Effect of nanocomposite-based packaging on microstructure and energy metabolism of Agaricus bisporus.

Rapid deterioration of postharvest Agaricus bisporus is a serious problem. Measures to improve the shelf-life are of great importance. Therefore, we used a nanocomposite-based packaging material (Nano-PM) containing nano-TiO2, nano-SiO2, nano-Ag and attapulgite to study its effect on microstructure and energy metabolism of A. bisporus. Nano-PM reduced the oxidation of lipids and proteins by activating antioxidant enzyme activities and inhibiting the accumulation of ROS, thereby maintained high level of energy status. Meanwhile, ATP content and energy charge of A. bisporus in Nano-PM increased through the energy metabolism system. This was attributed to the lower respiration rate and higher mitochondrial respiratory enzyme activities. These results indicated that Nano-PM could maintain the dense and intact microstructure of A. bisporus thus improve its shelf-life.

Evaluation of the infection process by Lecanicillium fungicola in Agaricus bisporus by scanning electron microscopy.

BACKGROUND: Lecanicillium fungicola causes dry bubble disease in Agaricus bisporus mushrooms leading to significant economic losses in commercial production. AIMS: To monitor the infection process of L. fungicola in Brazilian strains of A. bisporus. METHODS: The interaction between the mycelium of L. fungicola (LF.1) and three strains of A. bisporus (ABI 7, ABI 11/14 and ABI 11/21) was studied. Electron microscopy and X-ray microanalyses of vegetative growth and basidiocarp infection were evaluated. RESULTS: Micrographs show that the vegetative mycelium of the Brazilian strains of A. bisporus is not infected by the parasite. The images show that the pathogen can interlace the hyphae of A. bisporus without causing damage, which contributes to the presence of L. fungicola during the substrate colonization, allowing their presence during primordial formation of A. bisporus. In the basidiocarp, germ tubes form within 16h of infection with L. fungicola and the beginning of penetration takes place within 18h, both without the formation of specialized structures. CONCLUSIONS: Scanning electron microscopy enabled the process of colonization and reproduction to be observed within the formation of phialides, conidiophores and verticils of L. fungicola. The formation of calcium oxalate crystals by the pathogen was also visible using the X-ray microanalysis, both at the hyphae in the Petri plate and at basidiocarp infection site.

Electron microscopy studies of basidiosporogenesis in Agaricus brasiliensis.

The objective of this work was to study the basidiosporogenesis and the intraspecific variation in the number of basidiospores produced per basidium in Agaricus brasiliensis with transmission (TEM) and scanning electron microscopy (SEM). A. brasiliensis produces predominantly tetrasporic basidia, but this trait may vary depending on the strain. For certain strains, such as CS2 and CS7, the frequency of bisporic and trisporic basidia was similar to, or greater than, that of tetrasporic strains. These results suggest that some strains of A. brasiliensis may be amphithallic; however, this behavior is variable and strain dependent. The development of basidia and basidiospores occurs asynchronously during basidiocarp production. The basidiospore cell wall is composed of three distinct layers and presents variable thickness. The conspicuous presence of lipid bodies also was observed in the basidiospores, while nuclei, mitochondria, vacuoles and dolipore septa could be visualized only in the basidia. The basidiospores generally are produced free but also may be enveloped by an extracellular matrix with unknown chemical composition. The presence of connection hyphae linking the basidia was observed for the first time in A. brasiliensis. This characteristic, so far not reported for other fungi, may represent a specific strategy of A. brasiliensis for exchanging nuclei and other cell material between basidial cells during basidiosporogenesis.

[Morphogenesis and ultrastructure of basidiomycetes Agaricus and Pleurotus mitochondria].

Mitochondrial morphogenesis in 31 strains of 9 species of Agaricus--A. arvensis Schaeff., A. bisporus (Lange) Imbach, A. bitorquis (Quel.) Sacc., A. campestris L., A. excellens (F. H. Moller) F. H. Moller, A. macrocarpus (F. H. Möller) F. H. Möller, A. silvaticus Schaeff., A. silvicola (Vittad.) Peck, A. xanthodermus Genev--and 2 strains of Pleurotus--P. ostreatus (Jacg.) P. Kumm., P. pulmonarius (Fr.) Quel.--has many common features in mitochondria distribution under favorable growth conditions (type 1) and in reconstruction of chondriom (fission or fragmentation) under unfavorable growth conditions and aging (type 2). The first type of mitochondria distribution was observed in heterokaryotic mycelium of some Agaricus strains and Pleurotus grown in agar medium during 7-14 days, and also in submerged mycelium of some Agaricus strains and Pleurotus. The second type of mitochondria distribution was observed in homokaryotic Agaricus strains under condition of starvation, in aging mycelium (28 days of growth), and in submerged mycelium of most of Agaricus strains. The first type of chondriom consists of small granular mitochondria in the apical cells and long snake-like network in subapical cells, and restores almost completely the mitochondrial network in the aging mycelium cells. The second type of chondriom consists of small granular mitochondria in all cells of mycelium. The surface of chondriom type 2 mitochondrial membrane was usually closely associated with ribosomes and changed crists. Such mycelium cells in A. bisporus strain Bs94 were TUNEL positive. So, the types of mitochondria morphogenesis in the Agaricus and Pleurotus mycelium cells are similar at different time and growth conditions and depend on complex of physiological and biochemical process in the mycelium cells.

[Abnormal meiosis in bisporic mutants of white button mushroom Agaricus bisporus (Lange) Imbach].

A formerly developed method of obtaining spread preparations of mushroom basidial nuclei was applied to study of meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The phenotypes of disruptions in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After indusium breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.

Agaricus subrufescens, a cultivated edible and medicinal mushroom, and its synonyms.

Agaricus subrufescens Peck was cultivated first in the late 1800s in eastern North America. The type consists partly of cultivated material and partly of field-collected specimens. Once a popular market mushroom, the species faded from commerce in the early 20th century. More recently, a mushroom species growing wild in Brazil has been introduced into cultivation in Brazil, Japan and elsewhere. This Brazilian mushroom has been referred to by various names, most commonly as A. blazei Murrill (sensu Heinemann) and most recently as A. brasiliensis Wasser et al. The author first cultivated A. subrufescens in 1981 and has grown and studied Brazilian isolates since 1992. The species has an amphithallic pattern of reproduction. Based on DNA sequence analysis of the rDNA ITS region and on mating studies and genetic analysis of hybrid progeny, there is a strong case for conspecificity of the Brazilian mushrooms with A. subrufescens. Based on a study of the type and other data, the recent lectotypification of A. subrufescens is accepted. Data are presented on mushrooms of diverse geographical origins, including A. rufotegulis Nauta from western Europe, another apparent conspecific. A possible role for interpopulational hybridization in current populations of A. subrufescens is proposed. The agronomic history of the species is reviewed.

[Ultrastructure of the cell wall in vegetative mycelia of Agaricus bisporus (Lange) Imbach]. / Osobennosti ul'trastruktury kletochnoi stenki vegetativnogo mitseliia Agaricus bisporus (Lange) Imbach.

A comparative description of the cell wall ultrastructure was made for vegetative mycelia in Agaricus bisporus, other species of Agaricus, and Pleurotus ostreatus. Apart from well known ultrastructural features of cell walls of the examined strains, inclusions of different kinds were observed. The cell wall thickness varied in different Agaricus strains. Maximum differences between the examined strains were 1.4 times. The cell wall thickness in P. ostreatus strains was by 3 to 4 times less than that in Agaricus strains. Using electron micrographs, a brief description of mycelia and A. bisporus basidia ultrastructure was composed.

Aspects of the pathology and etiology of 'drippy gill' disease of the cultivated mushroom Agaricus bisporus.

Agaricus bisporus sporocarps exhibiting characteristic 'drippy gill' symptoms from a natural outbreak were examined. Discrete bacterial droplets on the hymenial lamellae often coalesced to form ribbons of bacterial ooze. Longitudinal splits on the stipe were lined with a similar bacterial ooze. Bacteria isolated from both the hymenium and stipe were identified as Pseudomonas agarici, and were confirmed to be the causal organism by satisfying Koch's postulates. By light and transmission electron microscopy, the causal bacteria were found to colonize the extrahyphal spaces and degrade the extracellular matrix within affected sporocarps. Degradation of the extracellular matrix was shown to reduce the integrity of the sporocarp, and result in stipe splitting and hymenium disruption. In artificial inoculations of the pileus, bacteria were shown to exist predominantly in sporocarp tissue below the point of inoculation and above affected areas of the hymenium, indicating an approximately vertical passage through the sporocarp via the extracellular spaces. The dissolution of the extracellular matrix, and the observed failure of the bacterium to produce a toxin active against A. bisporus, allow the bacteria to pass through protective membranes unnoticed, and infect the stipe and hymenium prior to veil break. These observations dispel previous assumptions of intrahyphal existence and transmission. In the few instances in which the bacteria were observed to be intrahyphal, the host fungal cell wall was often broken, suggesting intrahyphal existence was opportunistic rather than obligatory. The taxonomic position of a bacterium isolated previously from sporocarps exhibiting symptoms similar to those of drippy gill was determined by examining the biochemical and nutritional profiles of the bacterium, and comparing them with other Pseudomonas agarici isolates.

Algunos aspectos estructurales y funcionales de la pared celular de Agaricus bisporus y sus aplicaciones más inmediatas / Some structural and functional aspects on Agaricus bisporus cell wall and their more inmediate applications

Después de una breve introducción en la que se describe la trayectoria científica de la autora, previa a su presentación en esta Academia, se abordan algunos aspectos de la estructura y función de la pared celular de Agaricus bisporus, hongo Basidomiceto superior cultivado industrialmente para la alimentación humana, más conocido como champiñón común. Se resalta la importancia de ciertos componentes estructurales de esta envoltura celular externa, por sus aplicaciones como marcadores bioquímicos para la protección legal de cepas de interés comercial, y como barrera a vencer previamente a la necesaria mejora genética de este organismo, e igualmente por su papel no sólo estructural sino también funcional en el control de la verticiliosis, la enfermedad más dañina en los cultivos industriales del champiñón (AU)

Chemical analysis of the lamella walls of Agaricus bisporus fruit bodies.

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus.

Cell water balance of white button mushrooms (Agaricus bisporus) during its post-harvest lifetime studied by quantitative magnetic resonance imaging.

A combination of quantitative water density and T2 MRI and changes therein observed after infiltration with 'invisible' Gd-DTPA solution was used to study cell water balances, cell water potentials and cell integrity. This method was applied to reveal the evolution and mechanism of redistribution of water in harvested mushrooms. Even when mushrooms did not lose water during the storage period, a redistribution of water was observed from stipe to cap and gills. When the storage condition resulted in a net loss of water, the stipe lost more water than the cap. The water density in the gill increased, probably due to development of spores. Deterioration effects (i.e. leakage of cells, decrease in osmotic water potential) were found in the outer stipe. They were not found in the cap, even at prolonged storage at 293 K and R.H.=70%. The changes in osmotic potential were partly accounted for by changes in the mannitol concentration. Changes in membrane permeability were also indicated. Cells in the cap had a constant low membrane (water) permeability. They developed a decreasing osmotic potential (more negative), whereas the osmotic potential in the outer stipe increased, together with the permeability of cells.

An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies.

The SDS-insoluble protein fraction of Agaricus bisporus fruiting bodies was solubilized with trifluoroacetic acid. On SDS-PAGE this fraction was found to contain one abundant protein with an apparent M(r) of 16 kDa. The N-terminal amino acid sequence of this protein was determined and RT-PCR used to isolate a cDNA clone which upon sequencing identified the protein as a typical class I hydrophobin (ABH1). The gene (ABH1) was isolated and sequenced, and a second hydrophobin gene (ABH2) was found about 2.5 kbp downstream of ABH1. Purified ABH1 self-assembled at hydrophobic-hydrophilic interfaces, producing the typical rodlet layer known from other hydrophobins. Similar rodlets were observed on the surface of the fruiting body, while immunological localization showed the hydrophobin to be particularly abundant at the outer surface of fruiting bodies, in the veil and in the core tissue of the stipe. Transcripts of ABH1 were found only in fruiting-body hyphae. The ABH1 hydrophobin is probably solely responsible for the hydrophobicity of the fruiting-body surface but may also line air channels within fruiting bodies.

NMR imaging of white button mushroom (Agaricus bisporis) at various magnetic fields.

Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) have been applied to visualize physiological phenomena in plants and agricultural crops. Imaging sequences that result in contrast of a combination of parameters (e.g., proton density, T1, T2, T2*) cannot be used for a correct and unique interpretation of the results. In this study multiecho imaging together with monoexponential T2 decay fitting was applied to determine reliable proton density and T2 distributions over a mushroom. This was done at three magnetic field strengths (9.4, 4.7, and 0.47 T) because susceptibility inhomogeneities were suspected to influence the T2 relaxation times negatively, and because the influences of susceptibility inhomogeneities increase with a rise in magnetic field strength. Electron microscopy was used to understand the different T2's for the various tissue types in mushrooms. Large influences of the tissue ultrastructure on the observed T2 relaxation times were found and explained. Based on the results, it is concluded that imaging mushrooms at low fields (around or below 0.47 T) and short echo times has strong advantages over its high-field counterpart, especially with respect to quantitative imaging of the water balance of mushrooms. These conclusions indicate general validity whenever NMR imaging contrast is influenced by susceptibility inhomogeneities.

Phenotypic variation of Pseudomonas putida and P. tolaasii affects attachment to Agaricus bisporus mycelium.

The effect of phenotypic variation on attachment of Pseudomonas tolaasii and P. putida to Agaricus bisporus mycelium was investigated. Quantitative studies demonstrated the ability of each isolate to attach rapidly and firmly to A. bisporus mycelium and significant differences in attachment of wild-type and phenotypic variant strains were observed. This was most pronounced in P. tolaasii, where the percentage attachment of the wild-type form was always greater than that of the phenotypic variant. The medium upon which the bacteria were cultured, prior to conducting an attachment assay, had a significant effect on their ability to attach. Attachment of the wild-type form of P. putida was enhanced when the assay was performed in the presence of CaCl2, suggesting the involvement of electrostatic forces. No correlation was observed between bacterial hydrophobicity and ability to attach to A. bisporus mycelium. Scanning electron microscopy confirmed the results obtained from the quantitative studies and provided further evidence for marked differences in the ability of the pseudomonads to attach to mycelium. Fibrillar structures and amorphous material were frequently associated with attached cells and appeared to anchor bacteria to each other and to the hyphal surface. A time-course study of attachment using transmission electron microscopy revealed the presence of uneven fibrillar material on the surface of cells. This material stained positive for polysaccharide and may be involved in ensuring rapid, firm attachment of the cells.

The structure of the mycelial wall of Agaricus bisporus.

The chemical composition of fractions extracted from the mycelial walls of Agaricus bisporus was studied. Attempts were made to locate these materials "in situ" by comparing electron micrographs of shadowed and sectioned cell walls. There are at least two chemically distinct species of carbohydrate polymers: one yielding mainly glucose with lower amounts of mannose, galactose and xylose and amino acids, and the other yielding chiefly N-acetylglucosamine with some glucose and amino acids. It was shown that none of these polymers constituted a completely physically distinct layer, but there do seem to be two regions at the outer and inner surfaces with apparently different properties. Materials which react with MnO4K to produce an electron-dense material are located throughout the wall, but tend to be concentrated in the outer and inner regions. It was shown that fibers constitute one important fraction of the original wall material.