Production Of Basidiospores Research Articles

Optimization of the Solid-State Culture Conditions and Chemical Component Analysis of Poria cocos (Agaricomycetes).

The optimal cultivation conditions and chemical components of Poria cocos fruiting bodies were examined by employing the single factor and response surface methods to screen for optimal conditions for artificial cultivation. The differences in chemical composition among the fruiting bodies, fermented mycelium, and sclerotia of P. cocos were compared using UV spectrophotometry and high-performance liquid chromatography (HPLC). The optimal growth conditions for P. cocos fruiting bodies were 28.5°C temperature, 60% light intensity, and 2.5 g pine sawdust, which resulted in the production of numerous basidiocarps and basidiospores under microscopic examination. Polysaccharides, triterpenoids, and other main active components of P. cocos were found in the fruiting bodies, sclerotia, and fermented mycelium. The triterpenoid components of the fruiting bodies were consistent with those of the sclerotia. The content of pachymic acid in the fruiting bodies was significantly higher than that in the sclerotia, with a value of 33.37 ± 0.1902 mg/g. These findings provide novel insights into the sexual breeding and comprehensive development and utilization of P. cocos.

Tissue characterization of the medical fungus Hericium coralloides by focus-variation microscopy

ABSTRACT In this study, the potential of focus-variation microscopic imaging was evaluated in a study of morphological patterns of the potential medicinal fungus Hericium coralloides (Basidiomycota). We created three-dimensional reconstructions and visualizations using the imaging technique on a fresh H. coralloides basidioma. The aim was to approximate the spore dispersal efficiency of this basidiomata type regarding the investment of tissue biomass and its reproductive output (production of basidiospores). Results were correlated with published data gained from magnetic resonance imaging and micro-computed tomography. It is demonstrated that focus-variation microscopic imaging results in a more distinct picture of the morphology of the edible and potentially medicinal H. coralloides basidiomata. However, a direct measurement of spore production was not possible. Spore production could only be estimated in combination with a mathematical model because the surface was not directly measurable due to the cellular heterogeneity. However, focus-variation microscopic imaging allows a better and faster estimation of spore production compared with the published methods. Furthermore, it was found that a scanning resolution of 5× is sufficient for determining the fungal surface precisely because at a higher resolution artifacts occur, resulting in adulteration of the image.

Comparative transcriptomics of Gymnosporangium spp. teliospores reveals a conserved genetic program at this specific stage of the rust fungal life cycle

BackgroundGymnosporangium spp. are fungal plant pathogens causing rust disease and most of them are known to infect two different host plants (heteroecious) with four spore stages (demicyclic). In the present study, we sequenced the transcriptome of G. japonicum teliospores on its host plant Juniperus chinensis and we performed comparison to the transcriptomes of G. yamadae and G. asiaticum at the same life stage, that happens in the same host but on different organs.ResultsFunctional annotation for the three Gymnosporangium species showed the expression of a conserved genetic program with the top abundant cellular categories corresponding to energy, translation and signal transduction processes, indicating that this life stage is particularly active. Moreover, the survey of predicted secretomes in the three Gymnosporangium transcriptomes revealed shared and specific genes encoding carbohydrate active enzymes and secreted proteins of unknown function that could represent candidate pathogenesis effectors. A transcript encoding a hemicellulase of the glycoside hydrolase 26 family, previously identified in other rust fungi, was particularly highly expressed suggesting a general role in rust fungi. The comparison between the transcriptomes of the three Gymnosporangium spp. and selected Pucciniales species in different taxonomical families allowed to identify lineage-specific protein families that may relate to the biology of teliospores in rust fungi. Among clustered gene families, 205, 200 and 152 proteins were specifically identified in G. japonicum, G. yamadae and G. asiaticum, respectively, including candidate effectors expressed in teliospores.ConclusionsThis comprehensive comparative transcriptomics study of three Gymnosporangium spp. identified gene functions and metabolic pathways particularly expressed in teliospores, a stage of the life cycle that is mostly overlooked in rust fungi. Secreted protein encoding transcripts expressed in teliospores may reveal new candidate effectors related to pathogenesis. Although this spore stage is not involved in host plant infection but in the production of basidiospores infecting plants in the Amygdaloideae, we speculate that candidate effectors may be expressed as early as the teliospore stage for preparing further infection by basidiospores.

The rust fungus Puccinia arechavaletae, a potential biological control agent of balloon vine (Cardiospermum grandiflorum) in South Africa. I: Biology

Studies on the rust fungus Puccinia arechavaletae, were conducted to determine its potential for use as a classical biological control agent against the alien environmental weed Cardiospermum grandiflorum in South Africa. The production of basidiospores is a crucial stage in the life cycle of this microcyclic rust species, and was determined to optimally occur at 20 °C, under high relative humidity (above 90%), in the presence of free water. The first basidiospores were released from mature telia after being incubated for 4 h under optimum conditions, reaching a peak of production after 24 h. Germination of basidiospores optimally occurred at 20 °C, and they were extremely vulnerable to desiccation. Germinating basidiospores directly penetrated epidermal cells of South African C. grandiflorum within which a vesicle formed, followed by rapid radial colonization of the leaf tissue by fungal mycelium. The first symptoms observed were small chlorotic spots on the leaves from 14 days onwards, with telial pustules appearing between 14 and 21 days post inoculation. The rust P. arechavaletae was shown to be highly pathogenic towards South African C. grandiflorum, causing severe disease symptoms with a detrimental effect on plant health and vigor.

Effect of temperature, leaf wetness and the developmental stage of host tissue on infection of Acacia mearnsii by Uromycladium acaciae (Pucciniales)

Uromycladium acaciae causes a serious rust disease in plantations of non-native Acacia mearnsii (black wattle) in South Africa. Little is known about the biology of U. acaciae, making disease control difficult. Germination studies and artificial inoculations were conducted to identify the optimal environmental conditions for infection by U. acaciae. Germination of teliospores, basidiospores, and urediniospores was assessed at seven temperatures, with or without light. The effect of temperature on infection was also assessed. As was the effect of dew period length on germination of teliospores, production of basidiospores and infection. Teliospores and urediniospores germinated between 5 and 30 °C, with an optimum at 15–25 °C. Basidiospores were produced and germinated at temperatures between 5 and 25 °C, with an optimum at 15–20 °C. The optimum temperature for infection by basidiospores was 15–20 °C. All spore types germinated in 6–24 h under optimal conditions. However, production of basidiospores was severely reduced if teliospore germination was interrupted by dry periods, even if teliospores were re-wetted. Symptoms and telia developed on only one plant exposed to a dew period of less than 12 h, with a dew period of 48 h found to be optimal. Artificial inoculation experiments showed that U. acaciae was only able to infect young, growing tissues. Results of this research can be used to develop an artificial inoculation protocol for resistance screening and in disease risk modelling and forecasting.

Mycelial growth, starch degradation ability and aggressiveness of Moniliophthora perniciosa isolates towards cacao

The variability of Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao (Theobroma cacao), was assessed based on measures of (i) mycelial growth rates (MGR), (ii) area of starch degradation (ASD) and (iii) aggressiveness towards susceptible and resistant clones. Ninety-six isolates were obtained from vegetative and flower cushion brooms from five resistant clones and one susceptible clone at two experimental sites at CEPEC, BA, Brazil. Seven isolates varying in the production of basidiospores, MGR and ASD were inoculated into six cacao clones being one susceptible, (SIC17) and five resistant (CCN10, Sca 6, TSH 1188, TSH 565, and TSH 516) to the disease. Disease incidence (SYMP) and severity (TNB, total number of brooms and DI, disease index) were assessed. Isolates obtained from TSH565, TSH516 and SIC17 VB were the most aggressive towards the cacao genotypes. MGR was not associated with aggressiveness, but ASD was significantly associated, at moderate levels, with disease incidence (r = 0.44; p = 0.018), TNB (r = 0.44; p = 0.018), and DI (r = 0.47; p = 0.012). Our results suggest that ASD is a promising biochemical marker to select for aggressiveness within M. perniciosa based on DI and TNB. On the other hand, MGR is a poor predictor of aggressiveness.

Application of micro-computed tomography to microstructure studies of the medicinal fungus Hericium coralloides

The potential of 3-D nondestructive imaging techniques such as micro-computed tomography (micro-CT) was evaluated to study morphological patterns of the potential medicinal fungus Hericium coralloides (Basidiomycota). Micro-CT results were correlated with histological information gained from scanning electron microscopy (SEM) and light microscopy (LM). It is demonstrated that the combination of these imaging methods results in a more distinct picture of the morphology of the edible and potentially medicinal Hericium coralloides basidiomata. In addition we have created 3-D reconstructions and visualizations based on micro-CT imagery from a randomly selected part of the upper region of a fresh H. coralloides basidioma: Analyses for the first time allowed an approximation of the evolutionary effectiveness of this bizarrely formed basidioma type in terms of the investment of tissue biomass and its reproductive output (production of basidiospores).

Morphological and tissue characterization of the medicinal fungus Hericium coralloides by a structural and molecular imaging platform

In this study the potential of new imaging techniques such as Magnetic Resonance Imaging (MRI), Matrix-Assisted Laser Desorption/Ionization (MALDI) profiling mass spectrometry ("MALDI Profiling") and Fourier Transform Infrared (FTIR) spectroscopic imaging was evaluated to study morphological and molecular patterns of the potential medicinal fungus Hericium coralloides. For interpretation, the MALDI profiling, FTIR imaging and MRI results were correlated with histological information gained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM). Additionally we tested several evaluation processes and optimized the methodology for use of complex FTIR images to monitor molecular patterns. It is demonstrated that the combination of these spectroscopic methods enables to gain a more distinct picture concerning morphology and distribution of active ingredients. We were able to obtain high quality FTIR imaging and MALDI-profiling results and to distinguish different tissue types with their chemical ingredients. Beside this, we have created a 3-D reconstruction of a mature Hericium basidioma, based on the MRI dataset: analyses allowed, for the first time, a realistic approximation of the "evolutionary effectiveness" of this bizarrely formed basidioma type, concerning the investment of sterile tissue and its reproductive output (production of basidiospores).

Composition of the organic phosphorus fraction in basidiocarps of saprotrophic and mycorrhizal fungi

In our screening, we aimed to detect phosphonates and other forms of organic phosphorus in basidiocarps and vegetative mycelia of six common basidiomycetes. Organic phosphorus-containing compounds were extracted in alkali and analysed using 31P NMR. Monoesters, diesters, pyrophosphates and polyphosphates detected in high amounts reflected the high metabolic activity in basidiocarps (growth, production of basidiospores). Phosphonates were present in all samples, in concentrations ranging from 14 mg kg −1 of the extracted phosphorus in Boletus badius basidiocarp to 140 mg kg −1 in Amanita muscaria vegetative mycelium. Detection of phosphonates in basidiocarps together with our previous evidence from laboratory experiments support the fungal production of natural phosphonates in forest ecosystems.

Virulence Attributes and Hyphal Growth of C. neoformans Are Quantitative Traits and the MATα Allele Enhances Filamentation

Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both α and a mating type. Monokaryotic fruiting is a modified form of sexual reproduction that involves cells of the same mating type, most commonly α, which is the predominant mating type in both the environment and clinical isolates. However, some a isolates can also undergo monokaryotic fruiting. To determine whether mating type and other genetic loci contribute to the differences in fruiting observed between α and a cells, we applied quantitative trait loci (QTL) mapping to an inbred population of F2 progeny. We discovered that variation in hyphal length produced during fruiting is a quantitative trait resulting from the combined effects of multiple genetic loci, including the mating type (MAT) locus. Importantly, the α allele of the MAT locus enhanced hyphal growth compared with the a allele. Other virulence traits, including melanization and growth at 39 °C, also are quantitative traits that share a common QTL with hyphal growth. The Mac1 transcription factor, encoded in this common QTL, regulates copper homeostasis. MAC1 allelic differences contribute to phenotypic variation, and mac1Δ mutants exhibit defects in filamentation, melanin production, and high temperature growth. Further characterization of these QTL regions will reveal additional quantitative trait genes controlling biological processes central to fungal development and pathogenicity.

Chlamydospore formation during hyphal growth in Cryptococcus neoformans.

Cryptococcus neoformans, a basidiomycetous fungal pathogen, infects hosts through inhalation and can cause fatal meningoencephalitis in individuals if untreated. This fungus undergoes a dimorphic transition from yeast to filamentous growth during mating and monokaryotic fruiting, which leads to the production of hyphae and airborne infectious basidiospores. Here we characterized a novel morphological feature associated with the filamentous stages of the life cycle of C. neoformans which resembles resting or survival structures known as chlamydospores in other fungi. The C. neoformans chlamydospore-like structure is rich in glycogen, suggesting that it might have a role as an energy store. However, characterization of mutants with decreased or increased levels of glycogen production showed that glycogen levels have little effect on filamentous growth, sporulation, or chlamydospore formation. These results suggest that the formation of chlamydospores is independent of glycogen accumulation level. We also show that chlamydospore formation does not require successful sporulation and that the presence of chlamydospores is not sufficient for sporulation. Although the biological functions of chlamydospores remain to be established for this pathogenic fungus, their formation appears to be an integral part of the filamentation process, suggesting that they could be necessary to support sexual sporulation under adverse conditions and thereby facilitate the production of infectious basidiospores or long-term survival propagules in harsh environments.

A yeast under cover: the capsule of Cryptococcus neoformans.

Few fungi are pathogenic to humans. Of these, Cryptococcus neoformans has emerged as an important cause of mortality in immunocompromised patients, especially those with AIDS. As a result, extensive research efforts have addressed the pathogenesis and virulence of this organism. C. neoformans is a basidiomycetous fungus that is ubiquitous in the environment, where it is found in soil, in association with certain trees, and in bird guano (16). Because of its ubiquity, it has been suggested that most people are exposed to C. neoformans early in life (41). The fungus is heterothallic, with mating types MATa and MAT. Asexual reproduction takes place either by budding or, in the case of MAT cells, by haploid fruiting in response to nutrient deprivation or exposure to the mating pheromone a factor (106). Sexual reproduction occurs when cells of opposite mating types come together to form a heterokaryon that ultimately leads to the production of basidia and basidiospores (64). Desiccated cells and the spores formed by haploid fruiting or sexual reproduction have all been suggested to serve as infective particles, which must be less than 2 m in diameter to penetrate the lung parenchyma (44, 86). Infection occurs when the fungal particles are inhaled and enter the alveolar space. In most immunocompetent individuals, this infection is either cleared or remains dormant until an immune imbalance leads to further development. In the setting of compromised immune function, however, the fungus disseminates, with particular tropism for the central nervous system. In severe cases, cryptococcal infection progresses to a meningoencephalitis that is fatal if left untreated. C. neoformans virulence is mediated predominantly by a polysaccharide capsule that surrounds its cell wall and has multiple effects on the host immune system. This structure provides a physical barrier that interferes with normal phagocytosis and clearance by the immune system. Capsule components inhibit the production of proinflammatory cytokines, deplete complement components (by efficiently binding them), and reduce leukocyte migration to sites of inflammation (11). The capsule also constitutes the major diagnostic feature of cryptococcosis, because its components can be detected in the bloodstream and it can be visualized with light microscopy by using India ink staining. The capsule excludes the ink particles and forms characteristic halos (Fig. 1A) whose diameters are often several times that of the cell. The elaborate structure of the capsule may also be appreciated by electron microscopy (Fig. 1B and C). Given the importance of the capsule in cryptococcal disease, tremendous effort has been applied in recent years to understanding its biology. This review focuses on the resulting advances in our understanding of the structure and synthesis of the capsular components, the incorporation of these components into the existing capsular network, the association between the capsule and the cell wall, and the regulation of capsule growth.

Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus

Double-stranded RNA (dsRNA) has been isolated from Agaricus bisporus fruit bodies exhibiting a wide range of disease symptoms. The symptoms which occurred singularly or in combination included; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield. All symptoms were associated with loss of yield and/or product quality. Collectively, these symptoms are described as mushroom virus X (MVX) disease. The dsRNA titre was much lower than that previously encountered with the La France viral disease of mushrooms and a modified cellulose CF11 protocol was used for their detection. A broad survey of cultivated mushrooms from the British industry identified dsRNA elements ranging between 640 bp and 20.2 kbp; the majority have not previously been described in A. bisporus. 26 dsRNA elements were identified with a maximum of 17, apparently non-encapsidated dsRNA elements, in any one sample. Three dsRNAs (16.2, 9.4 and 2.4 kbp) were routinely found in mushrooms asymptomatic for MVX. Previously, La France disease was effectively contained and controlled by minimising the on-farm production and spread of basidiospores. Our on-farm observations suggest that MVX could be spread by infected spores and/or mycelial fragments.

Cloning and sequence analysis of the basidiomycete Lentinus edodes ribonucleotide reductase small subunit cDNA and expression of a corresponding gene in L. edodes

We have previously isolated the uck1 gene encoding UMP-CMP kinase from the basidiomycete Lentinus edodes (Kaneko et al., 1998). It was shown to be most actively transcribed in hymenophores of mature fruiting bodies of L. edodes. The reduction of NDPs produced by the nucleoside monophosphate kinase to dNDPs has been known to be catalyzed by ribonucleotide reductase (RNR) which consists of a heterodimer of large and small subunits. So we attempted to isolate the L. edodes cDNA(s) of RNR and study the expression in L. edodes of the corresponding gene(s), resulting in an isolation of the small subunit cDNA from a mature fruiting-body cDNA library of the fungus. This cDNA, named Le.rnr2c, was shown to encode a 418 amino acids (aa) protein, named Le.RNR2, of which the deduced aa sequence shows an overall identity of 71.9% to that of Schizosaccharomyces pombe RNR small subunit. The Le.rnr2 gene was found to be most actively transcribed in hymenophores of mature fruiting body of L. edodes. The in situ RNA-RNA hybridization analysis showed the presence of markedly large amount of the Le.rnr2 transcript in both hymenium and outer region of trama in the hymenophore. The same experiment was done for the uck1 gene, obtaining a similar result. The hymenium contains many basidia in which fusion of two nuclei, meiosis, replication, etc. essential for production of basidiospores occur. The outer region of trama is the region branching out into subhymenium. These imply that Le.rnr2 gene (and uck1 gene) play a role mainly in the nucleotide biosynthesis essential both for production of basidiospores and for divergence of trama cells into subhymenium cells in the hymenophore.

Mastigobasidium, a new teleomorphic genus for the perfect state of ballistosporous yeast Bensingtonia intermedia.

A new genus, Mastigobasidium, is proposed for teliospore-forming, xylose-lacking, ballistosporogenous, glucuronate-positive yeasts. The distinguishing features of the genus are: germination of the teliospore by several long aseptate hyphae; curved phragmometabasidia development on the apices of these hyphae; and production of basidiospores on a peg in clusters. The type strain of heterothallic, nitrate-negative species Mastigobasidium intermedium is VKM Y-2720T (Bullera intermedia type strain) and the allotype strain is VKM Y-2727AL (Sporobolomyces weijmanii type strain).

Teliospore germination in Tilletia opaca and T. sumatii and the nature of the tilletiaceous basidium

The process is described of teliospore germination in Tilletia opaca and T. sumatii leading to the production of basidiospores on basidia, and later the development of ballistospores and blastospores on agar. In both species the basidium shows some evidence of being a condensed dichotomously branched structure. The situation in these two smuts is discussed in relation to that in several other species of Tilletia , and some consideration is given to the origirt of the basidium and evolutionary trends within the genus.

Alternative methods for production and staining of Phanerochaete chrysosporium basidiospores.

Homokaryotic isolates of Phanerochaete chrysosporium are generally obtained by stimulating the production of basidiospores. The most commonly used method requires a special incubator that is maintained at 28 degrees C with continuous illumination. Here we report an alternate method which permits the production of basidiopores with common laboratory incubators and requires no special illumination conditions. This alternate method gives reproducible results and yields basidiospores that are not contaminated with conidia. We also report a detailed optimized method for staining basidiospores for visualizing nuclei.

Effects of Environmental Factors on Teliospore Germination, Basidiospore Formation, and Infection ofXanthium occidentalebyPuccinia xanthii

The effects of temperature, dew, high relative humidity, and light regimes on teliospore germination, basidiospore production and germination, and infection of Noogoora burr (Xanthium occidentale) by Puccinia xanthii were examined. The optimum temperature for the germination of teliospores was between 20 and 30 C, and the optimum temperature for the production and germination of basidiospores was 20 C. The total number of basidiospores produced increased as the period under conditions of high relative humidity was increased. The highest levels of infection were observed at temperatures of 20 and 25 C []

In vitro production of basidiospores of Crinipellis perniciosa, the causative agent of witches' broom disease of cocoa

The report is the first account of the production of basidiospores from in vitro mycelial cultures of C. perniciosa. It is concluded that specific light and temperature regimes may be responsible for stimulating spore production.

The effect of cane management techniques on raspberry yellow rust (Phragmidium rubi‐idaei)

SUMMARYThe effects of various cane removal treatments on the epidemiology of Phragmidium rubi‐idaei were studied in four experiments in infected raspberry plantations in Wales and Scotland. In 1981, removal of 15 cm high young canes either by cutting or by spraying dinoseb‐in‐oil reduced infection of a second flush of canes (replacement canes) compared to that of the first flush of canes on untreated plots of cv. Mailing Delight in Wales and of cv. Glen Clova in Scotland. In 1982, removal of 20 cm high young canes reduced infection of cv. Mailing Delight in Wales but in Scotland only removal of 60 cm high canes by cutting reduced infection of replacement canes. Removal of all fruiting canes and old cane stubs reduced rust infection of young canes, but removal of successive flushes of replacement canes did not reduce infection of lateral shoots on the fruiting canes.Dinoseb‐in‐oil applied to the bases of fruiting canes before emergence of young canes had no effect on infection of young canes in Wales or Scotland. The production of basidiospores was inhibited in vitro by dinoseb‐in‐oil at concentrations over 1.0 μg a.i./ml.