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

Erika Nascimento1, Sergio H. da Silva1, Everaldo dos Reis Marques1, Donald W. Roberts2 andGilberto U. L. Braga*1

1Departamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de CienciasFarmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil

2Department of Biology, Utah State University, Logan, UT

Received 31 March 2010, accepted 26 July 2010, DOI: 10.1111/j.1751-1097.2010.00793.x

ABSTRACT

Conidia are responsible for reproduction, dispersal, environmen-

tal 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 frequen-

cies 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.

INTRODUCTION

In contrast to vegetative mycelium, which has high-metabolic

activity, the conidia of most fungi are dormant structures (1–3).Processes such as transcription and protein synthesis do notoccur in Aspergillus and Metarhizium conidia until germina-

tion, which limits their physiological adaptation and responseto environmental changes (4–6). Conidia also differ from thevegetative cells both in their transcriptome and proteome (7–

11). For example, we have established conidial and mycelialproteome reference maps forMetarhizium acridum; and of 1130and 1200 proteins spots detected in ungerminated conidia and

fast-growing mycelia, respectively, only 35% of the proteinspots were common to both developmental stages (9). Also,over-represented proteins in resting conidia (compared with

mycelium) in Aspergillus fumigatus and M. acridum includes

stress-protector proteins such as heat-shock proteins (HSP),proteins involved in reactive oxygen intermediates detoxifica-tion, and in pigment biosynthesis (9,10). The presence of

pre-existing pools of mRNA and proteins in conidia arepresumably required for their tolerance to environmentalstresses and to their ability to respond to an environmental

stimulus by immediately resuming numerous metabolic activ-ities (7–13). Conidial germination is a complex process involv-ing morphological, biochemical and physiological changes that

vary between different species (4–8,11). The sublethal damageto the conidia caused by exposure to stress-generating envi-ronmental agents, such as desiccation, high temperatures andsolar radiation is probably repaired only at the beginning of

germination (14–18).The UV spectrum is conventionally divided into three

wavelength intervals: UVA (400–320 nm), UVB (320–280 nm)

and UVC (280–100 nm). The UV fraction of the solar spectrumthat reaches the Earth’s surface is composed exclusively ofUVAand UVB, since atmospheric ozone drastically reduces the

penetration of radiation with wavelengths of less than 320 nmand excludes wavelengths below 290 nm (19). Adverse effects ofsolar UV irradiation on fungal conidia, including those ofentomopathogenic fungi, such as Metarhizium spp., are well

recognized (14,16,17,20–30). Solar radiation can limit thesurvival and dispersal of important plant and animal patho-genic fungi, and its deleterious effects represent a serious

impediment to the use of entomopathogenic fungi as bioinsec-ticides. Direct exposure to solar radiation for a few hours cankill Metarhizium conidia, which is the fungal stage most

commonly used as field inoculum. Conidia are killed both bysolar UVA and UVB radiations (14). In addition to killingconidia, exposure to sublethal doses of UV radiation can cause

other adverse effects on conidia that reduce the efficacy of fungias a bioinsecticide. Surviving conidia usually have reducedgermination speed and virulence (14,24–28).

Of the fractions of the solar spectrum that reach the Earth’s

surface, UVB is the most harmful to biological systems, as isdemonstrated by the action spectra developed for variousorganisms (31–34). The nature of the DNA damage induced by

UV radiation depends strongly on the wavelength of theincident photons that strike the cell (35,36). The direct UVB

*Corresponding author email: gbraga@fcfrp.usp.br (Gilberto U. L. Braga)� 2010TheAuthors. JournalCompilation.TheAmericanSociety ofPhotobiology 0031-8655/10

Photochemistry and Photobiology, 2010, 86: 1259–1266

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radiation absorption by DNA results mainly in dimerizationbetween adjacent pyrimidine bases. Cyclobutane pyrimidinedimers (CPD) and pyrimidine (6-4)-pyrimidone photoproducts[(6-4) PPs] at a ratio that varies from 4:1 to 10:1 are the main

lesions induced by UVB radiation (36–39). CPD are formedbetween bonds 5 and 6 of two adjacent pyrimidine bases,whereas 6-4PPs are characterized by a stable bond between

positions 6 and 4 of two neighboring pyrimidines. Pyrimidinedimers are also induced by UVA radiation, although lessefficiently and through a mechanism different from that

triggered by UVB (36,40–42).Fungal tolerance to solar radiation is a quantitative and

complex trait that involves both protective mechanisms that

prevent or reduce the occurrence of damage to intracellularcomponents, as well as several systems that repair damagecaused by radiation during cell recovery (27,29,30). Theactions of these mechanisms sometimes partially overlap.

Among the major protective systems are melanin-like pigmentslocated in the cell wall and small noncromogenic cytoplasmaticmetabolites that act as sunscreens (20,27,30,43–45), and

enzymes and metabolites that inactivate reactive oxygenspecies induced by solar UV radiation (46,47). Among theseveral DNA repair mechanisms described in fungi, nucleotide

excision repair and photoreactivation (PR) are important inCPD repair (16,17,29,48).

The deleterious effects of UV radiation on conidial DNAhave been estimated mainly indirectly, i.e. based on analysis of

survival and on kinetics of germination (14,16,21–30). Studiesthat quantify and ⁄ or characterize UV-induced damage inconidial DNA are rare, and none are reported for the four

species used in this study with an environmentally realisticspectral fraction (16,29). Alkaline agarose gel electrophoresis isused to detect single-strand breaks generated in DNA either

directly by UV treatment or as a result of endonuclease V orendonuclease IV cleavage at CPD or apurinic sites (49–51).Treatment of DNA samples with an enzymatic probe such as

T4 Endo V followed by quantitative alkaline gel electropho-resis has been used to quantify UV-induced CPD in animal,plant, fungal and bacteria cells (16,29,31,49–51). Quantifica-tion of UV-induced CPD in conidia will allow the correlation

between damage intensity and other biological processes ofinterest, such as conidial survival, germination, pathogenicityand virulence. Additionally, it will enable the estimation of the

relative importance of the different factors involved in conidialprotection (such as pigments) and repair.

The principal goals of this study were to (1) estimate the

frequency of CPD induced by sublethal doses of UVBradiation in conidial DNA of selected ascomycetes:A. fumigatus, Aspergillus nidulans and M. acridum; (2) examinethe effects of CPD frequencies on germination speed; and (3)

estimate the protective effect of the wild-type green conidialpigmentation on DNA in Metarhizium robertsii.

MATERIALS AND METHODS

Fungal strains. M. acridum ARSEF 324 and M. robertsii ARSEF 23wild-type strains (both with dark green conidia) and the ARSEF 23-derived albino mutant strain DWR 180 (ARSEF 6998) (with whiteconidia) were obtained from the United States Department ofAgriculture (USDA)-Agricultural Research Service (ARS) Collectionof Entomopathogenic Fungal Cultures (ARSEF) (U.S. Plant, Soil andNutrition Laboratory, Ithaca, NY). The mutant DWR 180 was

obtained from M. robertsii ARSEF 23 as previously described (27).A. nidulans wild-type strain ATCC 10074 (dark green conidia) wasobtained from ATCC (American Type Culture Collection, Manassas,VA). A. fumigatus mutant strain CEA 17 (pyrG)) (dark green conidia)was obtained from the wild-type strain CEA 10 (CBS 144-89) aspreviously described (52).

Note: A recent taxonomic revision (53) elevated Metarhiziumanisopliae var. acridum to new-species status, viz., M. acridum; andspecified that strain ARSEF 324 is an M. acridum. M. anisopliae var.anisopliae was split into several species, and strain ARSEF 23 is nowM. robertsii.

Irradiation chamber, lamps and filter. Irradiation experiments wereconducted in a temperature-controlled growth chamber (Nova Etica,Sao Paulo, Brazil) at 28 ± 1�C. The UV irradiance was provided bytwo UVB fluorescent lamps (TL 20 W ⁄ 12 RS; Philips, Eindhoven,Holland), with primarily UVB (peak at 313 nm) and minimal UVAradiation output. The samples (conidial suspensions; see ‘‘Productionof conidia’’ and ‘‘UV exposure’’) were covered with a 0.13 mm thickcellulose diacetate film (JCS Industries, Le Mirada, CA) that blockedradiation below 290 nm. Spectral irradiance was measured at 1 nmincrements with a double-monochromator spectroradiometer (Optron-ic Model 742, Orlando, FL). The spectroradiometer was calibrated forabsolute responsivity against a 1 kW tungsten-halogen standard lamptraceable to the National Institute of Standards and Technology. Thespectral distribution of the filtered lamps is shown in Braga et al. (24).The action spectrum for CPD induction in DNA developed by Quaiteet al. (31) was used to calculate weighted UV irradiance (mW m)2).

Production of conidia. Strains were grown on 23 mL potatodextrose agar medium (Difco Laboratories, Detroit, MI) supple-mented with 1 g L)1 yeast extract (PDAY; Technical; Difco) in petridishes (polystyrene, 100 · 15 mm) in the dark at 28�C for 5 days(A. nidulans and A. fumigatus) or 12 days (Metarhizium). Conidia werecarefully scraped from the agar surface; suspended in 250 mL Tween80 (Sigma-Aldrich Chemie, St. Louis, MO) solution (0.01% vol ⁄ vol);filtered through gauze; centrifuged (20 min at 5000 g), the conidialpellet resuspended in phosphate-buffered saline (PBS; 10 mMM potas-sium phosphate, 150 mMM NaCl, pH 7.4); and adjusted to a concen-tration of 1 · 108 conidia mL)1.

UV exposure. Fifty milliliter aliquots of conidial suspension(108 conidia mL)1) were placed in the bottoms of Petri dishes(150 · 15 mm) on a turn table (TS-2000 A type VDRL; Biomixer,China) and exposed to 1000 mW m)2 UV-Quaite-weighted irradiancefor 15, 30, 60 and 90 min, with shaking at 60 rpm. Total doses were0.9, 1.8, 3.6 and 5.4 kJ m)2, respectively. After exposure, 1 mL of thesuspension was harvested to measure the effects of UV exposures onconidial germination. The remaining 49 mL was centrifuged at 5000 gfor 20 min at 4�C. The conidial pellets were frozen in liquid nitrogenand stored at )80�C until DNA extraction.

Effect of UV radiation on conidial germination. Germination exper-iments were conducted on plates (60 · 15 mm) containing 7 mLPDAY + 0.0015% benomyl (Sigma-Aldrich). This concentration ofbenomyl allowed germination of the three fungal species to bemonitored for extended periods of time because it inhibited post–germination-hyphal growth that otherwise would obscure late-germi-nating conidia (27). Conidia (two 20 lL drops of a 105 conidia mL)1

suspension) were placed on the medium, the plates were incubated inthe dark at 28�C and germination was observed at 400· magnificationafter 6, 12, 24, 36, 48 and 72 h. A total of 300 conidia per treatment ineach trial were evaluated. Conidia that had not germinated by 72 hwere considered dead. Three independent experiments were performed.

Relative percent germination after each period of incubation wascalculated by the equation:

Relative germination (%) ¼ ðWt=WcÞ � 100; ð1Þ

where Wt is the number of germlings at exposure time t, and Wc is thenumber of germlings on the non–UV-exposed control plate.

DNA extraction. Five milliliter of glass beads (average diame-ter = 2 mm) and 5 mL of extraction buffer (200 mMM Tris–HCl, pH8.5, 250 mMM NaCl, 25 mMM EDTA pH 8 and 0.5% SDS) were added to50 mL tubes containing the conidial pellets. The tubes were shakenvigorously for 10 min, the liquid phase was transferred to 15 mL tubesand DNA extracted and purified with phenol-chloroform (54). RNAwas removed with RNAse A (GE Healthcare, Piscataway, NJ)following the enzyme manufacturer’s protocol.

1260 Erika Nascimento et al.

Pyrimidine dimer quantification in conidial DNA. DNA solutionswere incubated with T4 endonuclease V (Epicentre Biotechnology,Madison, WI; 1 U of enzyme per lg of DNA) in a final volume of20 lL at 37�C for 60 min following the enzyme manufacturer’sprotocol. DNAs were then denatured by adding of 10 lL alkalinestop mixture (100 mMM NaOH, 4 mMM EDTA) and incubating for 30 minat 37�C. Five microliter of loading buffer (0.25% of bromphenol blue[Sigma-Aldrich], 0.25% xylene cianol FF [Sigma-Aldrich], 25%glycerol [Synth, Brazil]) were added and samples were electrophoresedthrough 1% alkaline agarose (UltraPure TM Agarose; Invitrogen,Carlsbad, CA) gel in an alkaline buffer (30 mMM NaOH [Sigma], 4 mMM

EDTA [Merck, Germany]) at 1.4 V cm)2 for 3 h. PstI digested k DNA(14.1, 5.08, 2.84, 1.15 kb) was used as molecular-length markers. Afterelectrophoresis, the gels were neutralized in 200 mL of 0.5 MM Tris–HCl,pH 8 for 30 min, stained with ethidium bromide (1 lg mL)1) for10 min, destained for 16 h in 200 mL of ultra pure water andphotographed. Frequencies of CPD were calculated as previouslydescribed (50). Digital images of the gels were analyzed using thesoftware Quantity One 4.6.1 software (Bio-Rad). The midpoint of themass of the DNA was determined by calculating the median migrationdistance of each sample, which was converted into the medianmolecular length (Lmed) of the DNA fragments in that sample relativeto the migration patterns of the molecular length standards. Theaverage molecular length (Ln) of the DNA was determined by theequation of Veatch and Okada (55):

Ln ¼ 0:6� Lmed: ð2Þ

The frequency of CPD was estimated as follows (51,56):

U ¼ ðLntÞ�1 � ðLn0Þ�1; ð3Þ

where Lnt is the average length in kb of the population of DNAmolecules from conidia exposed to UV radiation for t min, and Ln0 isthe average length in kb of DNA from nonexposed control conidia. AsLn is expressed in units of kb, U is in units of CPD ⁄ kb.

Pigmentation impact study. The impact of conidial pigmentation ongermination and CPD induction following UVB exposure wasestimated using the M. robertsii wild-type strain ARSEF 23 and itsderived albino mutant strain DWR 180. Only the intermediate dose of1.8 kJ m)2 was used. This dose was chosen because preliminaryexperiments showed that it was sublethal both for the wild-type andthe albino mutant.

Statistical analysis. The effects of UVB exposure time (15, 30, 60and 90 min) and germination periods (12, 24, 36, 48 and 72 h) wereassessed using a two-way factorial analysis of variance. Data fromeach strain were analyzed separately. No data transformations wererequired for statistical analysis. Significance levels of pairwisecomparisons between treatment means were performed using Student’st-test. P < 0.05 was considered significant. All computations weredone with PROC GLM in SAS ⁄ STAT Version 9.0 (57).

RESULTS

Conidial morphotype

Cell sizes and shapes are among the characteristics that caninfluence UV tolerance and the intensity of UVB-induceddamage. A. fumigatus, A. nidulans,M. acridum andM. robertsii

conidia differ in size and ⁄ or shape.Metarhizium conidia are rod-shaped and A. nidulans and A. fumigatus are spherical. Theaverage length ofM. acridum conidia is 8.8 ± 0.7 lm (n = 30),and the average length of M. robertsii ARSEF 23 and ARSEF

180 are 7.8 ± 1.2 and 8.1 ± 0.9 lm, respectively. The averagediameters of A. nidulans and of A. fumigatus conidia are3.5 ± 0.4 and 1.7 ± 0.4 lm, respectively.

Effect of UV radiation on conidial germination of three fungi

Conidial germination of A. fumigatus, A. nidulans andM. acridum strain ARSEF 324 after exposure to UVB for 0,

15, 30, 60 and 90 min was evaluated after incubation periodsof 6, 12, 24, 36, 48 and 72 h. Most of the doses were sublethalfor all three fungi. Exceptions were doses of 3.6 kJ m)2

(60 min) and 5.4 kJ m)2 (90 min) that killed approximately

7% and 20%, respectively, of the A. fumigatus conidia(P = 0.01 and <0.001, respectively; Fig. 1). In addition todose-related killing of A. fumigatus conidia by exposure to

UVB radiation, germination kinetics of surviving conidia alsowas negatively influenced in that their germination was delayed(interaction between exposure time and germination periods,

P < 0.0001). Delays were directly related to UVB doses. Forexample, relative germination (=germination of the conidiaexposed to UV radiation compared with nonexposed control

conidia) at dosages of 0.9 and 1.8 kJ m)2 were 50% and 29%after 12 h incubation on agar medium, increased to 62% and50% after 24 h and to 91% and 84% after 48 h, respectively.Exposures to UVB also changed the germination kinetics of

A. nidulans and M. acridum conidia (interaction betweenexposure time and germination periods, P < 0.001 for bothstrains); but the delay was much less pronounced for

A. nidulans, and almost no delay was observed for M. acridum.For example, with doses of 0.9 and 5.4 kJ m)2, A. nidulansrelative germination levels were 71% and 26% after 6 h

Figure 1. Conidial germination of (A) Aspergillus fumigatus; (B)Aspergillus nidulans; and (C) Metarhizium acridum after exposure toUVB irradiance of 1000 mW m)2 for 0, 15, 30, 60 and 90 min withevaluation of each after incubation periods of 6, 12, 24, 6, 48 and 72 h.Error bars are standard deviations of three independent experiments.

Photochemistry and Photobiology, 2010, 86 1261

incubation, increased to 99% and 81% after 12 h and to 100%and 95% after 48 h, respectively. For M. acridum at doses of3.6 and 5.4 kJ m)2, relative germination was 84% and 83%after 12 h, and increased to 100% and 98% after 24 h,

respectively.Conidial germination of M. robertsii ARSEF 23 and its

derived albino mutant strain DWR 180 was evaluated after

incubation periods of 12, 24, 36, 48 and 72 h following exposureof a UVB dosage of 1.8 kJ m)2 (30 min; Fig. 2). The dose wassublethal for both strains (P = 0.47 and 0.21 for strains

ARSEF 23 and DWR 180, respectively). Exposures to UVBchanged the germination kinetics in that germination of boththe wild-type and the albino conidia was delayed (interaction

between exposure time and germination periods, P < 0.001 forboth strains). Relative germination levels were 70% and 68%after 12 h incubation, increased to 91% and 80% after 36 h, to85% and 95% after 48 h and to 98% and 96% after 72 h for

wild-type and albino mutant, respectively.

UVB-induced CPD

One effect of UVB radiation on conidial DNA was a dose-

related induction of CPD. Typical results from the densito-metric analyses of conidial DNA from the different treatmentsare shown in Fig. 3. Frequencies of CPD (CPDs ⁄ 10 kb) in

conidial DNA of A. fumigatus, A. nidulans and M. acridumwere estimated after exposure to UVB doses of 0.9, 1.8, 3.6 and5.4 kJ m)2 (Fig. 4). The frequencies of dimers were linear anddirectly proportional to the doses (r2 = 0.961, 0.997 and 0.994

for A. fumigatus, A. nidulans and M. acridum, respectively)but the levels of induced CPD differed among species.CPD induction by the different UVB doses were greater with

Figure 2. Conidial germination of (A) Metarhizium robertsii wild-typestrain ARSEF 23 and (B) its albino mutant DWR 180 followingexposure for 0 or 30 min to UVB irradiance of 1000 mW m)2.Germination was evaluated at incubation periods of 12, 24, 36, 48 and72 h. Error bars are standard deviations of three independentexperiments.

Figure 3. (A) DNA extracted from conidia of Aspergillus nidulanselectrophoresed in 1% alkaline agarose gel. Lanes from left to right:M = molecular length standards (k PstI); 0 = DNA from conidia notexposed to UVB and not treated with T4 endo V before electropho-resis; 0 + T = DNA from conidia not exposed and treated with T4endo V; and 15, 30, 60, 90 = DNAs from conidia exposed to1000 mW m)2 UVB for 15, 30 60 and 90 min and treated with T4 endoV, respectively. (B) Distribution patterns of DNA extracted fromconidia of A. nidulans exposed to different UVB doses and treated withT4 endo V in relation to molecular length standards. (B1) DNA fromnonexposed conidia, (B2) 15 min UVB exposure, (B3) 30 min expo-sure, (B4) 60 min exposure and (B5) 90 min exposure.

1262 Erika Nascimento et al.

A. fumigatus than the other two species, viz.: 0.215, 0.455,0.803 and 1.628 CPD ⁄ 10 kb detected at the doses of 0.9, 1.8,3.6 and 5.4 kJ m)2 in A. fumigates; 0.037, 0.077, 0.142 and

0.202 CPD ⁄ 10 kb in A. nidulans; and 0.041, 0.085, 0.155 and0.255 CPD ⁄ 10 kb in M. acridum. Correlations between CPDfrequencies and relative germination for the three species are

presented in Fig. 5. Relative germination decreased withincrease in CPD frequencies.

Effect of wild-type pigmentation on UVB-induced CPD in

M. robertsii conidial DNA

CPD frequencies in conidial DNA of M. robertsii wild-typestrain ARSEF 23 and in its albino mutant DWR 180 wereestimated after exposure to UVB dose of 1.8 kJ m)2 (30 min;

Fig. 6). CPD generated in the albino mutant were 10-foldgreater than in the green wild-type conidia suggesting that thewild-type pigmentation of ARSEF 23 protects its conidial

DNA against UVB-induced damage. Based on 1.8 kJ m)2

dose, CPD-induction in M. robertsii ARSEF 23 was similar tothat of M. acridum and A. nidulans, and the albino mutant had

a CPD-induction level similar to A. fumigatus.

DISCUSSION

We used the action spectrum for DNA damage developed byQuaite et al. (31) and normalized to unity at 300 nm tocalculate weighted UV irradiance. We selected this biological

spectral weighting function for our fungus studies because Paulet al. (34) compared the most commonly used UV-weightingfunctions in relation to the UV responses of several fungalspecies and concluded that the Quaite action spectrum for

DNA damage most closely approximated the fungal responses.Both the Quaite-weighted UV irradiance (1000 mW m)2) andthe doses (from 0.9 to 5.4 kJ m)2) used in this study are

environmentally realistic for temperate-latitude locations (26).Tolerance to solar radiation varies widely among conidia of

different species. In general, species with larger and pigmented

conidia are more tolerant to solar radiation than species withsmaller and hyaline conidia (20,21,24,29). Variability intolerance to solar radiation is also great among isolates of

the same species, and these variations can present continuousvariation and a normal distribution at the population level(22,24,28). This variability reflects natural adaptation to

Figure 4. Cyclobutane pyrimidine dimer frequencies (CPD ⁄ 10 kb) in conidial DNA of Aspergillus nidulans, Metarhizium acridum and Aspergillusfumigatus after exposure to UVB irradiance of 1000 mW m)2 for 15, 30, 60 and 90 min. Error bars are standard deviations of three independentexperiments.

Figure 5. Relative percent germination for each cyclobutane pyrimi-dine dimer (CPD) frequency of (A) Aspergillus fumigatus, (B)Aspergillus nidulans and (C) Metarhizium acridum. Percent germina-tion after each period was calculated in relation to nonirradiatedcontrols. Error bars are standard deviations of three independentexperiments.

Photochemistry and Photobiology, 2010, 86 1263

different environmental conditions. Metarhizium isolates from

sites where the environmental levels of UV radiation are higherbecause of the lower latitude or the type of vegetation is moretolerant to UVB radiation (24,58).

As previously described forM. acridum and for other fungalspecies, this study has shown that exposure to UVB radiationdelays the germination of surviving conidia. The delaysdiffered between species. The three species of ascomycetes

also showed different yields of induced CDP lesions. Thefrequency of UVB-induced damage observed ranged from 0.04(dose of 0.9 kJ m)2 in A. nidulans) to 1.62 CPD ⁄ 10 kb (dose of

5.4 kJ m)2 in A. fumigatus). It is important to point out thatmost of the UVB doses used were sublethal to conidia of thefungi examined. Chelico et al. (16) observed frequencies up to

28 CPD ⁄ 10 kb in conidial DNA of the entomopathogenicfungus Beauveria bassiana after UVC exposures (dose of480 J m)2). This UVC dose killed approximately 100% of theconidia.

In this study, the frequency of UVB-induced CPD inA. nidulans DNA was similar to that in M. acridum; and bothwere lower than that in A. fumigatus. The small size of

A. fumigatus conidia (one-eighth of the volume of A. nidulansconidia) and consequently the low cytoplasmatic absorption ofUVB-radiation before reaching the A. fumigatus nucleus may

be one factor responsible for the higher frequency of CPD. Inaddition to shape and size of the conidia, other cellular factorsalso influence UVB-induced CPD frequencies and UVB

tolerance (27,29,30). We estimated the effects of UVB ondormant conidia of the three species on the basis of CPDquantification, conidial survival analysis and evaluation of thegermination kinetics of the surviving conidia. To quantify

CPD, DNA was extracted from dormant conidia immediatelyafter UVB exposures. Differences between species in CPDfrequencies are because of both differences in conidial

morphotypes and in mechanisms, such as UV-absorbingcompounds that reduce UV penetration into the cell. UVBexposure effects evaluated on conidial survival or germination

kinetics suggested that other mechanisms involved in repair ofDNA can act during cell recovery and may account forconidial tolerance to solar radiation (29). Both the presence

and the relative importance of the different DNA repairmechanisms vary between fungal species (29,48). Conditions inwhich conidia are maintained after UV exposure (i.e. temper-

ature, lighting, culture media) also affect their recovery (29).For example, in this study, conidia were maintained in thedark after exposure, thus avoiding the action of light-depen-dent DNA repair systems, such as PR. The deleterious effects

of UVB radiation are not restricted to DNA; proteins andlipids are also damaged (59). HSP (mostly chaperones)are important in protein protection and repair and are

differentially expressed and ⁄ or are over-represented in conidialproteomes (9–13,59). Quantitative and qualitative variationsin proteins stored in conidia also can affect stress tolerance

and may explain, at least partially, differences betweenspecies (10–13). Unlike other dormant cells, such as bacterialspores whose photochemistry and photobiology are well

known (60–62), these topics are still largely unknown indormant conidia. To date, only the dark green pigments inMetarhizium and Aspergillus conidia have been implicatedin protection against solar UVB and UVA in dormant conidia

(20,27,30)All the species used in this study produce greenish conidia.

The olive-green coloration of melanized fungi arises when

melanin is complexed with proteins and other compounds (63).Aspergillus fumigatus conidia are known to produce greenishpigments by using the dihydroxynaphthalene (DHN)-melanin

pathway (64). The chemical identity and the synthesis pathwayof the greenish pigment in Metarhizium conidia are stillunknown. The DHN-melanin synthesis pathway is probablyabsent in M. anisopliae, since the fungus lacks scytalone

dehydratase activity (a central enzyme in this pathway) (30).Also, tricyclazole, kojic acid and glufosinate ammonium donot inhibit conidial color formation, suggesting that DHN-

melanin, DOPA-melanin and carotenoid pathways are notcontributing to pigmentation in M. anisopliae (65). Likewise,M. anisopliae did not generate pyomelanin on medium-

containing LL-tyrosine with or without sulcotrione, indicatingthat it probably does not use the pyomelanin-synthesispathway (65). We have not detected carotenoids and xantho-

phylls in Metarhizium conidia (45). Fang et al. (65) reported aclass 1 laccase (MLAC1) involved in conidial pigmentation inM. anisopliae. Information about the presence and the impor-tance of noncromogenic small metabolites to Metarhizium

conidial tolerance to solar radiation is very limited. Recently,we isolated a novel UV-absorbing metabolite named tyrosinebetaine that accumulates exclusively in Metarhizium conidia

(45). We have demonstrated the importance of pigmentationof Metarhizium conidia, especially M. robertsii ARSEF 23, totolerance against solar-simulated radiation (27,30). Mutants of

this strain with white conidia were more sensitive to simulatedUV radiation than purple mutants, which were more sensitivethan yellow ones, which in turn were more sensitive than thegreen wild-type strain ARSEF 23 (27). A yellow and purple

mutant of ARSEF 23 that were very sensitive to UVBradiation were reverted to green conidia, after which theyboth exhibited wild-type ARSEF 23 UVB tolerance; again

signifying green pigmentation is a UVB-protection system inthisM. robertsii strain (27). Conidia of the white mutant DWR180 (the mutant of ARSEF 23 used in the present study) had a

10-fold lower survival rate than the wild strain after exposureto UV dose of 6.5 kJ m)2 (Quaite-weighted dose) (27). In thisstudy, we observed that the UVB-induced damage to the dose

of 1.8 kJ m)2 was approximately 10-fold higher in DWR 180than in the wild strain, explaining, at least in part, the lower

Figure 6. Cyclobutane pyrimidine dimer frequencies (CPD ⁄ 10 kb) inconidial DNA of Metarhizium anisopliae var. anisopliae wild-typestrain ARSEF 23 and in its albino mutant DWR 180 after exposure for0 or 30 min to UVB irradiance of 1000 mW m)2. Error bars arestandard deviations of three independent experiments.

1264 Erika Nascimento et al.

tolerance to solar radiation of the mutant. As far as we know,this is the first direct evidence showing that conidial pigmen-tation protects DNA against UVB-induced damage. Despitethe difference between the DNA damage intensities, no

proportional difference was observed between the germinationdelays of the wild-type and albino mutant. This discrepancymay be explained by both the multifactorial nature of the

tolerance, which does not depend only on the intensity of theDNA lesions, and the UVB dose (which was chosen because itwas sublethal for both strains).

As far as we know, no attempt has been made tocharacterize or quantify fungal photoproducts other thanCPD induced by UV radiation in conidial DNA (16,17). Thus,

knowledge about conidial DNA photochemistry is still largelyunknown, and additional studies are needed to identify andquantify other types of DNA lesions. Conidia are veryspecialized cells with different structural, physiological and

biochemical characteristics compared with metabolically activecells (1). These differences may affect conidial DNA photo-chemistry. Other specialized structures involved in tolerance,

such as bacterial spores, have marked differences in their DNAphotochemistry compared with vegetative cells (49,60–62). Inaddition, the deleterious effects of UVA radiation and visible

light on conidia also need to be better understood. We havenoted previously that both UVA and visible light at ecolog-ically relevant intensities inactivated conidia of not onlyMetarhizium spp. but also other entomopathogenic fungal

species, such as Verticillium lecanii (now: Simplicillium lanos-oniveum) and Aphanocladium album (now: Lecanicilliumaphanocladii) (26).

Acknowledgements—This work was supported by grant 03 ⁄ 07702-9from The State of Sao Paulo Research Foundation (FAPESP) and

grant 47.6990 ⁄ 2004-1 from The Brazilian National Council for

Scientific and Technological Development (CNPq). We sincerely

thank FAPESP for a PhD fellowship to E.N.

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