Influence of light on growth, conidiation and fumonisin production by Fusarium verticillioides

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Influence of light on growth, conidiation and fumonisin production by Fusarium verticillioides Francesca FANELLIa, Markus SCHMIDT-HEYDTb, Miriam HAIDUKOWSKIa,
a,* Antonia SUSCAa, Rolf GEISENb, Antonio LOGRIECOa, Giuseppina MULE a

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

b

article info

abstract

Article history:

Light is a very important signal for fungi since it influences many different physiological

Received 1 August 2011

responses. We analyzed the influence of light of varying wavelength and intensity on

Received in revised form

growth, conidiation and biosynthesis of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3) by Fusarium

11 November 2011

verticillioides ITEM 10027. Wavelengths across the visible spectrum, from red (627 nm) to

Accepted 14 November 2011

blue (470e455 nm), stimulated the growth and increased the fumonisin production, by

Available online 22 November 2011

up to 150 % over dark incubation. If the intensity of the 455 nm blue light increased from

Corresponding Editor:

200 to 1700 lx, the fumonisin biosynthesis decreased. Incubation under a short wave

Nicholas P. Money

blue light (390 nm) showed reduced fungal growth and fumonisin production by up to 85 %. White pulsing light had no effect on growth but reduced fumonisin production to

Keywords:

half of what observed during dark incubation. Real time reverse transcriptase (RT)-PCR

Fumonisin

was used to measure the expression level of Fum1, Fum21 and FvVE1 transcripts, which en-

Fum1

code proteins involved in fumonisin biosynthesis. There was a significant correlation be-

Fum21

tween gene expression and fumonisin production.

Fusarium verticillioides

ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

FvVE1 Light

Introduction Fusarium verticillioides belongs to the Liseola section of the Fusarium genus (Leslie & Summerell 2006), and its teleomorph, Gibberella moniliformis, belongs to the Gibberella fujikuroi complex. Fusarium verticillioides is a widely distributed pathogen and is the most commonly reported fungal species infecting maize (Leslie 1996; Munkvold & Desjardins 1997; Logrieco et al. 2002). It is associated with disease at all stages of plant development infecting the roots, stalk and kernels and symptomless infection can exist throughout the plant in leaves, stems, roots and grains (Bacon et al. 1992; Munkvold et al.

1997). Fusarium verticillioides is the main source of fumonisins (Desjardins et al. 1995; Leslie & Summerell 2006), a group of mycotoxins associated with several mycotoxicoses, including equine leukoencephalomalacia, porcine pulmonary oedema and experimental kidney and liver cancer in rats (Howard et al. 2001). Fumosins are divided into four series: A, B, C and G. With respect to toxicity the B-series is the most important one. Generally FB1 makes up 70 % of the total fumonisins, and FB2 and FB3 each make up the rest (Nelson et al. 1993). Genetic analysis of F. verticillioides has identified a fumonisin biosynthetic gene (FUM) cluster that consists of 17 genes (Proctor et al. 2003, 2008; Brown et al. 2007).

* Corresponding author. Tel.: þ39 080 5929329; fax: þ39 080 5929374. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.11.007

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The cluster gene Fum1 encodes for a polyketide synthase (FUM1P) essential for fumonisin production (Proctor et al. 1999). This enzyme would catalyze the synthesis of a linear polyketide that forms carbon atoms 3 (C-3) through 20 (C-20) of the backbone and adds the methyl groups at C-12 and C-16 (Bojja et al. 2004). Fum21 is located adjacent to Fum1 and encodes a transcriptional factor with a Zn(II)2Cys6 DNA-binding domain that positively regulates FUM gene expression and is required for fumonisin production (Brown et al. 2007). Other genes not belonging to the FUM cluster are involved in fumonisin biosynthesis pathway. These genes include FvVE1, the veA homologue in F. verticillioides which it regulates cell integrity, morphogenesis, cell surface hydrophobicity, hyphal polarity and conidiation pattern (Li et al. 2006) and mycotoxin production. FvVE1 is necessary for the expression of the pathway-specific regulatory gene Fum21 and the structural genes in the FUM cluster (Myung et al. 2009). Many environmental and abiotic factors affect fumonisin biosynthesis like temperature, water activity and solute potential (Marin et al. 1995, 2010; Samapundo et al. 2005; Frisvad et al. 2007; Mogensen et al. 2009), and may lead to high level of variation in the fumonisin biosynthetic phenotype. Light is a very important signal for fungi: it influences many different physiological responses such as pigmentation, sexual development asexual conidiation, the circadian clock and secondary metabolism. Increasing analysis of fungal genomes has identified numerous genes encoding proteins involved in light detection and has driven scientific efforts towards the discovery of the mechanisms by which the fungus activates physiological and morphological responses (Corrochano 2007; Herrera-Estrella & Horwitz 2007). Though the response to light has been analyzed in many fungal species, most of these studies have focused on the effects that constant or circadian illumination by white light have on fungal growth and metabolism. Recently, light wavelength and intensity have been shown to alter secondary metabolism in toxin producing species (Schmidt-Heydt et al. 2011; Fanelli et al. in press). In this study we analyzed the influence of light wavelength and light intensity on growth, conidiation and fumonisin biosynthesis by F. verticillioides ITEM 10027 and determined the expression levels of Fum1, Fum21 and FvVE1.

Material and methods Fungal strains and growth conditions We used the fumonisin producer Fusarium verticillioides ITEM 10027 (ITEM: Agri-Food Toxigenic Fungi Culture Collection of the Institute of Sciences of Food Production, CNR, Bari, Italy), which was isolated from maize kernels harvested from Italy. A spore suspension of the strain was prepared in sterile distilled water; 100 ml of a 106 ml1 conidial suspension was single point inoculated on MG (Malt extract agar (MEA, Merck, Darmstadt, Germany) plus 2 % glucose) to determine the influence of light on growth and toxin biosynthesis.

The light incubation equipment Four light boxes were constructed to enable incubation of the cultures under different wavelengths of light and different

F. Fanelli et al.

intensities of royal blue light. Light box 1 was subdivided into six chambers. Each chamber was equipped with 5 Luxeon high power 5 W Light Emitting Diodes (LEDs) (Philips Lumileds Lighting Company, San Jose California, USA) with the following different emitting wavelengths: chamber 1, royal blue low intensity (RBL 455 nm, 3350 lx); chamber 2, blue (B 470 nm, 2357 lx); chamber 3, green (G 530 nm, 7250 lx); chamber 4, yellow (Y 590 nm, 6400 lx); chamber 5, red (R 627 nm, 7700 lx); and chamber 6, white light (W 17 750 lx). The distance between the LEDs and the agar plates was 18 cm. Light box 2 was equipped with 25 LEDs of the same type as used in the royal blue high intensity (RBH) chamber of Light box 1; the irradiance was 1700 lx at a distance of 5 cm between the LEDs and the agar plates. Light box 3 was equipped with four Sera Deep Sea special neon tubes (Sera GmbH, Heinsberg, Germany) with the following technical specifications: 36 W, 390 nm, 400 lx. The distance between the neon tubes and the agar plates was 10 cm. The 390 nm wavelength belongs to the visible light spectrum (short wave blue light: SW) but is near the threshold of UV light. Light box 4 was equipped with an Eurolite-15 Strobe with a pulsating white light (PS) xenon lamp with 25 000 lx (Steinigke GmbH, Waldbuettelbrunn, Germany). A pulse rate of 1 s1 was used for the incubation of the cultures. The distance between lamp and agar plates was 20 cm. Two dark chambers were used as control in each experiment, one for the light boxes 1-2-3, and one for the light boxes 4, which were located in different places. The light intensities of the boxes were measured with a Testo 435 luminometer (Testo AG, Lenzkirch, Germany). No heating effect by the LEDs could be detected.

Growth assessment Plates of MG agar inoculated as described in Section 2.1 were incubated for 10 d under the tested light conditions and then photographed. Colony diameters were measured in two directions at right angles to each other. Each experiment was performed in triplicate.

Conidiation study Conidiation was assessed using cultures grown for 3 d at the respective experimental conditions on MG agar medium. Mycelia were scraped from the plate and resuspended in 1 ml of glycerol/water 1:1 v:v solution. The conidia were counted in a Thoma chamber following appropriate dilution. Each experiment was performed in triplicate.

Extraction and HPLC analysis To assess mycotoxin production, colonies were grown for 10 d under appropriate experimental conditions. FB1, FB2 and FB3 were analyzed according to Frisvad et al. (2007) with slight modifications (De Girolamo et al. 2010). One gram of agar was extracted with 1 ml of a solution of 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) by incubating it for 30 min at room temperature (20e25  C) on a rotary shaker; the agar plugs were discarded and the extract was evaporated to dryness in

Influence of light on growth, conidiation

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) at 1200 rpm and then filtered through RC 0.2 mm filters (Phenomenex, USA). 1:10 dilutions of samples were made in the same solvent; 110 mL of the extract were derivatized with 110 mL of o-phthaldialdehyde (OPA) (Sigmae Aldrich, Milan, Italy) mixed for 30 s. An HPLC autosampler (Varian Inc., Palo Alto, CA, USA) was used to inject 50 ml of the sample 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 mm (Waters, Milford, MA, USA) with a guard column inlet filter (0.5 mm$3 mm diameter, Rheodyne Inc., CA, USA). The mobile phase consisted of a binary gradient applied as: the initial composition of the mobile phase 60 % of (A) acetonitrileewatereacetic acid (30/69/1, v/v/v)/40 % of (B) acetonitrileewatereacetic acid (60/39/1, v/v/v) was kept constant for 5 min, then the 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 thermostated to 30  C. The flow rate of the mobile phase was 1.0 ml min1. The fluorometric detector was set at wavelengths ex ¼ 335 nm, em ¼ 440 nm. FB1, FB2 and FB3 were quantified by measuring peak areas, and comparing them with a calibration curve obtained with standard solutions. The detection limit for fumonisins was 0.05 mg g1 based on a signal-to-noise ratio of 3:1. Each experiment was performed in triplicate.

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RNA isolation and reverse transcription Total fungal 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 removed genomic DNA contamination from the samples (RNase-free DNase I, Qiagen), by following the manufacturer’s instructions. First strand cDNA was synthesized with the reverse transcriptase (RT) omniscript reverse transcription (Qiagen). Each 20 ml reaction contained 1 mg of total RNA, 2 ml of oligo d(T)16 (10 mM), 2 ml of 10 RT-PCR buffer, 2 ml dNTPs (10 mM), 1 ml (4 U) of RNase inhibitor, 1 ml (4 U) of Omniscript RT and RNase-free water to the final volume. Synthesis of cDNA was performed at 37  C for 1 h.

Real time RT-PCR Real time RT-PCR assays were used to quantify Fum1 and b-Tubulin (Tub2) expression in the Fusarium verticillioides strain with the primer pairs PQF1-F (50 -GAGCCGAGTCAGCAAG GATT-30 ) and PQF1-R (50 -AGGGTTCGTGAGCCAAGGA-3) for Fum1 and PQTUB-F (50 -CCCCGAGGACTTACGATGTC-30 ) and  pezPQTUB-R (50 -CGCTTGAAGAGCTCCTGGAT-30 ) for Tub2 (Lo Errasquın et al. 2007). The primer pairs FUM21_F3 (50 -ATGCAG ATCCGGAAGGTGTTC-30 ) and FUM21_R3 (50 -TGTAATCTCG TCTGCAATCAAATCC-30 ) for the amplification of Fum21 transcript and FvVe1-for (50 -CGGTTCTGGTTCAAAAGCCA-30 ) and FvVe1-rev (50 -TTGGTCCCTCGATAATCCGA-30 ) for the FvVE1

Fig 1 e Light boxes growth assessment. Morphology (A) and growth (B) of F. verticillioides ITEM 10027 colonies after incubation under different light conditions on MG medium: D, dark; W, white; R, red; G, green; Y, yellow; B, blue; RBL, royal blue low intensity (200 lx); RBH, royal blue high intensity (1700 lx); SW, short wave blue light (390 nm).

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transcript were designed in our laboratory using the Primer Express program (Applied Biosystems, Foster City, CA). Real time PCR reactions were performed on iQ5 Real Time PCR Detection System (BIO-RAD Laboratories, Hercules, CA). The PCR thermal cycling conditions were: 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 (BIO-RAD) was used as reaction mixture, adding 0.5 ml of sterile water, 5 ml of each primer (5 mM) and 2 ml of template cDNA (dilution 1:20), in a final volume of 25 ml. Each sample was run in triplicate in each in every experiment. The results were normalized using the Tub2 amplifications run on the same plate. Each experiment was performed in triplicate.

Results

F. Fanelli et al.

Fig 3 e Light boxes conidiation. Conidiation in F. verticillioides ITEM 10027 colonies after incubation under different light conditions on MG medium: D, dark; W, white; R, red; G, green; Y, yellow; B, blue; royal blue low intensity (200 lx); RBH, royal blue high intensity (1700 lx); SW, short wave blue light (390 nm) (*P < 0.05).

Growth assessment To gain an overview of the influence of various light wavelengths on the growth of Fusarium verticillioides, ITEM 10027 was inoculated on MG and incubated at 20  C for 10 d under different light wavelengths. Only minor changes in the morphology of the colonies were observed (Fig 1A). More growth occurred under R (long wavelength, 627 nm) or B (short wavelength, 470e455 nm) light than in the dark (Fig 1B). Incubation under the SW (390 nm) inhibited fungal growth and resulted in a colony with an intense orange colour to the mycelium. Similar pigmentation was observed in colonies incubated under PS (Fig 2) although the amount of colony growth by these colonies was similar to those grown in the dark.

Conidiation Incubation under long wavelength light (red to yellow) increased conidiation by Fusarium verticillioides ITEM 10027 (Fig 3) relative to cultures incubated into the dark. Incubation under B and RBL wavelengths (200 lx) reduced conidiation by 22e67 % respectively relative to the D, but at higher intensity (RBH, 1700 lx) the number of conidia was comparable to those produced in the dark. Under SW (390 nm) conidiation was six fold greater than in the D. Thus, visible light of very short

wavelength (390 nm) induces high levels of sporulation, whereas the effect of light with a somewhat longer wavelength (455 nm) is intensity dependent, and may increase or decrease sporulation relative to the amount observed in the dark.

Fumonisin production Total fumonisins were extracted from colonies of Fusarium verticillioides ITEM 10027 grown on MG medium for 10 d under various light conditions (Table 1). Light from across the whole spectrum stimulated the production of fumonisin relative to the amount produced in the D (Fig 4A): from R to B wavelengths there was at least a two fold increase of total FB production. This increase was higher (from three to five fold) with respect to FB2 and FB3 production. Incubation under W light had a slightly inhibitory effect, and reduced FB1 levels by 15 %. When incubated under SW light (390 nm) FB1 production was reduced by about 80 % and FB2 and FB3 were not detected. White PS also inhibited fumonisin production

Table 1 e HPLC quantification of FB1, FB2 and FB3 biosynthesis of F. verticillioides ITEM 10027 after incubation under different light conditions on MG agar medium. Growth condition

Fig 2 e Pulsing light growth assessment. Morphology of F. verticillioides ITEM 10027 colonies after incubation under different light conditions on MG medium: D, dark; PS, pulsing light.

Dark White Red Green Yellow Blue Royal blue low intensity (200 lx) Royal blue high intensity (1700 lx) SW (390 nm) n.d. ¼ not detectable.

FB1 (mg g1)

FB2 (mg g1)

FB3 (mg g1)

 1.1  1.4  0.2  0.3  0.9  0.8  1.7

1.5  0.2 1.4  0.3 5.3  0.8 6.3  0.3 4.6  0.8 4.8  0.5 5.6  0.4

1.5  0.0 1.1  0.2 3.9  0.4 4.4  0.3 4.0  0.6 3.3  0.4 3.0  0.5

11.3  0.8

5.2  0.8

4.1  1.2

n.d.

n.d.

6.3 5.4 12.6 12.2 10.1 10.7 12.6

1.4  0.5

Influence of light on growth, conidiation

245

Fig 4 e Light boxes fumonisin biosynthesis and expression analysis. FB1, FB2 and FB3 biosynthesis (A) and level of expression (B) of Fum1 and Fum21 of F. verticillioides ITEM 10027 colonies after incubation under different light conditions on MG medium: D, dark; W, white; R, red; G, green; Y, yellow; B, blue; RBL, royal blue low intensity (200 lx); RBH, royal blue high intensity (1700 lx) (*P < 0.05, **P < 0.01).

(Table 2). As shown in Fig 5A it reduced FB1 production by 55 %, FB2 production by 45 % and FB3 production of about 20 %, for a total mean reduction of 50 % relative to the dark.

Expression analysis We conducted RT-PCR analysis of Fum1 and Fum21 expression of Fusarium verticillioides strain ITEM 10027 in 10 d-old cultures incubated in the different wavelength light boxes (Fig 4B) relative to expression in the dark. In W light the expression of both genes decreased by 60 % relative to the dark. Under the R and Y lights the expression level of both genes increased slightly. Under the G light Fum1 expression was reduced slightly and Fum21 expression was increased slightly. Under the B wavelength

Table 2 e HPLC quantification of FB1, FB2 and FB3 biosynthesis of F. verticillioides ITEM 10027 after incubation under pulsing light on MG agar medium. Growth condition Dark Pulsing light

FB1 (mg g1)

FB2 (mg g1)

FB3 (mg g1)

2.8  0.6 1.3  0.1

1.00  0.1 0.5  0.0

0.7  0.1 0.5  0.0

lights, expression was dependent on the intensity of the illumination and the particular wavelength used: under the B light (470 nm) Fum1 expression was comparable to that of the D incubation, while Fum21 expression was reduced slightly; Fum1 expression was increased under RBL light (455 nm) at low intensity (200 lx), whereas at high intensity (RBH, 1700 lx) Fum1 levels are reduced by 40 % relative to the dark. In contrast the expression of the Fum21 gene was only slightly affected by RBL (200 lx) compared to the dark incubation; however under high intensity RBH (455 nm, 1700 lx) it was reduced by 70 % compared to the dark. Under SW light (390 nm) there was a reduction in both Fum1 and Fum21 comparable to the expression levels of high intensity RBH (455 nm, 1700 lx). There is congruence between expression data and phenotypic production of fumonisin, but not absolute concordance. Under W light expression levels were reduced compared to the situation in the dark. With R, Y and RBL light at 200 lx the expression level of both genes increased in parallel to the amount of fumonisin produced. With G, B (470 nm) and RBH light at 1700 lx gene expression levels were lower, but fumonisin production was still high. Thus, other factors, beside transcript levels, may be important in fumonisin biosynthesis. Alternatively, the time window of transcription may not be coincident with toxin production.

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Fig 5 e Pulsing light fumonisin biosynthesis and expression analysis. FB1, FB2 and FB3 biosynthesis (A) and level of expression (B) of Fum1, Fum21 and FvVE1 of F. verticillioides ITEM 10027 colonies after incubation under different light conditions on MG medium: D, dark; PS, pulsing light (*P < 0.05, **P < 0.01).

Based on real time RT-PCR analysis of Fum1, Fum21, and FvVE1 expression, the PS light significantly decreased the expression of all genes analyzed (Fig 5A). Fum1 expression was reduced by 80 %, Fum21 by 75 % and FvVE1 by 45 %.

Discussion Light is an abiotic factor that influences on secondary metabolite production. In Alternaria alternata the production of toxins can be reduced by blue light, and light pulses can € ggblom & Unestam 1979; reduce polyketide biosynthesis (Ha € ggblom & Niehaus 1986). Ha Incubation of Aspergillus parasiticus under continuous light and continuous darkness can alter aflatoxin and anthraquinones production by Bennett et al. (1981). Recently SchmidtHeydt et al. (2011) had reported that light has an influence on growth, morphology and ochratoxin A (OTA) biosynthesis in Aspergillus and Penicillium. Depending on light wavelength and intensity a complete cessation of growth and the biosynthesis of OTA was observed. However during that analysis, light did not have a general reducing effect on secondary metabolite levels. The production of citrinin, a mycotoxin produced by Penicillium verrucosum also is influenced by light incubation of cultures: blue light led to an increased production of citrinin in P. verrucosum. Citrinin biosynthesis in Penicillium expansum, a typical citrinin producing species, was also elevated by light and especially blue light. The results reported by Schmidt-Heydt et al. (2011) show that blue light may have either an inhibiting or an enhancing effect on secondary metabolite biosynthesis. The impact of light seems to be more dependent on the specific secondary metabolite, than on the

F. Fanelli et al.

species. For example in Fusarium verticillioides (this work), in Fusarium proliferatum, and in Aspergillus niger (Fanelli et al. in press) the production of fumonisin was increased under blue light treatment; in contrast the production of OTA was reduced in the same strain of A. niger (Fanelli F., pers. comm.), but also in Aspergillus carbonarius, P. verrucosum and Penicillium nordicum (Schmidt-Heydt et al. 2011). Light sensing has been studied in depth in Neurospora crassa, where both the perception and the response to blue light have been characterized. These responses require the WC-1 and WC-2 (Ballario et al. 1996; Ballario & Macino 1997) proteins to interact and form the White Collar Complex (WCC), which upon light exposure binds transiently to the promoters of light inducible genes to activate their transcription. Orthologs of WC have been found in Aspergillus nidulans (Purschwitz et al. 2008) and several other fungi. Another light regulatory protein was identified in A. nidulans as VeA, which is involved in sexual development and acts as a key regulator of the biosynthesis of many secondary metabolites. In concert with VelB and LaeA, VeA forms the velvet complex: under light exposure VeA is mostly retained in the cytoplasm; in the dark VeA is functionally active and is imported in the nucleus, where the complex can activate the sexual development and the secondary metabolite cluster expression (Bayram et al. 2008). As in other fungal genomes, there are numerous genes in Fusarium that encode for proteins that can detect light and trigger responses to light exposure, but the only wellcharacterized photo response in Fusarium spp. is the light induction of carotenoid biosynthesis (Prado et al. 2004; Thewes et al. 2005; Prado-Cabrero et al. 2007). The response of F. verticillioides ITEM 10027 to light is different than observed in ochratoxin producing species (SchmidtHeydt et al. 2011). The morphology of ITEM 10027 was almost unaffected by light. Growth was activated by light, with white light and the blue spectrum lights (455e470 nm) that were the most effective in promoting the growth. SW (390 nm), however, inhibited the growth of F. verticillioides ITEM 10027 while promoting conidiation. Under this condition, in which the vegetative growth of the mycelium is not supported, the conidiation is promoted. The same conclusion emerged from a work from Kumagai (1989), who reported an inductive effect of near-UV light irradiation on conidiation in Alternaria tomato. Although near-UV light has the opposite effect on the growth on F. verticillioides (reduced) and A. niger (increased) (Fanelli F., pers. comm.), conidiation is increased in both species. This information is important to understand the epidemiology of the pathogen and could be a reflection of an ecological strategy to warrant the fungal survival under stressing condition exerted by this light exposure and allow the fungal diffusion in the environment. Fumonisin production increased under individual wavelengths from both sides of the spectrum, from red to blue. White light had a slightly inhibitory effect on FB synthesis, and this effect was more pronounced when the light was pulsing too. The pulsing light did not inhibit fungal growth. Thus, FB production is not correlated with fungal growth rate but is affected by light quality and intensity. The meaning of this behaviour must be related to the ecology of F. verticillioides. Fusarium verticillioides is mainly a field fungus, which is more tolerant to high temperature than other Fusarium spp. (Marin et al. 1995). Fumonisin production by

Influence of light on growth, conidiation

F. verticillioides is increased under high water activities (Samapundo et al. 2005) and other environmental factors such as temperature, pH, nitrogen limitation and carbon nutrient availability (Shim & Woloshuk 1999; Bluhm & Woloshuk 2005). The ability to detect light and to regulate growth and fumonisin biosynthesis in vitro, as we have shown in this paper, could be a mean to trigger a response to changes in field environmental conditions. Many studies have demonstrated that the expression of Fum1 is correlated with fumonisin production (Proctor et al.  pez-Errasquın et al. 2007; Jurado et al. 1999; Seo et al. 2001; Lo 2008). In our study we found variation in the expression of Fum1 transcripts, with a maximum increase under red, yellow and royal blue (200 lx) light, and a decrease under white, green and SW. These variations correlate quite well with fumonisin production. When this correlation is weak we could hypothesize an incomplete correspondence between the time window of transcription measurement and the production. A similar hypothesis can be formulated to explain Fum21 expression. Brown et al. (2007) reported that in Dfum21 mutants Fum1 and Fum8 transcripts were detected in early stages of growth, indicating that Fum21 is not the only factor that regulates FUM expression. This study is the first work that correlates the expression of Fum21 gene with fumonisin production by real time RT-PCR. The differences in Fum21 expression pattern were similar to the Fum1 expression pattern, suggesting that both genes are regulated by the same pathway in response to light. Pulsing light affected all the parameters measured. A strong reduction in FB production was associated with a significant decrease in the expression of Fum1, Fum21 and FvVE1. FvVE1 is a major regulator of fumonisin biosynthesis, since deletion DfvVE1 mutants produce neither fumonisins nor fusarins on natural substrates (Myung et al. 2009). In conclusion this work has further characterized the influence of light on growth and fumonisin production by the F. verticillioides, indicating a clear ability of this strain to detect and trigger a response to different light incubations. Further studies will be necessary to clarify the role of light in regulating mycotoxin production in vitro and in vivo and to elucidate the molecular pathway that modulates fumonisin production.

Acknowledgement We thank Michaela Ebli for skillful technical assistance. This work was financially supported by EC KBBE-2007-222690-2 MYCORED.

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