Dual effect of blue light on Fusariumsolani clinical corneal isolates in vitro.

The purpose was to investigate the effect of daylight-intensity blue light on F. solani isolated from the cornea of patients with fungal keratitis. Spore suspensions of 5 F. solani strains (one standard strain and 4 clinical corneal isolates) were prepared in 6-well plates. Blue light groups were irradiated by a light-emitting diode (LED) device with a peak wavelength of 454 nm at 0.5 mW/cm2 for 0 to 48 h, while the controls were maintained in darkness. Hyphal morphology in the 6-well plates was recorded at 0, 12, 24, 36, 48 h. One hundred microliters of spore suspensions of each strain at these five time points was transferred to SGA plates and cultured for 36 h at 29 °C; the number of colonies formed was counted as a measure of conidia quality and viability. Blue light has dual effects on F. solani. The hyphal length of F. solani exposed to blue light was significantly shorter than that of the control (P < 0.01), indicating that fungal growth was inhibited. Meanwhile, instead of reducing the viability of spores, blue light significantly enhanced the conidia quality and viability after at least 24 h irradiation. Daylight-intensity blue light exposure will inhibit the hyphal growth of F. solani but promote conidiation, which would be more harmful to fungal keratitis. Eliminating the influence of blue light for these patients should be taken into account.

Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light.

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

Curcumin-based photosensitization inactivates Aspergillus flavus and reduces aflatoxin B1 in maize kernels.

Different methods have been applied in controlling contamination of foods and feeds by the carcinogenic fungal toxin, aflatoxin, but nevertheless the problem remains pervasive in developing countries. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa L.) that has been identified as an efficient photosensitiser for inactivation of Aspergillus flavus conidia. Curcumin mediated photoinactivation of A. flavus has revealed the potential of this technology to be an effective method for reducing population density of the aflatoxin-producing fungus in foods. This study demonstrates the influence of pH and temperature on efficiency of photoinactivation of the fungus and how treating spore-contaminated maize kernels affects aflatoxin production. The results show the efficiency of curcumin mediated photoinactivation of fungal conidia and hyphae were not affected by temperatures between 15 and 35 °C or pH range of 1.5-9.0. The production of aflatoxin B1 was significantly lower (p < 0.05), with an average of 82.4 µg/kg as compared to up to 305.9 µg/kg observed in untreated maize kept under similar conditions. The results of this study indicate that curcumin mediated photosensitization can potentially be applied under simple environmental conditions to achieve significant reduction of post-harvest contamination of aflatoxin B1 in maize.

Pro-Inflammatory Responses in Human Bronchial Epithelial Cells Induced by Spores and Hyphal Fragments of Common Damp Indoor Molds.

Damp indoor environments contaminated with different mold species may contribute to the development and exacerbation of respiratory illnesses. Human bronchial epithelial BEAS-2B cells were exposed to X-ray treated spores and hyphal fragments from pure cultures of Aspergillus fumigatus, Penicillum chrysogenum, Aspergillus versicolor and Stachybotrys chartarum. Hyphal fragments of A. fumigatus and P. chrysogenum induced expression and release of the pro-inflammatory cytokine interleukin (IL)-6 and the chemokine IL-8, while none of the other hyphal preparations had effects. Hyphal fragments from A. fumigatus and P. chrysogenum also increased the expression of IL-1α, IL-1ß and tumor necrosis factor (TNF)-α, but these cytokines were not released. X-ray treated spores had little or no inflammatory potential. Attenuating Toll-like receptor (TLR)-2 by blocking antibodies strongly reduced the A. fumigatus and P. chrysogenum hyphae-induced IL-6 and IL-8 release, whereas TLR4 antagonist treatment was without effects. Untreated A. fumigatus spores formed hyphae and triggered expression of pro-inflammatory genes with similarities to the effects of hyphal fragments. In conclusion, while X-ray treated spores induced no pro-inflammatory responses, hyphal fragments of A. fumigatus and P. chrysogenum enhanced a TLR2-dependent expression and release of IL-6 and IL-8.

Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth, but not pathogenicity, in Sclerotinia sclerotiorum.

The fungus Sclerotinia sclerotiorum is a necrotrophic plant pathogen causing significant damage on a broad range of crops. This fungus produces sclerotia that serve as the long-term survival structures in the life cycle and the primary inoculum in the disease cycle. Melanin plays an important role in protecting mycelia and sclerotia from ultraviolet radiation and other adverse environmental conditions. In this study, two genes, SCD1 encoding a scytalone dehydratase and THR1 encoding a trihydroxynaphthalene reductase, were disrupted by target gene replacement, and their roles in mycelial growth, sclerotial development and fungal pathogenicity were investigated. Phylogenetic analyses indicated that the deduced amino acid sequences of SCD1 and THR1 were similar to the orthologues of Botrytis cinerea. Expression of SCD1 was at higher levels in sclerotia relative to mycelia. THR1 was expressed at similar levels in mycelia and sclerotia at early stages, but was up-regulated in sclerotia at the maturation stage. Disruption of SCD1 or THR1 did not change the pathogenicity of the fungus, but resulted in slower radial growth, less biomass, wider angled hyphal branches, impaired sclerotial development and decreased resistance to ultraviolet light.

Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum.

This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism.

Employment of methylene blue irradiated with laser light source in photodynamic inactivation of biofilm formed by Candida albicans strain resistant to fluconazole.

A promising approach for the eradication of biofilm formed by the yeast Candida albicans seems to be photodynamic inactivation (PDI). This work presents a use of methylene blue (MB, 1 mM) irradiated with a red laser (output power 190 mW/cm2, wavelength 660 nm) for the eradication of a biofilm formed by the fluconazole-resistant (FLC-resistant) strain C. albicans CY 1123 compared to the standard strain C. albicans SC5314. The periods of irradiation corresponded to the fluence of 15, 23 and 57 J/cm2. Effectiveness of PDI was evident with following percentage of survived biofilm cells: 24.57, 23.46, and 22.29% for SC5314 and 40.28, 17.91, and 5.89% for CY 1123, respectively, compared to the samples without irradiation. Light and confocal laser scanning microscopy confirmed the effectiveness of PDI. However, the morphological form of C. albicans seems to play an important role as well, since prolonged duration of irradiation did not increase efficiency of PDI on C. albicans SC5314. An experiment with the yeast-to-hyphae transition revealed that the FLC-resistant strain expressed a markedly reduced capacity to form hyphae compared to SC5314. We summarized that PDI was effective on biofilm formed by the FLC-resistant strain, but resistance most likely did not play significant role in PDI. Additionally, we observed differences in susceptibility to PDI between biofilms composed of the mycelia and only of the yeasts, and finally, the employment of a laser in PDI enabled a decreasing period of irradiation while maintaining the high effectiveness of PDI.

Direct accumulation pathway of radioactive cesium to fruit-bodies of edible mushroom from contaminated wood logs.

This paper presents the accumulation process of radioactive Cs in edible mushrooms. We here first report the direct accumulation pathway of radioactive Cs from contaminated wood logs to the fruit-bodies of shiitake mushrooms through the basal portion of the stipe. In this pathway, radioactive Cs is not transported through the hyphae. This pathway results in a high accumulation of radioactive Cs in the fruit-body, more by the excess accumulation of radioactive Cs from the wood logs than that through the hyphae. We grew the fruit-bodies of Shiitake mushroom from radioactive-Cs-contaminated wood logs. The spatial distributions of radioactive Cs and Prussian blue as a tracer of interstitial water in the cross section of the wood log measured after the harvest of the fruit-body from the inoculated sawdust spawn area indicated that some fraction of the radioactive Cs and Prussian blue were transported directly to the basal portion of the stipe during the growth of the fruit-bodies.

Inhibitory effect of gamma irradiation and its application for control of postharvest green mold decay of Satsuma mandarins.

Gamma irradiation has been shown to be effective for the control of postharvest fungi in vitro, but little is known regarding antifungal action, responses to gamma irradiation, and its application to fresh produce. Gamma irradiation was evaluated for its in vitro and in vivo antifungal activity against Penicillium digitatum on Satsuma mandarin fruits. Green mold was inhibited in a dose-dependent manner. Gamma irradiation showed a complete inhibition of spore germination, germ tube elongation, and mycelial growth of P. digitatum, particularly at 1.0kGy. To further investigate the mechanisms by which gamma irradiation inhibits fungal growth, the membrane integrity and cellular leakage of conidia were tested, indicating that gamma irradiation results in the loss of plasma membrane integrity, causing the release of intracellular contents such as soluble proteins. In vivo assays demonstrated that established doses can completely inhibit the growth of fungal pathogens, but such high doses cause severe fruit damage. Thus, to eliminate the negative impact on fruit quality, gamma irradiation at lower doses was evaluated for inhibition of P. digitatum, in combination with a chlorine donor, sodium dichloro-s-triazinetrione (NaDCC). Interestingly, only a combined treatment with 0.4kGy of gamma irradiation and 10ppm of NaDCC exhibited significant synergistic antifungal activity against green mold decay. The mechanisms by which the combined treatment decreased the green mold decay of mandarin fruits can be directly associated with the disruption of cell membrane of the fungal pathogen, which resulted in a loss of cytoplasmic material from the hyphae. These findings suggest that a synergistic effect of combining treatment with gamma irradiation with NaDCC has potential as an antifungal approach to reduce the severity of green mold in mandarin fruits.

The microcyclic conidial stage of Coniochaeta pulveracea and its effect on selected biological interactions.

Coniochaeta pulveracea is a dimorphic lignicolous fungus that has mostly been isolated from decaying wood. However, relatively little work was conducted on the conditions for the dimorphic switch or the biological interactions of the fungus in its yeast-like microcyclic growth phase. Therefore, in this study, the microcyclic conidiation of C. pulveracea strains and representatives of the closely related species, Coniochaeta boothii and Coniochaeta subcorticalis, was studied under different environmental conditions. The strains were found to exhibit hyphal growth on solid substrates and underwent a dimorphic switch to produce microcycle conidiation upon transfer to a liquid medium which differed in physico-chemical composition compared to the original solid medium. Factors that were found to contribute to this dimorphic switch were temperature, pH and the presence of complex nitrogen sources such as casamino acids and peptone in the medium. However, C. pulveracea showed intraspecific differences with regard to its response to changes in the physico-chemical environment. The interactions of microcyclic Coniochaeta strains with selected yeasts, such as representatives of Meyerozyma guilliermondii and Cryptococcus neoformans, were subsequently studied in complex liquid media and it was found that, depending on medium composition, the microcyclic Coniochaeta exerted different effects on the different yeasts strains. In some co-cultures, a positive effect on yeast growth was observed, whilst in other cases microcyclic Coniochaeta inhibited yeast growth.

Cochliobolus lunatus colonizes potato by adopting different invasion strategies on cultivars: New insights on temperature dependent-virulence.

Extreme temperature fluctuations affect the interaction dynamics of Cochliobolus lunatus through temperature-dependent virulence, virulence differentiation and induced-virulence which poses a major threat to global food security. The relationship between higher temperature and pathogenicity of C. lunatus on reported hosts are poorly understood. In this study, temperature stress was applied on C. lunatus to investigate the correlation among the different types of conidia. Additionally, a comparative dissection of the invasion process, infection structures and conidial germination pattern on four different Solanum tuberosum L. (potato) cultivars were performed. Based on microscopic examination, it was found that C. lunatus adopts different hyphae morphology and septation pattern at different temperature regimes and produce different types of conidia. The study showed that four-celled conidia are overproduced at elevated temperature (>30 °C) than one, two, three and five-celled conidia. Our finding revealed that C. lunatus conidia exhibit bipolar germination (>14.67%, P<0.05), unipolar germination (>35.33%, P<0.05), penetrate subcutaneously via epidermal anticlinal cell wall (>0.33%, P < 0.05) and differentially form appressoria-like structures during colonization of four different potato cultivars. Importantly, it is shown that unipolar germination and bipolar germination in C. lunatus are independently occurring phenomenon irrespective of the host. It is confirmed that C. lunatus adopt different but highly successful strategies on four different potato cultivars to incite brown-to-black leaf spot disease. Altogether, our data showed that increase in temperature enhances C. lunatus virulence on different potato cultivars irrespective of their inherent thermotolerant traits.

Exploring the transfer of recent plant photosynthates to soil microbes: mycorrhizal pathway vs direct root exudation.

Plants rapidly release photoassimilated carbon (C) to the soil via direct root exudation and associated mycorrhizal fungi, with both pathways promoting plant nutrient availability. This study aimed to explore these pathways from the root's vascular bundle to soil microbial communities. Using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging and (13) C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of (13) CO2 -exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly (13) C-enriched compared to other root-cortex areas after 8 h of labelling. Immature fine root areas close to the root tip, where AM features were absent, showed signs of passive C loss and co-location of photoassimilates with nitrogen taken up from the soil solution. A significant and exclusively fresh proportion of (13) C-photosynthates was delivered through the AM pathway and was utilised by different microbial groups compared to C directly released by roots. Our results indicate that a major release of recent photosynthates into soil leave plant roots via AM intraradical hyphae already upstream of passive root exudations. AM fungi may act as a rapid hub for translocating fresh plant C to soil microbes.

Increased mycelial biomass production by Lentinula edodes intermittently illuminated by green light emitting diodes.

Fungi possess a range of light receptors to regulate metabolism and differentiation. To study the effect of light on Lentinula edodes (the shiitake mushroom), mycelial cultures were exposed to blue, green, and red fluorescent lights and light-emitting diodes, as well as green laser light. Biomass production, morphology, and pigment production were evaluated. Exposure to green light at intervals of 1 min/d at 0.4 W/m(2) stimulated biomass production by 50-100 %, depending on the light source. Light intensities in excess of 1.8 W/m(2) or illumination longer than 30 min/d did not affect biomass production. Carotenoid production and morphology remained unaltered during increased biomass production. These observations provide a cornerstone to the study of photoreception by this important fungus.

The observation of plcA mutation and localization in Aspergillus nidulans.

To know the function of the plcA gene, which encodes a putative phosphoinositide-specific phospholipase C, in a model filamentous fungus Aspergillus nidulans, it was disrupted thorough homologous recombination and examined. The germination rate of ΔplcA was reduced by approximately 65% and germination of ΔplcA at a lower temperature (25°C) was much slower than germination under normal conditions (37°C), suggesting the plcA is responsible for cold-sensitivity. The hyphal growth of ΔplcA was slightly reduced at 37°C and conspicuously reduced at 25°C. While germinating ΔplcA formed giant swollen spores, and generated short and thick hyphae. The results of the nuclear examination of ΔplcA showed nuclear division with missegregation, and the rate of nuclear division was lower than that of wild type at both 25°C and 37°C. The results of this study showed that plcA is localized to the nucleus through intracellular calcium signaling in A. nidulans. The abnormal nuclear division, resulting from plcA gene deletion, affects conidiation in asexual development. Taken together, these results suggested that plcA is required for normal vegetative growth, morphogenesis, conidiation, and nuclear division in A. nidulans.

Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast.

Selective damage to hyphal form through light-induced delivery of nitric oxide to Candida albicans colonies.

Candida albicans, an opportunistic fungal pathogen, causes severe to life-threatening infections in immunocompromised hosts (e.g. HIV patients, burn victims). Conversion of the commensal yeast form to the invasive hyphal form, triggered by environmental cues, initiates such episodes. Although the antifungal activity of nitric oxide (NO) has been established, very few convenient NO-donating systems for treating C. albicans infection have been reported. In this work, a biocompatible NO-donating material that delivers NO upon illumination with visible light has been employed to eradicate C. albicans in a dose-dependent way. Careful studies on the yeast and hyphal forms with this NO donor have revealed that the hyphal form is more susceptible to NO exposure than the yeast variety. Results of this work suggest that materials of this type could find use in thwarting invasion of the hyphal form of the fungus in cases of invasive C. albicans infection.

The fungal pathogen Aspergillus fumigatus regulates growth, metabolism, and stress resistance in response to light.

Light is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology.

Effect of 300 mT static and 50 Hz 0.1 mT extremely low frequency magnetic fields on Tuber borchii mycelium.

The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.

In Vitro effect of low-level laser therapy on typical oral microbial biofilms.

The aim of this study was to evaluate the effect of specific parameters of low-level laser therapy (LLLT) on biofilms formed by Streptococcus mutans, Candida albicans or an association of both species. Single and dual-species biofilms--SSB and DSB--were exposed to laser doses of 5, 10 or 20 J/cm(2) from a near infrared InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm, 0.04 W). After irradiation, the analysis of biobilm viability (MTT assay), biofilm growth (cfu/mL) and cell morphology (SEM) showed that LLLT reduced cell viability as well as the growth of biofilms. The response of S. mutans (SSB) to irradiation was similar for all laser doses and the biofilm growth was dose dependent. However, when associated with C. albicans (DSB), S. mutans was resistant to LLLT. For C. albicans, the association with S. mutans (DSB) caused a significant decrease in biofilm growth in a dose-dependent fashion. The morphology of the microorganisms in the SSB was not altered by LLLT, while the association of microbial species (DSB) promoted a reduction in the formation of C. albicans hyphae. LLLT had an inhibitory effect on the microorganisms, and this capacity can be altered according to the interactions between different microbial species.