The Biochemical Mechanisms of Antimicrobial Photodynamic Therapy†.

The unbridled dissemination of multidrug-resistant pathogens is a major threat to global health and urgently demands novel therapeutic alternatives. Antimicrobial photodynamic therapy (aPDT) has been developed as a promising approach to treat localized infections regardless of drug resistance profile or taxonomy. Even though this technique has been known for more than a century, discussions and speculations regarding the biochemical mechanisms of microbial inactivation have never reached a consensus on what is the primary cause of cell death. Since photochemically generated oxidants promote ubiquitous reactions with various biomolecules, researchers simply assumed that all cellular structures are equally damaged. In this study, biochemical, molecular, biological and advanced microscopy techniques were employed to investigate whether protein, membrane or DNA damage correlates better with dose-dependent microbial inactivation kinetics. We showed that although mild membrane permeabilization and late DNA damage occur, no correlation with inactivation kinetics was found. On the other hand, protein degradation was analyzed by three different methods and showed a dose-dependent trend that matches microbial inactivation kinetics. Our results provide a deeper mechanistic understanding of aPDT that can guide the scientific community toward the development of optimized photosensitizing drugs and also rationally propose synergistic combinations with antimicrobial chemotherapy.

Photoantimicrobials in agriculture.

Classical approaches for controlling plant pathogens may be impaired by the development of pathogen resistance to chemical pesticides and by limited availability of effective antimicrobial agents. Recent increases in consumer awareness of and/or legislation regarding environmental and human health, and the urgent need to improve food security, are driving increased demand for safer antimicrobial strategies. Therefore, there is a need for a step change in the approaches used for controlling pre- and post-harvest diseases and foodborne human pathogens. The use of light-activated antimicrobial substances for the so-called antimicrobial photodynamic treatment is known to be effective not only in a clinical context, but also for use in agriculture to control plant-pathogenic fungi and bacteria, and to eliminate foodborne human pathogens from seeds, sprouted seeds, fruits, and vegetables. Here, we take a holistic approach to review and re-evaluate recent findings on: (i) the ecology of naturally-occurring photoantimicrobials, (ii) photodynamic processes including the light-activated antimicrobial activities of some plant metabolites, and (iii) fungus-induced photosensitization of plants. The inhibitory mechanisms of both natural and synthetic light-activated substances, known as photosensitizers, are discussed in the contexts of microbial stress biology and agricultural biotechnology. Their modes-of-antimicrobial action make them neither stressors nor toxins/toxicants (with specific modes of poisonous activity), but a hybrid/combination of both. We highlight the use of photoantimicrobials for the control of plant-pathogenic fungi and quantify their potential contribution to global food security.

Thiopyridinium phthalocyanine for improved photodynamic efficiency against pathogenic fungi.

The emergence of opportunistic pathogens and the selection of resistant strains have created a grim scenario for conventional antimicrobials. Consequently, there is an ongoing search for alternative techniques to control these microorganisms. One such technique is antimicrobial photodynamic therapy (aPDT), which combines photosensitizers, light, and molecular oxygen to produce reactive oxygen species and kill the target pathogen. Here, the in vitro susceptibilities of three fungal pathogens, namely Candida albicans, Aspergillus nidulans, and Colletotrichum abscissum to aPDT with zinc(II) phthalocyanine (ZnPc) derivative complexes were investigated. Three ZnPc bearing thiopyridinium substituents were synthesized and characterized by several spectroscopic techniques. The Q-band showed sensitivity to the substituent with high absorptivity coefficient in the 680-720 nm region. Derivatization and position of the rings with thiopyridinium units led to high antifungal efficiency of the cationic phthalocyanines, which could be correlated with singlet oxygen quantum yield, subcellular localization, and cellular uptake. The minimum inhibitory concentrations (MIC) of the investigated ZnPc-R complexes against the studied microorganisms were 2.5 µM (C. albicans) and 5 µM (A. nidulans and C. abscissum). One ZnPc derivative achieved complete photokilling of C. albicans and, furthermore, yielded low MIC values when used against the tolerant plant-pathogen C. abscissum. Our results show that chemical modification is an important step in producing better photosensitizers for aPDT against fungal pathogens.

Potential cannabidiol (CBD) repurposing as antibacterial and promising therapy of CBD plus polymyxin B (PB) against PB-resistant gram-negative bacilli.

This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD + polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Time-kill assays results showed that the combination CBD + PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD + PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. pneumoniae, is particularly promising.

Photobiology of the keystone genus Metarhizium.

Metarhizium fungi are soil-inhabiting ascomycetes which are saprotrophs, symbionts of plants, pathogens of insects, and participate in other trophic/ecological interactions, thereby performing multiple essential ecosystem services. Metarhizium species are used to control insect pests of crop plants and insects that act as vectors of human and animal diseases. To fulfil their functions in the environment and as biocontrol agents, these fungi must endure cellular stresses imposed by the environment, one of the most potent of which is solar ultraviolet (UV) radiation. Here, we examine the cellular stress biology of Metarhizium species in context of their photobiology, showing how photobiology facilitates key aspects of their ecology as keystone microbes and as mycoinsectides. The biophysical basis of UV-induced damage to Metarhizium, and mechanistic basis of molecular and cellular responses to effect damage repair, are discussed and interpreted in relation to the solar radiation received on Earth. We analyse the interplay between UV and visible light and how the latter increases cellular tolerance to the former via expression of a photolyase gene. By integrating current knowledge, we propose the mechanism through which Metarhizium species use the visible fraction of (low-UV) early-morning light to mitigate potentially lethal damage from intense UV radiation later in the day. We also show how this mechanism could increase Metarhizium environmental persistence and improve its bioinsecticide performance. We discuss the finding that visible light modulates stress biology in the context of further work needed on Metarhizium ecology in natural and agricultural ecosystems, and as keystone microbes that provide essential services within Earth's biosphere.

Timing and duration of light exposure during conidia development determine tolerance to ultraviolet radiation.

Metarhizium is an important genus of soil-inhabiting fungi that are used for the biological control of insects. The efficiency of biocontrol is dependent on the maintenance of inoculum viability under adverse field conditions such as solar ultraviolet (UV) radiation. Therefore, increasing the tolerance of Metarhizium to UV radiation is necessary. It was previously established that, in mycelium, exposure to visible light increases tolerance to UV radiation. Similarly, growth under visible light for 14 days induces the production of tolerant conidia. However, a study evaluating if and how visible light affects conidia and their relationship with UV radiation was never performed. Here, we report that a relatively short and timed exposure to light around the time of conidiation is sufficient to induce the production of conidia with increased photoreactivating capacity and UV tolerance in Metarhizium acridum. Conidia produced by this method retain their characteristic higher tolerance even after many days of being transferred to the dark. Furthermore, we show that mature conidia of M. acridum and Metarhizium brunneum can still answer to light and regulate UV tolerance, suggesting that gene expression is possible even in dormant spores. Being able to respond to light in the dormant stages of development is certainly an advantage conferring improved environmental persistence to Metarhizium.

Antimicrobial photodynamic treatment as an alternative approach for Alicyclobacillus acidoterrestris inactivation.

Alicyclobacillus acidoterrestris is a cause of major concern for the orange juice industry due to its thermal and chemical resistance, as well as its spoilage potential. A. acidoterrestris spoilage of orange juice is due to off-flavor taints from guaiacol production and some halophenols. The present study aimed to evaluate the effectiveness of antimicrobial Photodynamic Treatment (aPDT) as an emerging technology to inactivate the spores of A. acidoterrestris. The aPDT efficiency towards A. acidoterrestris was evaluated using as photosensitizers the tetracationic porphyrin (Tetra-Py+-Me) and the phenothiazinium dye new methylene blue (NMB) in combination with white light-emitting diode (LED; 400-740 nm; 65-140 mW/cm2). The spores of A. acidoterrestris were cultured on YSG agar plates (pH 3.7 ± 0.1) at 45 °C for 28 days and submitted to the aPDT with Tetra-Py+-Me and NMB at 10 µM in phosphate-buffered saline (PBS) in combination with white light (140 mW/cm2). The use of Tetra-Py+-Me at 10 µM resulted in a 7.3 ± 0.04 log reduction of the viability of A. acidoterrestris spores. No reductions in the viability of this bacterium were observed with NMB at 10 µM. Then, the aPDT with Tetra-Py+-Me and NMB at 10 µM in orange juice (UHT; pH 3.9; 11°Brix) alone and combined with potassium iodide (KI) was evaluated. The presence of KI was able to potentiate the aPDT process in orange juice, promoting the inactivation of 5 log CFU/mL of A. acidoterrestris spores after 10 h of white light exposition (140 mW/cm2). However, in the absence of KI, both photosensitizers did not promote a significant reduction in the spore viability. The inactivation of A. acidoterrestris spores artificially inoculated in orange peels (105 spores/mL) was also assessed using Tetra-Py+-Me at 10 and 50 µM in the presence and absence of KI in combination with white light (65 mW/cm2). No significant reductions were observed (p < .05) when Tetra-Py+-Me was used at 10 µM, however at the highest concentration (50 µM) a significant spore reduction (≈ 2.8 log CFU/mL reductions) in orange peels was observed after 6 h of sunlight exposition (65 mW/cm2). Although the color, total phenolic content (TPC), and antioxidant capacity of orange juice and peel (only color evaluation) seem to have been affected by light exposition, the impact on the visual and nutritional characteristics of the products remains inconclusive so far. Besides that, the results found suggest that aPDT can be a potential method for the reduction of A. acidoterrestris spores on orange groves.

Efficient photodynamic inactivation of Candida albicans by porphyrin and potassium iodide co-encapsulation in micelles.

Photodynamic inactivation of bacterial and fungal pathogens is a promising alternative to the extensive use of conventional single-target antibiotics and antifungal agents. The combination of photosensitizers and adjuvants can improve the photodynamic inactivation efficiency. In this regard, it has been shown that the use of potassium iodide (KI) as adjuvant increases pathogen killing. Following our interest in this topic, we performed the co-encapsulation of a neutral porphyrin photosensitizer (designated as P1) and KI into micelles and tested the obtained nanoformulations against the human pathogenic fungus Candida albicans. The results of this study showed that the micelles containing P1 and KI displayed a better photodynamic performance towards C. albicans than P1 and KI in solution. It is noteworthy that higher concentrations of KI within the micelles resulted in increased killing of C. albicans. Subcellular localization studies by confocal fluorescence microscopy revealed that P1 was localized in the cell cytoplasm, but not in the nuclei or mitochondria. Overall, our results show that a nanoformulation containing a photosensitizer plus an adjuvant is a promising approach for increasing the efficiency of photodynamic treatment. Actually, the use of this strategy allows a considerable decrease in the amount of both photosensitizer and adjuvant required to achieve pathogen killing.

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.

The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Combining Transcriptomics and Proteomics Reveals Potential Post-transcriptional Control of Gene Expression After Light Exposure in Metarhizium acridum.

Light is an important stimulus for fungi as it regulates many diverse and important biological processes. Metarhizium acridum is an entomopathogenic fungus currently used for the biological control of insect pests. The success of this approach is heavily dependent on tolerance to environmental stresses. It was previously reported that light exposure increases tolerance to ultraviolet radiation in M. acridum There is no information in the literature about how light globally influences gene expression in this fungus. We employed a combination of mRNA-Sequencing and high-throughput proteomics to study how light regulates gene expression both transcriptionally and post-transcriptionally. Mycelium was exposed to light for 5 min and changes at the mRNA and protein levels were followed in time-course experiments for two and four hours, respectively. After light exposure, changes in mRNA abundance were observed for as much as 1128 genes or 11.3% of the genome. However, only 57 proteins changed in abundance and at least 347 significant changes at the mRNA level were not translated to the protein level. We observed that light downregulated subunits of the eukaryotic translation initiation factor 3, the eIF5A-activating enzyme deoxyhypusine hydroxylase, and ribosomal proteins. We hypothesize that light is perceived as a stress by the cell that responds to it by reducing translational activity. Overall, our results indicate that light acts both as a signal and a stressor to M. acridum and highlight the importance of measuring protein levels in order to fully understand light responses in fungi.

Survival variability of 12 strains of Bacillus cereus yielded to spray drying of whole milk.

The variability in spore survival during spray drying of 12 Bacillus cereus strains was evaluated. B. cereus spores were inoculated on whole milk (7.2 ±â€¯0.2 log10 spores/g dry weight) and processed in a spray-dryer. Twelve independent experiments were carried out in triplicate. The spore count was determined before and after each drying process, based on the dry weight of whole milk and powdered milk. Then, the number of decimal reductions (γ) caused by the spray drying process was calculated. B. cereus strains presented γ values ranging from 1.0 to 4.7 log10 spores/g dry weight, with a high coefficient of variation (CV) of 46.1%. Cluster analysis allowed to group B. cereus as sensitive (strains 511, 512, 540, 432 and ATCC 14579), intermediate (strains B18, B63, and B86) and resistant strains (strains B3, B94, B51 and 436). Three strains (one of each group) were selected for further investigation and characterization of their physicochemical and molecular (proteomics) differences. Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) were used to determine physicochemical characteristics and glass transition temperature (Tg), respectively. No differences in signs among the three strains were found in spectra ranging from 900 to 4000 cm-1. The endothermic peak ranged between 54 and 130 °C for strain 540; between 81 and 163 °C for strain B63; and between 110 and 171 °C for strain 436. However, they showed different Tg: 88.82 °C for strain 540; 114.32 °C for strain B63; and 122.70 °C for strain 436. A total of eleven spots were identified by mass spectrometry, with the spore coat protein GerQ, sporulation protein YtfJ (GerW), and peptidyl-prolyl cis-trans isomerase being found in at least two strains. Altogether, the results suggested that the high survival variability of B. cereus spores to the spray drying process seems to be mainly associated with different Tg and protein content. The study highlights the importance of quantifying the effects of this unit operation over the target microorganisms. These data may be relevant for the development of effective measures aiming to control the occurrence of B. cereus in milk powder as well as to reduce spoilage or safety issues associated with the presence of this bacterium in foods, particularly those formulated with milk powder.

The second International Symposium on Fungal Stress: ISFUS.

The topic of 'fungal stress' is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Species of the Metarhizium anisopliae complex with diverse ecological niches display different susceptibilities to antifungal agents.

Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots. There is little information available about the susceptibility of Metarhizium species to clinical and non-clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole, griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the generalist Metarhizium species suggest that this characteristic is associated with their different ecological niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by Metarhizium species.

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence.

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m-2); 3) PDA medium under blue light (4.8 W m-2); 4) PDA medium under red light (2.8 W m-2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.

The Xenon Test Chamber Q-SUN® for testing realistic tolerances of fungi exposed to simulated full spectrum solar radiation.

The low survival of insect-pathogenic fungi when used for insect control in agriculture is mainly due to the deleterious effects of ultraviolet radiation and heat from solar irradiation. In this study, conidia of 15 species of entomopathogenic fungi were exposed to simulated full-spectrum solar radiation emitted by a Xenon Test Chamber Q-SUN XE-3-HC 340S (Q-LAB® Corporation, Westlake, OH, USA), which very closely simulates full-spectrum solar radiation. A dendrogram obtained from cluster analyses, based on lethal time 50 % and 90 % calculated by Probit analyses, separated the fungi into three clusters: cluster 3 contains species with highest tolerance to simulated full-spectrum solar radiation, included Metarhizium acridum, Cladosporium herbarum, and Trichothecium roseum with LT50 > 200 min irradiation. Cluster 2 contains eight species with moderate UV tolerance: Aschersonia aleyrodis, Isaria fumosorosea, Mariannaea pruinosa, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, and Torrubiella homopterorum with LT50 between 120 and 150 min irradiation. The four species in cluster 1 had the lowest UV tolerance: Lecanicillium aphanocladii, Beauveria bassiana, Tolypocladium cylindrosporum, and Tolypocladium inflatum with LT50 < 120 min irradiation. The QSUN Xenon Test Chamber XE3 is often used by the pharmaceutical and automotive industry to test light stability and weathering, respectively, but it was never used to evaluate fungal tolerance to full-spectrum solar radiation before. We conclude that the equipment provided an excellent tool for testing realistic tolerances of fungi to full-spectrum solar radiation of microbial agents for insect biological control in agriculture.

Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide.

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC50 and IC90, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC50 > 4.7 µM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC50 < 0.2 µM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC50 between 0.4 and 0.9 µM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC50 between 1.0 and 1.2 µM). In cluster 4 were species with moderate to high tolerance (IC50 between 1.3 and 1.6 µM). Cluster 5 contained the species with high tolerance (IC50 between 1.9 and 4.0 µM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.

Exposing Metarhizium acridum mycelium to visible light up-regulates a photolyase gene and increases photoreactivating ability.

Metarhizium acridum is an entomopathogen currently used against acridids. We have previously reported that exposing mycelium to visible light increases M. acridum tolerance to ultraviolet-B (UV-B) radiation. Here we evaluated if light could also increase tolerance to ultraviolet-C (UV-C) radiation. We observed that, as opposed to UV-B radiation, light did not increase tolerance to UV-C radiation under dark repair conditions. However, light did increase tolerance to UV-C radiation if photoreactivating light was present after UV-C exposure. Quantitative PCR experiments revealed that light up-regulates a photolyase gene. This is the first report showing that light regulates photoreactivating ability in M. acridum.

Photodynamic inactivation of conidia of the fungus Colletotrichum abscissum on Citrus sinensis plants with methylene blue under solar radiation.

Antimicrobial photodynamic treatment (APDT) is a promising light based approach to control diseases caused by plant-pathogenic fungi. In the present study, we evaluated the effects of APDT with the phenothiazinium photosensitizer methylene blue (MB) under solar radiation on the germination and viability of conidia of the pathogenic fungus Colletotricum abscissum (former Colletotrichum acutatum sensu lato). Experiments were performed both on petals and leaves of sweet orange (Citrus sinensis) in different seasons and weather conditions. Conidial suspensions were deposited on the leaves and petals surface, treated with the PS (25 or 50µM) and exposed to solar radiation for only 30min. The effects of APDT on conidia were evaluated by counting the colony forming units recovered from leaves and petals and by direct evaluating conidial germination on the surface of these plant organs after the treatment. To better understand the mechanistic of conidial photodynamic inactivation, the effect of APDT on the permeability of the conidial plasma membrane was assessed using the fluorescent probe propidium iodide (PI) together with flow cytometry and fluorescence microscopy. APDT with MB and solar exposure killed C. abscissum conidia and prevented their germination on both leaves and petals of citrus. Reduction of conidial viability was up to three orders of magnitude and a complete photodynamic inactivation was achieved in some of the treatments. APDT damaged the conidial plasma membrane and increased its permeability to PI. No damage to sweet orange flowers or leaves was observed after APDT. The demonstration of the efficacy of APDT on the plant host represents a further step towards the use of the method for control phytopathogens in the field.