycorrhiza partially protect chicory roots against oxidative stress induced by two fungicides, fenpropimorph and fenhexamid

The present work examined the oxidative stress induced by different concentrations (0.02 and 0.2 mg l-1) of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) in non-target chicory root colonised or not by Glomus intraradices in a monoxenic system. The fungicides were found to cause oxidative damage by increasing lipid peroxidation measured by malondialdehyde production in non-colonised roots. Detoxification of the H₂O₂ product was measured at 0.2 mg l-1 of fenpropimorph by an increase in peroxidase activities suggesting an antioxidant capacity in these roots. Moreover, this study pointed out the ability of arbuscular mycorrhiza to alleviate partially the oxidative stress in chicory roots, probably by lowering reactive oxygen species concentrations, resulting from increases in antioxidant defences. Our results suggest that the enhanced fungicide tolerance in the AM symbiosis could be related to less cell membrane damage.     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

Growth, reproductive and photosynthetic responses to copper in the yellow-horned poppy, Glaucium flavum Crantz.

Glaucium flavum Crantz. is found in an anthropized coastal grassland at the joint estuary of the Tinto and Odiel rivers (SW Spain), growing under the influence of high levels of copper contamination derived from nearby petrochemical industries, with no obvious adverse affects on the performance of the plant. In addition, this species exhibits a series of ecological characteristics which may render it appropriate for use in the phytoremediation of contaminated areas. Nonetheless, the response of G. flavum to elevated copper concentrations has not been studied. A greenhouse experiment was conducted to investigate the effects of a range of Cu concentrations (0 to 47 mmol l⁻¹) on the growth, reproduction and photosynthetic performance of G. flavum, by measuring relative growth rate, fruit and seed production, chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. We also determined total copper, nitrogen, phosphorous, sulphur, calcium and magnesium concentrations. G. flavum survived with concentrations of up to 730 mg Cu kg⁻¹ DW in the leaves, when treated with 30 mmol Cu l⁻¹ (2000 mg l⁻¹). Quantum efficiency of PSII, net photosynthesis rate, as well as leaf Ca and Mg concentrations were all negatively affected by Cu concentrations greater than 9 mmol l⁻¹ in the nutrient solution. Our results indicate that the reduction in photosynthetic performance may be attributed to the adverse effect of excess Cu on the photosynthetic apparatus of the plant, both directly, via a decrease in pigment concentrations, and indirectly, via interference of Cu with Ca ions of PSII. Growth and seed production were only slightly affected by leaf tissue concentrations as high as 230 mg Cu kg⁻¹ dry mass, which suggests that this species could play an important role in phytoremediation of Cu-contaminated soils.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

Calcareous impact on arbuscular mycorrhizal fungus development and on lipid peroxidation in monoxenic roots

The present work underlined the negative effects of increasing CaCO₃ concentrations (5, 10 and 20 mM) both on the chicory root growth and the arbuscular mycorrhizal fungus (AMF) Glomus irregulare development in monoxenic system. CaCO₃ was found to reduce drastically the main stages of G. irregulare life cycle (spore germination, germinative hyphae elongation, root colonization, extraradical hyphae development and sporulation) but not to inhibit it completely. The root colonization drop was confirmed by the decrease in the arbuscular mycorrhizal fungal marker C16:1ω5 amounts in the mycorrhizal chicory roots grown in the presence of CaCO₃. Oxidative damage evaluated by lipid peroxidation increase measured by (i) malondialdehyde (MDA) production and (ii) the antioxidant enzyme peroxidase (POD) activities, was highlighted in chicory roots grown in the presence of CaCO₃. However, MDA formation was significantly higher in non-mycorrhizal roots as compared to mycorrhizal ones. This study pointed out the ability of arbuscular mycorrhizal symbiosis to enhance plant tolerance to high levels of CaCO₃ by preventing lipid peroxidation and so less cell membrane damage.     

Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8 mg/50 ml culture) and exposure time (24, 48, 72, 96 and 120 h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89 mg g⁻¹ DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24 h in which the silymarin content was 2.42 times higher than the control (0.78 mg g⁻¹ DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863 mg g⁻¹ DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24 h after treatment by ∼1.57- fold (0.21 Δ OD₂₃₄/mg protein min⁻¹). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26 mg g⁻¹ DW) were also elevated after 24 h, in which the linoleic acid content was 2.37 times higher than the control (16.1 mg g⁻¹ DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.     

Biodegradation of phenanthrene and pyrene by Ganoderma lucidum

Biodegradation of two polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by a white rot fungus, Ganoderma lucidum, in broth cultures was investigated. It was found that the biomass of the organism decreased with the increase of PAH concentration in the cultures. In the cultures with 2 to 50 mg l⁻¹ PAHs, the degradation rate constants (k₁) increased with the PAH concentration, whereas, at the level of 100 mg l⁻¹, the degradation rate constants decreased. In the presence of 20 mg l⁻¹ PAHs, the highest degradation rates of both PAHs occurred in cultures with an initial pH of 4.0 at 30 °C. The addition of CuSO₄, citric acid, gallic acid, tartaric acid, veratryl alcohol, guaiacol, 2,2′-azino-bis-(3- ethylbenzothazoline-6-sulfonate) (ABTS) enhanced the degradation of both PAHs and laccase activities; whereas the supplement of oxalate, di-n-butyl phthalate (DBP), and nonylphenol (NP) decreased the degradation of both PAHs and inhibited laccase production. In conclusion, G. lucidum is a promising white rot fungus to degrade PAHs such as phenanthrene and pyrene in the environment.     

Accumulation of Δ8,14-sterols in suspension cultures of bramble cells cultured with an azasterol antimycotic agent (A25822B)

Bramble suspension cultures normally contain Δ⁵-sterols (sitosterol, campesterol and isofucosterol). When the cells were grown in a medium supplemented with 15-aza-24-methylene-d-homocholesta-8,14-dien- 3β-ol, Δ⁵-sterols disappeared almost completely whereas Δ^8,14-sterols accumulated strongly. Five Δ^8,14-sterols, including two new compounds, (24R)-24-ethyl-5α-cholesta-8,14-dien-3β-ol and 4α-methyl-5α-stigmasta-8,14, Z-24(28)-trien-3β-ol, were identified. The 15-azasterol probably inhibited the reduction of the Δ¹⁴-bond. Cell lines growing permanently in an azasterol-supplemented medium were obtained.     

Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1

Harvesting biohydrogen from inhibiting wastewaters is of practical interest since the toxicity of compounds in a wastewater stream commonly prevents the bioenergy content being recovered. The isolated Clostridium sp. R1 is utilized to degrade cellobiose in sulfide or nitrite-containing medium for biohydrogen production. The strain can effectively degrade cellobiose free of severe inhibitory effects at up to 200 mg l⁻¹ sulfide or to 5 mg l⁻¹ nitrite, yielding hydrogen at >2.0 mol H₂ mol⁻¹ cellobiose. Principal metabolites of cellobiose fermentation are acetate and butyrate, with the concentration of the former increases with increasing sulfide and nitrite concentrations. The isolated strain can yield hydrogen from cellobiose in sulfide-laden wastewaters. However, the present of nitrite significantly limit the efficiency of the biohydrogen harvesting process.     

Actinoplanes utahensis ZJB-08196 fed-batch fermentation at elevated osmolality for enhancing acarbose production

Acarbose, a potent α-glucosidase inhibitor, is as an oral anti-diabetic drug for treatment of the type two, noninsulin-dependent diabetes. Actinoplanes utahensis ZJB-08196, an osmosis-resistant actinomycete, had a broad osmolality optimum between 309 mOsm kg⁻¹ and 719 mOsm kg⁻¹. Utilizing this unique feature, an fed-batch culture process under preferential osmolality was constructed through intermittently feeding broths with feed medium consisting of 14.0 g l⁻¹ maltose, 6.0 g l⁻¹ glucose and 9.0 g l⁻¹ soybean meal, at 48 h, 72 h, 96 h and 120 h. This intermittent fed-batch culture produced a peak acarbose titer of 4878 mg l⁻¹, increased by 15.9% over the batch culture.     

Competition between Salvinia minima and Spirodela polyrhiza mediated by nutrient levels and herbivory

We investigated the effects of initial biomass, nutrients, herbivory, and competition with Spirodela polyrhiza (L.) Schleid on Salvinia minima Baker biomass and density. S. minima populations were subjected to two levels of herbivory (control vs. two adults per plant) from the weevil Cyrtobagous salviniae Calder and Sands and eight levels of competition from S. polyrhiza, while growing in high (5 mg N l⁻¹) or low (0.5 mg N l⁻¹) nutrient conditions. Herbivory was the most important factor in S. minima biomass production while competition or fertility had no measurable impact. In contrast, S. polyrhiza biomass was mediated primarily by nutrients, not competition. There was no herbivory treatment for this plant. S. polyrhiza was superior to S. minima at converting nutrients to biomass but this did not give it a competitive advantage since S. minima biomass was unchanged when herbivory was absent. S. minima can generally overtop S. polyrhiza which, in turn, can form multiple layers within its mat. These characteristics may act to lessen competition between these species, thereby permitting their habitat sharing.     

H2O2参与AM真菌与烟草共生过程

以烟草((Nicotiana tabacum,品种CF90NF)为寄主,苗期接种丛枝菌根(AM)真菌摩西球囊霉(Glomus mosseae,G.m),测定G.m与烟草共生过程中烟草根部H2O2含量以及多胺氧化酶(PAO)和过氧化物酶(POD)活性;研究外源H2O2对G.m侵染烟草的影响以及H2O2清除剂和合成抑制剂对烟草侧根H2O2含量及烟草侧根和菌丝中H2O2荧光强度的影响,以探究H2O2在AM真菌侵染烟草过程中的作用。结果表明,接种G.m 20d后烟草侧根中出现H2O2含量的猝发,一定浓度的外源H2O2促进G.m对烟草的侵染,而H2O2清除剂抗坏血酸(AsA)显著削弱烟草侧根和菌丝中的H2O2荧光强度,降低G.m对烟草的侵染率,表明H2O2参与G.m与烟草共生过程;在G.m与烟草共生过程中,PAO和POD活性显著升高,PAO抑制剂二氨基十二烷(DADD)和POD抑制剂水杨羟肟酸(SHAM)显著降低烟草侧根中H2O2荧光强度,对菌丝中H2O2荧光强度无显著影响,表明烟草根部和G.m均可产生H2O2,PAO和POD参与烟草侧根中H2O2的合成,菌丝中可能存在其他来源的H2O2。     

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.     

Phytotoxic and genotoxic effects of silver nanoparticles exposure on germinating wheat seedlings

We investigated the effects of 1 and 10 mg L⁻¹ AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L⁻¹ AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs.     

Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum

Antifungal activities of zinc oxide nanoparticles (ZnO NPs) and their mode of action against two postharvest pathogenic fungi (Botrytis cinerea and Penicillium expansum) were investigated in this study. ZnO NPs with sizes of 70 ± 15 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ were used. Traditional microbiological plating, scanning electron microscopy (SEM), and Raman spectroscopy were used to study antifungal activities of ZnO NPs and to characterize the changes in morphology and cellular compositions of fungal hyphae treated with ZnO NPs. Results show that ZnO NPs at concentrations greater than 3 mmol l⁻¹ can significantly inhibit the growth of B. cinerea and P. expansum. P. expansum was more sensitive to the treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra indicate two different antifungal activities of ZnO NPs against B. cinerea and P. expansum. ZnO NPs inhibited the growth of B. cinerea by affecting cellular functions, which caused deformation in fungal hyphae. In comparison, ZnO NPs prevented the development of conidiophores and conidia of P. expansum, which eventually led to the death of fungal hyphae. These results suggest that ZnO NPs could be used as an effective fungicide in agricultural and food safety applications.