A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus

Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, alpha1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.     

Effect of Chlorine Dioxide Gas on Fungi and Mycotoxins Associated with Sick Building Syndrome

The growth of indoor molds and their resulting products (e.g., spores and mycotoxins) can present health hazards for human beings. The efficacy of chlorine dioxide gas as a fumigation treatment for inactivating sick building syndrome-related fungi and their mycotoxins was evaluated. Filter papers (15 per organism) featuring growth of Stachybotrys chartarum, Chaetomium globosum, Penicillium chrysogenum, and Cladosporium cladosporioides were placed in gas chambers containing chlorine dioxide gas at either 500 or 1,000 ppm for 24 h. C. globosum was exposed to the gas both as colonies and as ascospores without asci and perithecia. After treatment, all organisms were tested for colony growth using an agar plating technique. Colonies of S. chartarum were also tested for toxicity using a yeast toxicity assay with a high specificity for trichothecene mycotoxins. Results showed that chlorine dioxide gas at both concentrations completely inactivated all organisms except for C. globosum colonies which were inactivated an average of 89%. More than 99% of ascospores of C. globosum were nonculturable. For all ascospore counts, mean test readings were lower than the controls (P < 0.001), indicating that some ascospores may also have been destroyed. Colonies of S. chartarum were still toxic after treatment. These data show that chlorine dioxide gas can be effective to a degree as a fumigant for the inactivation of certain fungal colonies, that the perithecia of C. globosum can play a slightly protective role for the ascospores and that S. chartarum, while affected by the fumigation treatment, still remains toxic.     

Colony development in Streptomyces carpinensis: a streptomycete with substrate mycelium spores

In this paper we present an ultrastructural study of spore formation in aerial vs. substrate mycelia of Streptomyces carpinensis. Both mycelia initiated spore formation at nearly the same time of colony development but exhibited different patterns of spatial localization of sporulation: spore formation took place throughout the aerial mycelium whereas in the substrate mycelium was confined to a narrow zone at the bottom of the colony. The ultrastructural changes leading to spore formation, however, were quite similar in both mycelia, differing only with respect to the outer components of the sporal wall. Spores formed in the aerial mycelium were covered by a thin sheath whereas the spores formed in the substrate mycelium were covered by an amorphous electron-dense material.     

Lipid content during sexual development in the homothallic mucor Zygorhynchus moelleri

In Zygorhynchus moelleri, a homothallic Mucor, triglycendes are the main components of chloroform/methanol extractable lipids. The triglycerides accumulate in the aerial hyphae, particularly in the developing zygospores and in the lateral suspensors, but only after zygospore maturation. They are probably transported from the submerged mycelium to the aerial hyphae. Most of the fatty acid synthetase activity is found in the submerged mycelium. The fatty acid composition of the triglycerides does not change appreciably during sexual development. No influence of trisporic acids has been found on triglyceride synthesis or transport.     

Performance of two isolates of Isaria fumosorosea from hot climate zones in solid and submerged cultures and thermotolerance of their propagules

Isaria fumosorosea frequently causes mycosis of agricultural pests in the hot semiarid and dry tropical regions of Mexico. Because temperature tolerance restricts the use of fungal biopesticides, we investigated two isolates from these areas for possible development into mycoinsecticides for use in hot weather agricultural zones. We studied the effects of culture system (solid or submerged cultures) and temperature on the fungal growth, extracellular enzyme production, pathogenicity, and thermotolerance of the produced propagules. Between 20 and 28 °C, the specific growth rates of the isolate PCC were higher on solid media, but in the submerged culture, the isolate P43A grew faster even at temperatures of up to 34 °C. On solid media, P43A produced 1.5-fold more proteases than PCC, but in the submerged culture, both strains had similar activities. Under the same culture conditions, PCC produced a blastospore:conidia ratio of 1:2, and P43A produced a ratio of 1:5. PCC aerial conidia had the shortest Lethal Time 50 (LT₅₀, the time to reach 50 % mortality) against Galleria mellonella larvae, but LT₅₀ was equal for the aerial conidia and the submerged propagules of P43A and PCC. The submerged and aerial propagules of P43A were more thermotolerant than those of PCC. Each isolate performed differently in each culture system, and we concluded that the intended production method should be included as a criterion for screening of entomopathogenic fungus. We found that thermotolerance is a specific characteristic of an isolate from a given species. Because of its specific characteristics, P43A shows more promise for the development of a submerged conidia-based mycoinsecticide for foliar application in aqueous form in hot climate regions.     

Fungi and Bacteria in or on Leaves of Eelgrass (Zostera marina L.) from Chesapeake Bay

Samples of green and brown leaves of eelgrass (Zostera marina L.) were incubated in seawater without an additional carbon source. Parallel leaf samples were used for acridine orange bacterial counting and water-soluble aniline blue estimation of fungal biovolume. The incubations produced no evidence that there is an eelgrass counterpart for the chytridialean symbiont which is very common in turtlegrass (Thalassia testudinum König). Sterile mycelium (i.e., living mycelium without identifiable propagules) was the most prevalent fungal form on incubated samples from submerged sites, whereas Dendryphiella salina and Sigmoidea sp. (marina?) were prevalent on brown leaves from the wrack line. Attempts to assay fungal biovolume in field samples indicated that the sterile mycelium observed after incubation represented the outgrowth of formerly dormant propagules or weakly established microcolonies. It was calculated that fungal biomass could not account for more than 0.5% of leaf mass, and it was probably much smaller than this, for no fungal structures were observed even in concentrated leaf homogenates. Bacterial densities fell within the range reported for other particulate substrates. A speculative estimate of bacterial productivity was 1.4× the standing stock per day.     

The Influence of Temperature on Phytophthora infestans (Mont.) de Bary

The influence of temperature on the growth rate, sporulation density and zoospore release of Phytophthora infestans, cultivated on rye agar, has been studied. Temperature significantly influenced all the features of the fungus mentioned above. The highest yield of sporangia per 1 cm² of aerial mycelium occurred at 24°C while the highest percentage of sporangia releasing zoospores was observed when the fungus was grown at 15 °C. When considering the size of the fungal colony the highest production of sporangia was obtained at 20°C. It was concluded that the temperature at which the fungus was cultured predetermined the way it germinated.     

Endophytic blastospores of Beauveria bassiana provide high resistance against plant disease caused by Botrytis cinerea

The entomopathogenic fungus Beauveria bassiana is widely used for insect pest control and can produce three distinct infective unit types under different nutritional and environmental conditions: aerial conidia, blastospores, and submerged conidia. Here, we identified endophytic colonization ability and existing forms of the three types of B. bassiana infective units after inoculating Arabidopsis plants via soil drenching, and tested their effects on their presence mold disease caused by Botrytis cinerea. We found that all B. bassiana infective unit types colonized Arabidopsis leaves, with germinating and producing hyphae by hydrophilic blastopores and submerged conidia; further, we showed that blastospores were more effective in defending against B. cinerea, compared with aerial conidia. These findings suggest that in addition to aerial conidia, the colonization of other two types of entomopathogenic fungal infective units could also have important impacts on plant resistance. This study contributes to better understanding on the function of B. bassiana as fungal endophytes, which could lead to a new paradigm for how to successfully use these organisms in biological control against plant diseases.     

Scaling from mycelial growth to infection dynamics: a reaction diffusion approach

Numerous models have been proposed for the dynamics of fungal growth, and also for the dynamics of infection. Few models, however, have combined the mechanistic interpretation of mycelial growth with epidemiological models for the transmission of infection. Many of the mechanistic models seek to include considerable biological detail, which necessarily leads to a proliferation of state variables and parameters. Including such models within an epidemiological framework makes interpretation of underpinning processes difficult. A simple reaction diffusion model for the growth and spread of fungal mycelium is introduced and analysed, scaling from the small-scale parameters for mycelial dynamics to the large-scale properties of the colony. By coupling the output to a parsimonious epidemiological model for the dynamics of primary infection, we analyse the sensitivity of the probability of successful infection of a host to the colony dynamics associated with local bulking-up, extension, growth and nutrient consumption by the mycelium. In particular we identify optimal trade-offs in bulking-up versus dispersal in controlling infection dynamics.     

The kinetics of mycelial growth.

Based on the assumption that mycelial growth follows the logistic growth law, formulae have been developed to express the growth of fungal colonies under a variety of geometric constraints. Analysis was done of Deppe's (1973) results on surface colony growth, where the mass of the colony grew exponentially during most colonial growth, and of Trinci's (1970) results on submerged "pellet" growth, where the mass of the colony increased as the cube of time during most colony growth. In both cases, the linear dimensions of the colony were increasing linearly while the mass was changing in these quantitatively different manners. It is concluded that these disparate growth behaviours result from different habits of growth; in two-dimensional colony growth a new region of space if invaded by an amount of mycelium small in proportion to the final "carrying capacity" of the region, and in three-dimensional colony growth a region is invaded with an amount of mycelium almost equal to the region's final limiting mycelial mass. Thus, the types of growth law for colony mass which are applicable for a particular organism in a particular physical environment depend critically on the degree to which the invading hyphae initially occupy the space.     

The effect of copper ions on the production of laccase by the fungus Lentinus (Panus) tigrinus

The basidiomycete Lentinus tigrinus was cultured in media containing copper ions added at different growth stages. Copper ions at increased concentrations decelerated of the fungal biomass accumulation. The later Cu²⁺ ions were added, the better the fungal mycelium developed, and the toxic effect of Cu²⁺ was less pronounced. The maximum laccase activity (47 U/ml) was observed in the presence of 1.5–2.0 mM Cu²⁺ added on day 4 of cultivation.     

Effects of submergence on the growth of Phragmites australis seedlings

Colonisation by reed seedlings, Phragmites australis (Cav.) Trin. ex Steud. is rare and usually occurs after drawdown and when shallow water prevails. P. australis seeds have high rates of germination but successful colonisation is dependent upon subsequent water depths. We investigated the capacity of young reed plants to resist a 4 weeks submergence stress within a 5 months period, and their subsequent recovery. A pond experiment examined the interactions between submergence depth and the age of the seedlings at submergence. Four submergence treatments were used. In two partial submergence treatments, 50 and 80% of the initial leaf area was submerged. In two total submergence treatments, plants were either submerged at 125% of their initial height with possible subsequent development of emerged leaves, or the water was deepened as they grew to maintain total submergence for 4 weeks. The ages at submergence were 40, 60 and 80 days. Plants were harvested at 5 months. Shoot elongation, biomass allocations to aerial biomass, roots and rhizomes, and photosynthetic activity of aerial leaves were measured. Redox potential was measured for a subsample.Mortality (18.7%) occurred only in the permanent submergence treatment for 40-day-old seedlings. In all treatments, submerged leaves senesced, except the terminal (youngest) leaves of permanently submerged plants. Submergence differentially affected shoot length and biomass, depending upon the intensity of the treatment and the seedling age. The major differences were found between the two partial and two total submergence treatments. Partial submergence (50 and 80%) significantly enhanced biomass accumulation and growth, whereas total submergence largely decreased biomass production and growth in length, with less effect on shoot numbers. The 80-day-old seedlings tolerated submergence better but growth was poorest in medium-aged plants (60-day-old). Increased elongation of the growing internodes of up to 140% was caused by submergence, and photosynthetic activity was enhanced by 85% in emergent leaves of plants initially submerged but allowed to produce emerged leaves during the treatment period.Young P. australis plants require shallow water levels without long lasting submergence to grow and survive. Tolerance to submergence increases with age. These processes contribute to define the conditions for colonisation via seeds in P. australis.     

Growth inhibition and intracellular distribution of Pb ions by the white-rot fungus Abortiporus biennis

When Abortiporus biennis was grown on PbO-amended media, Pb (II) ions accumulated near fungal membrane structures, in the cell wall, and in the cytoplasm of the fungal cells, and their presence caused cell vacuolization. Increased concentration of PbO in the growth media caused a decline in fungal accumulation capacity. Neither PbO solubilization nor an increased level of organic acids underneath the mycelium was found.     

Regulation of hyphal morphogenesis by Ras and Rho small GTPases

The fungal kingdom is extremely diverse – comprised of over 1.5 million species including yeasts, molds and mushrooms. Essentially, all fungi have cell walls that contain chitin and the cells of most fungi grow as tube-like filaments called hyphae. These filamentous fungi, such as the mold Neurospora crassa, develop branched radial networks of hyphae referred to as mycelium. In contrast, non-filamentous fungi do not form radial mycelia, but grow as single cells, which reproduce by either budding or fission such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, respectively. Finally, there are fungi that are capable of switching between single cell, yeast form growth and filamentous growth such as Candida albicans. The switch from yeast to filamentous growth in these so-called dimorphic fungi is a virulence trait in many human and plant pathogens. Highly conserved master regulators of all three fungal growth modes – filamentous, non-filamentous and dimorphic – are the Ras and Rho small GTPases, which spatially and temporally control cell polarity establishment and maintenance. This review summarizes the key roles of the Ras and Rho GTPases during hyphal morphogenesis in a range of fungi.     

Age-dependent differential responses ofSaprolegnia hyphal tips to a helical growth-inducing factor in the agar substitute,gellan

Saprolegnia ferax produces more-or-less straight, subapically branched, hyphae when growing in liquid or agar-solidified media, with abundant aerial mycelium on the latter. In Contrast, the same medium solidified with gellan gum induced helical growth with reduced branching and almost no aerial mycelium. Helical growth induction was gellan concentration-dependent, peaking at 0.4–0.6% (w/v), when about 60% of tips were helical. Gellan-induced helices showed concentration-dependent inhibition by agarose and polyethylene glycol. Colonies on gellan-agarose, where helices were inhibited, reverted to having aerial mycelium, whereas those on gellan-polyethylene glycol did not. Branches on helical hyphae were initially linear, but converted to helical growth after about 2 h of extension. This transition was often marked by a branch, thus branch and helix competency appeared to be related. Germinating cysts took twice as long as hyphal inocula before producing helical hyphae, reinforcing the suggestion that helix competence was age-related.Achlya, but notPhytophthora, also showed gellan-induced helical growth and aerial mycelium suppression. These results showed (a) that morphogenic regulators of hyphal growth responded to gelling agents, probably high-molecular-weight polysaccharides, (b) that all growing hyphal tips were not equivalent, and (c) that hyphal tips underwent age-related changes in their response to the environment. The gellan-related differences in aerial mycelium mimic hydrophobin-based mycelium behavior and may thus indicate environmental regulation of hydrophobin production.     

Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application

Streptomyces transglutaminase (TGase) is an important industrial enzyme that catalyzes cross-linking of proteins. It is secreted as a zymogene and then is activated by proteases under physiological conditions. Although the activation process of TGase has been well investigated, the physiological function of TGase in Streptomyces has not been revealed. In this study, physiological function of TGase from Streptomyces hygroscopicus was found to be involved in differentiation by construction of a TGase gene interruption mutation strain (Δtg). The mutant Δtg showed an absence of differentiation compared with the parent strain. Furthermore, the production of TGase was found to be increased with the extending growth arrest phase of mycelium in submerged cultures. Thus, to enhance yield of TGase, the mycelium differentiation of Streptomyces was regulated via low temperature stress in a 3-L stirred-tank fermenter. The production of TGase increased by 39 % through extending the growth arrest phase for 4 h. This study found that TGase is involved in Streptomyces differentiation and proposed an approach to improve TGase production by regulation of mycelium differentiation in submerged cultures.     

Effect of Plasterboard Composition on Stachybotrys chartarum Growth and Biological Activity of Spores

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-α production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.     

The biology and immunogenicity of a 34-kDa antigen of Stachybotrys chartarum sensu lato

We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials. Fungal spores contain a modest number of proteins present in high concentrations and mycelia can contain large numbers of such proteins. Their distribution in mycelium when grown on laboratory media often does not reflect their occurrence in nature. Stachybotrys chartarum occurs on wet cellulose based building materials including paper-faced gypsum board and wood. To find potentially characteristic antigenic proteins from this fungus, we screened mycelium and spores from geographically representative strains using sera from a large number of patients who had been identified as having antibodies to fungi that grow in the built environment. Proteins were sought that were common across strains and for which antibodies were present in many sera. The use of human sera as the source of antibodies permitted the conclusion that any proteins of interest were produced in nature. The target proteins were shown to be present in spores/mycelium produced on natural substrata including straw and paper. This report concerns the isolation and partial characterization of a 34-kDa exoantigen. It is a glycoprotein that appears to comprise two subunits with molecular weights of 34 and 21 kDa which are not bound together through disulfide bonds. Both proteins have pIs in the acidic range. The sequences indicate that the protein is related to an extracellular DNase from Nectria haematococca. The partial sequences of the larger subunit are similar to that of a hypothetical protein from Gibberella zeae.     

Production of an Antifungal Chitinase from Enterobacter sp. NRG4 and its Application in Protoplast Production

Summary In this study flake chitin, crab shell chitin, mushroom stalk, fungal cell wall, wheat bran and rice bran were used as substrate for chitinase production by Enterobacter sp. NRG4 under submerged and solid state fermentation (SSF) conditions. Enterobacter sp. NRG4 produced 72 and 49.7 U/ml of chitinase in presence of cell walls of Candida albicans and Fusarium moniliforme in submerged fermentation. Under SSF, maximum chitinase production was 965 U/g solid substrate with flake chitin and wheat bran (1:3 ratio) at 75% moisture level after 144 h. The purified chitinase inhibited hyphal extension of Fusarium moniliforme, Aspergillus niger, Mucor rouxi and Rhizopus nigricans. The chitinase was effective in release of protoplasts from Trichoderma ressei, Pleurotus florida, Agaricus bisporus and Aspergillus niger. Protoplasts yield was maximum with 60 mg of 24 h old fungal mycelium incubated with 60 U of chitinase and 60 U of cellulase.