Invasion of wood by basidiomycetous fungi. V. The role of fungal elicitors of plant cell necrobiosis in the expression of pathovirulence and kratovirulence

Within the intact tree the live cell is the exclusive carrier of phytopathological defence reactions against microbial attack. In a comprehensive search for fungal virulence factors capable of weakening the living tissues in timber, 48 species of wood-destroying basidiomycetous fungi (WBF)¹) with known degree of pathosism and the corresponding degree of saprophytism were grown on liquid still cultures. Droplets of the filtered culture fluids were then applied to pin-prick wounds in the interveinal area of leaves from several tree and one herb species. Within 48 hours of development circular spots of brown necrotic tissue expanded around the pin pricks to indicate and approximately quantify the presence of fungal elicitors of necrobiosis. The resulting necrobiosis index was, with certain exceptions, not correlated with the antimicrobial potency (kratovirulence) of the fungus, its pathosism against standing timber, and the activity of exoenzymes in white-rot fungi. The necrobiosis index was, in part, positively correlated with the activity of those brown-rot fungi that dispose of an effective wood-decay capacity. Characteristic of the fungal species as well as of the fungal strain the necrobiosis index ranked from 0 to 1,421.2 mm² necrotic net area per 300 pin pricks but there was no fungus to be unable of eliciting browning in at least a few of the leaf species. The most severe necrosis was permanently observed on leaves of several generally oversusceptible tree species rather than on the notorious host species of the test fungus. It is therefore concluded that the elicitors of plant cell necrobiosis in WBF are predominantly host-nonspecific stress factors that may sometimes increase fungal pathovirulence. Their purification should help characterize their action as phytotoxic, necrobiotic, enzymatic, or even antimicrobial.     

The development of species-specific immunodiagnostics for Stachybotrys chartarum: the role of cross-reactivity

Mold contamination and exposure to fungi in indoor environments has been associated with various adverse health effects but little is known about the significance of individual fungal species in the initiation or exacerbation of such effects. Using Stachybotrys chartarum as a model fungus we sought to demonstrate that monoclonal antibodies (mAbs) can provide species-specific diagnostic reagents and also be used to investigate immunological cross-reactivity patterns among fungi. Mice were immunized with S. chartarum spore walls and monoclonal antibodies were screened against 60 fungal species and 24 different isolates of S. chartarum using an indirect ELISA. One species-specific mAb (IgG(1)) reacted only with spore preparations but not mycelium of S. chartarum or propagules of any other fungus. Five cross-reactive mAbs (IgM) documented extensive cross-reactivity among nine related Stachybotrys species and several non-related genera including several species of Cladosporium, Memnoniella, Myrothecium and Trichoderma. We also found that the ELISA reactivity for cross-reactive antigens and different isolates of S. chartarum differed considerably for normalized total amounts of mycelial antigen. We demonstrate that mAbs and immunoassays have the potential to detect S. chartarum species-specifically. The observed reactivity patterns with cross-reactive mAbs suggest that several fungi may share common antigens and that the majority of antigens are expressed by spores and mycelia. The observed cross-reactivity patterns need to be considered for accurate interpretations of environmental and serological analyses.     

“Definitive senescence” in stock cultures of basidiomycetous wood-decay fungi

For 7 cultures of basidiomycetous wood-decay fungi the decline of the following vitality marks were recorded over a 6-year period: (i) Consumption of sterilized beech wood substrate within a time unit as an indicator of the overall vigorousness of the fungus, (ii) decline of the competitive saprophytic ability (kratovirulence), which enables the fungus to colonize natural nonsterile substrates, (iii) capacity for sexual reproduction, (iv) several morphological features of the fungal colony. The reference strains kept growing at + 1 … 2°C on fertilized wood dust substrate retained in 5 of 7 cases all of their juvenility marks, although the wood-decay capacity gradually dropped back to 65 to 34% that of the initial value. The substrains maintained on liquid still cultures at 23°C lost their vitality marks after a period of continuous decline rather abruptly. With the loss of kratovirulence and fruiting capacity the strains were no longer viable under field conditions, and with the drop of the sterile wood-decay capacity to less than 3% by dry weight the formation of aerial mycelium and melanin pigments virtually ceased. In several fungal species the colonies segregated into coexisting sectors of extreme senescence and comparative juvenility, and a spread of senescence to the juvenile sectors did not occur. It is concluded that stock cultures of wood-decay fungi are subject to progressive senescence even under optimum conditions of strain preservation. Their current vitality should be examined in a sterile wood decay test before they are used in biotechnology or research.     

Phycomyces blakesleeanus BURGEFF and several wood-decay fungi in a bioassay for growth factor resources in fresh and microbially precolonized timber

The presence of the essential growth factor thiamine in timber was ascertained with the determination of the mycelial growth figures of several indicator fungi on the respective timber substrates. The behaviour of the absolutely thiamine-dependent phycomycete, Phycomyces blakesleeanus BURGEFF (Pb), as well as of 6 strains of basidiomycetous wood-decay fungi (WBF) that did or did not depend on exogenous vitamin supply proved in accordance that (i) both sapwood and heartwood material of 14 native timber species examined were able to furhish fungi with any degree of vitamin dependence with the essential growth factor(s); (ii) both fresh timber and timber pre-colonized by bacteria, ascomycetous discolouration fungi, and WBF contained thiamine resources above the saturation level of 50 μg per kg; (iii) neither a thiamine supply of 2 to 20 mg per kg total weight nor the residues of microbial pre-colonizers added to the vitamin pool of timber in that the growth of the test fungi could be further stimulated; (iv) the depressive growth of WBF on vitamin-free liquid media was not observed on vitamin-free solid media such as fertilized builder's sand. A vitamin supply did not improve the vigour of WBF of any degree of vitamin dependence, but it was a prerequisite for the growth of Pb on these substrates. It is concluded that vitamins in timber have, on their permanent excessive presence, no ecological significance. They do not add to host wood specificity, to the election of the fungus' host range, to the specialization for gymnosperm wood, to the preference for sapwood or heartwood within the stem, to the establishment of pathogenesis on standing timber, and to the preference for fresh or pre-colonized wood material.     

Development of an Oligonucleotide Array for Direct Detection of Fungi in Sputum Samples from Patients with Cystic Fibrosis

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.     

Drugs and drug intermediates from fungi: Striving for greener processes

Abstract

There is an ever-increasing demand of newer and improved drugs from biological sources to cater to the bio-pharmaceutical sector. Among various other resources, fungal species have an immense contribution owing to their potential to carry out the bio-transformations and drug synthesis in diverse conditions and in an eco-friendly manner. Advancement in the biotechnological processes has accelerated the process. Genome sequence information of various fungal species has opened newer avenues for improved and faster drug targeting and designing. The review highlights the production of pharmaceutical drugs and drug intermediates like antibiotics, anti-cancer, anti-cholesterol, anti-diabetic, immunosuppressant, anti-anxiety, anti-virals and many other drugs from fungus. Many of these have been commercialized and there are many more which are either in research or in clinical trial phase. There is a need to exploit and explore the vast biota of fungi in the hope of discovering untapped therapeutic uses of the earth’s countless species of fungus.     

Study of fungal and bacterial infections of the diabetic foot

Microbiological study for aerobic organisms, anaerobic organisms and fungi from 105 cases of diabetic foot ulcers was carried out to determine the aetiological agents and their antibiograms. Out of 265 microbial isolates obtained, 160 were aerobes, 50 anaerobes and 55 fungal strains. Polymicrobial infection was observed in 73 (69.5%) cases. The most frequently isolated aerobic microorganisms were Staphylococcus aureus and Pseudomonas aeruginosa. Among the anaerobes Bacteroides melaninogenicus and Bacteroides fragilis were most common. Candida species were preponderant among the fungal isolates. Antimicrobial sensitivity pattern of the isolates is discussed in detail.     

Hormones and the resistance of women to paracoccidioidomycosis

Paracoccidioidomycosis, one of the most important endemic and systemic mycoses in Latin America, presents several clinical pictures. Epidemiological studies indicate a striking rarity of disease (but not infection) in females, but only during the reproductive years. This suggested a hormonal interaction between female hormones and the etiologic dimorphic fungus Paracoccidioides brasiliensis. Many fungi have been shown to use hormonal (pheromonal) fungal molecules for intercellular communication, and there are increasing numbers of examples of interactions between mammalian hormones and fungi, including the specific binding of mammalian hormones by fungal proteins, and suggestions of mammalian hormonal modulation of fungal behavior. This suggests an evolutionary conservation of hormonal receptor systems. We recount studies showing the specific hormonal binding of mammalian estrogen to proteins in P. brasiliensis and an action of estrogen to specifically block the transition from the saprophytic form to the invasive form of the fungus in vitro. This block has been demonstrated to occur in vivo in animal studies. These unique observations are consistent with an estrogen-fungus receptor-mediated effect on pathogenesis. The fungal genes responsive to estrogen action are under study.     

Extraction and optimization of Penicillium sclerotiorum strain AK-1 pigment for fabric dyeing

Recently, the demand for fungal pigments has increased due to their several benefits over synthetic dyes. Many species of fungi are known to produce pigments and a large number of fungal strains for pigment production are yet to be extensively investigated. The natural pigment from sustainable natural sources has good economic and industrial value. Many synthetic colorants used in textile and various industries have many harmful effects on the human population and environment. Pigments and coloring agents may be extracted from a wide range of fungal species. These compounds are among the natural compounds having the most significant promise for medicinal, culinary, cosmetics, and textile applications. This study attempts to isolate and optimize the fermentation conditions of Penicillium sclerotiorum strain AK-1 for pigment production. A dark yellow-colored pigment was isolated from the strain with significant extractive value and antioxidant capacity. This study also identifies that the pigment does not have any cytotoxic effect and is multicomponent. The pigment production was optimized for the parameters such as pH, temperature, carbon and nitrogen source. Fabric dyeing experiments showed significant dyeing capacity of the pigment on cotton fabrics. Accordingly, the natural dye isolated from P. sclerotiorum strain AK-1 has a high potential for industrial-scale dyeing of cotton materials.     

Quorum sensing in fungi--a review

Quorum sensing (QS) is a mechanism of microbial communication dependent on cell density that can regulate several behaviors in bacteria such as secretion of virulence factors, biofilm formation, competence and bioluminescence. The existence of fungal QS systems was revealed ten years ago after the discovery that farnesol controls filamentation in the pathogenic polymorphic fungus Candida albicans. In the past decade, farnesol has been shown to play multiple roles in C. albicans physiology as a signaling molecule and inducing detrimental effects on host cells and other microbes. In addition to farnesol, the aromatic alcohol tyrosol was also found to be a C. albicans QS molecule (QSM) controlling growth, morphogenesis and biofilm formation. In Saccharomyces cerevisiae, two other aromatic alcohols, phenylethanol and tryptophol were found to be QSMs regulating morphogenesis during nitrogen starvation conditions. Additionally, population density-dependent behaviors that resemble QS have been described in several other fungal species. Although fungal QS research is still in its infancy, its discovery has changed our views about the fungal kingdom and could eventually lead to the development of new antifungal therapeutics.     

Dynamics of asexual transmission of a mitochondrial plasmid in Cryphonectria parasitica

In the chestnut blight fungus Cryphonectria parasitica, as in most fungi, little is known about the efficiency of the asexual transmission of optional mitochondrial plasmids, vertically through conidia, and horizontally through hyphal anastomoses. In this paper, we show that pCRY1, a circular mitochondrial plasmid, is transmitted vertically with 100%-efficiency through conidia. Moreover, the plasmid is transmitted horizontally through hyphal contact from donor strains to vegetatively compatible and most incompatible strains. An allelic difference between the donor and recipient strain, at only one of the five nuclear incompatibility genes that were tested strongly inhibited, but did not absolutely prevent, the transfer of pCRY1 through hyphal fusions. In contrast, allelic differences in any one or several of the other four heterokaryon-compatibility loci suppressed the transmission of the plasmid only partially or not at all. The plasmid was also transmitted among incompatible strains by protoplast fusion without the concomitant transfer of mitochondrial DNA (mtDNA). A comparison of plasmid-bearing with plasmid-free isogenic strains revealed that pCRY1 significantly diminishes the pathogenic potency of some strains of the fungus, but does not affect the virulence of others. Collectively, the observations indicate that the introduction of deleterious mitochondrial genetic elements into natural populations may be a means for managing fungal pathogens. Received: 22 August 1999 / 9 December 1999     

Strategies for the identification of virulence determinants in human pathogenic fungi

The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.     

Susceptibility of Trichophyton quinckeanum and Trichophyton rubrum to products of oxidative metabolism.

Two dermatophyte strains, Trichophyton quinckeanum and Trichophyton rubrum, were highly susceptible to in vitro killing by components of the H2O2-peroxidase-halide system. Both strains were, however, resistant to relatively high concentrations of reagent H2O2 or H2O2 enzymatically generated by glucose and glucose oxidase, KI, or lactoperoxidase (LPO) alone. Resistance to hydrogen peroxidase killing was found to be in part due to the presence of endogenous catalase in the fungi; susceptibility was increased by pretreatment of the fungi with a catalase inhibitor. Kinetic studies using small quantities of reagent or enzymatically generated H2O2 and LPO-KI showed that the system was lethal for both fungal strains within 1 min. Furthermore, using the glucose-glucose oxidase-LPO-KI system, it was shown that catalase, superoxide dismutase and histidine scavengers of H2O2, superoxide anion and singlet oxygen, respectively, prevented the killing of fungus, whereas scavengers of hydroxyl radicals such as benzoate and mannitol had no effect. T. quinckeanum was found to contain large quantities of superoxide anion, as judged by the nitroblue-tetrazolium test. Consequently, the xanthine (or hypoxanthine) and xanthine oxidase system in which the main product is superoxide anion had no toxic effect on the fungus. The high sensitivity of dermatophytes to killing by the H2O2-peroxidase-halide system active in polymorphonuclear neutrophils and macrophages may account in part for fungal toxicity in vivo.     

Genomics of the fungal kingdom: insights into eukaryotic biology

The last decade has witnessed a revolution in the genomics of the fungal kingdom. Since the sequencing of the first fungus in 1996, the number of available fungal genome sequences has increased by an order of magnitude. Over 40 complete fungal genomes have been publicly released with an equal number currently being sequenced--representing the widest sampling of genomes from any eukaryotic kingdom. Moreover, many of these sequenced species form clusters of related organisms designed to enable comparative studies. These data provide an unparalleled opportunity to study the biology and evolution of this medically, industrially, and environmentally important kingdom. In addition, fungi also serve as model organisms for all eukaryotes. The available fungal genomic resource, coupled with the experimental tractability of the fungi, is accelerating research into the fundamental aspects of eukaryotic biology. We provide here an overview of available fungal genomes and highlight some of the biological insights that have been derived through their analysis. We also discuss insights into the fundamental cellular biology shared between fungi and other eukaryotic organisms.     

Fungal allergens.

Airborne fungal spores occur widely and often in far greater concentrations than pollen grains. Immunoglobulin E-specific antigens (allergens) on airborne fungal spores induce type I hypersensitivity (allergic) respiratory reactions in sensitized atopic subjects, causing rhinitis and/or asthma. The prevalence of respiratory allergy to fungi is imprecisely known but is estimated at 20 to 30% of atopic (allergy-predisposed) individuals or up to 6% of the general population. Diagnosis and immunotherapy of allergy to fungi require well-characterized or standardized extracts that contain the relevant allergen(s) of the appropriate fungus. Production of standardized extracts is difficult since fungal extracts are complex mixtures and a variety of fungi are allergenic. Thus, the currently available extracts are largely nonstandardized, even uncharacterized, crude extracts. Recent significant progress in isolating and characterizing relevant fungal allergens is summarized in the present review. Particularly, some allergens from the genera Alternaria, Aspergillus, and Cladosporium are now thoroughly characterized, and allergens from several other genera, including some basidiomycetes, have also been purified. The availability of these extracts will facilitate definitive studies of fungal allergy prevalence and immunotherapy efficacy as well as enhance both the diagnosis and therapy of fungal allergy.     

科玛嘉念珠菌显色培养基在诊断侵袭性真菌感染中的应用

目的探讨CHROMagar显色培养基检测侵袭性真菌感染的应用价值。方法将2007年7月至2008年6月湖南省肿瘤医院肿瘤病人的呼吸道标本采用CHROMagar显色培养基及沙保罗培养基进行真菌检测,CHROMagar显色培养基上生长的真菌根据菌落的颜色及形态进行鉴别,在沙保罗培养基上生长的真菌经VITEK2-Compact全自动细菌鉴定仪进行鉴定。结果660份标本在沙保罗培养基上分离出383株真菌,经鉴定其中白色假丝酵母菌为262株、热带假丝酵母菌为51株、克柔假丝酵母菌15株、光滑假丝酵母菌34株,其他真菌21株。CHROMagar显色培养基上检出360株真菌,检出率与前者的符合率为93．99％。结论科玛嘉显色培养基能作为临床快速鉴定酵母样真菌感染的方法,对侵袭性真菌感染的早期诊断有意义;但鉴定菌种有限,有一定的局限性。     

Dermatophytes isolated from asymptomatic dogs in Adana,Turkey: A preliminary study

Recent studies demonstrate that zoophilic dermatophyte fungi, e.g., Trichophyton mentagrophytes and Microsporum canis, where emergent, are responsible for human scalp infections and the carrier state in Adana, Turkey. The aim of this study was to determine which fungi predominated the carrier state of dermatophytes and their prevalence among dogs without lesions. The fungal flora of the coats of 154 asymptomatic dogs were examined. Four (2.6%) dermatophyte strains were isolated; three T. mentagrophytes var. mentagrophytes (75%) and one M. gypseum (25%). M. canis was not isolated from any dog during the study. A detailed survey of this keratinophilic fungus, with a particular emphasis on cats, is needed to determine the exact dermatophyte flora.