Mycelial Fragments Research Articles

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5′ strains by sequencing of the 59′ end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10–12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Secondary metabolites from Penicillium corylophilum isolated from damp buildings

Indoor exposure to the spores and mycelial fragments of fungi that grow on damp building materials can result in increased non-atopic asthma and upper respiratory disease. The mechanism appears to involve exposure to low doses of fungal metabolites. Penicillium corylophilum is surprisingly common in damp buildings in USA, Canada and western Europe. We examined isolates of P. corylophilum geographically distributed across Canada in the first comprehensive study of secondary metabolites of this fungus. The sesquiterpene phomenone, the meroterpenoids citreohybridonol and andrastin A, koninginin A, E and G, three new alpha pyrones and four new isochromans were identified from extracts of culture filtrates. This is the first report of koninginins, meroterpenoids and alpha pyrones from P. corylophilum. These secondary metabolite data support the removal of P. corylophilum from Penicillium section Citrina and suggest that further taxonomic studies are required on this species.

First Report of Ramularia Leaf Spot Caused by Ramularia collo-cygni on Leaves and Seeds of Barley in Estonia.

Ramularia leaf spot (RLS) is a disease of barley (Hordeum vulgare) caused by the fungus Ramularia collo-cygni Sutton & Waller (Rcc). Rcc causes necrotic lesions, premature senescence of leaves, and yield reduction. Under Estonian conditions, there are usually no leaf spots on the upper leaves of barley prior to flowering. In 2009, 2010, and 2012, symptoms similar to those of RLS were observed on leaves of spring and winter barley in several Estonian agricultural regions. Approximately 30% of the plants in affected fields were symptomatic. Symptoms were not observed in 2011, which was a dry and hot year. Initial symptoms were small brown spots, beginning on the upper leaves (flag leaf, F-1 leaf) at the flowering growth stage (4). Later, the spots spread to the sheaths, stems, and awns and became necrotic. The lateral margins of the spots were delimited by the leaf veins and spots are surrounded by a chlorotic halo. During summer 2012, two samples of 15 F-1 leaves were collected from spring barley cv. Maali and line SJ111609 from the Estonian Crop Research Institute in eastern Estonia in late July at growth stage 71 (4). In addition, six grain samples, containing 200 seeds each of the cv. Maali, were collected from different agricultural regions in Estonia, along with one grain sample of SJ111609 from Jõgeva. All samples were collected from untreated plots and leaves were observed under a dissecting microscope, revealing white clusters of conidiophores in rows on the undersides of the leaves. Conidia and conidiophores were scraped aseptically from the leaf surface using a sterile needle under a dissecting microscope and transferred to potato dextrose agar (PDA) containing ampicillin sodium salt (50 mg l-1). Plates were incubated at 18°C in the dark for 20 days until fungal mycelia were produced. The fungus was initially identified as Rcc on the basis of morphological characteristics (3). Colorless, 0- to 3-septate conidiophores were 15 to 17 × 2 to 5 μm, with a strongly curved end. Conidia were 7 to 11 × 3 to 6 μm, solitary, subglobose, single-celled, and of a darkish color. To confirm the presence of Rcc, DNA was extracted from the original barley leaf material, milled seeds, and positive control mycelia of Rcc grown on PDA using DNeasy Plant Mini Kit (Qiagen Gmbh, D-40724 Hilden, Germany) following manufacturer's guides. Rcc specific primers RC3 and RC5 (1) were used. A positive control consisting of 1 ng of purified Rcc DNA was included in the PCR. Standard PCR was conducted in a SEE AMP Seegene cycler. PCR were carried out in 20 μl volumes, containing 2 μl of DNA, 10 μl PCR mix, 0.4 μl each of forward and reverse Rcc primers, and 7.2 μl H2O. Qualitative detection analyzed by standard PCR with primers RC3 and RC5 revealed the presence of Rcc in symptomatic leaves and seeds. To complete Koch's postulates, a pathogenicity test was performed. Twenty-five barley seedlings were grown under controlled conditions (15°C/48 h dark, 16 h light/8 h dark, 70% RH) and spray-inoculated with a suspension of Rcc mycelium fragments as described by Macepeace et al. (2). The pathogen was re-isolated from leaves with necrotic lesions similar to those observed in the field, thus fulfilling Koch's postulates. To our knowledge, this is the first confirmed report of Ramularia leaf spot caused by Ramularia collo-cygni on barley in Estonia.

First Report of Phytophthora Taxon Walnut in Lombardy, North Italy.

The park Boscoincittà, Milan, North Italy (136 m a.s.l., 45°29'06″ N, 9°5'32″ E), has an area of 110 ha and includes tree stands, wood clearings, trails, and watercourses. Recently, common walnut (Jugland regia) trees in the park have begun to suffer from a progressive dieback that has caused roughly 90% mortality. Aerial symptoms were stunted growth, loss of vigor, crown thinning, and bark cankers with tarry exudates on the lower stem. The xylem tissue of trees showed large necrosis and flame-shaped discoloration below the bark. Since the dieback seemed caused by Phytophthora, samples were taken from three symptomatic trees and, by baiting, from the nearby soil and watercourses. Isolations from apple baits were carried out after a week. Isolations taken from tissue at the edge of active lesions of the trees were transferred on the selective medium V8A-PARPNH (1) and incubated at 24°C. Cottony colonies appeared after 3 days and single hyphal tip derivatives were transferred to V8A for a further 4 to 7 days. Fragments (1 cm2) of mycelium of the subcultured colonies were then placed in filtered (Ø 0.22 μm nitrocellulose filters, Millipore) pond water. Three isolates were retrieved within 24 h, two from tree tissue and one from water. These produced ovoid, non-papillate sporangia, of which 30 per isolate were measured. Sporangia averaged 52.5 ± 9.6 × 32.9 ± 4.7 μm (range 30.8 to 67 × 22.5 to 42.8) with a l:b ratio of 1.59 ± 0.19 (range 1.27 to 2.05), and exit pores of 11.1 ± 1.7 μm (range 7.31 to 14.21). External proliferation from previously emptied sporangia and hyphal swellings were observed on V8A. On V8A, colonies had optimal growth at 32°C (5.7 ± 0.8 mm d-1) with a maximum at 37°C. Colonies had a chrysanthemum-shaped, scanty fluffy aspect on PDA. Isolates were identified as Phytophthora taxon walnut on the basis of macro- and micro-morphology and sequence information from the ITS-rDNA region, that was amplified with primers ITS6 and ITS4 (3) after DNA extraction with a commercial kit (Sigma Aldrich). A region of the cox1 gene of isolate B164 was also amplified with primers OomCoxILevup and Fm85mod (4) and sequenced (GenBank Accession No. KC291584), but this was irrelevant for identification purposes because that gene region has not been sequenced for other isolates of this taxon. A BLAST search in GenBank and the Phytophthora database revealed a 99% identity of the ITS-rDNA from our isolate B164 (KC291550) with the P. taxon walnut isolate P532 (AF541910) (2). Inoculation trials were conducted on 10 detached leaves. A little lesion was produced with a sterile scalpel on the lower leaf surfaces and a 0.5 cm Ø agar plug was placed over the wounds. Necrotic lesions averaged 3.7 ± 1.6 × 2.0 ± 0.5 cm after 1 week of incubation at 20°C in the dark. Control leaves showed no symptoms. Re-isolations on V8-PARPNH agar confirmed Phytophthora taxon walnut as the causal agent. Members of the Phytophthora genus grouping with the Phytophthora taxon walnut in clade 6 are mainly reported as saprophytes or pathogens from riparian ecosystems and forests (2). To our knowledge, this is the first report of Phytophthora taxon walnut from Italy. Since the oomycete proved in our growth trial to be distinctly thermophilic, we hypothesize that its spread is being favored by the rising temperatures observed during the last decades in the area.

In vitro susceptibility testing of dermatophytes with their fragmented mycelia as inoculum

377 clinical isolates and seven dermatophyte culture collection strains were tested in vitro by microdilution against a panel of 18 antifungal agents. A newly introduced method for rapid and reliable inoculum preparation for moulds using the fragmented mycelia of freshly grown isolates as inoculum was firstly applied to dermatophytes. The performance of the method was tested with two different culture media which are recommended by different standardized testing methods for moulds. The standardized fragmented mycelia provided both, countable single colonies (viable units) on solid culture media and distinct readable endpoints (minimum inhibitory concentrations) in the microdilution wells for all tested topical and systemic drugs, including the echinocandins. As with moulds, and as shown by the culture collection strains, reproducible minimum inhibitory concentrations were obtained with an essential agreement (± 1 log2-dilution) of 97% to 100%, by a significant reduction of the overall testing time. As exemplarily shown for the microdilution technique, this inoculum method should also provide a solid basis for improved agar-based susceptibility testing methods such as disc-, tablet-, or strip-tests.

Tools for advanced and targeted genetic manipulation of the β-lactam antibiotic producer Acremonium chrysogenum

Acremonium chrysogenum is the major producer of the β-lactam antibiotic cephalosporin C and therefore of great importance for the pharmaceutical industry. However, this filamentous fungus is known to reproduce solely by asexual means, shows only sporadic conidiospore production, and has gradual fragmentation of the vegetative mycelium into arthrospores. Due to these peculiar growth characteristics and life style, strain improvement by recombinant technologies is much more challenging than for other biotechnologically relevant fungi. Here, we describe several molecular tools for genetic engineering of A. chrysogenum, including a ΔAcku70 deletion strain for homologous recombination. No physiological or morphological changes occurred due to deletion of the ku70 gene or integration of the nat1 cassette in this recipient strain. We also used a xylose-inducible promoter from Sordaria macrospora (Smxyl) to demonstrate induction of the gfp reporter gene in A. chrysogenum. The Smxyl promoter was used for construction of a vector molecule to develop a one-step FLP/FRT recombination system in A. chrysogenum. This system was then used in the ΔAcku70 deletion strain to construct a marker-free recipient strain for targeted DNA insertion into genomic DNA. The applicability of our tools was demonstrated by construction of a marker-free transgenic strain, lacking any foreign genes.

Generation and Evaluation of High β-Glucan Producing Mutant Strains of Sparassis crispa

A chemical mutagenesis technique was employed for development of mutant strains of Sparassis crispa targeting the shortened cultivation time and the high β-glucan content. The homogenized mycelial fragments of S. crispa IUM4010 strain were treated with 0.2 vol% methyl methanesulfonate, an alkylating agent, yielding 199 mutant strains. Subsequent screening in terms of growth and β-glucan content yielded two mutant strains, B4 and S7. Both mutants exhibited a significant increase in β-glucan productivity by producing 0.254 and 0.236 mg soluble β-glucan/mg dry cell weight for the B4 and S7 strains, respectively, whereas the wild type strain produced 0.102 mg soluble β-glucan/mg dry cell weight. The results demonstrate the usefulness of chemical mutagenesis for generation of mutant mushroom strains.

First Report of Gray Mold Caused by Botrytis cinerea on Greenhouse-Grown Zucchini in Korea.

In the winter of 2011, greenhouse-grown zucchini (Cucurbita pepo) in Andong City, Korea, showed severe disease symptoms on fruits and dying leaves of zucchini plants that resembled gray mold disease with about 20% yield loss. Symptoms included extensive growth of mycelia and gray conidia on stem and fruit lesions. Lesions expanded rapidly under cool, humid conditions. As the disease progressed, leaves, stems, and fruits became necrotic and were covered by an abundant, soft, gray, sporulating mycelium. Diseased fruit tissue was excised and surface sterilized by immersion in 2% NaOCl for 1 min, placed on PDA (potato dextrose agar), and incubated at 22°C. Fungal colonies were initially white and became gray to brown after 72 h. Analysis of light micrographs showed the presence of elliptical conidia on PDA that was 7.5 to 16.0 μm long and 5 to 10.5 μm wide. In culture, a few, black, small and large irregular sclerotia were produced. Microsclerotia were round, spherical or irregular in shape, and ranged from 1.0 to 3.3 and 1.2 to 3.4 mm (width and length). Conidiophores were slender and branched with enlarged apical cells bearing smooth, ash-colored conidia. These morphological characteristics identified the fungus as Botrytis cinerea (1). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 (forward) and ITS4 (reverse) primer set (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3') and sequenced (2). BLAST analysis of the PCR product showed that the sequence had 100% identity with the nucleotide sequences for B. cinerea. Pathogenicity tests were performed by placing mycelium fragments (1 cm2) of PDA cultures on zucchini fruits. Controls were treated with PDA alone. Five replicates for the inoculated and control plants were used. All fruits were covered with plastic bags and incubated in a growth chamber to maintain 90 to 100% relative humidity at 22°C. Typical symptoms appeared 2 to 6 days after inoculation. The inoculated plants developed typical gray mold symptoms with gray sporulating lesions, while controls remained healthy with no lesions. B. cinerea reisolated from the inoculated tissues was morphologically identical to the original isolates. In a cold outside (below 0°C), wet greenhouse, plants are likely to be exposed to resident Botrytis populations and if the gray mold disease occurs, it can spread on zucchini plants very fast, in 2 days to a week inside a 100 m2 greenhouse. Therefore, gray mold disease could have a significant impact on greenhouse production of zucchini. To our knowledge, this is the first report of B. cinerea causing gray mold of greenhouse-grown zucchini in Korea.

Induction of Dectin-1 and asthma-associated signal transduction pathways in RAW 264.7 cells by a triple-helical (1, 3)-β-d glucan, curdlan

People living in damp buildings are typically exposed to spore and mycelial fragments of the fungi that grow on damp building materials. There is experimental evidence that this exposure to triple-helical (1, 3)-β-D glucan and low molecular weight toxins may be associated with non-atopic asthma observed in damp and moldy buildings. However, the mechanisms underlying this response are only partially resolved. Using the pure (1, 3)-β-D glucan, curdlan, and the murine macrophage cell line, RAW 264.7, there were two objectives of this study. The first was to determine whether signal transduction pathways activating asthma-associated cell signaling pathways were stimulated using mouse transduction Pathway Finder(®) arrays and quantitative real-time (QRT) PCR. The second objective was to evaluate the dose and temporal responses associated with transcriptional changes in asthma-associated cytokines, the signal transduction receptor gene Dectin-1, and various transcription factor genes related to the induction of asthma using customized RT-PCR-based arrays. Compared to controls, the 10(-7) M curdlan treatment induced significant changes in gene transcription predominately in the NFkB, TGF-β, p53, JAK/STAT, P13/AKT, phospholipase C, and stress signaling pathways. The 10(-8) M curdlan treatment mainly induced NFkB and TGF-β pathways. Compared to controls, curdlan exposures also induced significant dose- and time-dependent changes in the gene translations. We found that that curdlan as a non-allergenic potentiator modulates a network of transduction signaling pathways not only associated with TH-1, TH-2, and TH-3 cell responses including asthma potentiation, but a variety of other cell responses in RAW 264.7 cells. These results help provide mechanistic basis for some of the phenotypic changes associated with asthma that have been observed in in vitro, in vivo, and human studies and open up a hypothesis-building process that could explain the rise of non-atopic asthma associated with fungi.

Breeding of new high-temperature-tolerant strains of Flammulina velutipes

Flammulina velutipes is a typical low-temperature mushroom that requires low-temperature to induce fruiting body formation. The temperature requirement causes high-energy consumption and cultivation cost. To solve these problems, strains adapted to higher temperatures must be developed. This article reported the treatments to mycelial fragments of F. velutipes with ultraviolet irradiation mutagenesis for 6h, 1%EMS mutagenesis for 3h, 0.8% LiCl mutagenesis, and combined mutagen (UV+LiCl+EMS) mutagenesis. “Double stage” screening, i.e., screening at 33°C during mycelial growth and at 20°C during fruiting body development, was performed. The results showed that the efficiencies of EMS and UV mutagenesis were higher than that of LiCl mutagenesis. Three high-temperature-tolerant strains, namely, F1U4, F1E6, and F1C4 were successfully obtained through mutagenesis. The mycelia of three mutants grew at 33°C and underwent fruiting at 20°C whereas FLA1 failed to grow at 33°C and undergo fruiting at 20°C. The results of antagonist test indicated that there is a strong antagonism resistance between 3 mutant strains and FLA1. In conclusion, the strains F1U4, F1E6, and F1C4 were all new strains.

A new immunoassay to quantify fungal antigens from the indoor mould Aspergillus versicolor

Aspergillus versicolor is among the most commonly found moulds in moisture-damaged buildings and can be associated with adverse health effects in humans. This paper reports the development, validation and application of an enzyme immunoassay to quantify A. versicolor antigens. A sandwich ELISA was developed using polyclonal antibodies that recognize a broad range of A. versicolor proteins present in fungal spores and in mycelia fragments. To validate the new method, A. versicolor antigens were quantified in samples collected from homes with visible mould growth, including dust from vacuumed walls and bulk samples of building materials. Antigen concentrations were compared to the results of a commercial ELISA based on monoclonal antibodies (AveX ELISA, Indoor Biotechnologies, Charlottesville, USA) and correlated with colony forming units (CFU) of A. versicolor. The A. versicolor ELISA was very sensitive with a lower detection limit of 120 pg ml(-1). The assay also showed some reactivity to other moulds with strongest reactions with other Aspergillus species (1-3% reactivity). The new assay detected A. versicolor antigens in a much higher percentage of dust samples (88% vs. 27%) and bulk samples (89% vs. 24%) than the AveX assay. A significant correlation (r = 0.67, and p < 0.0001) was found between antigen concentrations and CFU of A. versicolor. Based on its low detection limit and good correlation with the culture-based method, this new immunoassay seems to be a useful tool for the measurement of A. versicolor exposure levels and a reliable complement to the traditional monitoring techniques, such as mould cultivation or microscopy.

First Report of Sclerotinia Stem Blight Caused by Sclerotinia trifoliorum on Trifolium ambiguum in Poland.

Kura clover (Trifolium ambiguum M. Bieb.), a rhizomatous, persistent legume native to the Caucasus region, has received recent attention in North America and New Zealand as a pasture and silage crop. It is reported to be resistant to most pathogens affecting other clovers, including Sclerotinia trifoliorum Eriks. (3,4), one of the most destructive pathogens of clovers in northern Europe. Kura clover (cv. KTA202) was established in May 2009 near Mochełek, Poland (53° 13' N, 17° 51' E) on a Luvisol soil. By May 2011, 70% of plants grown in an experimental field (350 m2) had died, and 20% of the remaining plants were yellow and wilted. At crowns and the lower stem regions, wet, brown lesions with delicate white mycelium were observed. Lesion development was followed by death of the entire plant in a few days. By early June, only a few asymptomatic plants per square meter remained in the field. Tissue fragments of 20 symptomatic plants were surface-sterilized with 1% NaOCl for 1 min and plated on potato dextrose agar (PDA). A fungus with morphological characteristics of S. trifoliorum was consistently isolated. DNA isolation from sclerotia was performed with the DNeasy Plant Mini Kit (Qiagen, USA). Amplification and sequencing of the ITS region of rDNA was performed with primers ITS1/ITS4. NCBI Blast analysis of the 542-bp segment showed a 99% homology with most of S. trifoliorum and S. sclerotiorum strains in GenBank (e.g., AY547267.1 and EU082466.1). Sequence of isolate St0211TA was deposited in GenBank (Accession No. JQ743329). To determine growth rate of hyphae, morphology, and dimensions of sclerotia, colonies were grown in three replications on PDA at 20 ± 1°C in the dark. S. trifoliorum (CBS 122377) and S. sclerotiorum from our local collection were used as controls. Mean growth rate of S. trifoliorum isolates (20.5 mm/day) was slower compared to S. sclerotiorum (32.3 mm/day). Sclerotia began to form on delicate and smooth mycelium of S. trifoliorum on the entire surface of the plate in 7 to 8 days. Sclerotial size on day 28 was 2.0 to 9.0 × 2.0 to 7.0 mm (average 4.2 × 3.6 mm). Ultimately, the identification of S. trifoliorum was confirmed on the basis of ascospore morphology. Apothecia grew from sclerotia in wet sand at 12°C after 12 weeks. Asci contained dimorphic ascospores: four larger 13.0 to 16.0 × 6.0 to 9.0 mm (average 14.1 × 7.4 μm) and four smaller 10.0 to 12.0 × 5.4 to 6.0 mm (average 10.6 × 6.0 μm), typical for this species (1). Isolate St0511TA, which most intensively produced apothecia, was deposited in CBS (No. 133234). Koch's postulates were fulfilled by pathogenicity tests carried out on 2-week-old T. ambiguum seedlings grown in pots (6 × 30 plants), sprayed with a mycelial fragment suspension, and incubated at 15°C (2). Brown, wet spots with delicate white mycelium were observed on cotyledons after 3 days. After 5 days, approximately 10% of cotyledons were killed and mycelium appeared on stems and leaves, and after 10 days, 73% of seedlings were dead. S. trifoliorum was reisolated from all symptomatic tissues. To our knowledge, this is the first report of S. trifoliorum stem blight on T. ambiguum in the field.

A mitochondrial respiratory mutant of Podospora anserina obtained by short-term submerged cultivation of senescent mycelium

A spontaneous long-lived isolate of Podospora anserina obtained by relatively short-term submerged cultivation of the wild-type senescent culture and conventionally termed “immortal” was shown to be a cox1 mutant. As a respiratory mutant, the isolate in question is characterized by dysfunction of the cytochrome respiratory chain, activation of alternative respiration leading to a low level of reactive oxygen species production, and the lack of accumulation of α-senDNA, the specific factor of P. anserina senescence. Absence of visible vegetative incompatibility was shown in the fungal mutants carrying respiratory defects. It was discovered that the P. anserina female sex organs could be fertilized not only by microconidia but also by the fragments of vegetative mycelium. Partial nonobservance of monoparental female inheritance of mitochondria associated with fertilization by vegetative mycelium was also revealed.

Exploration of two epimerase homologs in Streptomyces peucetius ATCC 27952

Streptomyces peucetius ATCC 27952 is a potent producer of the therapeutically important antitumor drug, doxorubicin. S. peucetius contains two deoxythymidine diphospho (dTDP)-4-keto-6-deoxyglucose 3,5-epimerase-encoding genes, dnmU and rmbC, in its genome. While dnmU from the doxorubicin biosynthesis gene cluster is involved in the biosynthesis of dTDP-L-daunosamine, rmbC is involved in the biosynthesis of dTDP-L-rhamnose, a precursor of cell wall biosynthesis. The proteins encoded by dnmU and rmbC share 47 % identity and 64 % similarity with each other. Both enzymes converted the same substrate, dTDP-4-keto-6-deoxy-D-glucose, into dTDP-4-keto-L-rhamnose in vitro. However, when disruption of dnmU or rmbC was carried out, neither gene in S. peucetius compensated for each other's loss of function in vivo. These results demonstrated that although dnmU and rmbC encode for similar functional proteins, their native roles in their respective biosynthetic pathways in vivo are specific and independent of one other. Moreover, the disruption of rmbC resulted in fragmented mycelia that quickly converted into gray pigmented spores. Additionally, the production of doxorubicin, a major product of S. peucetius, appeared to be abolished after the disruption of rmbC, demonstrating its pleiotropic effect. This adverse effect might have switched on the genes encoding for spore formation, arresting the expression of many genes and, thereby, preventing the production of other metabolites.

First Report of Leaf Spot Caused by Phoma costarricensis on Delphinium malabaricum in Western Ghats of India.

Delphinium L. is a genus of more than 300 species of perennial flowering plants belonging to the family Ranunculaceae and is native throughout the Northern Hemisphere. In India, 24 species are found mainly in the Himalayan regions. Delphinium malabaricum (Huth) Munz is the only species of the genus endemic to northern Western Ghats. Its mediumsized violet-to-metallic blue spurred flowers have considerable ornamental value as a floriculture crop (3). There is no report of serious diseases of this genus in India. Since 2008, a severe foliar disease was observed each year on D. malabaricum cultivated on the experimental plots located at Shivaji University, Kolhapur, India. Symptoms were small, necrotic spots on the abaxial and adaxial sides of infected leaves, with spots enlarging to form round areas that were 6 to 9 mm in diameter and well defined by a dark black margin. Severely infected leaves wilted without abscising. No symptoms were observed on other plant parts. From infected leaves, a fungus was isolated on Czapek Dox agar (CDA) amended with 25 mg/liter of streptomycin sulfate. The fungus was grown on CDA and cultures were maintained at 4°C for further studies. After 6 days, black pycnidia developed, which were variable in size, releasing abundant hyaline, elliptical conidia measuring from 3 to 4 × 1.5 to 2 μm. On the basis of cultural and morphological characteristics, the fungus was identified as Phoma costarricensis (1,2). The identification was verified by sequencing a region of 28S ribosomal RNA with the geneOmbio LSU gene sequencing primers and ABI BigDye Terminator v3.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Carlsbad, CA). The sequence was deposited as Accession No. HE608244 in EMBL-Bank. Blast analysis of the sequence obtained showed a 99% homology with several isolates of P. costarricensis in the GenBank database (Accession No. GU238058.1). Pathogenicity tests were performed by spraying leaves of 10 healthy plants with spores (~10,000 spores or mycelial fragments per ml) on the abaxial and adaxial surface of leaves. Noninoculated plants served as control. Symptoms identical to those on field samples developed on all inoculated plants 1 week after inoculation but controls remained asymptomatic during the observation period. P. costarricensis was reisolated from inoculated symptomatic plants and the identity was confirmed, which completed Koch's postulates. This experiment was repeated three times in a greenhouse, confirming the pathogenicity of P. costarricensis on D. malabaricum. P. ajacis (Thum.) Aa & Boerema, P. delphinii (Rabenh.) Cooke, P. aquilegiicola M. Petrov, and P. xanthina Sacc. are reported to cause leaf spot and stem rot in Delphinium spp. (1). However, to our knowledge, there are no previous reports of leaf spot of D. malabaricum caused by P. costarricensis. Leaf spot severity induced premature defoliation, leading to reduction in flower setting and ultimately the yield. The disease could lead to an economic crisis in the farmer community of western India. The only other known host of P. costarricensis is Coffea arabica (2).

Establishment of cultivating strategy for highly aggregated mycelia of Morchella esculenta in a stirred-tank bioreactor

Mycelia of Morchella esculenta were found to aggregate rapidly in a submerged culture, which caused the decrease in dispersed mycelia and the problem of diffusion limitation. The effect of different agitation schemes on the growth of mycelia was investigated in a stirred-tank bioreactor. At the constant speed of 100 or 300 rpm, rapid aggregation caused the biomass concentration to drop to zero in 30 h, which was even worse than achieved under static culture. Intermittent agitation maintained a higher mycelium fragment concentration for 48 h and enhanced the biomass concentration to 4.73 g/L at 120 h. The operation with a polytron connection disrupted effectively mycelium aggregation, thus increasing the specific growth rate, biomass concentration and maximum productivity to 0.0613 1/h, 7.73 g/L and 0.0878 g/L h at 88 h, respectively. Moreover, logistic equations and genetic algorithm (GA) were used for the simulation of biomass growth and estimation of all kinetic coefficients. The operating strategy developed in this study could be used for the production of highly aggregated mycelia, which could also achieve a high cell-density culture in a stirred tank reactor.

Effects of Phanerochaete chrysosporium on biologic activity of soil amended with olive mill wastewaters

Effects of the untreated olive mill wastewater (UOMW) and the bioaugmented olive mill wastewater (BOMW) with the fungus Phanerochaete chrysosporium (P. chrysosporium ) for the amendment of soil were investigated. Results showed that UOMW inhibited the soil respirometric activity, while BOMW exhibited significantly higher respiration levels compared to the unamended and the UOMW amended soils. The ratio of C-CO 2/C tot decreased from 6.7 in the unamended soil to 6.34, to 2.74 and to 1.6 in soils amended consecutively with 50, 100 and 200 m 3 ha -1 of UOMW. Based on the first results, the UOMW dose of 50 m 3 ha -1 showed the elevated C-CO 2/C tot ratio in comparison with the two other doses (100 and 200 m 3 ha -1 ) so that it was chosen to test the effects of its bioaugmentation with P. chrysosporium on the soil biodegradation activities. The fungus P. chrysosporium was added at three different forms; spores suspension (10 6 spores g -1 ), mycelium form (cultivated in liquid medium) and colony form (mycelium fragments from solid medium).The soil amended with 50 m 3 ha -1 of BOMW showed higher CCO 2/C tot ratio in comparison with control soil (unamended) and with soil amended with 50 m 3 ha -1 of

Current Trends in Fungal Genomics and Biology

The deliberate decision in the early 1940s to use Neurospora crassa in the Nobel Prize winning work that demonstrated an association between genes and enzymes initiated an avalanche of research now known as Molecular Genetics. Among the reasons Beadle and Tatum chose a fungus for that seminal work included the facts that while Neurospora is a eukaryote, it can be rapidly propagated from asexual conidia or mycelial fragments, with each culture providing millions of genetically identical haploid nuclei. Further, strains of opposite mating type can readily be crossed in order to analyze the inheritance pattern of mutant traits. When added to the fact that many fungi are pathogenic to plants and animals and contain much less DNA per genome than higher eukaryotes, it is somewhat surprising that the only fungal genome sequenced before the human genome was released in 2001 was that from a strain of Saccharomyces cerevisiae However, as rapid developments in technology have greatly reduced the expense of sequencing, fungal genomics has literally exploded. As of this writing, 353 fungal and 20 oomycete species are listed in the ‘genome completed’ section of The National Center for Biologic Information (NCBI). A ‘1,000’ Fungal Genomes Project initiated at the end of 2010.

Influence of 17β-estradiol on gene expression of Paracoccidioides during mycelia-to-yeast transition.

Background Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17β-estradiol, E2) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E2 inhibits M-to-Y transition.MethodologyWe assessed temporal gene expression in strain Pb01 in the presence or absence of E2 at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E2-regulated clones were sequenced to identify genes and biological function.Principal FindingsE2-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E2 belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E2-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E2, whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E2.ConclusionThis study characterizes the effect of E2 at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E2 may be working through signaling genes that regulate dimorphism.

First Report of Wilt on Alfalfa in China Caused by Verticillium nigrescens.

Alfalfa (Medicago sativa Linn.), widely grown throughout the world, is an important perennial forage crop. It is high in protein and digestible fiber and is an excellent source of several vitamins (A, D, E, and K) and minerals for beef cattle, horses, sheep, goats, and even humans (2). Wilt symptoms on alfalfa were observed during a disease survey in Yangling, Shaanxi, China in 2009. Symptoms included discoloration, shortened internode, and plant death. However, the vascular tissue of diseased alfalfa plants did not exhibit discoloration and typical "V" symptoms of Verticillium albo-atrum infection. Eleven fungal isolates were obtained from diseased alfalfa plants in Yangling by a tissue isolation method (1). Isolates were cultured on Czapek Dox Agar (CDA; pH 7.2) slants at 22 ± 1°C in darkness. Colonies on CDA plates were whitish and cream-white when viewed from the underside, later becoming dark gray due to the formation of gray or dark brown chlamydospores in single or in short chains. DNA was extracted from each isolate and the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA) was amplified and sequenced using primers ITS-1F and ITS4. The 11 isolates were divided into five groups based on their in vitro morphological characters. A single isolate from each of the five groups was chosen for ITS sequencing. All five isolates had the same ITS sequence (GenBank Accession No. AB551216). On the basis of the ITS sequence and morphology (4), these isolates were identified as V. nigrescens Pethyhr. (recently renamed as Gibellulopsis nigrescens). Five representative isolates were used to fulfill Koch's postulates. Alfalfa seeds (cv. Cossack) were surface sterilized with 75% ethanol for 5 min, allowed to dry, and planted into cow dung compost that had been autoclaved at 160°C for 2 h. Plants were cultivated under controlled greenhouse conditions at 23 to 25°C with a photoperiod of 14 h. Inoculum was prepared by comminuting 15-day-old cultures and sterile deionized water into a suspension of mycelial fragments and conidia (105 to 106 CFU/ml) in a blender. Seedlings (four-leaf stage) were inoculated by immersing roots in the inoculum suspension for 60 min (3). Each isolate was inoculated onto 30 seedlings, six in each pot; another 30 seedlings were soaked with sterile deionized water for 60 min as a control. After 20 days in the greenhouse, all inoculated plants exhibited wilt symptoms similar to the original wilt symptoms observed on diseased alfalfa plants. In contrast, none of the control plants showed wilt symptoms. The pathogen was reisolated from all diseased plants and confirmed to the original ones. To our knowledge, this is the first report of V. nigrescens infecting M. sativa in China, indicating V. nigrescens as one possible important pathogen of alfalfa.