Ability of extracellular proteinases of micromycetes <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>, <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>, and <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> to affect proteins of the human haemostatic system

The effect of extracellular proteinases of A. flavipes A17, A. fumigatus D1, and A. sydowii 1 on proteins of the human haemostasis system was studied. It was shown that A. fumigatus D1 proteinases are able to hydrolyze a wide range of chromogenic peptide substrates of specific human proteinases of the haemostatic system. Proteinases formed by A. flavipes A17 and A. sydowii 1 have a narrow specificity, mainly to thrombin and plasmin substrates. It was first shown that proteinase of A. flavipes A17 is capable to activate protein C and Factor X. Extracellular proteinase produced by A. sydowii 1 has greater fibrinolytic activity as compared with proteinases produced by A. flavipes A17 and A. fumigatus D1.     

New sterigmatocystin-producing species of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Versicolores</Emphasis> from indoor air in Croatia

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Protein nutritional value,polyphenols and antioxidant properties of corn fermented with <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis> and <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Solid-state fermentation (SSF) with Agaricus brasiliensis and Agaricus bisporus on corn was carried out. The results showed that SSF with the two fungi made up the deficiency of tryptophan in corn and improved the protein nutritional value of corn. The conjugated polyphenols contents in fermented corn decreased and free polyphenols (FPP) contents increased. FPP contents in corn fermented with the two fungi reached respectively 25 and 88 times of control, total polyohenols contents reached respectively 1.4 and 3.3 times of control. The antioxidant properties (i.e. 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity, reducing power, ferrous ion chelating ability and superoxide anion radical scavenging ability) of fermented corn were improved significantly. SSF with A. bisporus was more favorable to the enhancement in protein nutritional value and antioxidant properties of corn than that of A. brasiliensis. The results indicated that SSF with the two fungi could upgrade the protein nutritional value, FPP content and antioxidant properties of corn.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

The Role of <Emphasis Type="Italic">sho1</Emphasis> in Polarized Growth of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Aspergillus fumigatus is an opportunistic pathogen that may cause severe invasive disease in immunocompromised patients. The filamentous fungi undergo polarized growth, searching for nutrients in the environment and causing invasive growth in tissue. Sho1 is a sensor of the high osmolarity glycerol pathway, and the sho1 mutant showed a decrease in growth rate. We found that sho1 is involved in the polarized growth of A. fumigatus. The sho1 mutation resulted in extended isotropic growth of germinating conidia followed by multiple germ tubes and wide hyphae with short intercalary cells by calcofluor white staining. The mechanism by which sho1 gene affected polarized growth is investigated. A reduced number of apical vesicles with greater dispersion were observed by transmission electron microscopy in the Spitzenkörper body of the sho1 mutant. Actin patches were distributed randomly at low density at early stages of mutant strain fungal development and reaggregated to the hyphal tip of later stages when long filamentous fungi formed. Actin patches located at the tip of polarized wild-type cells. RNA levels of polarized growth-related genes Rho GTPases were detected by real-time PCR. The sho1 gene did not affect the RNA expression when strains were cultured at 37°C for 6 h. At 17 h, the RNA expression of rho1, rho3 and CDC42 in the sho1 mutant were 0.18-, 0.18- and 0.33-fold of that in the wild type. The sho1 gene affected the polarized growth through affecting the expression of Rho GTPases, the distribution of actin cytoskeleton, vesicle quantity and distribution.     

Taxonomic and nomenclatural notes concerning <Emphasis Type="Italic">Ormopteris</Emphasis> and <Emphasis Type="Italic">Pellaea</Emphasis> (Pteridaceae) in Brazil

We typify and clarify the nomenclature and identity of three species originally described in the fern genus Pellaea. We find Pellaea bongardiana to be illegitimate and recognize it as a synonym of Ormopteris riedelii (= P. riedelii). We also consider Pellaea brasiliensis to be a synonym of O. riedelii. The phylogenetic position of Pellaea flavescens, recently combined in Ormopteris (as O. flavescens), is discussed, and a key to all species of Ormopteris in Brazil is provided.     

Role of Host Glycosphingolipids on <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Adhesion

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor^®488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

Age-Specific Functional Response of <Emphasis Type="Italic">Aphidius matricariae</Emphasis> and <Emphasis Type="Italic">Praon volucre</Emphasis> (Hymenoptera: Braconidae) on <Emphasis Type="Italic">Myzus persicae</Emphasis> (Hemiptera: Aphididae)

The green peach aphid, Myzus persicae (Sulzer), is one of the most important aphid pests on pepper. Aphidius matricariae Haliday and Praon volucre (Haliday) are known as biological control agents for aphids in vegetable crops. In this research, age-specific functional responses of these two parasitoids were evaluated on different densities of 2, 4, 8, 16, 32, and 64 green peach aphids. Type of functional response varied from type II to type III for different ages of A. matricariae, but type of functional response was not affected by female age for P. volucre. The functional response of P. volucre was determined as type II in the whole parasitoid lifetime. The searching efficiency (a), b, and handling time (T _h ) were estimated using the Rogers equations. The highest searching efficiency (a) and lowest handling time were observed during the first half of lifetime of A. matricariae and P. volucre. Aphidius matricariae and P. volucre caused reasonable mortality of the green peach aphid by parasitism of 52.17 and 47.05 host aphids, respectively, in 24 h. Therefore, they are suggested as suitable candidates for control of M. persicae in pepper greenhouses.     

Toxic metals accumulation in <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> and <Emphasis Type="Italic">T. harzianum</Emphasis>

Heavy metal contamination represents an important environmental issue due to the toxic effects of metals on different organisms. Filamentous fungi play an important impact in the bioremediation of heavy metal-contaminated wastewater and soil. The purpose of this investigation was to observe fungal uptake behavior toward heavy metal. For this aim Trichoderma asperellum TS141 and T. harzianum TS103 at growth period were screened for their tolerance and uptake capability of cadmium (Cd), lead (Pb) and nickel (Ni) at different concentrations (0, 25, 50, 100, and 200 mg/L) in PDB media (potato dextrose broth as a complex medium). Results showed that both fungi were able to survive at the maximum concentration of 200 mg/L of the heavy metals, and remove them. T. asperellum had a better uptake capacity for Cd compared to Pb and Ni in the highest metal concentration in media. Maximum removal efficiency of Pb (68.4%) at 100 mg/L and Ni (78%) at 200 mg/L was performed by T. asperellum. For Cd, the highest removal efficiency (82.1%) was recorded by T. harzianum at 200 mg/L Cd in aqueous solution. The uptake of Cd was highly dependent on pH of solution than Pb and Ni so that the optimal pH of Cd uptake was 9 for T. asperellum and 4 for T. harzianum. Also, optimal temperature was 35°C for Cd and Pb uptake in both fungi, whereas for Ni uptake was 30 and 35°C in T. harzianum and T. asperellum, respectively. We propose that T. asperellum TS141 and T. harzianum TS103 can be used as a bioremediation agent for metal remediation from wastewater and heavy metal-contaminated soils.     

Mycorrhization between <Emphasis Type="Italic">Cistus ladanifer</Emphasis> L. and <Emphasis Type="Italic">Boletus edulis</Emphasis> Bull is enhanced by the mycorrhiza helper bacteria <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Migula

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for aflatoxin generation fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ～ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (～ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.     

Biodegradation of phenol, <Emphasis Type="Italic">o-</Emphasis>cresol, <Emphasis Type="Italic">m-</Emphasis>cresol and <Emphasis Type="Italic">p-</Emphasis>cresol by indigenous soil fungi in soil contaminated with creosote

Seven non-basidiomycetic fungi, Aspergillus, Candida, Cladosporium, Fusarium, Monicillium, Trichoderma and Penicillium, and two basidiomycetic fungi, Pleurotus and Phanerochaete were isolated from a creosote-contaminated soil by using mineral salts medium and soil extract broth containing antibiotics. Soil contaminated with phenol, o-cresol, m-cresol and p-cresol was collected from the yard of a wood treatment plant in South Africa and inoculated with the strains of Aspergillus, Cladosporium, Fusarium, Monicillium, Penicillium and Phanerochaete, selected from the isolate. The soil in some of the treatment reactors was amended with nutrient supplements to give a C:N:P ratio of 25:5:1. A total of 18 duplicate treatments were established and incubated in the dark at 25°C for 70days. The soil in all the reactors was tilled weekly and moisture was maintained at 70% field capacity. Soil samples were collected every 2weeks for analysis of residual concentrations of the phenols tested, pH measurement and moisture content determination. The nutrient-supplemented treatments were more effective in degrading the phenols (between 84 and 100%) than those that were not supplemented. Barley, which was used as bulking agent enhanced the growth of the fungi and subsequently the degradation of the phenols. Inoculation with a mixture of the six fungal isolates promoted more phenol degradation than with single isolates.     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.