Gilberto U.L. Braga

Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation

Abstract The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek’s medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects.

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.

Molecular and physiological effects of environmental UV radiation on fungal conidia

Conidia are specialized structures produced at the end of the asexual life cycle of most filamentous fungi. They are responsible for fungal dispersal and environmental persistence. In pathogenic species, they are also involved in host recognition and infection. Conidial production, survival, dispersal, germination, pathogenicity and virulence can be strongly influenced by exposure to solar radiation, although its effects are diverse and often species dependent. UV radiation is the most harmful and mutagenic waveband of the solar spectrum. Direct exposure to solar radiation for a few hours can kill conidia of most fungal species. Conidia are killed both by solar UV-A and UV-B radiation. In addition to killing conidia, which limits the size of the fungal population and its dispersion, exposures to sublethal doses of UV radiation can reduce conidial germination speed and virulence. The focus of this review is to provide an overview of the effects of solar radiation on conidia and on the major systems involved in protection from and repair of damage induced by solar UV radiation. The efforts that have been made to obtain strains of fungi of interest such as entomopathogens more tolerant to solar radiation will also be reviewed.

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.

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.

Susceptibilities of the dermatophytes Trichophyton mentagrophytes and T. rubrum microconidia to photodynamic antimicrobial chemotherapy with novel phenothiazinium photosensitizers and red light

Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative to conventional chemotherapy that can be used to treat localized mycosis. The development of PACT depends on identifying effective and selective PS for the different pathogenic species. The in vitro susceptibilities of Trichophyton mentagrophytes and Trichophyton rubrum microconidia to PACT with methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer S137 were investigated. The efficacy of each PS was determined based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of PACT with NMBN and S137 on the survival of the microconidia of both species. S137 showed the lowest MIC. MIC for S137 was 2.5 μM both for T. mentagrophytes and T. rubrum, when a light dose of 5 J cm(-2) was used. PACT with NMBN (10 μM and 20 J cm(-2)) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. PACT with S137 at 1 μM and 20 J cm(-2) resulted in a reduction of approximately 3 logs in the survival of both species. When a S137 concentration of 10 μM was used, no survivor was observed for both species at all light doses (5, 10 and 20 J cm(-2)).

In vitro photodynamic inactivation of Candida species and mouse fibroblasts with phenothiazinium photosensitisers and red light

In the present study, the in vitro susceptibilities of five Candida spp. to photodynamic antimicrobial chemotherapy (PACT) with four phenothiazinium derivatives, methylene blue (MB), new methylene blue N (NMBN), toluidine blue O (TBO) and the novel pentacyclic phenothiazinium photosensitiser S137, in combination with red light were investigated. The efficacy of each PS was determined, initially, based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of the photodynamic treatment with NMBN and S137 on Candida survival and on the mouse fibroblast cell line L929. MICs varied both among PS and species and decreased with light dose increase. For most treatments (species and fluences) NMBN and S137 showed the lowest MICs. MICs for NMBN and S137 were <2.5 μM for all the Candida species when a fluence of 25 J cm⁻² was used. PACT with NMBN (fluence of 15 J cm⁻²) resulted in reductions in survival from 0.3 log (Candida krusei) to 3 logs (C. parapsilosis). PACT with S137 was more effective than with NMBN. Fluence of 15 J cm⁻² resulted in reductions in survival from 1 log (C. krusei) to 3 logs (C. parapsilosis) and fluence of 25 J cm⁻² resulted in a reduction of approximately 2 logs (C. krusei) and between 3 and 4 logs in survival of the other 4 species of Candida. In vitro relative toxicities of the phenothiazinium PS to mammalian cells exhibited a similar trend to the antifungal data, i.e. greater toxicity and phototoxicity with NMBN and S137 compared to the established PS.

Tolerance of entomopathogenic fungi to ultraviolet radiation: a review on screening of strains and their formulation

Ultraviolet radiation from sunlight is probably the most detrimental environmental factor affecting the viability of entomopathogenic fungi applied to solar-exposed sites (e.g., leaves) for pest control. Most entomopathogenic fungi are sensitive to UV radiation, but there is great inter- and intraspecies variability in susceptibility to UV. This variability may reflect natural adaptations of isolates to their different environmental conditions. Selecting strains with outstanding natural tolerance to UV is considered as an important step to identify promising biological control agents. However, reports on tolerance among the isolates used to date must be analyzed carefully due to considerable variations in the methods used to garner the data. The current review presents tables listing many studies in which different methods were applied to check natural and enhanced tolerance to UV stress of numerous entomopathogenic fungi, including several well-known isolates of these fungi. The assessment of UV tolerance is usually conducted with conidia using dose-response methods, wherein the UV dose is calculated simply by multiplying the total irradiance by the period (time) of exposure. Although irradiation from lamps seldom presents an environmentally realistic spectral distribution, laboratory tests circumvent the uncontrollable circumstances associated with field assays. Most attempts to increase field persistence of microbial agents have included formulating conidia with UV protectants; however, in many cases, field efficacy of formulated fungi is still not fully adequate for dependable pest control.

Furocoumarins and coumarins photoinactivate Colletotrichum acutatum and Aspergillus nidulans fungi under solar radiation

The increasing tolerance to currently-used fungicides is a major problem both in clinical and agricultural areas leading to an urgent need for the development of novel antifungal strategies. This study investigated the in vitro antimicrobial photo treatment (APT) of conidia of the plant-pathogenic fungus Colletotrichum acutatum and the ascomycete Aspergillus nidulans with the furocoumarins 8-methoxypsoralen (8-MOP) and isopimpinellin, and a mixture of two coumarins (7-methoxy coumarin and citropten). Subcellular localization of the photosensitizer 8-MOP was also determined in C. acutatum conidia. Additionally, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP (50μM) led to a reduction of approximately 4 logs in the survival of the conidia of both species, and the mixture of the two coumarins (12.5mgL(-1)) resulted in a reduction of approximately 4 logs for A. nidulans and 3 logs for C. acutatum. Isopimpinellin (50μM) displayed a reduction of 4 logs for A. nidulans but less than 2 logs for C. acutatum. Washing the conidia to remove unbound photosensitizers before light exposure reduced the photodynamic inactivation of C. acutatum both with 8-MOP and the mixture of the two coumarins. The reduction was smaller for A. nidulans. 8-MOP spread throughout the cytoplasm and accumulated in structures such as lipid bodies of C. acutatum conidia. No damage to orange tree leaves was observed after APT with any of the photosensitizers.

In vitro photodynamic inactivation of plant-pathogenic fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium photosensitizers.

ABSTRACT The increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum, Colletotrichum gloeosporioides, and Aspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS in C. acutatum conidia was determined. The effects of photodynamic treatments on leaves of the plant host Citrus sinensis were also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 μM for the three fungal species when a fluence of 25 J cm−2 was used. APDT with NMBN (50 μM) and S137 (10 μM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm−2. Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, of C. acutatum conidia. No damage to orange tree leaves was observed after APDT.