Imperfect match between radiation exposure times required for conidial viability loss and infective capacity reduction attenuate UV-B impact on Beauveria bassiana.

BACKGROUND: UV-B radiation represents a significant challenge for the widespread use of entomopathogenic fungi in pest management. This study focused on research of the asynchronous response between virulence and conidial viability against Ceratitis capitata adults using specific statistical models. Moreover, it was also investigated whether the observed differences in susceptibility to UV-B radiation in in vitro assays among three selected isolates of Beauveria bassiana were reflected in the above-mentioned asynchrony. RESULTS: While the irradiation of the three isolates of B. bassiana was associated with a significant loss of conidial viability, their virulence was not significantly affected compared to nonirradiated treatments when exposed to 1200 mW m-2 for 6 h before or after the inoculation of C. capitata. In fact, the irradiation time needed to reduce the mortality to 50% compared to the controls was 34.69 h for EABb 10/225-Fil, 16.36 h for EABb 09/20-Fil, and 24.59 h for EABb 09/28-Fil. Meanwhile, the irradiation time necessary to reduce conidial viability to 50% was 9.89 h for EABb 10/225-Fil, 8.74 h for EABb 09/20-Fil, and 4.71 h for EABb 09/28-Fil. CONCLUSION: These results highlight the importance of modeling the response of entomopathogenic fungi virulence and conidial susceptibility when exposed to UV-B radiation for the selection of environmentally competent isolates, regardless of the results obtained in previous in vitro assays on conidial germination. This strategic approach is critical in overcoming the challenges posed by UV-B radiation and holds the key to realizing the full potential of entomopathogenic fungi in pest management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Optional strategies for low-risk and non-risk applications of fungal pesticides to avoid solar ultraviolet damage.

BACKGROUND: Solar ultraviolet (UV) irradiation is harmful to formulated conidia as active ingredients of fungal pesticides and hence restrains their field application in sunny days of summer, a season requiring frequent pest controls. This conflict makes it necessary to explore optimal strategies for the application of fungal pesticides to suppress pest populations but avoid solar UV damage during summer. RESULTS: The conidia of Beauveria bassiana, a wide-spectrum fungal pesticide, were tolerable to UVB (major solar UV wavelengths) damage of ≤0.5 J cm-2 . The damage of this upper limit caused a loss of conidial viability and infectivity if not photoreactivated by light exposure after irradiation. Intriguingly, the light exposure resulted in a high photoreactivation rate of UVB-inactivated conidia and an insignificant or marginal difference in insecticidal activity between normal conidia and those photoreactivated. Modeling analysis of solar UVB intensity recorded hourly over the daylight of five sunny summer days from 5:00 am to 7:00 pm at 30° 17'57'' N and 120°5'7'' E revealed a variation of daily accumulated UVB dose from 2.07 to 2.78 J cm-2 , which was far beyond the upper limit. A more tolerable dose of ~0.2 J cm-2 appeared between 3:00 pm and 5:00 pm, and no harmful dose accumulated between 5:00 pm and 7:00 pm. CONCLUSION: Fungal UVB tolerance, fungal photoreactivation capability and the daily accumulation pattern of solar UV irradiation are based to propose an optional strategy for low-risk or non-risk application of fungal pesticides after 3:00 or 5:00 pm during summer. © 2022 Society of Chemical Industry.

Low- or high-white light irradiance induces similar conidial stress tolerance in Metarhizium robertsii.

White light during mycelial growth influences high conidial stress tolerance of the insect-pathogenic fungus Metarhizium robertsii, but little is known if low- or high-white light irradiances induce different stress tolerances. The fungus was grown either in the dark using two culture media: on minimal medium (Czapek medium without sucrose = MM) or on potato dextrose agar (PDA) or PDA medium under five different continuous white light irradiances. The stress tolerances of conidia produced on all treatments were evaluated by conidial germination on PDA supplemented with KCl for osmotic stress or on PDA supplemented with menadione for oxidative stress. Conidia produced on MM in the dark were more tolerant to osmotic and oxidative stress than conidia produced on PDA in the dark or under the light. For osmotic stress, growth under the lower to higher irradiances produced conidia with similar tolerances but more tolerant than conidia produced in the dark. For oxidative stress, conidia produced under the white light irradiances were generally more tolerant to menadione than conidia produced in the dark. Moreover, conidia produced in the dark germinated at the same speed when incubated in the dark or under lower irradiance treatment. However, at higher irradiance, conidial germination was delayed compared to germination in the dark, which germinated faster. Therefore, growth under light from low to high irradiances induces similar conidial higher stress tolerances; however, higher white light irradiances cause a delay in germination speed.

Inhibition of Aspergillus oryzae Mycelium Growth and Conidium Production by Irradiation with Light at Different Wavelengths and Intensities.

Aspergillus oryzae is a safe filamentous fungus widely used in the food, medicine, and feed industries, but there is currently not enough research on the light response of A. oryzae. In this study, 12 different light conditions were set and A. oryzae GDMCC 3.31 was continuously irradiated for 72 h to investigate the effect of light on mycelial growth and conidium production. Specifically, each light condition was the combination of one light wavelength (475, 520, or 630 nm) and one light intensity (20, 40, 60, or 80 µmol photon m-2 s-1). The results show that mycelium growth was inhibited significantly by green light (wavelength of 520 nm and intensities of 20 and 60 µmol photon m-2 s-1) and blue light (wavelength of 475 nm and intensity of 80 µmol photon m-2 s-1). The production of conidia was suppressed only by blue light (wavelength of 475 nm and intensities of 40, 60, and 80 µmol photon m-2 s-1), and those levels of inhibition increased when the intensity of blue light increased. When the strain was irradiated by blue light (80 µmol photon m-2 s-1), the number of conidia was 57.4% less than that of the darkness group. However, within our set range of light intensities, A. oryzae GDMCC 3.31 was insensitive to red light (wavelength of 630 nm) in terms of mycelium growth and conidium production. Moreover, interaction effects between light wavelength and intensity were found to exist in terms of colony diameter and the number of conidia. This research investigated the light response of A. oryzae, which may provide a new method to regulate mixed strains in fermented foods by light. IMPORTANCE Studies on the monochromatic light response of Aspergillus nidulans and Neurospora crassa have gone deep into the molecular mechanism. However, research methods for the light response of A. oryzae remain in the use of white light sources. In this study, we first demonstrated that A. oryzae GDMCC 3.31 was sensitive to light wavelength and intensity. We have observed that blue light inhibited its growth and sporulation and the inhibitory effect increased with intensity. This research not only adds new content to the study of the photoreaction of Aspergillus but also brings new possibilities for the use of light to regulate mixed strains and ultimately improve the flavor quality of fermented foods.

Inactivation of Aspergillus species in real water matrices using medium pressure mercury lamps.

The aim of this work is to understand the inactivation efficiency of medium pressure mercury lamps, measured in terms of growth inhibition as well as cell death, damage and response, using three strains from three different Aspergillus species (A. fumigatus, A. niger and, A. terreus) spiked in filtered surface water. A complete characterization of the effect of the treatment on each strain of the fungal species was assessed considering spores' morphology, cell wall integrity and enzymatic activity, the formation of pyrimidine dimers in the DNA and proteome analysis. Results showed that, when subjected to medium pressure mercury lamps, A. niger is the most resistant to inactivation, that both A. fumigatus and A. niger suffer more morphological changes and present a higher number of damaged spores and A. terreus presented more dead spores. DNA damages detected in A. niger were able to be repaired to some extent, under both light and dark conditions. Finally, proteome analysis showed that the UV radiation treatment triggered different types of stress response, including cell wall reorganization and DNA repair in A. fumigatus and A. terreus, and oxidative stress responses like the increase in production of citric acid and itaconic acid in A. niger and A. terreus, respectively.

Vital roles of Pks11, a highly reducing polyketide synthase, in fungal conidiation, antioxidant activity, conidial cell wall integrity, and UV tolerance of Beauveria bassiana.

Fungal polyketide synthases play important and differential roles in synthesizing secondary metabolites and regulating several cell events, including asexual development, environmental adaptation, and pathogenicity. This study shows the important functions of a highly reducing polyketide synthase, Pks11, in Beauveria bassiana, a filamentous fungal insect pathogen used worldwide for pest biocontrol. The deletion of pks11 led to severe defects in conidial yields on different media and a decrease of 36.27% in the mean thickness of conidial cell wall under normal conditions. Compared with the wild-type, Δpks11 showed higher tolerance to oxidation and increased sensitivity to high temperature during colony growth. Moreover, the lack of pks11 caused a decrease in conidial germination after exposure to UV radiation but did not affect the virulence of B. bassiana against Galleria mellonella larvae via typical cuticle infection. These findings concurred with the alteration in the transcript levels of some phenotype-related genes. These data suggested that pks11 played vital roles in the asexual development, cell wall integrity, and fungal responses to oxidation, high temperature, and UV irradiation of B. bassiana.

UV-B tolerances of conidia, blastospores, and microsclerotia of Metarhizium spp. entomopathogenic fungi.

The aim of the present study was to analyze ten native Metarhizium spp. isolates as to their UV-B tolerances. Comparisons included: different fungal propagules (conidia, blastospores, or microsclerotia [MS]); conidia in aqueous suspensions or in 10% mineral oil-in-water emulsions; and conidia mixed with different types of soil. The UV-B effect was expressed as the germination of conidia or culturability of blastospores and MS relative to nongerminated propagules. Metarhizium anisopliae LCM S05 exhibited high tolerance as blastospores and/or MS, but not as conidia; LCM S10 and LCM S08 had positive results with MS or conidia but not blastospores. The formulations with 10% mineral oil did not always protect Metarhizium conidia against UV-B. Conidia of LCM S07, LCM S08, and LCM S10 exhibited the best results when in aqueous suspensions, 24 h after UV-B exposure. In general, conidia mixed with soil and exposed to UV-B yielded similar number of colony forming units as conidia from unexposed soil, regardless the soil type. It was not possible to predict which type of propagule would be the most UV-B tolerant for each fungal isolate; in conclusion, many formulations and propagule types should be investigated early in the development of new fungal biocontrol products.

Novel inactivation of the causative fungal pathogen of white-nose syndrome with methoxsalen plus ultraviolet A or B radiation.

White-nose syndrome is a fungal disease responsible for the rapid decline of North American bat populations. This study addressed a novel method for inactivating Pseudogymnoascus destructans, the causative agent of WNS, using ultraviolet A (UVA) or B (UVB) radiation in combination with methoxsalen, a photosensitizer from the furanocoumarin family of compounds. Fungal spore suspensions were diluted in micromolar concentrations of methoxsalen (50-500 µM), then exposed to fixed doses of UVA radiation (500-5000 mJ/cm2), followed by plating on germination media. These plates were examined for two to four weeks for evidence of spore germination or inactivation, along with resultant growth or inhibition of P. destructans colonies. Pretreatment of fungal spores with low doses of methoxsalen resulted in a UVA dose-dependent inactivation of the P. destructans spores. All doses of methoxsalen paired with 500 mJ/cm2 of UVA led to an approximate two-log10 (~99%) reduction in spore viability, and when paired with 1000 mJ/cm2, a four-log10 or greater (>99.99%) reduction in spore viability was observed. Additionally, actively growing P. destructans colonies treated directly with methoxsalen and either UVA or UVB radiation demonstrated UV dose-dependent inhibition and termination of colony growth. This novel approach of using a photosensitizer in combination with UV radiation to control fungal growth may have broad, practical application in the future.

Modeling the inactivation of Aspergillus fischeri and Paecilomyces niveus ascospores in apple juice by different ultraviolet light irradiances.

The present work aimed to evaluate and to model the influence of UV-C light treatments with different irradiances (6.5, 13, 21, and 36 W/m2) on Aspergillus fischeri and Paecilomyces niveus ascospores inactivation in clarified apple juice. Approximately 5.0 and 6.0 log CFU/mL spores of P. niveus and A. fischeri, respectively, were suspended in 30 mL of clarified apple juice (pH 3.8, 12 ± 0.1°Brix) and exposed to UV-C light at different irradiances (as above) and exposure times (0 to 30 min). The first-order biphasic model was able to describe the experimental data with good statistical indices (RMSE = 0.296 and 0.308, R2 = 0.96 and 0.98, for P. niveus and A. fischeri respectively). At the highest irradiance level tested (36 W/m2), the UV-C light allowed the reduction of 5.7 and 4.2 log-cycles of A. fischeri and P. niveus ascospores, respectively, in approximately 10 min. P. niveus was the most UV-C resistant mould. The results showed that, to a defined UV-C fluence, a change in the level of either time or UV-C irradiance did not affect the effectiveness of UV-C light for A. fischeri and P. niveus inactivation. Thus, the modeling of the inactivation as a function of the UV-C fluence allowed the estimation of the primary model parameters with all experimental data and, consequently, no secondary models were needed. The model parameters were validated with experiments of variable UV-C fluences. Accordingly, experimental results allowed to conclude that UV-C treatment at the irradiances tested is a promising application for preventing A. fischeri and P. niveus spoilage of juices.

Differential susceptibility of blastospores and aerial conidia of entomopathogenic fungi to heat and UV-B stresses.

We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m-2 under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors.

Outcome of blue, green, red, and white light on Metarhizium robertsii during mycelial growth on conidial stress tolerance and gene expression.

Fungi sense light and utilize it as a source of environmental information to prepare against many stressful conditions in nature. In this study, Metarhizium robertsii was grown on: 1) potato dextrose agar medium (PDA) in the dark (control); 2) under nutritive stress in the dark; and 3) PDA under continuous (A) white light; (B) blue light lower irradiance = LI; (C) blue light higher irradiance = HI; (D) green light; and (E) red light. Conidia produced under these treatments were tested against osmotic stress and UV radiation. In addition, a suite of genes usually involved in different stress responses were selected to study their expression patterns. Conidia produced under nutritive stress in the dark were the most tolerant to both osmotic stress and UV radiation, and the majority of their stress- and virulence-related genes were up-regulated. For osmotic stress tolerance, conidia produced under white, blue LI, and blue HI lights were the second most tolerant, followed by conidia produced under green light. Conidia produced under red light were the least tolerant to osmotic stress and less tolerant than conidia produced on PDA medium in the dark. For UV tolerance, conidia produced under blue light LI were the second most tolerant to UV radiation, followed by the UV tolerances of conidia produced under white light. Conidia produced under blue HI, green, and red lights were the least UV tolerant and less tolerant than conidia produced in the dark. The superoxide dismutases (sod1 and sod2), photolyases (6-4phr and CPDphr), trehalose-phosphate synthase (tps), and protease (pr1) genes were highly up-regulated under white light condition, suggesting a potential role of these proteins in stress protection as well as virulence after fungal exposure to visible spectrum components.

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.

Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains.

Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress.IMPORTANCE Fungal spores contain secondary metabolites that can protect them from a multitude of abiotic and biotic stresses. Conidia (asexual spores) of the human pathogen Aspergillus fumigatus synthesize several metabolites, including melanin, which has been reported to be important for virulence in this species and to be protective against UV radiation in other fungi. Here, we investigate the role of melanin in diverse isolates of A. fumigatus and find variability in its ability to protect spores from UV-C radiation or impact virulence in a zebrafish model of invasive aspergillosis in two clinical strains and one ISS strain. Further, we assess the role of other spore metabolites in a clinical strain of A. fumigatus and identify fumiquinazoline as an additional UV-C-protective molecule but not a virulence determinant. The results show differential roles of secondary metabolites in spore protection dependent on the environmental stress and strain of A. fumigatus As protection from elevated levels of radiation is of paramount importance for future human outer space explorations, the discovery of small molecules with radiation-protective potential may result in developing novel safety measures for astronauts.

GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

Red- and Blue-Light Sensing in the Plant Pathogen Alternaria alternata Depends on Phytochrome and the White-Collar Protein LreA.

The filamentous fungus Alternaria alternata is a common postharvest contaminant of food and feed, and some strains are plant pathogens. Many processes in A. alternata are triggered by light. Interestingly, blue light inhibits sporulation, and red light reverses the effect, suggesting interactions between light-sensing systems. The genome encodes a phytochrome (FphA), a white collar 1 (WC-1) orthologue (LreA), an opsin (NopA), and a cryptochrome (CryA) as putative photoreceptors. Here, we investigated the role of FphA and LreA and the interplay with the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway. We created loss-of function mutations for fphA, lreA, and hogA using CRISPR-Cas9 technology. Sporulation was reduced in all three mutant strains already in the dark, suggesting functions of the photoreceptors FphA and LreA independent of light perception. Germination of conidia was delayed in red, blue, green, and far-red light. We found that light induction of ccgA (clock-controlled gene in Neurospora crassa and light-induced gene in Aspergillus nidulans) and the catalase gene catA depended on FphA, LreA, and HogA. Light induction of ferA (a putative ferrochelatase gene) and bliC (bli-3, light regulated, unknown function) required LreA and HogA but not FphA. Blue- and green-light stimulation of alternariol formation depended on LreA. A lack of FphA or LreA led to enhanced resistance toward oxidative stress due to the upregulation of catalases and superoxide dismutases. Light activation of FphA resulted in increased phosphorylation and nuclear accumulation of HogA. Our results show that germination, sporulation, and secondary metabolism are light regulated in A. alternata with distinct and overlapping roles of blue- and red-light photosensors.IMPORTANCE Light controls many processes in filamentous fungi. The study of light regulation in a number of model organisms revealed an unexpected complexity. Although the molecular components for light sensing appear to be widely conserved in fungal genomes, the regulatory circuits and the sensitivity of certain species toward specific wavelengths seem different. In N. crassa, most light responses are triggered by blue light, whereas in A. nidulans, red light plays a dominant role. In Alternaria alternata, both blue and red light appear to be important. In A. alternata, photoreceptors control morphogenetic pathways, the homeostasis of reactive oxygen species, and the production of secondary metabolites. On the other hand, high-osmolarity sensing required FphA and LreA, indicating a sophisticated cross talk between light and stress signaling.

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.

Inactivation of conidia from three Penicillium spp. isolated from fruit juices by conventional and alternative mild preservation technologies and disinfection treatments.

Fungi are able to grow on diverse food products and contribute to food spoilage worldwide causing food loss. Consumers prefer freshly squeezed fruit juices, however, the shelf life of these juices is limited due to outgrowth of yeast and fungi. The shelf life of pulsed electric field (PEF) treated juice can be extended from 8 days up to a few weeks before spoilage by moulds becomes apparent. Conidia produced by three Penicillium ssp. (Penicillium expansum, Penicillium buchwaldii and Penicillium bialowiezense), previously isolated from spoiled PEF treated fruit juice and smoothie, were characterized for resistance towards selected mild physical processing techniques in orange juice and toward sanitizers on surfaces. The results show that Penicillium spp. conidia are susceptible to mild heat, high pressure pasteurization (HPP), PEF, cold atmospheric plasma (CAP), UV, and chemical sanitizers chlorine dioxide and hypochlorite albeit with different susceptibility. Treatment with mild heat, HPP, PEF, or chlorine dioxide reduced conidia by more than 5 log. For hypochlorite, UV, and CAP the reduction was between 1 and 3 log. Together, this study provides data for the development of intervention strategies to eliminate spoilage mould conidia in fruit juices.

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.

UV-C irradiation compromises conidial germination, formation of appressoria, and induces transcription of three putative photolyase genes in the barley powdery mildew fungus, Blumeria graminis f. sp. hordei.

UV-C irradiation is known to compromise germination of Blumeria graminis conidia and to reduce powdery mildew infestation. However, only scarce information is available on the effects of UV-C irradiation on B. graminis appressorium formation. Applying a Formvar® resin-based in vitro system allowed for analyzing B. graminis germination and appressorium formation in absence of plant defense. UV-C irradiation more strongly affected the differentiation of appressoria than conidial germination. In vivo and in vitro, a single dose of 100 J m-2 UV-C was sufficient to reduce germination to less than 20 % and decrease appressorium formation to values below 5 %. UV-C irradiation negatively affected pustule size and conidiation. White light-mediated photoreactivation was most effective immediately after UV-C irradiation, indicating that a prolonged phase of darkness after UV-C treatment increases the efficacy of B. graminis control. UV-C irradiation increased transcript levels of three putative B. graminis photolyase genes, while mere white light or blue light irradiation did not contribute to the transcriptional up-regulation. Thus, UV-C irradiation effectively controls B. graminis infestation and proliferation by restricting prepenetration processes. Nevertheless, photoreactivation plays an important role in UV-C-based powdery mildew control in crops and hence has to be considered for planning specific irradiation schedules.

Two Photolyases Repair Distinct DNA Lesions and Reactivate UVB-Inactivated Conidia of an Insect Mycopathogen under Visible Light.

Fungal conidia serve as active ingredients of fungal insecticides but are sensitive to solar UV irradiation, which impairs double-stranded DNA (dsDNA) by inducing the production of cytotoxic cyclobutane pyrimidine dimers (CPDs) and (6-4)-pyrimidine-pyrimidine photoproducts (6-4PPs). This study aims to elucidate how CPD photolyase (Phr1) and 6-4PP photolyase (Phr2) repair DNA damage and photoreactivate UVB-inactivated cells in Beauveria bassiana, a main source of fungal insecticides. Both Phr1 and Phr2 are proven to exclusively localize in the fungal nuclei. Despite little influence on growth, conidiation, and virulence, singular deletions of phr1 and phr2 resulted in respective reductions of 38% and 19% in conidial tolerance to UVB irradiation, a sunlight component most harmful to formulated conidia. CPDs and 6-4PPs accumulated significantly more in the cells of Δphr1 and Δphr2 mutants than in those of a wild-type strain under lethal UVB irradiation and were largely or completely repaired by Phr1 in the Δphr2 mutant and Phr2 in the Δphr1 mutant after optimal 5-h exposure to visible light. Consequently, UVB-inactivated conidia of the Δphr1 and Δphr2 mutants were much less efficiently photoreactivated than were the wild-type counterparts. In contrast, overexpression of either phr1 or phr2 in the wild-type strain resulted in marked increases in both conidial UVB resistance and photoreactivation efficiency. These findings indicate essential roles of Phr1 and Phr2 in photoprotection of B. bassiana from UVB damage and unveil exploitable values of both photolyase genes for improved UVB resistance and application strategy of fungal insecticides.IMPORTANCE Protecting fungal cells from damage from solar UVB irradiation is critical for development and application of fungal insecticides but is mechanistically not understood in Beauveria bassiana, a classic insect pathogen. We unveil that two intranuclear photolyases, Phr1 and Phr2, play essential roles in repairing UVB-induced dsDNA lesions through respective decomposition of cytotoxic cyclobutane pyrimidine dimers and (6-4)-pyrimidine-pyrimidine photoproducts, hence reactivating UVB-inactivated cells effectively under visible light. Our findings shed light on the high potential of both photolyase genes for use in improving UVB resistance and application strategy of fungal insecticides.