Photodynamic Inactivation of Conidia of the Fungi Metarhizium anisopliae and Aspergillus nidulans with Methylene Blue and Toluidine Blue

Antimicrobial photodynamic treatment (PDT) is a promising method that can be used to control localized mycoses or kill fungi in the environment. A major objective of the current study was to compare the conidial photosensitization of two fungal species (Metarhizium anisopliae and Aspergillus nidulans) with methylene blue (MB) and toluidine blue (TBO) under different incubation and light conditions. Parameters examined were media, photosensitizer (PS) concentration and light source. PDT with MB and TBO resulted in an incomplete inactivation of the conidia of both fungal species. Conidial inactivation reached up to 99.7%, but none of the treatments was sufficient to achieve a 100% fungicidal effect using either MB or TBO. PDT delayed the germination of the surviving conidia. Washing the conidia to remove unbound PS before light exposure drastically reduced the photosensitization of A. nidulans. The reduction was much smaller in M. anisopliae conidia, indicating that the conidia of the two species interact differently with MB and TBO. Conidia of green and yellow M. anisopliae mutants were less affected by PDT than mutants with white and violet conidia. In contrast to what occurred in PBS, photosensitization of M. anisopliae and A. nidulans conidia was not observed when PDT was performed in potato dextrose media.State of Sao Paulo Research Foundation (FAPESP)[03/07702-9]Brazilian National Council for Scientific and Technological Development (CNPq)[47.6990/2004-1

Effect of UV-b on Conidia and Germlings of the Entomopathogenic Hyphomycete Metarhizium Anisopliae

The effects of two uv irradiances (920 and 1200 mW m−2 weighted irradiance) on the conidia and germlings of the ARSEF 2575 and ARSEF 23 strains of M. anisopliae were studied. Conidia were exposed to the two irradiance levels for 1, 2, 3, 4, 6, or 8 h. The 30% increase (from 920 to 1200 mW m−2) in uv irradiance caused a significant decrease in culturability following all periods of exposure and reduced the 50% lethal time (TL50) 36% for strain 2575 and 48% for strain 23. Exposure to uv radiation of only 1 h caused a delay of several hours in the germination of surviving conidia. Longer periods of exposure delayed germination for days, demonstrating that, depending on the dose, the fungus may require a long period of time to recover and to resume germination. The results demonstrated the inability of the fungus to germinate during direct exposure to the uv-B portion of simulated sunlight. Both strains showed a transitory increase in uv tolerance during germination. The beginning of germination increased uv tolerance. However, starting on the 6th hour of germination, a decrease in tolerance was observed, indicating that uv tolerance varies as a function of physiological state and cell-cycle phase

Effects of UVB Irradiance on Conidia and Germinants of the Entomopathogenic Hyphomycete Metarhizium Anisopliae: a Study of Reciprocity and Recovery

We tested the effects of irradiances of 920 and 1200 mW m−2 (weighted irradiance) on the conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae. The conidia were exposed to the two irradiances for 1, 2, 4, 6, 7 or 8 h. Increased exposure decreased relative percent culturability. The inactivation provoked by the irradiance of 1200 mW m−2 was higher than for the 920 mW m−2, with a reduction in the 50% lethal time (LT50) from 6 h 40 min to 4 h 26 min. Reciprocity was not observed when conidia in water suspension and germinants in different stages of the germinative process were exposed to a 17.3 kJ m−2 total dose at both irradiance levels. Although nonreciprocity was observed in all situations, its magnitude varied as a function of metabolic state and/or cell-cycle phase in which the conidia were at the exposure time. The least difference between the effects of the two irradiance levels was observed when nongerminating conidia in suspension were exposed, and the greatest was observed when conidia were exposed during an advanced germination phase. Doses of 6.6 and 17.3 kJ m−2 supplied through the two irradiance levels delayed the germination of the surviving conidia. At both doses, delay was greater during exposure to the higher irradiance. Nonreciprocity was higher for the 17.3 kJ m−2 dose. Nonreciprocity magnitude, in addition to depending on the conidial physiological state, also depended on dose. The results demonstrate the importance of evaluating the impact of the increase in irradiance during the different stages of the fungal life cycle, especially during the stages which are more sensitive to UV, and not simply in dormant conidia

Visible Light During Mycelial Growth and Conidiation of Meterhizium Robertsii Produces Conidia With Increased Stress Tolerance

Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m−2). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions

Visible light during mycelial growth and conidiation of Metarhizium robertsii produces conidia with increased stress tolerance

Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m-2). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions.Brazilian National Council for Scientific and Technological Development (CNPq)[GDE 200382/02-0]Brazilian National Council for Scientific and Technological Development (CNPq)[SWE 2006412005-0]Utah Department of Agriculture and Foo

Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61° N to 54° S

The effects of irradiances of 920 and 1200 mW m−2 (biologically effective weighted irradiance) were examined in 2 Metarhizium album strains, 26 M. anisopliae strains, 1 M. flavoviride strain, and 1 M. taii strain isolated from sites located at latitudes from 61°N to 54°S. Conidia were exposed to UV-B from 1 to 6 h and subsequently examined for relative percentage culturability. Total dosage received at the end of the exposure periods ranged from 3.3 to 19.9 kJ m−2 for the lower irradiance and from 4.3 to 25.9 kJ m−2 for the higher irradiance. Both the irradiance values and the doses are environmentally realistic and can be observed even in temperate regions. The relationships between latitude of origin and UV-B tolerance were compared for the two levels of irradiance for the data from 1 and 2 h exposure. Exposure to both irradiances drastically reduced the relative percentage culturability of all strains. Tolerance to UV-B varied widely among strains and high variation was observed for both irradiances after all periods of exposure. After 1 h of exposure, a difference between the two irradiance levels was detectable, and this difference was magnified at longer irradiations. A significant quadratic relationship of decreasing UV-B tolerance with increasing latitude was observed after exposure of 1 and 2 h. The shape of the relationship did not differ for the two levels of irradiance. Also, we studied the effect of 1200 mW m−2 irradiance on conidial germination time in 1 M. album strain, 7 M. anisopliae strains, and 1 M. taii strain. Exposure to UV-B delayed the germination of surviving conidia of all strains. In general, the delay in germination was directly proportional to the dose

Both Solar UVA and UVB Radiation Impair Conidial Culturability and Delay Germination in the Entomopathogenic Fungus Metarhizium Anisopliae

The entomopathogenic hyphomycete Metarhizium anisopliae has been used in programs of agricultural pest and disease vector control in several countries. Exposure to simulated solar radiation for a few hours can completely inactivate the conidia of the fungus. In the present study we determined the effect of exposures to full-spectrum sunlight and to solar ultraviolet A radiation at 320–400 nm (UVA) on the conidial culturability and germination of three M. anisopliae strains. The exposures were performed in July and August 2000 in Logan, UT. The strains showed wide variation in tolerance when exposed to full-spectrum sunlight as well as to UVA sunlight. Four-hour exposures to full-spectrum sunlight reduced the relative culturability by approximately 30% for strain ARSEF 324 and by 100% for strains ARSEF 23 and 2575. The relative UV sensitivity of the two more sensitive strains was different under solar UV from that under ultraviolet B radiation at 280–320 nm (UVB) in the laboratory. Four-hour exposures to solar UVA reduced the relative culturability by 10% for strain ARSEF 324, 40% for strain ARSEF 23 and 60% for strain ARSEF 2575. Exposures to both full-spectrum sunlight and UVA sunlight delayed the germination of the surviving conidia of all three strains. These results, in addition to confirming the deleterious effects of UVB, clearly demonstrate the negative effects of UVA sunlight on the survival and germination of M. anisopliae conidia under natural conditions. The negative effects of UVA in sunlight also emphasize that the biological spectral weighting functions for this fungus must not neglect the UVA wavelengths

Quantification of Cyclobutane Pyrimidine Dimers Induced by UVB Radiation in Conidia of the Fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m-2. CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m-2) of UVB radiation.State of Sao Paulo Research Foundation (FAPESP)[03/07702-9]Brazilian National Council for Scientific and Technological Development (CNPq)[47.6990/2004-1

Variability in UVB Tolerances of Melanized and Nonmelanized Cells of Cryptococcus neoformans and C-laurentii

Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. Inactivation by UVA and UVB radiation is especially important for the control of fungi that disperse infective units through the air, including fungi such as Cryptococcus spp. that infect their vertebrate hosts by inhalation. Cryptococcus neoformans produces melanin in the presence of certain exogenous substrates such as l-3,4 dihydroxyphenylalanine and melanization may protect the fungus against biotic and abiotic environmental factors. In the present study, we investigated the effect of exposure to an UVB irradiance of 1000 mW m(-2) (biologically effective weighted irradiance) on the survival of melanized and nonmelanized cells of four strains of C. neoformans and four strains of C. laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of cells grown 2, 4, 6 or 8 days on medium with or without L-dopa was determined after exposure to UVB doses of 1.8 and 3.6 kJ m(-2). Both the irradiance spectrum and the intensities of those doses are environmentally realistic, and, in fact, occur routinely during summer months in temperate regions. Differences in tolerance to UVB radiation were observed between the C. neoformans and C. laurentii strains. The C. neoformans strains were more susceptible to UVB radiation than the C. laurentii strains. In C. neoformans, differences in tolerance to radiation were observed during development of both melanized and nonmelanized cells. For most treatments (strain, time of growth and UVB dose), there were virtually no differences in tolerances between melanized and nonmelanized cells, but when differences occurred they were smaller than those previously observed with UVC. In tests with two strains of C. laurentii, there was no difference in tolerance to UVB radiation between melanized and nonmelanized cells during 8 days of culture; and in tests with four strains for less culture time (4 days) there were no significant differences in tolerance between melanized and nonmelanized cells of any strain of this species.The State of Sao Paulo Research Foundation (FAPESP)[03/07702-9]Brazilian National Council for Scientific and Technological Development (CNPq)[47.6990/2004-1

Damage and Recovery From UV-b Exposure in Conidia of the Entomopathogens Verticillium Lecanii and Aphanocladium Album

We evaluated the effects of exposure to doses supplied at an environmentally realistic intensity of UV-B radiation (800 mW m(-2) weighted irradiance) on the culturability and germination of selected strains of the entomopathogenic Hyphomycetes Verticillium lecanii and Aphanocladium album. Increased UV-B exposure decreased relative percent culturability for all strains. Four hours of exposure to UV-B were sufficient to reduce the culturability close to zero. The LT(50) (50% lethal time) ranged from 120 ± 5 min for the V. lecanii strain ARSEF 6430 to 86 ± 14 min for the A. album strain ARSEF 6433. A strong delay in the germination of surviving conidia was observed. To determine the occurrence of photoreactivation in these two genera, we evaluated the effect of exposure to visible light after exposure to UV-B radiation. There was no significant difference in relative culturability between conidia exposed to visible light after UV-B exposure compared to those incubated in the dark after UV-B exposure. This indicates that photoreactivation, if it occurs, must have limited importance in the repair of the damage induced by UV-B radiation in these two genera