[Sinonasal fungal infections are not exclusively due to mucorales and Aspergillus!]. / Les infections fongiques nasosinusiennes ne sont pas uniquement liées aux mucorales et aux Aspergillus !

Rhino-sinusal infections are serious diseases and possibly lethal. When they are invasive, we easily discuss apergilloses and mucormycoses. The confirmation of the diagnosis of mucormycosis need an extensive surgery for precise histopathological and mycological evaluation. The pathologist may be faced to other rare mycoses such as phaeohyphomycoses, which present different morphological features than mucormycoses and Aspergillus. Once the diagnosis is established, an appropriate antifungal treatment is quickly started. The aim of our work is to report two observations of phaeohyphomycoses, to describe their histopathological features, to discuss complementary diagnostic methods and to present the main differential diagnoses.

Characterization of Alternaria infectoria extracellular vesicles.

Many fungi use membrane vesicles to transport complex molecules across their cell walls. Like mammalian exosomes, fungal vesicles contain lipids, proteins, and polysaccharides, many of which are associated with virulence. Here we identify and characterize extracellular vesicles (EVs) in Alternaria infectoria, a ubiquitous, environmental filamentous fungus that is also an opportunistic human pathogen. Examination of the A. infectoria EVs revealed a morphology similar to that of vesicles described in other fungal species. Of note, proteomic analysis detected a reduced number of vesicle-associated proteins. There were two prevalent categories among the 20 identified proteins, including the polysaccharide metabolism group, probably related to plant host invasion or biosynthesis/degradation of cell wall components, and the nuclear proteins, especially DNA repair enzymes. We also found enzymes related to pigment synthesis, adhesion to the host cell, and trafficking of vesicles/organelles/molecules. This is the first time EV secretions have been identified in a filamentous fungus. We believe that these vesicles might have a role in virulence.

In vitro and in vivo control of Alternaria alternata in cherry tomato by essential oil from Laurus nobilis of Chinese origin.

BACKGROUND: Many essential oils were reported to be used as natural, environmental friendly antimicrobial agents. The antifungal activity in vitro and in vivo of an essential oil extracted from Chinese local Laurus nobilis leaves against Alternaria alternata has been studied. RESULT: The main components of the essential oil were investigated by means of gas chromatography­mass spectrometry and 14 components were identified. The main ones were eugenol, caryophyllene and cinnamaldehyde. The antifungal test showed that at 800 µg mL−1 of L. nobilis oil completely inhibited the growth of A. alternata. In addition, the conidial germination of the pathogen was significantly inhibited at 200 µgmL−1, and the weight of mycelia efficiently decreased at 500 µgmL−1. The in vivo assay indicated that 500 µg mL−1 L. nobilis oil was effective in protecting cherry tomatoes (Lycopersicon esculentum)from infection of A. alternata, with an inhibition ratio of 33.9%. Scanning electron microscopy observations of the pathogen revealed significant morphological alterations in the hyphae. CONCLUSION: This work suggested that L. nobilis oil could be used as a potential fungicide to control the post-harvest disease caused by A. alternata.

Alternaria hungarica sp. nov., a minor foliar pathogen of wheat in Hungary.

During routine wheat disease surveys in Hungary in 2007 Alternaria was isolated from leaf samples collected in Debrecen. Macro- and micro-morphological examinations and ITS sequence analyses indicated that the isolates represented a new Alternaria species, which we described as A. hungarica. The usually solitary conidia of A. hungarica resemble those of A. mouchaccae and A. molesta. However growth and sporulation pattern are more like those of A. geniostomatis and A. soliaridae. Phylogenetic analysis of ITS sequences indicated that this new species can be distinguished from all other examined Alternaria and Embellisia species. Pathogenicity tests indicated that A. hungarica can be considered a minor pathogen of wheat.

A novel antifungal peptide from foxtail millet seeds.

BACKGROUND: Antifungal proteins (AFP) help plants to combat phytopathogenic fungi and thus protect plants from the devastating damage caused by fungal infections and prevent massive economic losses. To date, several proteins with antibacterial and/or antifungal properties have been isolated and characterized from different plant species and tissues; however, there are no reports concerning the antifungal peptide from foxtail millet seeds. RESULTS: An antifungal peptide with a molecular mass of 26.9 kDa was isolated from dry seeds of the foxtail millet (Setaria italica (L.) Beauv.), using a procedure that involved four chromatographic steps. The antifungal peptide was adsorbed on CM-Sepharose, Affi-gel blue gel and Superdex 75. It was further purified by C(18) reverse-phase high-performance liquid chromatography and submitted for analysis of peptide mass fingerprint. The Mascot peptide mass fingerprint of the isolated protein hit no existing protein (score >60), and it was proved to be a novel antifungal peptide. It inhibited mycelial growth in Alternaria alternate with an IC(50) of 1.3 µmol L(-1) , and it also exhibited antifungal activity against Trichoderma viride, Botrytis cinerea and Fusarium oxysporum. Transmission electron microscopy of mold forms of Alternaria alternate after incubation with 20 µg mL(-1) of the antifungal protein for 48 h revealed marked ultrastructural changes in the fungus. CONCLUSION: A novel antifungal peptide with high potency was isolated from foxtail millet seeds.

Inhibitory effects of antioxidant reagent in reactive oxygen species generation and penetration of appressoria of Alternaria alternata Japanese pear pathotype.

In the Japanese pear pathotype of Alternaria alternata, H2O2 is generated solely from penetration pegs and not from other portions of subcuticular hyphae within the pectin layers of host leaves. A close association between H2O2 generation and fungal aggressiveness is expected because the pegs are important for fungal penetration into the host epidermis. To determine the potential role of reactive oxygen species in microbial pathogenicity, we studied the inhibitory effects of the antioxidant reagent ascorbic acid and the NADPH oxidase inhibitor diphenylene iodonium on infection of the pathogen. In our study, we showed H2O2 generation to be inhibited by inoculation with the mixture of ascorbic acid or diphenylene iodonium and spores at the pegs in the spore-inoculated host leaves. The decrease of generation in the pegs resulted in penetration failure, indicating that H2O2 generation probably contributed to strengthening of the penetration and probably was recruited by NADPH oxidase.

Complexity of Brassica oleracea-Alternaria brassicicola Susceptible Interaction Reveals Downregulation of Photosynthesis at Ultrastructural, Transcriptional, and Physiological Levels.

Black spot disease, caused by Alternaria brassicicola in Brassica species, is one of the most devastating diseases all over the world, especially since there is no known fully resistant Brassica cultivar. In this study, the visualization of black spot disease development on Brassica oleracea var. capitata f. alba (white cabbage) leaves and subsequent ultrastructural, molecular and physiological investigations were conducted. Inter- and intracellular hyphae growth within leaf tissues led to the loss of host cell integrity and various levels of organelle disintegration. Severe symptoms of chloroplast damage included the degeneration of chloroplast envelope and grana, and the loss of electron denseness by stroma at the advanced stage of infection. Transcriptional profiling of infected leaves revealed that photosynthesis was the most negatively regulated biological process. However, in infected leaves, chlorophyll and carotenoid content did not decrease until 48 hpi, and several chlorophyll a fluorescence parameters, such as photosystem II quantum yield (Fv/Fm), non-photochemical quenching (NPQ), or plant vitality parameter (Rdf) decreased significantly at 24 and 48 hpi compared to control leaves. Our results indicate that the initial stages of interaction between B. oleracea and A. brassicicola are not uniform within an inoculation site and show a complexity of host responses and fungal attempts to overcome host cell defense mechanisms. The downregulation of photosynthesis at the early stage of this susceptible interaction suggests that it may be a part of a host defense strategy, or, alternatively, that chloroplasts are targets for the unknown virulence factor(s) of A. brassicicola. However, the observed decrease of photosynthetic efficiency at the later stages of infection is a result of the fungus-induced necrotic lesion expansion.

Identification of endophytes with biocontrol potential from Ziziphus jujuba and its inhibition effects on Alternaria alternata, the pathogen of jujube shrunken-fruit disease.

Endophytic strains were isolated from different parts of a healthy "Dongzao" jujube (Ziziphus jujuba Mill. 'Dongzao') to find biocontrol agents against jujube shrunken-fruit disease caused by Alternaria alternata. The strains were screened using A. alternata strain CN193 as the target pathogen. The nutrient competition for all isolates was studied using the dual culture, and their inhibitive capability was tested by measuring the inhibition width of filter paper disks with filtrate. Influence of filtrate from the selected strains with strong inhibition of mycelial growth on spore germination was studied with hanging drop method on concavity slides. Colonization in the jujube leaves was assayed using a rifampicin-resistant mutant of strain St-zn-34 as the screening marker. Strains were identified based on their morphological, physiological, and biochemical characteristics, 16S rDNA sequencing, and phylogenetic analysis. A total of 81 endophytic strains were isolated from the stems, leaves, flowers, and fruits of winter jujube. Among these isolates, 14 strains showed strong antagonism against A. alternata. Further study showed that the filtrate of strains St-zn-9 and St-zn-34 could inhibit the mycelial growth of A. alternata, and the widths of their inhibition zone reached 6.14±0.03 mm and 8.27±0.09 mm, respectively. However, strain St-zn-34 showed stronger inhibition on spore germination than strain St-zn-9. St-zn-34 could significantly reduce the spore germination rate of A. alternata, and the spore did not germinate at all or the germ tube was very short. A rifampicin resistant-derivative of wild-type strain St-zn-34, which was designated as St-zn-34r, was obtained by transferring the strains to media with stepwise-increased rifampicin. Colonization assays indicated that St-zn-34r could colonize in jujube leaves, and the population of St-zn-34r was 1.2×103 CFU/g FW after inoculation for 30 days. Except for its salt tolerance, St-zn-34 was the closest to those of Bacillus subtilis. Thus, the strain was identified as B. subtilis.

[Inhibition of hyphal growth of the fungus Alternaria alternata by chlorine dioxide gas at very low concentrations].

The efficacy of chlorine dioxide (ClO(2)) gas at very low concentrations for hyphal growth of Alternaria alternata related to fungal allergy was evaluated using a fungus detector. The fungus detector is a plastic sheet with a drop of spore-suspending medium, and it makes possible clear observations of hyphal growth with a light microscope. ClO(2) gas (average 0.075 ppm, 0.21 microg/l) inhibited hyphal growth of the fungus, but not germination of fungal spores. The hyphal length was more than 1780 mum under air conditions (control) and 49+/-17 microm under ClO(2) gas conditions for 72 h. According to the international chemical safety card, threshold limit values for ClO(2) gas are 0.1 ppm as an 8-h time-weight average and 0.3 ppm as a 15 min short-term exposure limit. From these data, we propose that treatment with ClO(2) gas at very low concentrations in space is a useful tool for the growth inhibition of fungi in the fields of food, medicine, etc. without adverse effects.

Detection of allergens from Alternaria alternata by gold-conjugated anti-human IgE and field emission scanning electron microscopy.

Fungal allergens are present in viable and non-viable conidia, hyphae and fungal fragments. It has been shown that large quantities of allergen are released from conidia during germination. We used a gold immunolabelling technique and field emission scanning electron microscopy to examine the allergen release from Alternaria alternata conidia. Immunolabelling was associated with the hyphal tip and amorphous matter associated with the emerging hyphae. Non-specific antibody controls showed no labelling associated with germinating fungi. This suggests that material released from hyphae may be an additional source of fungal allergens.

Further studies on the blue and near ultraviolet reversible photoreaction with an intracellular particulate fraction of the fungus, Alternaria tomato.

Further studies have been made on the blue and near ultraviolet-reversible photoreaction in a cell-free system of the fungus Alternaria tomato, prepared by two-layer sucrose density gradient centrifugation. A light-minus-dark difference spectrum of the supernatant fraction showed a dip in the near ultraviolet region, mainly at 280 nm, when irradiated with near ultraviolet light which could not be reversed by following the initial irradiation with blue light. The difference spectrum of the dialyzed particulate fraction, located in the 68% sucrose band, showed a peak absorbance in the blue region, mainly at 400 nm, when irradiated with near ultraviolet light, and also could not be reversed by blue light. When the particulate fraction plus the supernatant fraction was irradiated with near ultraviolet light, the increment rate of peak absorbance in the blue region increased compared to that of the particulate fraction lacking the supernatant, and both peak absorbance in the blue region and the dip in the near ultraviolet region were reversed by irradiation with blue light following the application of near ultraviolet light. Thus, it was concluded that two photoresponsive systems are involved in blue and near ultraviolet reversible photoreaction on an intracellular particulate fraction.

Circular DNA plasmid in the phytopathogenic fungus Alternaria alternata: its temperature-dependent curing and association with pathogenicity.

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an approximately 5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26 degrees showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29 degrees. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23 degrees, most of which maintained the plasmid, and from mycelia grown at 29 degrees, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (approximately 1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.

SEM study of water activity and temperature effects on the initial growth of Aspergillus ochraceus, Alternaria alternata and Fusarium verticillioides on maize grain. Scanning electron microscopy.

A scanning electron microscopy (SEM) study of the effect of water activity (a(w)), temperature and fungal interactions on the very early phases of Aspergillus ochraceus, Alternaria alternata and Fusarium verticillioides development on maize grains was carried out. Germination and growth of individual fungal strains were assayed at 0.92, 0.95 and 0.98 a(w), and 20 and 30 degrees C. Hyphal lengths were measured on micrographs taken by SEM at different periods of incubation. A. alternata had the highest linear growth at 0.98 a(w), and was the only species able to grow under all conditions tested, whereas A. ochraceus was not able to germinate at 0.92 a(w) at any temperature assayed. F. verticillioides demonstrated a different behaviour depending on growth temperature. Fungal interactions were studied at 0.95a(w) and 30 degrees C. A. ochraceus germination was inhibited when it was co-inoculated with one or two of the other species. A. alternata showed an increased growth rate when growing together with the other fungi, whereas growth of F. verticillioides was significantly inhibited when paired with A. ochraceus.

Fungitoxic activity of 12 essential oils against four postharvest citrus pathogens: chemical analysis of thymus capitatus oil and its effect in subatmospheric pressure conditions.

The fungitoxic activity against Penicillium digitatum, Penicillium italicum, Botrytis cinerea, and Alternaria citri of 12 essential oils (EOs) distilled from medicinal plants is reported. The results of the in vitro trials show strong fungitoxic activity of Thymus capitatus (L.) Hofmgg EOs, which inhibited the growth of the four fungi at a concentration of 250 ppm (vol/vol). The other 11 essences reduced the development of the fungi from 95 to 9% at 250 ppm (vol/vol). The fungitoxic activity of T. capitatus EOs (75, 150, and 250 ppm) on healthy orange fruits, inoculated with P. digitatum (10(8) conidia ml(-1)) by spraying and placed in 10-liter desiccators, was weak at atmospheric pressure (3 to 10% inhibition at all three concentrations), while in vacuum conditions (0.5 bar), conidial mortality on the exocarp was high (90 to 97% at all three concentrations). These data proved not to be statistically different from treatments with thiabendazole-TBZ (2,000 ppm). Scanning electron microscope observations showed that T. capitatus EO vapors altered the morphology of P. digitatum hyphae and conidia. The gas-chromatographic analyses of thyme EO indicated that carvacrol was present at 81 to 83%, p-cymene at 4.5 to 5%, gamma-terpinene at 2.6 to 3.3%, caryophyllene at 1.5 to 1.6%, beta-myrcene at 1.6%, and linalool at 1.1 to 1.2%. Carvacrol proved to be the most important fungitoxic compound among the thyme EO constituents, but, unlike thyme EO, it caused alterations to the fruit at the concentration of 75 ppm.

[Human cutaneous alternariosis. 3 cases. Review of the literature (author's transl)]. / Les alternarioses cutanées humaines. A propos de 3 cas. Revue de la littérature.

The authors present 3 new cases of dermal alternariosis, associated with one Hodgkin's disease and 2 kidney transplants. Review of the other cases of literature demonstrates the pathogenicity of this saprophytic fungus which becomes opportunistic mostly in case of immuno-deficiency. Clinical aspect is usually that of papulo-nodular, ulcerative or vegetating, unique or multiple lesions of limbs and head. Its course is very capricious, not infrequently chronic and extensive. The treatment is not yet well established.

Anadenanthera colubrina (Vell.) Brenan produces steroidal substances that are active against Alternaria alternata (Fr.) Keissler and that may bind to oxysterol-binding proteins.

BACKGROUND: In previous studies, the extract from Anadenanthera colubrina was active against Alternaria alternata in vitro and reduced the disease caused by this fungus on Murcott tangor fruits to levels that have been obtained using commercial fungicides. Therefore, the goal of the present work was to isolate and identify the active substances in this extract and identify in silico their protein target in the fungus. RESULTS: The bioguided fractionation of the methanol extract from the fruits of A. colubrina resulted in the isolation of ß-sitosterol and ß-sitosteryl linoleate, which had minimal inhibitory concentrations (MICs) of 250 and 500 µg mL(-1) , respectively, against A. alternata. Under the same conditions, the MICs for two commercial fungicides were 1250 and 19 µg mL(-1) . In silico studies showed that these steroidal substances bind well to oxysterol-binding proteins from Saccharomyces cerevisiae. CONCLUSION: ß-Sitosterol and ß-sitosteryl linoleate, produced by A. colubrina, are active against A. alternata. In silico studies suggest that these substances may act by binding to oxysterol-binding proteins. Therefore, both substances and these proteins have potential use in the development of new steroidal structures and analogues to control the disease caused by A. alternata.

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific, toxin-producing fungus Alternaria alternata†Japanese pear pathotype.

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant-pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.

Plant extracts to control Alternaria alternata in Murcott tangor fruits.

BACKGROUND: Alternaria alternata causes the Alternaria brown spot disease (ABS) in many tangerines and their hybrids worldwide. Plant extracts offer an alternative method for controlling this disease, which control is based on chemical fungicides. AIMS: To identify plant species with antifungal properties against A. alternata, the causal agent of the ABS. METHODS: Plant extracts prepared from leaves, barks, flowers, and stalks collected from 105 plant species in the State of Minas Gerais, Brazil, were tested for activity against the fungus A. alternata in vitro and in vivo. RESULTS: The most promising extract was obtained from Anadenanthera colubrina, which reduced the disease on Murcott tangor fruits to levels obtained with commercial fungicides. Artemisia annua, Cariniana estrelensis, Ficus carica, and Ruta graveolens presented moderate in vitro antifungal activity, but no effects were observed on the disease when the extracts were applied to fruits inoculated with the fungus. Besides, A. colubrina was the most active extract against A. alternata in the in vitro assay. CONCLUSIONS: The results obtained in the in vitro and in vivo assays suggested that the fungal growth test, which uses 96-well polypropylene plates, seems to be appropriate for selecting potential plant species for testing new methods to control ABS.