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Studies on the diversity of arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly alkaline anthropogenic sediment.

A field survey of the arbuscular mycorrhizal status of herbaceous plant species was conducted in a highly alkaline anthropogenic sediment resulting from the disposal of waste from an acetylene and polyvinyl chloride factory. Most plant species found at the site were mycorrhizal and the dominant mycotrophic plant species was Conyza bilbaoana. Fungal species richness was assessed by identification of spores extracted from the sediment and from continuously propagated trap pot cultures. All of the six species of arbuscular mycorrhizal fungi (AMF) found were from the genus Glomus. Glomus intraradices and G. mosseae were found in field-collected sediment samples and also occurred most frequently in trap cultures. To test the symbiotic effectiveness of these two fungi, seedlings of C. bilbaoana were inoculated with either native G. intraradices BEG163 or G. mosseae BEG198 and non-native G. intraradices BEG75 or G. mosseae BEG25 isolates in sterile and non-sterile sediment collected from the study site. All four isolates were able to colonise C. bilbaoana. However, AMF native to the target sediments were generally more effective than the non-native fungi in promoting plant establishment and growth under highly alkaline conditions. The non-native G. intraradices was, however, more effective than the non-native G. mosseae. The results of this study suggest the use of adapted AMF as inoculants for phytorestoration of alkaline anthropogenic-stressed sediments.

Biomass↗

Arbuscular mycorrhizal fungi and plant symbiosis in a saline-sodic soil.

The seasonality of arbuscular mycorrhizal (AM) fungi-plant symbiosis in Lotus glaber Mill. and Stenotaphrum secundatum (Walt.) O.K. and the association with phosphorus (P) plant nutrition were studied in a saline-sodic soil at the four seasons during a year. Plant roots of both species were densely colonized by AM fungi (90 and 73%, respectively in L. glaber and S. secundatum) at high values of soil pH (9.2) and exchangeable sodium percentage (65%). The percentage of colonized root length differed between species and showed seasonality. The morphology of root colonization had a similar pattern in both species. The arbuscular colonization fraction increased at the beginning of the growing season and was positively associated with increased P concentration in both shoot and root tissue. The vesicular colonization fraction was high in summer when plants suffer from stress imposed by high temperatures and drought periods, and negatively associated with P in plant tissue. Spore and hyphal densities in soil were not associated with AM root colonization and did not show seasonality. Our results suggest that AM fungi can survive and colonize L. glaber and S. secundatum roots adapted to extreme saline-sodic soil condition. The symbiosis responds to seasonality and P uptake by the host altering the morphology of root colonization.

Lotus↗

Nested multiplex PCR--a feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root.

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5' end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhiza fungal species in a same plant root system.

Astragalus Plant↗

Detailed sterol compositions of two pathogenic rust fungi.

Teliospores of cedar-apple rust Gymnosporangium juniperi-virginianae were collected from the eastern red cedar Juniperus virginiana, and aeciospores of quince rust G. clavipes were collected from the fruit of English hawthorn Crataegus laevigata. The sterol fractions were separated by HPLC, and their identities were determined by 600 MHz 1H NMR. Twenty-six sterols were isolated from G. juniperi-virginianae and 18 sterols were isolated from G. clavipes. The principal sterol of both fungi was (Z)-stigmasta-7,24(28)-dien-3beta-ol. Other major sterols were (24S)-ergost-7-en-3beta-ol, (24S)-stigmast-7-en-3beta-ol, and (24S)-stigmasta-5,7-dien-3beta-ol. The sterols of the hosts were found to be very different from those of the fungi. The 24-alkyl sterols of the fungi had the 24alpha-configuration, whereas those of the hosts had the 24beta-configuration. Similarities to the sterol composition of the AIDS pneumonia fungus Pneumocystis carinii are discussed.

Basidiomycota↗

Effects of crude drugs and berberine hydrochloride on the activities of fungi.

The effects of crude drugs on fungi have been used for a thousand years in China and Japan. These drugs include: Saussureae radix, Magnoliae cortex, Cinnamomi cortex, Hydrangeae dulcis folium, and Artemisiae capillarius flos. The activity of Coptidis rhizoma and Phellodendri cortex was stronger than other crude drugs against the three fungi. Berberine hydrochloride, which is a component of the two crude drugs, was investigated. Minimal inhibitory concentration values of berberine hydrochloride were 1, 0.125, and 0.5 mg/ml against Candida albicans, C. tropicalis, and C. glabrata, respectively. In C. glabrata, compared with C. albicans and C. tropicalis, berberine hydrochloride greatly inhibited the growth of fungi.

Acrylic Resins↗

The effect of zeolite on the toxicity of lead to fungi.

In order to determine whether clinoptilolite, a naturally occurring zeolite, had any ameliorative effect on lead (Pb) toxicity to fungi, a series of growth experiments were performed. Three fungi, Aspergillus niger, Botrytis cinerea, and Fusarium culmorum, were grown on appropriately amended solid agar media, and their linear extension rates determined. B. cinerea was 25% inhibited, as compared to a control, at 100 mg dm(-3) Pb, and completely inhibited at 1000 mg dm(-3) Pb. F. culmorum was completely inhibited, and A. niger 97% inhibited at 1000 mg dm(-3) Pb. The addition of 3% clinoptilolite partially removed this inhibition in the case of A. niger and B. cinerea and almost completely removed it for F. culmorum. At a constant 500 mg dm(-3) Pb, increasing concentrations of clinoptilolite increased the linear extension rate of F. culmorum and B. cinerea, close to the rates achieved by the untreated controls. A. niger was not inhibited markedly at this Pb concentration. The evidence suggests that the Pb is adsorbed by the clinoptilolite which reduces the availability, and hence toxicity, of the metal to the fungi.

Journal Article↗

Fungi as bioweapons.

Fungi cause disease directly by infection or indirectly through mycotoxins. Fungi that are used as weapons might be targeted against humans, livestock, or crops. Humans and animals encounter fungi and mycotoxins through inhalation, ingestion, and contact with skin and mucous membranes. Effective fungal bioweapons would require the ability to cause significant destruction and a means of delivery to target populations or farms. Effective counter measures against fungal bioweapons would be able to prevent or treat this damage. This article describes several potential biological warfare or bioterrorism fungal species and mycotoxins in regard to their biology, epidemiology, potential for weaponization, and the clinical features, prevention, and treatment of the diseases that they cause.

Biological Warfare↗

Analysis of the distribution and regulation of three representative subtilase genes in sapstaining fungi.

In order to grow in wood, sapstaining fungi produce multiple proteases. Previously we have shown that three groups of subtilases appear to be present in sapstaining fungi; however, it is unknown whether these groups have distinct physiological roles. A representative gene from each of the three groups was chosen and the copy number and presence of homologous genes in other sapstaining fungi were determined. As well, the expressional regulation of these genes was determined in response to available nutrients, exogenous pH, and culture age. Gene homologues in the Ofloc1 group were common in Ophiostoma species. However, homologues from the Opic group were found in only certain Ophiostoma species. Cr group homologues were found in all of the species tested, except for Ophiostoma piceae. The expression of opil1, an Ofloc1 group gene, was induced by BSA, regulated by pH, and expressed within 12h of induction by BSA. The expression of the opic gene, an Opic group gene, was induced by BSA but required the removal of either nitrogen or carbon repression, was also regulated by pH, and was expressed within 24h of BSA induction. The Cr group gene opil2 was expressed under all conditions tested.

Ascomycota↗

Evaluation of five essential oils from aromatic plants of Cameroon for controlling food spoilage and mycotoxin producing fungi.

Five essential oils (EO) extracted from Cymbopogon citratus, Monodora myristica, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale were investigated for their inhibitory effect against three food spoilage and mycotoxin producing fungi, Fusarium moniliforme, Aspergillus flavus and Aspergillus fumigatus. Five strains of each fungus were tested. The agar dilution technique was used to determine the inhibitory effect of each EO on the radial growth of the fungus, and a dose response was recorded. The EO from O. gratissimum, T. vulgaris and C. citratus were the most effective and prevented conidial germination and the growth of all three fungi on corn meal agar at 800, 1000 and 1200 ppm, respectively. Moderate activity was observed for the EO from Z. officinale between 800 and 2500 ppm, while the EO from M. myristica was less inhibitory. These effects against food spoilage and mycotoxin producing fungi indicated the possible ability of each essential oil as a food preservative. A comparative test on the preservative ability of the EO from O. gratissimum and potassium sorbate against A. flavus at pH 3.0 and 4.5 showed that the EO remained stable at both pH, whereas the efficacy of potassium sorbate was reduced at higher pH. We concluded that the EO from O. gratissimum is a potential food preservative with a pH dependent superiority against potassium sorbate, and these are novel scientific information.

Antifungal Agents↗

Inhibition of phytopathogenic fungi by essential oil, hydrosol, ground material and extract of summer savory (Satureja hortensis L.) growing wild in Turkey.

The antifungal activities of the essential oil, hydrosol, ground material and extract of summer savory (Satureja hortensis L.) on mycelial growth of Alternaria mali Roberts and Botrytis cinerea Pers. were determined. All doses of extract inhibited 100% the mycelial growth of both fungi, and exhibited a fungicidal effect. The 15% level of hydrosol and the 1.0% level of ground material had a 100% effect on B. cinerea. The other doses showed weak inhibition on mycelial growth of the fungi, and antifungal activity of the essential oil varied depending on concentrations. While the levels of essential oil show fungistatic effect, the increasing doses of hydrosol and ground material showed a fungicidal effect against B. cinerea and A. mali. While the ground material had not showed any fungicidal activity against mycelial growth of A. mali, the 1% and 1.5% levels of the ground material exhibited a fungicidal effect on B. cinerea. The results obtained from this study may contribute to the development of environmentally safer alternatives to protect the spoilage of food products from pathogenic and saprophytic fungi.

Alternaria↗

Diversity of entomopathogenic fungi near leaf-cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae.

We investigated the prevalence of entomopathogenic fungi associated with leaf-cutting ant colonies in a small area of tropical forest in Panama. There was a high abundance of Metarhizium anisopliae var. anisopliae near the colonies. Beauveria bassiana was also detected in the soil, Aspergillus flavus in dump material, and six Camponotus atriceps ants were found infected with Cordyceps sp. Based on a partial sequence of the IGS region, almost all of the M. anisopliae var. anisopliae isolates fell within one of the three main clades of M. anisopliae var. anisopliae, but with there still being considerable diversity within this clade. The vast majority of leaf-cutting ants collected were not infected by any entomopathogenic fungi. While leaf-cutting ants at this site must, therefore, regularly come into contact with a diversity of entomopathogenic fungi, they do not appear to be normally infected by them.

Animals↗

Arbuscular mycorrhizal fungi can contribute to maintain antioxidant and carbon metabolism in nodules of Anthyllis cytisoides L. subjected to drought.

The symbiosis legume-arbuscular mycorrhizal fungi-nitrogen fixing bacteria is of relevant interest in Mediterranean regions where Anthyllis cytisoides L. grows. In these areas, nitrogen is one of the nutrients that most limits plant growth. In addition, the long periods of water deficit decrease the diffusion rate of phosphorus and, consequently, also decrease the biological nitrogen fixation. It is well known that mycorrhizal fungi can improve phosphorus uptake and, recently, some authors have found that antioxidant activities in mycorrhizal plants can delay drought-induced nodule senescence. The objective of our work was to evaluate weather mycorrhizal fungi could preserve the nodule metabolism in A. cytisoides subjected to drought. Results showed that a low soil water content associated with an enhancement of soil compaction accelerated the senescence of nodules in both non-mycorrhizal and mycorrhizal plants. However, while total soluble protein, leghaemoglobin (Lb) content, as well as carbon and antioxidant metabolism significantly decreased in nodules from non-mycorrhizal A. cytisoides subjected to drought, nodules from stressed mycorrhizal plants maintained Lb levels, showed greater rates of carbon metabolism, and exhibited higher enzymatic activities related to the removal of reactive oxygen species. In addition to the greater activity of antioxidant enzymes, other mechanisms related or unrelated to enhanced nodule water status could also be implied in the better nodule functioning observed in mycorrhizal plants under stressful conditions.

Antioxidants↗

Cytoskeleton and motor proteins in filamentous fungi.

In filamentous fungi, the actin cytoskeleton is required for polarity establishment and maintenance at hyphal tips and for formation of a contractile ring at sites of septation. Recently, formins have been identified as Arp (actin-related protein) 2/3-independent nucleators of actin polymerization, and filamentous fungi contain a single formin that localizes to both sites. Work on cytoplasmic dynein and members of the kinesin and myosin families of motors has continued to reveal new information regarding the function and regulation of motors as well as demonstrate the importance of microtubules in the long-distance transport of vesicles/organelles in the filamentous fungi.

Aspergillus↗

Antifungal activity of Tagetes patula extracts on some phytopathogenic fungi: ultrastructural evidence on Pythium ultimum.

Methanol extract, obtained from Tagetes patula plant, was assayed against three phytopathogenic fungi: Botrytis cinerea, Fusarium moniliforme and Pythium ultimum. The antifungal activity was tested both in the dark and in the light, using two different lighting systems. The data showed that the extract proved to have a dose-dependent activity on all the fungi with a marked difference between treatments in the light than in the dark. Good growth inhibition was observed in fungi only when these were treated with the highest dose of the extract and irradiated, whereas the same dose gave only a modest inhibition when the experiment was conducted in the dark. At 5 and 10 microg/ml in the dark, growth increased. The results indicated that the presence of a luminous source enhances the antifungal activity, with small differences between UV-A and solar spectrum light. SEM and TEM observations on Pythium ultimum revealed that the Tagetes patula extract induced alterations on cell fungal membranes with a photoactivation mechanism possibly involving the production of free radicals and leading to a premature aging of the mycelium.

Antifungal Agents↗

A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi.

A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose-peptone-corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids.

Buffers↗

Application of hammerhead ribozymes in filamentous fungi.

Metabolic engineering in filamentous fungi is a emerging field of research as many fungi produce high value primary and secondary metabolites. Ribozyme technology can be used as a tool for metabolic engineering to influence metabolic pathways and to knock down the expression of specific genes of interest. Hammerhead ribozymes can target virtually any mRNA sequence of choice and prevent gene expression on the post-transcriptional level. They are thus a versatile tool for timed and spatial elimination of unwanted gene products. As current research has only investigated the application of ribozymes in bacteria, yeast and mammalian cells, we decided to carry out a study on whether this technology can also function with filamentous fungi. We employed a sensitive, quantitative reporter-based model system as a proof of concept, using the Escherichia coli beta-glucuronidase transcript (uidA) as the target mRNA and Aspergillus giganteus as the host. This system was used to validate the in vivo activities of seven different hammerhead ribozymes, which were selected by in silico analysis of the uidA mRNA. All ribozymes tested were able to reduce the reporter activity up to a maximum of 100%, demonstrating that ribozyme technology is indeed a useful tool in fungal metabolic engineering.

Aspergillus↗

Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest.

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high number of species on stems of trees in the Andean cloud forest, were unknown. Root samples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ultrastructural evidence of symbiotic interaction was combined with molecular sequencing of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analyses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from coding regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of the internal transcribed spacers, obtained directly from the roots and from the fungal isolates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were restricted to two Tulasnella sequence types while the other orchids were associated with up to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest.

Ecuador↗

In vitro degradation of equine keratin by dermatophytes and other keratinophilic fungi.

Keratinolytic properties of two dermatophytes (Microsporum gypseum, Trichophyton mentagrophytes) and three moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum) isolated from diseased equine hooves were examined to improve the understanding of pathogenic mechanisms leading to equine onychomycosis. Equine hoof horn material and skin, as well as hoof keratin and dermal keratin extracted from corresponding tissues, were used as sole carbon and nitrogen sources in five test tubes for each fungus. Within 18 days, supernatants of all tubes were repeatedly examined for keratinolytic activity by SDS-PAGE and Western blot analysis. In addition, fungal growth rates were determined to identify the preferred tissue of the individual fungi. Among the fungi examined, M. gypseum was the most keratinolytic species, followed by T. mentagrophytes and S. brevicaulis. In the concentration applied, the moulds A. alternata and G. candidum showed minimal keratinolytic activity. With respect to growth rates, M. gypseum favoured hoof horn material, S. brevicaulis and G. candidum preferred skin as a keratin source, whereas for the other two fungi no clear preference was detectable.

Animals↗