Autophagy During Conidiation,Conidial Germination and Turgor Generation in Magnaporthe grisea

Autophagy is a ubiquitous and evolutionarily conserved process found in all eukaryotic cells that allows for the degradation and recycling of old proteins and organelles. Starvation can induce autophagy, and autophagic pathway is an essential process for cellular function under starvation. In Magnaporthe grisea, starvation is one of the key induced factors for the germ tube tip to differentiate into an appressorium. Considering the importance of the rice blast fungus as a primary model for host-pathogen interaction, the role of autophagy in fungal development, appressorium turgor generation and pathogenicity of M. grisea via its role in organelle and protein turnover is a very significant subject.

Addendum to:

Involvement of a Magnaporthe grisea Serine/Threonine Kinase, MgATG1, in Appressorium Turgor and Pathogenesis

X.-H. Liu, J.-P. Lu, L. Zhang, B. Dong, H. Min and F.-C. Lin

Eukaryotic Cell 2007; In press     

Structural Features of Sugars That Trigger or Support Conidial Germination in the Filamentous Fungus Aspergillus niger

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.     

Biochemical and Histochemical Localization of Invertase in Neurospora crassa During Conidial Germination and Hyphal Growth

The intracellular localization of Neurospora invertase, an enzyme partially secreted and partially retained by Neurospora at the cell periphery, was investigated. A cell wall fraction was isolated, to which 24% of the cell-bound invertase was firmly attached. A sensitive osmiophilic stain for invertase was developed and used in conjunction with the technique of indirect immunofluorescence to follow the pattern of invertase localization during the development of Neurospora from the germination of conidia to the mature hypha. These studies revealed that: (i) conidial invertase was uniformly distributed along the cell periphery; (ii) growing hyphal tips of germinating conidia showed pronounced invertase activity as the rest of the conidial cell wall lost its peripheral activity; (iii) hyphae in early log-phase growth had strong enzyme activity associated with the cell wall, and in late log phase the activity became associated with the plasma membrane and points where new hyphal branches were being formed; and (iv) hyphae in early stationary phase had strong fluorescence at incipient branching points, in "dots" close to the plasma membrane, and in the cytoplasm.     

Filamentous Fungi Transported by Birds During Migration Across the Mediterranean Sea

The potential for the transport and diffusion of some pathogenic microorganisms by migratory birds is of concern. Migratory birds may be involved in the dispersal of microorganisms and may play a role of mechanical and biological vectors. The efficiency of dispersal of pathogenic microorganisms depends on a wide range of biotic and abiotic factors that influence the survival or disappearance of a given agent in a geographical area. In the present study, 349 migratory birds were captured in four sites (Mazara del Vallo, Lampedusa, Ustica and Linosa), representing the main stop-over points during spring and autumnal migration, and analyzed for the presence of filamentous fungi. A total of 2,337 filamentous fungi were isolated from 216 birds and identified by a combined phenotypic-genotypic approach to species level. Twelve species were identified in the study, with Cladosporium cladosporioides, Alternaria alternata, and Aspergillus niger as the most abundant. The transport of these fungal species isolated in this study is of considerable importance because some of these species can create dangers to human health.     

New Feather-Degrading Filamentous Fungi

Among 106 filamentous fungi isolated from poultry farm waste, 13 species belonging to seven genera (Aspergillus, Acremonium, Alternaria, Beauvaria, Curvularia, Paecilomyces, and Penicillium) were able to grow and produce keratinase in stationary cultures using poultry feather powder as the only substrate. The four most efficient keratinase producers were selected for a comparative study of keratinase production in submerged and stationary conditions. The highest keratinolytic activities were produced after 4-6 days of cultivation in submerged conditions: 53.8 +/- 6.1 U/mL (Alternaria tenuissima), 51.2 +/- 5.4 U/mL (Acremonium hyalinulum), 55.4 +/- 5.2 U/mL (Curvularia brachyspora), and 62.8 +/- 4.8 U/mL (Beauveria bassiana). These novel nondermatophytic keratinolytic fungi have potential use in biotechnological processes involving keratin hydrolysis. The results of this work contribute to show that keratinolytic activity is relatively widespread among common filamentous fungi and may have an important rule in feather decomposition in natural settings.     

The pcz1 Gene,which Encodes a Zn(II)2Cys6 Protein,Is Involved in the Control of Growth,Conidiation, and Conidial Germination in the Filamentous Fungus Penicillium roqueforti

Proteins containing Zn(II)₂Cys₆ domains are exclusively found in fungi and yeasts. Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)₂Cys₆ protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active Gαi protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by Gαi. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed.     

Otomycosis due to Filamentous Fungi

Otomycosis is common throughout the world but barely studied in Spain. Our objective was to determine the microbiological and epidemiological characteristics of this pathology in Cadiz (Spain) between 2005 and 2010. Samples from patients with suspicion of otomycosis underwent a direct microscopic examination and culture on different media for fungi and bacteria. Mycological cultures were incubated at 30°C for at least seven days. Identification of fungi was based on colonial morphology and microscopic examination of fungal structure. From a total of 2,633 samples, microbial growth was present in 1,375 (52.2%) and fungal isolation in 390 (28.4%). We identified 228 yeasts and 184 filamentous fungi (13.4% of positive cultures and 47.2% of otomycosis), associated with yeasts in 22 cases (5.6%). The most frequent species were Aspergillus flavus (42.4%), A. niger (35.9%), A. fumigatus (12.5%), A. candidus (7.1%), A. terreus (1.6%), and Paecilomyces variotii (0.5%). Infection was predominant in men (54.9%) and patients beyond 55 years old (46.8%). The most common clinical symptoms were itching (98.9%), otalgia (59.3%), and hypoacusis (56.0%). Fall season reported the lowest number of cases (20.1%). Incidence of otomycosis and fungi producing otomycosis vary within the distinct geographical areas. In Cadiz, this infection is endemic due to warm temperatures, high humidity, sea bathing, and wind, which contributes to disseminate the conidia. Despite Aspergillus niger has been reported as the main causative agent, A. flavus is predominant in Cadiz. Although infection is usually detected in warm months, we observed a homogeneous occurrence of otomycosis in almost all the seasons.     

Chitinolytic Activity of Filamentous Fungi

The chitinolytic activity of nine species of filamentous fungi, classified with seven genera (specifically, Aspergillus, Penicillium, Trichoderma, Paecilomyces, Sporotrichum, Beaueria, and Mucor), was studied. When cultured in liquid medium containing 1% crystalline chitin, all fungi produced extracellular chitosans with activity varying from 0.2 U/mg protein (Sporotrichum olivaceum, Mucorsp., etc.) to 4.0–4.2 U/mg protein (Trichoderma lignorum, Aspergillus niger).     

木质素降解菌Bax的筛选及特性研究

为了高效降解造纸污水中木质素类化合物,采用苯胺兰和鞣酸平板法从腐木分离、筛选得到一株具有高降解木质素活性的丝状真菌,经鉴定为绿色木霉,命名为Bax.最适碳源为葡萄糖和蔗糖,最适氮源为蛋白胨和尿紊,最适酸碱度为pH 5.0,最适温度为30℃.通过对木质素氧化酶系分析,主要起作用的是漆酶和木质素酶,为造纸污水的处理奠定了基础.     

Ethylene Glycol-Induced Alteration of Conidial Germination in Neurospora crassa

In nutrient medium containing 3.22 M ethylene glycol or glycerol, conidia of Neurospora crassa grow as single cells, without forming the germ tubes characteristic of normal morphological germination. Ethylene glycol is more effective than glycerol in producing this response. After growth in ethylene glycol medium for a suitable time, the cells are easily disrupted by an abrupt decrease in osmotic pressure. Osmotic disruption yields intact nuclei and mitochondria, although mitochondrial fractions obtained in this way show significantly reduced concentrations of cytochromes c + c(1), as compared to those observed for comparable fractions obtained from vegetative hyphae. Cell cultures gradually adapted to lower concentrations of the glycol show a much higher degree of synchrony in the formation of germ tubes than do untreated conidia.     

Antifungal Susceptibility Testing of Filamentous Fungi

Methods developed for testing filamentous fungi (molds) include standardized broth microdilution (Clinical and Laboratory Standards Institute [CLSI] and European Committee for Antimicrobial Susceptibility Testing [AFST-EUCAST]) methods and disk diffusion (CLSI) methods. Quality control limits also are available from CLSI for MIC (minimal inhibitory concentration), MEC (minimal effective concentration), and zone diameters. Although clinical breakpoints based on correlations of in vitro results with clinical outcome have not been established, epidemiologic cutoff values have been defined for six Aspergillus species and the triazoles, caspofungin, and amphotericin B. The link between resistance molecular mechanisms, elevated MICs, and clinical treatment failure has also been documented, especially for Aspergillus and the triazoles. Other insights into the potential clinical value of high MICs have also been reported. Various commercial methods (e.g., YeastOne, Etest, and Neo-Sensitabs) have been evaluated in comparison with reference methods. This review summarizes and discusses these developments.     

Filamentous Fungi and Bacteria in Macaroni and Spaghetti Products

Filamentous fungi, especially Aspergillus flavus-oryzae, and bacteria were present in all samples of macaroni and spaghetti products tested. No aflatoxin was detected in the three samples, all relatively high in A. flavus-oryzae, that were tested for it. Five of the nine isolates of A. flavus-oryzae from macaroni and sphaghetti grown in corn, incorporated into an otherwise balanced ration, and given to ducklings resulted in death of one to five ducklings within six days.     

丝状真菌基因工程进展

丝状真菌被广泛应用于生物工程来生产化学药品、药物制剂及酶。为了提高其生产能力,基因工程战略是一种较好的途径。为了使丝状真菌更好的用于工业生产,近年来出现许多新的转化技术。然而,由于缺乏有效的基因工程战略,许多真菌在生产新的、有商业价值的代谢产物上都存在着不足。在此总结了几种最新介导和控制基因表达的方法,并讨论了各自的优、缺点。此外,对丝状真菌今后的发展进行了展望。     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

Estimation of Mitotic Stability in Conidial Fungi: A Theoretical Framework

Mitotic stability refers to the probability that genetic elements are transmitted to both daughters during mitosis. This is of practical importance in molecular genetics because autonomous cloning vectors should be transmitted at high frequency during mitosis. In filamentous coencytic fungi it is difficult to quantify mitotic stability because a fluctuation test is not feasible. We show how to get around this problem by formulating a general model of the transmission of nuclear genetic elements through the course of conidiogenesis. We derive formulas by two different methods for the expected proportion of conidiospores that retain the element as a function of its mitotic stability and the number of generations of spore production. An important by-product yields the exact probability distributions for the number of conidiospores retaining elements at each stage of conidiophore development. We outline, and illustrate through specific numerical examples, how to use these formulas to estimate mitotic stability. Although we use Aspergillus nidulans as our biological paradigm, the same general framework can be extended to other fungal species, and possibly to less closely related systems as well.