Influence of light on growth, fumonisin biosynthesis and FUM1 gene expression by Fusarium proliferatum

International Journal of Food Microbiology 153 (2012) 148–153

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Influence of light on growth, fumonisin biosynthesis and FUM1 gene expression by Fusarium proliferatum Francesca Fanelli a, Markus Schmidt-Heydt b, Miriam Haidukowski a, Rolf Geisen b, Antonio Logrieco a, Giuseppina Mulè a,⁎ a b

Institute of Sciences of Food Production, CNR, via Amendola 122/0, 70126 Bari, Italy Max Rubner Institut, Department for Safety and Quality of Fruit and Vegetables, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany

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Article history: Received 5 July 2011 Received in revised form 26 September 2011 Accepted 23 October 2011 Available online 15 November 2011 Keywords: Fumonisin Fusarium proliferatum Light FUM1 Real time RT-PCR

a b s t r a c t Fumonisins are a group of mycotoxins, mainly found in maize and maize-based food and feed, associated with several diseases in animals. The impact of these toxins on the economy and health worldwide has driven several efforts to clarify the role of environmental factors that can influence fumonisin biosynthesis by the toxigenic species. We analyzed the influence of light of varying wavelength on growth and fumonisin biosynthesis by the fungus Fusarium proliferatum ITEM 1719. Light in general had a positive influence on growth, with a mean increase of the grow rate of about 40% under light exposure in comparison to the dark incubation. Wavelengths from both sides of the spectrum, from long (627 nm) to short wavelength (470–455 nm) had a stimulating effect on fumonisin biosynthesis compared to the dark incubation: fumonisins B1 (FB1) and B2 (FB2) production increased of about 40 fold under red, 35 fold under blue, 20 fold under royal blue, 10 fold under green, 5 fold under yellow and 3 fold under white light in comparison to the dark incubation. The transcriptional regulation of the FUM1 fumonisin biosynthesis gene was analyzed by Real time reverse transcriptase PCR quantification, revealing a correlation between fumonisin biosynthesis and gene expression. These findings show a role of light on the growth and the modulation of fumonisin biosynthesis and provide new information on the physiology of an important toxigenic maize pathogen. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Fusarium proliferatum belongs to the Liseola section of the Fusarium genus (Nelson et al., 1983), and its teleomorph, Gibberella intermedia, belongs to the G. fujikuroi complex, composed of at least 13 reproductively isolated biological species (mating populations) (Leslie and Summerell, 2006; Scauflaire et al., 2011; Van Hove et al., 2011). Fusarium proliferatum is a causal agent of diseases of various economically important plants such as maize, banana and other hosts including pine trees, asparagus, wheat and barley. The distribution on maize of this species is particularly significant in Southern Europe (Logrieco et al., 2002; Jurado et al., 2004) though it has been recorded also in Nepal (Desjardins et al., 2000) and Mexico (De Souza and Formento, 2004). Fusarium proliferatum produces different mycotoxins, including moniliformin (Marasas et al., 1984), beauvericin (Logrieco et al., 1998), fusaric acid (Bacon et al., 1996), fusaroproliferin (Ritieni et al., 1995) and fumonisins (Ross et al., 1990). Fumonisins are a group of mycotoxins associated with several mycotoxicoses, including equine leukoencephalomalacia, porcine pulmonary edema and experimental kidney and liver cancer in rats ⁎ Corresponding author. Tel.: + 39 080 5929329; fax: + 39 080 5929374. E-mail address: [email protected] (G. Mulè). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.10.031

(Howard et al., 2001). They are divided into four groups: A, B, C and G, with the B-type fumonisins being the most toxic. Generally, FB1 makes up approximately 70%, and FB2 and FB3 each make up about 10–20% of the total fumonisin content (Nelson et al., 1993). The fumonisin biosynthetic gene cluster (FUM) has been identified in several fumonisin producers (Brown et al., 2007; Pel et al., 2007; Proctor et al., 1999, 2008). A comparative genomic approach was used in F. proliferatum (Waalwijk et al., 2004) to determine the presence of the FUM cluster, revealing the same order and orientation of genes described for F. verticillioides and F. oxysporum (Proctor et al., 2003, 2008). The cluster gene FUM1 encodes a polyketide synthase which would catalyze synthesis of the linear polyketide of fumonisins. The encoded FUM1 (previously FUM5) polyketide synthase sequence of F. proliferatum shows 85% identity with F. verticillioides (Waalwijk et al., 2004). The low level of identity (77–89% at amino-acid level) of FUM genes and the different genomic locations of the cluster in F. verticillioides and F. proliferatum indicate that each species may have acquired the cluster independently (Waalwijk et al., 2004). In F. proliferatum many environmental and abiotic factors, such as temperature, water activity and solute potential, have been found to affect fumonisin biosynthesis (Kohut et al., 2009; Samapundo et al., 2005; Marín et al., 2010), even though a high variability in phenotypic fumonisin biosynthesis has been reported.

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Light is a very important signal for fungi since it influences many different physiological responses such as pigmentation, sexual development, asexual conidiation, circadian clock and secondary metabolism. The increasing knowledge about fungal genomes has revealed the occurrence of numerous genes encoding proteins involved in light detection and has directed scientific efforts toward the discovery of the mechanisms by which the fungus activates physiological and morphological responses (Corrochano, 2007; Herrera-Estrella and Horwitz, 2007). The molecular background of light perception and its influence on physiological functions, such as sexual and asexual development or secondary metabolism, has also been elucidated (Bayram et al., 2010; Atoui et al., 2010). Though the response to light has been analyzed in many fungal species, these studies were limited to show the effect that constant or circadian illumination by white light could have on fungal growth and metabolism. Only recently the effect of light wavelength and light intensity on secondary metabolism of toxin producing filamentous fungi has been studied systematically (Schmidt-Heydt et al., 2011). During that analysis it has been shown that light in general, and red and blue light in particular, has inhibiting properties on growth and toxin biosynthesis especially of ochratoxin A producing fungal species. In this study we analyzed the influence of light of varying wavelength on growth, fumonisin biosynthesis and FUM1 gene expression by the toxigenic fungus F. proliferatum ITEM 1719. 2. Material and methods 2.1. Fungal strains and growth conditions Fusarium proliferatum ITEM 1719 (ITEM: Agri-Food Toxigenic Fungi Culture Collection of the Institute of Sciences of Food Production, CNR, Bari, Italy, http://www.ispa.cnr.it/Collection) was isolated from Zea mays ear rot from Italy (Sardinia, Sassari) and was reported to produce FB1, moniliformin and beauvericin (Logrieco et al., 1995). The strain was grown for 5 days at 25 °C on yeast extract sucrose agar medium (YES: 20 g/L yeast extract, 150 g/L sucrose, 15 g/L agar). Then the conidia were harvested and a conidial suspension was prepared in sterilized distilled water; the conidia were counted in a Thoma chamber and the suspension was diluted to a final concentration of 106 conidia/mL (Oakley, 1999). An amount of 100 μL of the conidia suspension was single point inoculated on YES plates (20 mL for each plate) and used for the growth assessment and for fumonisin and molecular analysis. 2.2. The light incubation equipment The light box was constructed to enable the incubation of the cultures under different wavelengths of light (Schmidt-Heydt et al., 2011). The box was subdivided into 6 chambers; each chamber was equipped with 5 Luxeon high power 5 W Light Emitting Diodes (Philips Lumileds Lighting Company, San Jose California, USA) one placed in the middle of the top side of the box and the other 4 at right angles in respect to the central Led. The chamber had the following different emitting wavelengths: chamber 1, royal blue (455 nm, 3350 lx); chamber 2, blue (470 nm, 2357 lx); chamber 3, green (530 nm, 7250 lx); chamber 4, yellow (590 nm, 6400 lx); chamber 5, red (627 nm, 7700 lx); and chamber 6, white light (17750 lx). The distance between the LEDs and the agar plates was 18 cm. The plates were positioned directly under the central LED and at 45° degrees with respect to the other LEDs in order to guarantee the highest intensity and homogeneity of the irradiation. Each LED exhibit a typical Radiation Pattern (Lambertian). No heating effect by the LEDs could be detected at this distance. An irradiance of 38 mW/cm 2 was achieved under these conditions. A dark chamber was used as a control. The boxes had sufficient capacity for air exchange and were placed in

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a temperature and humidity controlled room. Inoculated agar plates were placed into the boxes and incubated at a constant temperature of 20 °C and a relative humidity of 85% for different time periods. 2.3. Growth assessment For analyzing growth and morphology, triplicate YES agar plates, prepared as described in 2.1, were inoculated and incubated for 5 days at 20 °C under the respective light conditions and photographed. The diameters of the colonies were measured in two directions at right angles to each other. Each experiment was performed in triplicate. 2.4. Fumonisin extraction and HPLC analysis For determination of mycotoxin production YES agar plates, prepared as described in 2.1, were grown for 10 days at 20 °C at the respective light conditions (see Section 2.2). FB1 and FB2 were analyzed according to Frisvad et al. (2007) with slight modifications (De Girolamo et al., 2010). Six agar plugs (D= 6 mm; 1 g) were taken from the region between center, corresponding to the inoculum point, and in a radius towards the edge of the colony, with the aid of a sterile corer (Frisvad et al., 2007). One gram of agar was extracted with 1 mL of a solution methanol/water (70:30, v/v) (HPLC-grade, Mallinckrodt Baker, Milan, Italy. Ultrapure water was produced by a Millipore Milli-Q system Millipore, Bedford, MA, USA) for 30 min at room temperature on a rotary shaker; the agar plugs were discarded and the extract was evaporated to dryness in a vacuum concentrator (Speed Vac, Savant Instruments, Farmingdale, USA). The residues were dissolved in 1 mL of acetonitrile/water (30:70, v/v) by shaking for 120 min (Thermo shaker TS100, Biosan) and then filtrated using RC 0.2 μm filters (Phenomenex, U.S.A.). Decimal dilutions of samples were made in the same solvent; 110 μL of the extract were derivatized with 110 μL of o-phtaldialdehyde (OPA) (Sigma-Aldrich, Milan, Italy) mixed for 30 s. Using the HPLC autosampler (Varian Inc., Palo Alto, CA, USA) 50 μL were injected by full loop at 3 min after adding the OPA reagent for fumonisin analysis. The analytical column was a SymmetryShield RP18 15 cm × 4.6 mm, 5 μm (Waters, Milford, MA, USA) with a guard column inlet filter (0.5 μm × 3 mm diameter, Rheodyne Inc. CA, USA) and the mobile phase consisted of a binary gradient was applied as follows: the initial composition of the mobile phase 60% of (A) acetonitrile–water–acetic acid (30/69/1, v/v/v) / 40% of (B) acetonitrile–water–acetic acid (60/39/1, v/v/v) was kept constant for 5 min, then B solvent was linearly increased to 88% in 21 min and kept constant for 4 min. Finally, to clean the column, the amount of acetonitrile was increased to 100% and kept constant for 4 min. The column was thermostatized to 30 °C. The flow rate of the mobile phase was 1.0 mL/min. The fluorometric detector was set at wavelengths ex = 335 nm, em = 440 nm. FB1 and FB2 were quantified by measuring peak areas and comparing them with a calibration curve obtained with standard solutions. The detection limit for fumonisin was 0.05 μg/g based on a signal-to-noise ratio of 3:1. Each experiment was performed in triplicate. 2.5. RNA isolation and reverse transcription For RNA isolation YES agar plates, prepared as described in Section 2.1, were grown for 10 days at 20 °C at the respective light conditions (see Section 2.2). Fungal total RNA was isolated using the RNeasy Plant Mini Kit for purification of total RNA from plants and fungi (Qiagen; Hilden, Germany), according to the manufacturer's instructions, and stored at − 80 °C. DNAse I treatment to remove genomic DNA contamination from the samples was performed using the RNase-free DNase I (Qiagen), following the manufacturer's instructions. First strand cDNA was synthesized using the RT Omniscript Reverse Transcription Kit (Qiagen). Each 20 μl reaction contained 1 μg

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of total RNA, 2 μl of oligo d(T)16 (10 μM), 2 μl of 10 × RT-PCR buffer, 2 μl of dNTPs (10 mM), 1 μl (4 U) of RNAse inhibitor, 1 μl (4 U) of Omniscript Reverse Transcriptase and RNase free water to the final volume. Synthesis of cDNA was performed at 37 °C for 1 h. 2.6. Real-time RT-PCR Real-time RT-PCR assays were used to quantify FUM1 and βtubulin (tub2) expression in the F. proliferatum strain using the primer pairs FUM1P2-F (5′-CCCCCATCATCCCGAGTAT-3′) and FUM1P2-R (5′TGGGTCCGATAGTGATTTGTCA-3′) for FUM1 transcript and PQTUB-F2 (5′-ACATCCAGACAGCCCTTTGTG-3′) and PQTUB-R2 (5′-AGTTTCCGATGAAGGTCGAAGA-3′) for tub2 transcript (Jurado et al., 2010). Real time PCR reactions were performed using iQ5 Real-Time PCR Detection System (BIO-RAD Laboratories, Hercules, CA). The PCR thermal cycling conditions were as follows: an initial step at 52 °C for 2 min, 95 °C for 10 min, and 40 cycles at 95 °C for 20 s (denaturalization), at 50 °C for 40 s (annealing) and at 72 °C for 1 min (extension). The iQ™ SYBR® Green Supermix Mix (BIORAD) was used as reaction mixture, adding 0.5 μl of sterile water, 5 μl of each primer (5 μM) and 2 μl of template cDNA (dilution 1:20), in a final volume of 25 μl. Each sample was amplified tree times in each experiment. The results were normalized using the tub2 amplifications run on the same plate. Each experiment was performed in triplicate. 2.7. Statistical analysis One-way analysis of variance (ANOVA) test was used to determine if different light exposure has a significant effect on the growth, the FB production or FUM1 gene expression. For each light condition the statistical significance was calculated with reference to the dark incubation. The test was performed by the Statistical Package for Social Science 16.0 (SPSS, IBM). The statistical significance was expressed at P b 0.05 or at P b 0.01.

3. Results 3.1. Growth assessment As shown in Fig. 1a, only slight changes in the morphology of the colonies occurred. Wavelength from both sides of the spectrum, from red (long wavelength, 627 nm) to blue (short wavelength, 470–455 nm), had a slight but significant (at P > 0.01) stimulating effect on the growth comparing to the dark incubation (Fig. 1b). This stimulating effect took place only after a lag phase of about 2 days, during which the growth of all cultures showed the same growth rate. The incubation under red, blue and royal blue light induced an intense orange color in the mycelium. 3.2. Fumonisin biosynthesis FB1 and FB2 were extracted from colonies grown on YES agar medium for 10 days under different light conditions. The results of the HPLC quantification are reported in Table 1. All light conditions promoted fumonisin biosynthesis. As shown in Fig. 2a, red and blue light had the most stimulating effect on fumonisin biosynthesis, with an increase respectively of 40 and 33 fold of FB1 and of 14 and 11 fold of FB2 in comparison to the dark incubation; the effect of green and royal blue light on fumonisin biosynthesis was less strong and led to an increase of about 16 and 19 fold of FB1 and about 5 and 8 fold of FB2. The effect of incubation under white and yellow light increased the FB1 production about 4 and 3 fold respectively, and the FB2 production about 3 and 2 fold, in comparison to the dark incubation. 3.3. Expression analysis The results of the real-time RT-PCR analysis of FUM1 gene expression of the F. proliferatum ITEM 1719 in 10 day-old cultures incubated in the different wavelength light boxes are shown in Fig. 2b. The dark

Fig. 1. Growth assessment. Morphology (a) and growth (b) of F. proliferatum ITEM 1719 after incubation under different light conditions on YES agar medium for 5 days at 20 °C: D, dark; W, white; R, red; G, green; Y, yellow; B, blue; RB, royal blue. Error bars represent the standard error measured between independent replicates.

F. Fanelli et al. / International Journal of Food Microbiology 153 (2012) 148–153 Table 1 HPLC quantification of FB1 and FB2 biosynthesis of F. proliferatum ITEM 1719 after incubation under different light conditions on YES agar medium for 10 days at 20 °C. Growth condition

FB1 (μg/g)

FB2 (μg/g)

Dark White Red Green Yellow Blue Royal blue

11 ± 1.3 48 ± 4.4 446 ± 52.1 179 ± 1.2 34 ± 13.1 368 ± 71.1 215 ± 4.4

2 ± 0.4 6 ± 0.6 29 ± 1.1 10 ± 0.2 5 ± 0.9 23 ± 0.6 17 ± 3.5

± indicates the dispersion measurements of independent replicates.

incubation was used as calibrator. The expression of the FUM1 gene was clearly activated under light exposure. The highest level of expression was revealed under green, red and blue light incubation, with an induction of 9, 7 and 6 fold respectively, which roughly fits with the phenotypic production under these conditions. The Pearson's correlation coefficient measured between fumonisin production and gene expression was 0.54. 4. Discussion Light is one of the abiotic factors that have an influence on secondary metabolite production. It has been demonstrated that the biosynthesis of Alternaria toxins can be reduced under blue light and that light pulses can reduce polyketide biosynthesis (Häggblom and Unestam, 1979; Häggblom and Niehaus, 1986). The incubation under continuous light and continuous darkness can affect the aflatoxin and anthraquinone biosynthesis by Aspergillus parasiticus (Bennett et al., 1981). Recently

Fig. 2. Fumonisins production and expression analysis. FB1 and FB2 production (a) and level of expression of FUM1 gene (b) of F. proliferatum ITEM 1719 after incubation under different light conditions on YES agar medium for 10 days at 20 °C: D, dark; W, white; R, red; G, green; Y, yellow; B, blue; RB, royal blue (* significant at P b 0.05, ** significant at P b 0.01). Error bars represent the standard error measured between independent replicates.

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Schmidt-Heydt et al. (2011) have reported that light has an influence on growth, morphology and ochratoxin A biosynthesis in Aspergillus and Penicillium. Depending on light wavelength and intensity a complete cessation of growth and biosynthesis of ochratoxin A, with respect to the species treated, was observed. Red and blue light were particularly inhibitory. The biosynthesis of citrinin by P. verrucosum was also influenced by light, but opposite to ochratoxin, blue light led to an increased biosynthesis of citrinin in P. verrucosum and in P. expansum, a typical citrinin producing species. Light sensing has been thoroughly studied in Neurospora crassa, where both the perception and the response to blue light have been characterized. These responses required the white collar gene products WC-1 and the WC-2 (Ballario et al., 1996; Ballario and Macino, 1997) that interact and form the White Collar Complex (WCC), that upon light exposure binds transiently to the promoters of light inducible genes to activate their transcription. Purschwitz et al. (2008) demonstrated that A. nidulans is able to sense red and blue light by receptors FphA (red light receptor) and lreA/B (blue light receptors, homologues of wc-1 and wc-2). The ability to detect light is ascribable to the light regulatory protein VeA; it is encoded by the velvet gene, is involved in sexual development and acts as a key regulator of the biosynthesis of many secondary metabolites. In concert with VelB and LeaA, VeA forms the velvet complex (Bayram et al., 2008): while under light exposure VeA is mostly retained in the cytoplasm, in the dark VeA is functionally active and is imported into the nucleus, where the complex can activate the sexual development and the secondary metabolite cluster expression (Bayram et al., 2010). Not too much is known about the influence of light on the physiology of Fusaria. Estrada and Avalos (2008) showed that a homologue of the white collar protein is not responsible for photocarotenogenesis, but for regulation of conidiation and secondary metabolism in F. fujikuroi. So far, to our knowledge, no study has been carried out on the influence of light on F. proliferatum. This is the first work in which the effect of light on growth and secondary metabolism has been studied in this fungal species. A similar analysis has been recently conducted on F. verticillioides (Fanelli et al., in press), indicating also in this species a clear response that varies under different light incubation. In F. proliferatum ITEM 1719 light exposure promoted growth and fumonisin biosynthesis. The most effective light wavelengths in promoting FB production were red and blue light. The induction of FB biosynthesis under these conditions appeared to be regulated at the transcriptional level: the FUM1 gene expression indeed followed the FB biosynthesis profile, being higher under red and blue light in comparison to the dark incubation. Supporting this hypothesis of a transcriptional regulation, Jurado et al. (2010) have reported a significant correlation between the FUM1 transcript and fumonisin content. Nevertheless, since the knowledge of the F. proliferatum FUM cluster is still incomplete, further studies will be needed in order to understand the molecular pathway of the biosynthesis of fumonisins and their regulation in this species. Exactly the conditions which are inhibiting for ochratoxin A biosynthesis in Aspergilli and Penicillia (e. g. red and blue; SchmidtHeydt et al., 2011), led to an increase of fumonisin biosynthesis in F. proliferatum. A similar situation was observed with citrinin in the case of P. verrucosum and P. expansum (Schmidt-Heydt et al., 2011): the biosynthesis of this mycotoxin also increased after treatment with red and blue light. It was suggested that citrinin may be a light protectant, because citrinin producing colonies grew better under these wavelengths than non-producing colonies. If this is also the case for fumonisin, it must be shown in further experiments. However the fact that this fumonisin producing strain grew better under light conditions after some time of adaptation, points in that direction. These results indicate that light of the same wavelength can either have a positive or a negative influence of secondary metabolite biosynthesis in different fungal species. This differential regulation surely must have an ecological basis.

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The fact that F. proliferatum especially reacts under red and blue treatment indicates also the presence of red and blue light receptors. Estrada and Avalos (2008) have described wcoA as a possible homologue of the N. crassa wc-1 gene in F. fujikuroi, however they did not find a clear correlation between the activity of this putative light receptor gene and carotenoid biosynthesis. In contrast to these results, in the analysis described here, red and blue light incubation induced an intense orange color in the mycelium; this suggested an increased production of carotenoid which indicates that, in the case of F. proliferatum, blue and red light receptors are involved. Estrada and Avalos (2008) also analyzed the influence of light on the biosynthesis of the secondary metabolites fusarin, bikaverin and gibberellin and they found no clear influence of light on gibberellin and bikaverin biosynthesis. In the case of fusarin, they could detect a higher biosynthesis in the dark, but only in wcoA mutant strains. Audhya and Russel (1974) described a 3 fold increase in enniatin biosynthesis by F. sambucinum under continuous illumination. However, other than these reports, no further systematic analysis on the influence of light on secondary metabolism biosynthesis in Fusaria is known. Although these fungi have been grown on agar medium, a solid substrate providing easier access for light than would apply to living plants, the effect of light on growth and on fumonisin production that we have reported in this study is significant. The results are relevant from an epidemiological point of view, providing new information about the behavior of this toxigenic fungus in response to environmental conditions from a practical point of view, enabling better evaluation of mycotoxin risk. During the different growth stages the intensity of the light hitting the plant changes, both because of changes in the exposed surfaces and from the shifting light conditions in the environment. Furthermore wounds caused by insects, acting as vectors of inoculum, may expose parts of the plant to light, favoring fungal infection and mycotoxin production. All this information could be used to improve monitoring and good agricultural practice for reducing mycotoxin contamination. Fusarium proliferatum, together with F. verticillioides, is the main species responsible for fumonisin accumulation in maize (Shephard et al., 1996). Although F. verticillioides is considered the major cause of Fusarium ear rot of maize, the prevalence of F. proliferatum appears to vary with environmental conditions and geographic region (see Marín et al., 2004 for a review on the ecophysiology of fumonisin-producing strains). F. proliferatum is isolated more frequently from crowns than roots, and the infection is principally due to airborne conidia or insects as vectors of inoculum. In this scenario light exposure assumes a relevant ecological role since exposure to light induces stress to the fungus. The influence of stress on the activation of mycotoxin biosynthetic genes has been reported several times (Jayashree and Subramanyam, 2000; Jurado et al., 2008; Kohut et al., 2009; Schmidt-Heydt et al., 2008). Also in the case of F. proliferatum, the increase of fumonisin biosynthesis reported in this work could be a response to a stress condition induced by light exposure and may support the adaptation of the fungus to these conditions. The reported data suggest also in F. proliferatum the presence of a light sensing system that can modulate a response to light. This response would include different features of fungal physiology from growth to secondary metabolism. These findings can open a new field of research, aimed towards understanding the light perception in this fungal species and discovering genes coding for different putative photoreceptors such as phytochrome, flavin binding proteins and opsin, already found in several fungal genomes, involved in this pathway (Corrochano, 2007; Herrera-Estrella and Horwitz, 2007). Acknowledgment We thank Michaela Ebli for skillful technical assistance. This work was financially supported by EC KBBE-2007-222690-2 MYCORED.

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