Fusarium asiaticum infects cereal crops and produces trichothecenes such as deoxynivalenol and nivalenol. To determine the trichothecene induction mechanism, effects of carbon sources on the production of deoxynivalenol, nivalenol, 3-acetyl deoxynivalenol (3ADON), and 4-acetyl nivalenol (4ANIV) were examined in liquid cultures incubated with various strains. Sucrose supported significantly higher levels of acetylated trichothecene production in all strains than did the other carbon sources. Structural isomers of sucrose did not induce trichothecene production. The inducing effect of sucrose on trichothecene production was lost after the carbon source in the culture medium changed from sucrose to maltose in the process of incubation. Tri4 and Tri5 expressions were specifically up-regulated in the sucrose-containing medium and down-regulated with sucrose exhaustion. These findings suggest that F. asiaticum recognizes sucrose molecules and regulates Tri gene expression and trichothecene production. Moreover, an accelerating effect on trichothecene production by acidification of the culture medium containing specific amines during fungal incubation was exhibited only in the presence of sucrose in the medium. F. asiaticum induces trichothecene production in the presence of sucrose and accelerates the production when the medium containing specific amines is acidified during incubation.
Fusarium head blight (FHB) is a destructive fungal disease of many small grains such as wheat and barley and can cause significant yield losses and reduce grain quality by contamination with mycotoxins such as deoxynivalenol and nivalenol. The primary etiological agents of FHB belong to the Fusarium graminearum species complex (Fg complex), which consists of at least 16 phylogenetically distinct species (Sarver et al., 2011). Fusarium graminearum sensu stricto (s. str.) is the dominant species in the Fg complex causing FHB worldwide, whereas F. asiaticum is mostly located in Asia.
Mycotoxins produced by the Fg complex belong to the type B group of trichothecenes including nivalenol, deoxynivalenol, and their acetylated derivatives (4ANIV, 3ADON, and 15ADON) (Miller et al., 1983; Miller & Greenhalgh, 1985). There are three types in strain-specific profiles of trichothecene produced by the Fg complex: strains producing deoxynivalenol and 3ADON, deoxynivalenol and 15ADON, and nivalenol and 4ANIV (Miller et al., 1991). Trichothecenes are nonspecific inhibitors of eukaryotic protein synthesis, and acute toxicity has been demonstrated in both animals and humans (Sugita-Konishi & Kumagai, 2005; Pestka, 2010). In addition, nivalenol is more toxic to animals than deoxynivalenol (Takahashi et al., 2008). Therefore, a better understanding of trichothecene production and its regulatory mechanisms is desirable for the development of new strategies to efficiently manage the risks posed by FHB.
The trichothecene production pathway from farnesyl pyrophosphate by the Tri genes has been established in F. graminearum s. str. (Kimura et al., 2003). The first committed step in the pathway is catalyzed by the sesquiterpene synthase (trichodiene synthase) encoded by Tri5 (Hohn & Beremand, 1989). The expression of Tri5 is correlated with the production of trichothecenes both in planta and in culture (Doohan et al., 1999). Tri4 encodes a cytochrome P450 monooxygenase involved in four oxygenation steps of the pathway after trichodiene synthesis by Tri5 (Tokai et al., 2007).
Production of trichothecenes by F. graminearum s. str. can be triggered by various factors in plants (Kazan et al., 2012). Carbon and nitrogen sources are the key factors that influence not only infected fungal growth but also secondary metabolites. We analyzed the effects of carbon sources on trichothecene production in liquid cultures incubated with 3ADON producing F. graminearum s. str. and showed that sucrose significantly induced Tri4 and Tri5 expression and then 3ADON production (Jiao et al., 2008). On the other hand, induction of trichothecene production by 15ADON producers of F. graminearum s. str. was not affected by any of the 190 carbon sources, including sucrose (Gardiner et al., 2009a). These data suggest that different chemotype strains of F. graminearum s. str. possessed different induction mechanisms of trichothecene production. Therefore, we were strongly interested in the effects of carbon sources on induction of trichothecene production by different chemotype strains of F. asiaticum. Gardiner et al. (2009a) screened nitrogen sources for the identification of inducers of trichothecene production and showed that several kinds of amines, including agmatine and putrescine, induced Tri5 expression and trichothecene production by a 15ADON producer of F. graminearum s. str. However, it is not clear whether sucrose and amines induced trichothecene production in F. asiaticum. Moreover, it is necessary to analyze the interaction between carbon sources and nitrogen sources for trichothecene production.
In this study, the effects of different carbon sources on production of trichothecenes during culture with different chemotype strains of F. asiaticum were examined. For analysis of interaction between the amount of sucrose in culture medium and Tri gene expression during incubation, time-dependent changes in fungal growth, sugar content, Tri gene expression, and trichothecene production during incubation were determined. We also investigated whether trichothecene biosynthesis was affected by sucrose isomers and specific amines.
Materials and methods
Fungal strains, media, and culture conditions
Thirteen strains of F. asiaticum isolated from wheat ears displaying FHB symptoms in Japan were used for the experiments. Nine strains (228-003 < MAFF240556 >, 229-003, 232-001, 239-010, 242-008, 242-021, 243-006, 244-014, and 244-024) produced deoxynivalenol and 3ADON, and 5 strains (NIV2, NIV7 < MAFF240552 >, NIV10 < MAFF240553 >, 212-011, and 224-091) produced nivalenol and 4ANIV. All strains were identified to the species level based on the colonial characterization on potato dextrose agar, spore morphology on synthetic nutrient agar (SNA), and PCR-amplified fragment length polymorphism (Suga et al., 2008). Modified Czapek liquid media (pH 7.0) consisted of 5.7 mM K2HPO4, 6.7 mM KCl, 2.0 mM MgSO47H2O, 29.0 μM Fe-ethylenediaminetetraacetic acid, 10.7 mM l-glutamic acid (monosodium salt), 2 mL trace elements stock solution (Correll et al., 1987), and 20 g of a carbon source (glucose, fructose, sucrose, 1-kestose, nystose, maltose, amylose, amylopectin, cellobiose, xylose, turanose, maltulose, or palatinose) per liter. The carbon sources selected comprised the main carbohydrates in plants and were purchased from Wako Chemical Industries (Osaka, Japan) and Tokyo Chemical Industry (Tokyo, Japan). Of the medium, 5 or 15 mL was dispensed in a glass tube or a 100-mL conical flask, respectively, inoculated with 4 × 102 conidia mL−1 medium, and incubated with shaking (120 r.p.m.) at 25 °C in the dark. For analyzing the effects on trichothecene production of a change in carbon source from sucrose to maltose in the culture medium, 228-003 or NIV7 strain had been incubated in a 100-mL conical flask with a sucrose-containing medium for 4 days, and the fungal mycelia were washed with sterilized water. The washed mycelia were resuspended in transfer medium containing sucrose or maltose and incubated for 2 or 4 days. For analyzing time-dependent change in mycelial growth, expression of Tri genes, and trichothecene accumulation and sugar levels in the culture medium during reculture after addition of sucrose or maltose, 228-003 strain was precultured in a 100-mL conical flask with 15 mL of a medium containing 3% (w/v) of maltose for 3 days and recultured after 10 mL of a medium containing 7.5% (w/v) sucrose or maltose had been added to the preculture medium. The culture filtrate and mycelia were time-dependently sampled after 0, 12, 18, 24, 36, 48, and 72 h of reculturing. For analyzing the accelerated effect of trichothecene production by specific amines such as putrescine and agmatine, the two strains (228-003 and 242-021) that produced 3ADON and the two strains (212-011 and NIV7) that produced 4ANIV were each incubated in a glass tube with 5 mL of modified Czapek liquid medium containing 10 mM of l-glutamic acid, putrescine, or agmatine as a sole nitrogen source for 8 days. Putrescine and agmatine were purchased from Tokyo Chemical Industry. All experiments were performed in triplicate.
Determination of fungal growth, medium pH, trichothecene, and sugar
For quantitative determination of fungal growth after incubation, the mycelium harvested on filter paper was dried at 60 °C overnight before measurement of dry weight. The pH value of the medium after incubation was measured with a pH meter (Twin pH, Horiba, Kyoto, Japan).
A 1.6-mL aliquot of the supernatant after fungal incubation was mixed with 8.4 mL acetonitrile and applied to an MF-T1500 multifunctional column (Showa Denko, Tokyo, Japan) without preconditioning. The first 3 mL of elute was discarded, and the following 5 mL was collected in a new test tube. A 4.0-mL aliquot of the collected solution was dried at 40 °C by a centrifugal evaporator with a vacuum pump. The residue was redissolved in 0.4 mL of acetonitrile/methanol (4 : 1). The supernatant was passed through a 0.45-μm-pore filter and served as the sample solution for direct injection into the HPLC-UV apparatus. A Prominence series HPLC system connected to a photodiode array detector (Shimadzu, Kyoto, Japan) was used, and data were acquired and integrated using LC solution software (Shimadzu). Isocratic HPLC conditions were adopted at a mobile phase of acetonitrile/methanol/water (1 : 1 : 18) for deoxynivalenol and nivalenol, acetonitrile/water (1 : 4) for 3ADON, and acetonitrile/methanol/water (1 : 3 : 16) for 4ANIV. A C18 column (250 × 4.6 mm in inside diameter; C18M-4E, Showa Denko) was used at 40 °C, and the injection volume was 10 μL. The detector was set at a wavelength of 220 nm. Detected peaks identified as deoxynivalenol, nivalenol, 3ADON, and 4ANIV were quantified using authentic standards (Wako Chemical Industries).
The supernatant after fungal incubation was passed through a 0.45-μm-pore filter and mixed in 3 times its volume of acetonitrile, and this served as the sample solution for direct injection into the HPLC refractive index (RI) apparatus. A Prominence series HPLC system connected to an RI (Shimadu) was used, and data were acquired and integrated using LC solution software (Shimazu). Isocratic HPLC conditions were adopted at a flow rate of 1 mL min−1 with a mobile phase of acetonitrile/water (3 : 1). An Asahipak NH2P-50 4E column (250 × 4.6 mm in inside diameter, Showa Denko) was used at 30 °C, and the injection volume was 10 μL. Detected peaks identified as sucrose and maltose were quantified using authentic standards (Wako Chemical Industries).
Extraction of total RNA and preparation of cDNA
After the F. asiaticum 228-003 strain had been incubated in modified Czapek liquid medium, the mycelium was collected by centrifugation and was ground for 20 s at 1500 r.p.m. several times with a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) in liquid nitrogen. Total RNA was extracted using RNAiso (Takara, Shiga, Japan) following the manufacturer's instructions. The extracted RNA pellet was dissolved in 70 μL of RDD buffer (Qiagen, Valencia, CA), incubated with 10 μL DNase I (Qiagen) for 30 min at 28 °C, and then purified using an RNeasy Plant mini kit (Qiagen) following the manufacturer's instructions for RNA cleanup. First-strand cDNA was synthesized using a Superscript VIRO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) from 1 μg total RNA primed with random hexamer primers.
Expression analysis by real-time PCR
Abundance of the transcripts of Tri4 and Tri5 was analyzed by an Mx3000P Real-Time QPCR System (Agilent Technologies, Santa Clara, CA) with 0.2 μL cDNA in a 20-μL PCR using a DyNAmo™ HS SYBR Green qPCR Kit (FINNZYMES, Vantaa, Finland) following the manufacturer's instructions. The expression of β-tubulin was used as an endogenous control. The PCR was carried out at 95 °C for 15 min for initial denaturation followed by 40 cycles of 94 °C for 10 s, 58 °C for 25 s, and 72 °C for 30 s. Results were obtained from three replicate experiments. Primer pairs for amplifying β-tubulin, Tri4, and Tri5 were designed as previously described (Jiao et al., 2008).
The relative expression method was used in the analysis according to the ABI User Bulletin (Applied Biosystems) after performing a validation experiment to demonstrate that amplification efficiencies of the two target Tri genes and the reference β-tubulin gene were approximately equal. Fold change in gene expression was normalized to β-tubulin and compared with the expression just after inoculation with cultures containing maltose using the method (Livak & Schmittgen, 2001).
Effects of carbon source on mycelial growth and trichothecene yield
All tested carbon sources supported sufficient mycelial growth for the two strains, although the growth differed depending on the carbon source (Fig. 1). A significantly higher production level of 3ADON by 228-003 strain or 4ANIV by NIV7 strain was evident when the medium was supplemented with sucrose, 1-kestose, or nystose. The 228-003 and NIV7 strains also produced deoxynivalenol and nivalenol, respectively, in sucrose-containing medium, but the amounts were much smaller than those of the acetylated forms. Similar results were obtained when 8 stains producing 3ADON and 4 strains producing 4ANIV were incubated with 10 carbon sources (Supporting Information, Table S1). The pH of the tested media after incubation ranged from 7.9 to 8.6 for 228-003 and from 7.9 to 8.3 for NIV7.
228-003 strain and NIV10 strain grew well in the medium containing sucrose or its isomeric α-d-glucosyl-d-fructoses, that is, turanose [α-(1.3)], maltulose [α-(1.4)], or palatinose [α-(1.6)], but did not produce trichothecenes in the medium containing sucrose isomers (Fig. 2).
Effects on trichothecene production of a change in carbon source from sucrose to maltose in the culture medium
By the initial incubation for 4 days in the medium containing sucrose, 228-003 strain and NIV7 strain induced trichothecene production (Fig. 3). Resuspended mycelia of all strains resulted in a significant increase in trichothecene production in the sucrose-containing medium over the incubation time but in a severe repression of trichothecene production in the maltose-containing medium.
Time-dependent change in mycelial growth, expression of Tri genes, and trichothecene accumulation and sugar levels in the culture medium during reculture after addition of sucrose or maltose
The levels of mycelial growth during reculturing were not greatly different in the media with the addition of maltose and sucrose (Fig. 4). 3ADON was not detected in the medium with the addition of maltose throughout the reculturing period. In contrast, 3ADON was detected in the medium with the addition of sucrose after 36 h of reculturing and the level had significantly increased by 72 h. Tri4 and Tri5 transcripts in the medium with the addition of maltose were detected, but their abundance did not increase throughout the reculturing period (Fig. 5). The expression levels of Tri4 and Tri5 in the medium with the addition of sucrose started increasing after 18 h of reculturing, reached a maximum during the period from 36 h to 48 h, and drastically dropped from 48 h to 72 h. In the medium with the addition of maltose, maltose content time-dependently decreased during the reculturing period. In the medium with the addition of sucrose, sucrose content time-dependently decreased to 13.5, 7.2, 2.3, and 0.7 mg mL−1 after 24, 36, 48, and 72 h of reculturing, respectively.
Accelerating effects of specific amines on trichothecene production
Almost all strains (228-003, 242-021, and NIV7 strains) grew best in the medium containing l-glutamic acid, and mycelial growth in putrescine-containing medium decreased by half compared with that in l-glutamic acid- or agmatine-containing medium (Fig. 6). The carbon sources contained in the media did not affect mycelium growth. The average pH values of the media containing putrescine, agmatine, and l-glutamic acid after incubation were 1.7, 1.9, and 7.8, respectively. The carbon sources contained in the media did not affect pH value. 3ADON or 4ANIV was detected in the sucrose-containing medium, but not in the maltose-containing medium. All strains produced significantly larger amounts of trichothecenes in the putrescine-containing medium than in the l-glutamic acid-containing medium. An accelerating effect of agmatine on trichothecene production when compared to culture in the medium containing l-glutamic acid was detected with only 2 strains (228-003 and 242-021).
3ADON producers of F. graminearum s. str. and 3ADON producers and 4ANIV producers of F. asiaticum are predominantly distributed in Japan as pathogens of FHB (Suga et al., 2008). A previous study of 3ADON producers of F. graminearum s. str. found that sucrose induced the production of significantly high levels of trichothecenes (Jiao et al., 2008). We analyzed the effects of different carbon sources on trichothecene production by 3ADON producers and 4ANIV producers of F. asiaticum. Sucrose was found to be more effective than the other carbon sources for 3ADON or 4ANIV production (Fig. 1, Table S1). Our results indicate that sucrose commonly induces trichothecene production in main species within the Fg complex that cause FHB in Japan. On the other hand, 15ADON producers of F. graminearum s. str. did not influence the trichothecene production by any carbon sources (Gardiner et al., 2009a). Although we could not test trichothecene productivity by 15ADON producers of F. asiaticum because 15ADON producers of F. asiaticum were rarely isolated in Japan (Suga et al., 2008), the mechanisms of trichothecene induction by carbon sources might differentiate between 3ADON producers and 15ADON producers in Fg complex, but not between F. graminearum s. str. and F. asiaticum. Further study is needed to determine the effects of carbon sources on induction of trichothecene production by 15ADON producers of F. asiaticum. Trichothecene production was also induced by 1-kestose and nystose, although trichothecene yields induced by 1-kestose and nystose were smaller than those induced by sucrose. 1-Kestose and nystose are fructooligosaccharides that bind one molecule and two molecules of the β-(2,1)-linked fructosyl unit to sucrose, respectively, and therefore have structures that are very similar to the structure of sucrose. Fusarium fujikuroi produced a large amount of bikaverin, one of the polyketides, in a medium containing sucrose compared with the other carbon sources (Rodríguez-Ortiz et al., 2010). Fusarium verticillioides has been found to induce the production of a large amount of fumonisin B1 by incubation with amylopectin (Bluhm & Woloshuk, 2005). These results indicate that fungi regulate secondary metabolism by specific carbon sources. Moreover, the induction of trichothecene production by sucrose but not by sucrose isomers raises the possibility that the specific α-glucoside structure included in sucrose might be a key factor for the induction of trichothecene production by F. asiaticum. Fusarium graminearum s. str. infects wheat spike tissues after anthesis and produces trichothecenes (Proctor et al., 1995; Jansen et al., 2005). The wheat spike tissues after anthesis contain large amounts of various carbon sources, and the amounts of sucrose and fructooligosaccharides drastically increase until the tenth day after anthesis and then gradually decreased with kernel maturation (Takahashi et al., 2001). Trichothecene production by F. graminearum s. str. is low during growth on harvested wheat kernels but high during infection in spike tissues after anthesis, indicating that specific factors present only during infection in spike tissues enhance trichothecene production and seem to be absent during colonization in harvested kernels (Voigt et al., 2007). The data indicate that F. asiaticum selectively recognizes sucrose in plant tissues containing various carbon sources and then produces trichothecenes, as is the case with F. graminearum s. str.
Expression of Tri5 and Tri4 was specifically induced at 18 h, and the expression levels drastically increased until 48 h of reculturing in the media containing sucrose (Fig. 5). With the increase in the expression levels of Tri5 and Tri4, 3ADON was specifically detected at 36 h and the amounts increased until 72 h after reculturing in the medium with the addition of sucrose. These results indicate that sucrose strongly induces expression of the Tri genes of F. asiaticum and that trichothecene production induced by sucrose is regulated by transcription levels of Tri genes. The expression levels of Tri5 and Tri4 greatly decreased from 48 h to 72 h. At the same time, sucrose content in the medium decreased from 2.3 mg mL−1 to 0.7 mg mL−1. After 72 h of reculturing, the medium with the addition of sucrose contained sufficient amounts of carbohydrates other than sucrose for fungal growth. Moreover, the change in carbon source from sucrose to maltose in the process of incubation severely reduced the effect of sucrose to induce trichothecene production (Fig. 3). The results indicate that Tri gene expression is greatly inhibited when sucrose concentration in the medium falls below the threshold. Fungi sense nutrients such as sugar and amino acids in the environment (Thevelein et al., 2005; Van Dijck, 2009). Fusarium asiaticum might possess a system that specifically identifies sucrose and regulates the trichothecene production pathway.
Low pH of the medium or a combination of low pH and amines such as agmatine and putrescine in the medium significantly enhanced the expression of the Tri5 gene and increased trichothecene production by 15ADON producers of F. graminearum s. str. (Gardiner et al., 2009b; Merhej et al., 2010). Gardiner et al. (2009b) suggested that acidification of a culture medium containing amines during fungal incubation was caused by the fungus and that the fungus-mediated acidification increased Tri5 expression and trichothecene production. We found that the pH value after incubation dropped to 2 or lower in the medium containing agmatine or putrescine, but not in the medium containing l-glutamic acid, and the accelerating effect of amines on trichothecene production when compared with l-glutamic acid was detected by putrescine, but not agmatine. These results suggest that acidification of the culture medium containing amines during fungal incubation does not necessarily correspond to acceleration of trichothecene production by F. asiaticum. Gardiner et al. (2009b) suggested that the drop in pH observed prior to the onset of trichothecene production was required to induce Tri5 expression. Mycelial growth of F. graminearum s. str. was significantly inhibited under low pH conditions in the culture medium (Merhej et al., 2010). Mycelial growth of F. asiaticum during incubation for 8 days decreased by half in the medium containing putrescine but was not greatly different in agmatine-containing medium compared with that in the medium containing l-glutamic acid. The results raise the possibility that acidification occurs from the early period of fungal incubation in the putrescine-containing medium but occurs gradually in the agmatine-containing medium. The difference in acidification rate during fungal incubation may affect fungal growth and the level of trichothecene production. Moreover, an accelerating effect of putrescine on trichothecene production when compared with l-glutamic acid was detected in the sucrose-containing medium, but not in the maltose-containing medium (Fig. 6). The results suggest that the initial step for trichothecene production of F. asiaticum is the recognition of sucrose, not acidification, in the medium. F. asiaticum will initially recognize sucrose in the medium, induce trichothecene production, and accelerate the production when the medium is acidified prior to the onset of trichothecene production. Therefore, it is thought that sucrose is an inducer and acidification by specific amines is an accelerator of trichothecene production by F. asiaticum. Further study is needed to determine the interactions between sucrose and the nitrogen sources other than amines for trichothecene production.