Manganese superoxide dismutase based phylogeny of pathogenic fungi

Superoxide dismutases (SODs), which provide protection against oxidative stress, exhibit an essential role for fungal cell survival, especially during host invasion. Here, 20 partial SOD sequences from 19 pathogenic fungi were determined and aligned with 43 homologous fungal sequences from databases. All sequences encoded tetrameric manganese (Mn)-containing SODs showing predicted cytosolic or mitochondrial subcellular localization. Numerous fungi possessed both cytosolic and mitochondrial MnSODs in addition to the mainly cytosolic copper/zinc isozyme. Moreover, MnSOD sequence variability was higher than SSU rRNA and similar to ITS rRNA, suggesting MnSOD could be used to identify closely related fungal species. MnSOD- and SSU rRNA-based phylogenetic trees were constructed and compared. Despite a more complex topology of the MnSOD tree, due to several gene duplication events, all the classic fungal classes and orders were recovered. A salient point was the existence of two paralogous cytosolic and mitochondrial MnSODs in some Ascomycota. A hypothetical evolutionary scenario with an early gene duplication of the "mitochondrial" gene is proposed.     

Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi.

Artemisia annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. Present investigation is aiming at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity against crop-threatening fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum, Gerlachia nivalis and Phytophthora capsici. These plant pathogens are still causing wheat take-all, sharp eyespot, common rot, scab, snow mould, and pepper phytophthora blight, respectively. Out of 39 endophytes investigated, 21 can produce in vitro substances that are inhibitory to all or a few of the tested phytopathogens whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with EtOAc and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were EtOAc-extractable.     

Lipases in autolysed cultures of filamentous fungi

Fifty-one fungi from different genera and strains were checked in plate to determine lipase activity in protein precipitates from their autolysed cultures. Each of them was then analysed at 3·5, 6·5 and 9·2 pH units and, as a consequence, basic lipases with high activity at 9·2 pH were found after 1 h of incubation. Only 25% of the studied fungi showed this lipase activity, among them the best producers were fungi from genus Fusarium (47% of fungi had lipase activity). In addition to lipase activity, Fusaria showed a low hydrolytic activity on cutin and suberin. The genus Aspergillus produced lipase and cutinase activity to a similar extent. Aspergillus nidulans 2544 also showed suberinase activity in a considerable amount. Penicillium species had very low activities. Other species and strains from genus Trichoderma , order Mucorales and class Basidiomycetes, did not show lipase activity in their degradative processes.     

Crystal formation in cultures of fungi II

Summary The present study offers the picture of crystal formation in cultures of dermatophytes photographed in white light. Isometric and anisometric crystal forms are profusely depicted. Pranks and mimics of crystal forms are pointed out. Photomicrographic data are given.

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Zusammenfassung In der vorliegenden Untersuchung wird das Bild der Krystallbildung in Dermatophytenkulturen in weissem Licht dargestellt. Isometrische und anisometrische Krystallformen werden reichlich illustriert. Auf Streiche und Nachahmungen der Krystallformen wird hingewiesen. Photomikrographische Daten sind gegeben.

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Résumé La présente étude offre le tableau des formations des crystaux des dermatophytes photographiées en lumière blanche. Des formes isométriques et anisométriques des crystaux ont été illustrées abondamment. Des coups et des mimiques des crystaux sont présentés. Les données photomicrographiques ont été élucidées.

     

Oxidative stress in submerged cultures of fungi

It has been known for many years that oxygen (O2) may have toxic effects on aerobically growing microorganisms, mainly due to the threat arising from reactive oxygen species (ROS). In submerged culture industrial fermentation processes, maintenance of adequate levels of O2 (usually measured as dissolved oxygen tension (DOT)) can often be critical to the success of the manufacturing process. In viscous cultures of filamentous cultures, actively respiring, supplying adequate levels of O2 to the cultures by conventional air sparging is difficult and various strategies have been adopted to improve or enhance O2 transfer. However, adoption of those strategies to maintain adequate levels of DOT, that is, to avoid O2 limitation, may expose the fungi to potential oxidative damage caused by enhanced flux through the respiratory system. In the past, there have been numerous studies investigating the effects of DOT on fungal bioprocesses. Generally, in these studies moderately enhanced levels of O2 supply resulted in improvement in growth, product formation and acceptable morphological changes, while the negative impact of higher levels of DOT on morphology and product synthesis were generally assumed to be a consequence of "oxidative stress." However, very little research has actually been focused on investigation of this implicit link, and the mechanisms by which such effects might be mediated within industrial fungal processes. To elucidate this neglected topic, this review first surveys the basic knowledge of the chemistry of ROS, defensive systems in fungi and the effects of DOT on fungal growth, metabolism and morphology. The physiological responses of fungal cells to oxidative stress imposed by artificial and endogenous stressors are then critically reviewed. It is clear that fungi have a range of methods available to minimize the negative impacts of elevated ROS, but also that development of the various defensive systems or responses, can itself have profound consequences upon many process-related parameters. It is also clear that many of the practically convenient and widely used experimental methods of simulating oxidative stress, for example, addition of exogenous menadione or hydrogen peroxide, have effects on fungal cultures quite distinct from the effects of elevated levels of O2, and care must thus be exercised in the interpretation of results from such studies. The review critically evaluates our current understanding of the responses of fungal cultures to elevated O2 levels, and highlights key areas requiring further research to remedy gaps in knowledge.     

Manganese and iron oxidation by fungi isolated from building stone

Acid and nonacid generating fungal strains isolated from weathered sandstone, limestone, and granite of Spanish cathedrals were assayed for their ability to oxidize iron and manganese. In general, the concentration of the different cations present in the mineral salt media directly affected Mn(IV) oxide formation, although in some cases, the addition of glucose and nitrate to the culture media was necessary. Mn(II) oxidation in acidogenic strains was greater in a medium containing the highest concentrations of glucose, nitrate, and manganese. High concentrations of Fe(II), glucose, and mineral salts were optimal for iron oxidation. Mn(IV) precipitated as oxides or hydroxides adhered to the mycelium. Most of the Fe(III) remained in solution by chelation with organic acids excreted by acidogenic strains. Other metabolites acted as Fe(III) chelators in nonacidogenic strains, although Fe(III) deposits around the mycelium were also detected. Both iron and manganese oxidation were shown to involve extracellular, hydrosoluble enzymes, with maximum specific activities during exponential growth. Strains able to oxidize manganese were also able to oxidize iron. It is concluded that iron and manganese oxidation reported in this work were biologically induced by filamentous fungi mainly by direct (enzymatic) mechanisms.Correspondence to: G. Gomez-Alarcon.     

Immuno-identification of cultures of fungi pathogenic to man

Because isolates of the fungal pathogensBlastomyces dermatitidis. Coccidioides immitis, Histoplasma capsulatum, andParacoccidioides brasiliensis frequently vary widely in gross and microscopic features and are often difficult or impossible to convert to their tissue forms, a simple diagnostic procedure not dependent upon sporulation is needed to identify them specifically and rapidly. The exoantigen technique has been found to meet this need effectively. On the basis of studies with 166Histoplasma spp. isolates, 128C. immitis isolates, 59B. dermatitidis isolates, 30P. brasiliensis isolates, and 181 saprophytes, we determined that the exoantigen test is valuable for the presumptive identification of the four fungal pathogens studied. All of the positive reactions have correlated with the cultural, histologic, or other available laboratory data, and we are unaware of any false positive or flase negative reactions.     

A new technique for the preparation of permanent reference cultures of fungi.

Fungal cultures were grown by a new microslide method and by the traditional slide-culture method. Sporulation results were similar by both methods. The microslide method has the advantages of increased safety to the user and ease of storage.     

Scanning electron microscopy of cultures of rare macromycetous fungi

Using scanning electron microscopy it was possible to describe structures on the mycelia of macromycetous fungi that possess both taxonomic and functional importance. For taxonomic characteristization of 11 studied species, the presence and distribution of clamps on hyphæ represent an, important feature; in the case ofLyophyllum decastes the form of clamps may be useful for identifying cultures. Formation and types of anamorhps inPleurotus calyptratus, Montagnea arenaria andLyophyllum decastes are also of taxonomic importance. For additional taxonomic characterization of cultures ofTricholoma mongolicum the formation of “coils” might be exploited. Formation of anastomoses and crystals has predominantly a physiological role. However, in our view, production of crystals under certain culture conditions may serve as a diagnostic parameter.     

Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage.

Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal fungi and 27 isolates of 15 species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 mm in diameter, were removed from the cultures and stored in sterile distilled water in test tubes at 5 degrees C. Sixty-four, 61, and 41 isolates of the symbiotic fungi were viable after 1, 2, and 3 years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, four fungi (three symbionts and one pathogen) were tested and found to have retained their original growth rates and root-infecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 degrees C and subcultured every 4 to 6 months, grew slower and did not infect as many feeder roots of pine as the water-stored isolates.     

Chemical and physiological changes in filamentous fungi during autolysis. X. Changes in the concentration of polyol in autolysing cultures of several fungi

Botrytis cinerea, Colletotrichum gloeosporioides, Aspergillus niger andPenicillium griseofulvum were grown in flasks as stationary cultures in the ordinary Czapek-Dox medium at 24° C in the dark for long periods of time. At convenient intervals during autolysis samples of mycelium were harvested and its polyol content determined. The loss of mycelial mannitol during autolysis reached to a little more than 60 %, more than 70 %, to nearly 80 % and 90 % forC. gloeosporioides, P. griseofulvum, A. niger andB. cinerea, respectively. Seventy per cent of the arabitol contained inP. griseofulvum and 85 % of the arabitol inC. gloeosporioides disappeared during autolysis. Mannitol remained present in appreciable amounts in these four fungi throughout the whole period of autolysis described, whereas arabitol disappeared fromB. cinerea and fromA. niger during the process.

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Zusammenfassung Botrytis cinerea, Colletotrichum gloeosporioides, Aspergillus niger undPenicillium griseofulvum sind als stationäre Kulturen in Czapek-Dox Nährboden bei 24° C lange Zeit in der Dunkelheit gezüchtet worden. In geeigneten Zwischenzeiten sind während der Autolyse Myzeliumproben entnommen und ihr Polyol-Inhalt bestimmt. Der Verlust vom myzelialen Mannitol während der Autolyse erreichte mehr als 60 %, mehr als 70 %, beinahe 80 % und 90 % inC. gloeosporioides, P. griseofulvum, A. niger, undB. cinerea, beziehungsweise. Siebzig Prozent von Arabitol inP. griseofulvum und 85 % von Arabitol inC. gloeosporioides verschwanden während der Autolyse. Mannitol verblieb in beträchtlicher Menge in diesen vier Pilzen während der ganzen Periode der Autolyse, während Arabitol vonB. cinerea und vonA. niger in derselben Zeit verschwand.

     

Cooling aspects of solid-state cultures of mesophilic and thermophilic fungi

Cooling air requirements in solid-state cultures of filamentous fungi were studied. The growth conditions of Trichonderma viride TS, Thermoascus aurantiacus and Sporotrichum (Chrysosporium) thermophile in sugar-beet pulp medium were estimated. Heat generation and heat removal in relation to water activity of the medium are discussed. Heat removal from the culture media was due to enthalpy changes and water vapourization. Changes in the water sorption properties in the solid media during the fermentation process are presented. It was estimated that in real solid-state conditions the requirement for cooling air in a mesophilic culture is 2-fold higher than that under thermophilic conditions.     

Interaction between embryogenic cultures of Scots pine and ectomycorrhizal fungi

 Embryogenic cell masses of three Scots pine (Pinus sylvestris) cell lines K779, K884 and K1009 were cultivated with the ectomycorrhizal (ECM) fungi Laccaria bicolor, L. proxima, Pisolithus tinctorius, Paxillus involutus and two strains of Suillus variegatus. The average growth ratio of the slowly proliferating cell line K1009 was improved by L. proxima and S. variegatus strain H, while of the rapidly proliferating lines K779 and K884 the non-mycorrhizal controls grew best. The fungi caused two distinct reactions in embryogenic cultures. In the positive reaction, the shape and light yellow colour of the cultures resembled the controls, while in the negative reaction the embryogenic cells became brown and necrotic and the fungi grew aggressively over them. These reactions to the fungi did not correlate completely with effects on the growth ratio. All the cell lines enhanced the radial growth of S. variegatus H and of P. tinctorius, while the Laccaria species and S. variegatus strain 1 thrived better alone. This study shows that early-stage embryogenic cells of Scots pine and ECM fungi are able to interact. As some fungi produced a positive reaction or even increased proliferation, they could be used to enhance somatic embryogenesis of Scots pine. Specific fungi might be used to induce the growth of slowly proliferating cell lines, and knowledge of positive cell line-fungus interactions could be useful in work with later stages of somatic embryogenesis, such as rooting. Accepted: 16 July 1998