Digital monitoring of mycelium growth kinetics and vigor of shiitake (Lentinula edodes (Berk.) Pegler) on agar medium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

RAPD analysis of the sexual state and sterile mycelium of the fungus cultivated by the leaf-cutting ant Acromyrmex hispidus fallax

That the symbiotic fungus of leaf-cutting ants only occasionally produces the sexual phase makes their identification confusing. This has occurred so rarely, either in laboratory nests, or in unbalanced field nests. that the possibility of contamination of the fungal garden by other fungi cannot be disregarded. In this paper we describe the formation of several basidiomata in a healthy and free-living nest of the leaf-cutting ant Acromyrmex hispidus fallax. The cultivation in vitro of the sterile mycelia (isolated from the fungal garden) with their typical inflated tips, and the similarity of both forms confirmed by RAPD analysis of their genomic DNA. The fungus was identified as Leucoagaricus gongylophorus.

Influence of solid wastes in the mycelium growth of Lentinula edodes (berk.) Pegler

In Brazil there was little research related to Shiitake axenic culture. The aim of this research was to understand the substratum effects in the kinetics of the Shiitake mycelium growth. It was used two Shiitake strains and two different base substrate (eucalyptus sawdust and sugar cane bagasse) varying in three proportions of the supplements. The supplements, a blend of rice and wheat brans, were added in the proportion of 0, 10 and 20% of the base substrate. The experiment was composed of six treatments. The mycelium growth kinetics in volume had no effect relation to the strains and substrate and it followed a mathematical model represented by logarithmic equation. Beta, gamma and delta parameters didn't show any correlation with the growth velocity in volume. The strain L55 was better adapted than L17.

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chrornatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and C-13 NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q(1A) that was a beta-(1 -> 6)-D-glucan with the following structure:[GRAPHICS]The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: KIP (insoluble) that comprised a beta-(1 -> 3)-D-glucan with beta-D-glucose branches at C-6 with the structure:[GRAPHICS]and K1SA (soluble) consisting of a backbone chain of alpha-(1 -> 4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues:[GRAPHICS](c) 2008 Elsevier Ltd. All rights reserved.

Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca 2+ levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca 2+ with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca 2+ accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca 2+ homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca 2+ channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca 2+/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca 2+ homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-β-glucosidase) in mycelium cells; and ags (an α-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

Mycelium tectonics: studio di comportamenti morfologico-costruttivi attraverso sistemi biologici e simulazioni al computer

Mycelium Tectonics è un lavoro multidisciplinare che interseca l’architettura con la biologia e con la tecnologia. Il concetto di tettonica - qui definito come il territorio in cui si costruiscono le relazioni tra l’organizzazione formale e i processi di funzionamento endogeni - viene indagato partendo da un punto di vista materico, dai limiti fisici e meccanici della materia e dalle differenze che ne possono emergere attraverso il cambio di scala. Procedendo dunque dal basso, sono stati studiati fenomeni quali l’auto-organizzazione e le intelligenze collettive, costituite da elementi con comportamenti autonomi, in cui l’organizzazione globale non è pianificata a priori ma emerge dalle interrelazioni degli elementi stessi. Si è tentato di descrivere una tettonica in cui fosse proprio la differenziazione e la variazione, di cui il sistema è intrinsecamente capace, a produrre una propria forma di organizzazione tettonica ed estetica su cui la funzionalità potesse essere mappata in modi non convenzionali. La biologia fornisce in questo diversi stimoli circa il concetto di costruire in termini di articolazione spaziale e adattabilità: in natura ogni struttura viene generata mediante processi di crescita intrinsecamente coerenti, e le relazioni che la regolano rendono impossibile scindere le parti dal tutto; una logica profondamente differente dai processi produttivi - e costruttivi – odierni, che racchiude in questo il potenziale per superarne i limiti. L’esperienza di laboratorio ha permesso un’ indagine approfondita sulle capacità esplorative e di morfogenesi del micelio: un organismo pluricellulare molto semplice formato da numerosi filamenti (ife), capaci di ramificarsi e riconnettersi tra loro per formare una rete biologica di trasporto. Le strategie messe in atto durante la crescita, poi simulate digitalmente, si sono evidenziate durante tutto il percorso di ricerca pratica, fornendo non solo motivo di dibattito teorico, quanto stimoli e possibilità a livello operativo. Partendo dagli esperimenti in vitro, lo studio si è poi soffermato sulla possibilità di far crescere il micelio (della specie Pleurotus Ostreatus) su strutture fibrose di canapa. Queste sono state simulate ed indagate digitalmente, al fine di costruire prototipi fisici da far colonizzare attraverso una crescita controllata del micelio. I modelli, lasciati essiccare, mostrano caratteristiche e performance emergenti, coerentemente alle premesse architettoniche. Considerando i risultati - seppur parziali - dell’attività teorico-sperimentale condotta, diviene necessario considerare un significato più esteso del termine sostenibilità, oltre ad un esame più approfondito delle ripercussioni a scala ecologica conseguenti l’applicazione di soluzioni qui soltanto ipotizzate.

Structural Analysis and Form-Finding of Mycelium-Based Monolithic Domes

As sustainability becomes an integral design driver for current civil structures, new materials and forms are investigated. The aim of this study is to investigate analytically and numerically the mechanical behavior of monolithic domes composed of mycological fungi. The study focuses on hemispherical and elliptical forms, as the most typical solution for domes. The influence of different types of loading, geometrical parameters, material properties and boundary conditions is investigated in this study. For the cases covered by the classical shell theory, a comparison between the analytical and the finite element solution is given. Two case studies regarding the dome of basilica of “San Luca” (Bologna, Italy) and the dome of sanctuary of “Vicoforte” (Vicoforte, Italy) are included. After the linear analysis under loading, buckling is also investigated as a critical type of failure through a parametric study using finite elements model. Since shells rely on their shape, form-found domes are also investigated and a comparison between the behavior of the form-found domes and the hemispherical domes under the linear and buckling analysis is conducted. From the analysis it emerges that form-finding can enhance the structural response of mycelium-based domes, although buckling becomes even more critical for their design. Furthermore, an optimal height to span ratio for the buckling of form-found domes is identified. This study highlights the importance of investigating appropriate forms for the design of novel biomaterial-based structures.

A prospective study on bioactive properties of wild mushrooms mycelium grown in vitro under different conditions

Wild mushrooms have been extensively studied for their value as sources of high quality nutrients and of powerful physiologically bioactive compounds [1,2]. The present study was designed to evaluate the in vitro development of two wild edible mushroom species: Pleurotus eryngii (DC.) Quél. and Suillus belinii (Inzenga) Watling, by testing different solid (Potato Dextrose Agar medium –PDA and Melin-Norkans medium- MMN) and liquid culture media (Potato dextrose broth- PDB and Melin-Norkans medium- MMN). Each strain of mushroom produces a special type of mycelium and this range of characteristics varies in form, color and growth rate. S. bellinii presents a pigmented and rhizomorphic mycelia, whereas, P. eryngii has depigmented and cottony mycelia. The mycelium isolated and grown in PDA showed a faster radial growth compared to the mycelium isolated and grown in both solid and liquid incomplete MMN medium. P. eryngii exhibited a rapid growth and a higher mycelia biomass in both medium compared to S. belinii. Moreover, the obtained mycelia will be characterized in terms of well-recognized bioactive compounds namely, phenolic acids and mycosterols (mainly ergosterol), by using high performance liquid chromatography coupled to diode array and ultraviolet detectors, respectively. These compounds will be correlated to mycelia bioactivity: i) antioxidant activity, evaluated through free radicals scavenging activity, reducing power and lipid peroxidation inhibition in vitro assays; ii) anti-inflammatory activity, assessed through nitric oxide production inhibition in murine macrophages (RAW 264.7 cell line); iii) cytotoxic activity, evaluated either in human tumor cell lines (MCF-7- breast adenocarcinoma, NCIH460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma) as also in a non-tumor porcine primary liver cells culture established in-house (PLP2). Overall, our expectation is that the bioactive formulations obtained by in vitro culture can be applied as nutraceuticals or incorporated in functional foods.

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag.

Ceratocystis atrox sp. nov. associated with Phoracantha acanthocera infestations on Eucalyptus grandis in Australia

Ceratocystis spp. include important pathogens of trees as well as apparently saprophytic species. Four species have been recorded on Eucalyptus grandis in Australia, of which only one, C. pirilliformis Barnes and M.J. Wingf., is known to be pathogenic. A recent survey of pests and diseases of Eucalyptus trees in northern Queensland revealed a species of Ceratocystis associated with the tunnels made by the aggressive wood-boring insect Phoracantha acanthocera (Macleay) (Cerambicydae: Coleoptera). The aim of the present study was to identify the fungus based on morphological characteristics and comparisons of DNA sequence data for three gene regions. The fungus peripherally resembles C. fimbriata Ell. and Halst. but differs from this species most obviously by having much darker mycelium, longer ascomatal necks, segmented hyphae and an absence of aleuroconidia. Comparisons of combined sequence data confirmed that the Ceratocystis sp. from P. acanthocera represents an undescribed taxon, which is provided with the name Ceratocystis atrox sp. nov. C. atrox appears to have a close relationship with P. acanthocera, although its role in the biology of the insect is unknown and its pathogenicity has not been considered.

Alkaloid production by isolates of the sorghum ergot pathogen (Claviceps africana) from Australia and other countries

Isolates of Claviceps africana from Australia, Africa, Asia, and America were tested for the production of dihydroergosine (DHES), and its biogenic precursors dihydroelymoclavine (DHEL) and festuclavine (FEST), in culture. Several growth media were evaluated to optimise alkaloid production with little success. The best of these involved 2-stage culturing on high-sucrose substrate. Australian C. africana isolates varied widely and inconsistently in alkaloid production, with DHES concentrations in mycelium ranging from: <0.1 to 9 mg DHES/kg; <0.1 to 1.6 mg DHEL/kg; and <0.1 to 0.4 mg FEST/kg. In a separate experiment using similar culturing techniques, DHES was produced by 2 of 3 Australian isolates, 1 of 3 USA isolates, 1 of 4 Indian isolates, the sole Puerto Rican isolate, the sole Japanese isolate, but not the sole South African isolate. In this experiment, DHES concentrations detected in mycelium of Australian isolates (0.1-1.0 mg DHES/kg) were of similar magnitude to isolates from other countries (0.2-1.8 mg DHES/kg). Three C. africana isolates, including one that produced only traces of alkaloid in culture after 8 weeks, were inoculated onto panicles of sterile male sorghum plants. After 8 weeks, all 3 isolates produced 10-19 mg DHES/kg in the panicles, demonstrating that the growing plant favoured more consistent alkaloid production than culture medium.

Taxonomic notes on two powdery mildews: Phyllactinia chorisiae and Ovulariopsis wissadulae (Erysiphaceae: Phyllactinieae)

The powdery mildew Phyllactinia chorisiae has been considered conspecific with P. guttata. A re-examination of the type material of P. chorisiae and another specimen showed that this fungus, unlike P. guttata, has dimorphic conidia and its anamorph does not belong to the genus Ovulariopsis, which is the typical anamorph for Phyllactinia species. This suggests that P. chorisiae is morphologically distinct from P. guttata and should no longer be accepted as a synonym. Re-evaluation of type material of Oidiopsis wissadulae revealed that it has monomorphic conidia (mostly lemon-shaped) and hemiendophytic mycelium, a combination of characters that clearly places this fungus in the genus Ovulariopsis. Emended descriptions of P. chorisiae and Ovulariopsis wissadulae are presented.

Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae (Erysiphaceae)

Some whole leaf-clearing and staining techniques are described for the microscopic observation of the origin of powdery mildew conidiophores, whether from external mycelium or internal mycelium, emerging through stomata. These techniques enable separation of the two genera, Oidiopsis and Streptopodium, in the Erysiphaceae.