Ann Microbiol (2012) 62:965–971 DOI 10.1007/s13213-011-0335-1

ORIGINAL PAPERS

Rapid macroconidia production in graminearum 3- and 15-acetyldeoxynivalenol (ADON) chemotypes using sucrose-water medium

Manel Ben Mansour & Yit Kheng Goh & Vladimir Vujanovic

Received: 23 May 2011 /Accepted: 25 July 2011 /Published online: 11 August 2011 # Springer-Verlag and the University of Milan 2011

Abstract Sucrose-water medium induced a high rate of Petch] is the most common and devastating fungal disease macroconidia formation in Fusarium graminearum 3- and of small grain cereals in North America (Bai and Shaner 15-acetyldeoxynivalenol (ADON) chemotypes. The F. grami- 1994, 2004). Moreover, produced by F. grami- nearum isolates tested produced uniform macroconidia in nearum 3- and 15-ADON chemotypes are major sources of large quantities and within a shorter incubation period in food contamination (Desjardins and Proctor 2007, 2011). sucrose-water medium compared to other assessed media. At The primary mycotoxins present in grain include zearale- the same time, high rates of accumulation none and trichothecene derivatives, such as deoxynivalenol (trichothecenes: DON, 3-ADON, 15-ADON, and : (DON), 3-acetyldeoxynivalenol (3-ADON) and 15- ) in macroconidia were detected using high performance acetyldeoxynivalenol (15-ADON) (Foroud and Eudes liquid chromatography. The proposed “saccharose-water” 2009; Gale et al. 2011; Von der Ohe et al. 2010; Ward method represents a valuable alternative for conidia produc- et al. 2002). tion in mycotoxigenic F. graminearum isolates, having In fungal plant pathogens, including food molds, asexual advantages over the potentially mutagenic UV light, and spore formation (sporulation or conidiation) is a regular potato-dextrose agar, which can degenerate macroconidia, and secondary reproductive cycle for massive generation of over expensive chemical compounds or natural substances for conidia or infective inocula (Ohara et al. 2004; Sutton which the preparation protocols and handling procedures are 1982). Macroconidia are used frequently in artificially relatively complex or time-consuming. inoculated field trials to test the level of isolate pathoge- nicity by measuring mycotoxin accumulation in cereal grain Keywords Fusarium graminearum . Chemotype . (Harris et al. 1999; Miedaner et al. 2010). The latter is an Macroconidia formation . HPLC . Mycotoxin important parameter in the assessment of food quality and safety. Fusarium graminearum 3-ADON isolates have higher Introduction macroconidia production and growth rates compared to 15- ADON strains (Ward et al. 2008). This feature might Fusarium head blight (FHB) caused by Fusarium grami- partially explain the rapid spread of 3-ADON over 15- nearum Schwabe [teleomorph: zeae (Schw.) ADON populations in North America (Gilbert et al. 2001; Ward et al. 2008). Under in vitro conditions, F. graminearum is unable to : : * M. B. Mansour Y. K. Goh V. Vujanovic ( ) generate macroconidia on commercially available, Department of Food and Bioproduct Sciences, University of Saskatchewan, carbohydrate-rich media such as potato dextrose agar Saskatoon SK S7N 5A8, Canada (PDA). Physical (near UV light), chemical [CMC: 1- e-mail: [email protected] cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p- toluenesulfonate] and natural (carnation leaf and M. B. Mansour Department of Plant Biotechnology, University of Tunis El Manar, bran) elements have been proposed to induce large-scale F. 1060 Tunis, Tunisia graminearum macroconidia production in the laboratory 966 Ann Microbiol (2012) 62:965–971

(Eudes et al. 2001;Fisheretal.1982; Hassan and Oakville, ON); (2) BM with 7 g/L CMC; (3) BM with Bullerman 2009). UV light and PDA medium are poten- 15 g/L CMC; (4) BM with 7 g/L CMC and without yeast tially mutagenic and degenerative, respectively (Leslie and powder; (5) 1% sucrose (saccharose) in sterile distilled

Summerell 2006; Seifert 2006; Wing et al. 1995). In water(SDW);and(6)SDWonly.BMwasMgSO4·7H2O, particular, PDA medium has been shown to induce the 0.5 g/L; NH4NO3 1g/L;KH2PO4, 1 g/L; yeast powder, formation of irregularly sized and shaped conidia. More- 1 g/L (Eudes et al. 2001). Liquid media inoculated with F. over, CMC based-medium is relatively costly, while natural graminearum mycelial plugs were incubated at 23°C on a products require complex steps in media preparation and shaker (230 rpm) for up to 2 weeks. The number of have only a slight effect on conidia production (Leslie and macroconidia per milliliter produced in the different media Summerell 2006). The objectives of this study were (1) to was assessed with a hemocytometer under Carl Zeiss use a sucrose-water suspension as an environmentally Axioscop2 with a Carl Zeiss AxioCAm IcC1 camera 3, 7, friendly and inexpensive method of stimulating rapid 10 and 14 days after inoculation (Zeiss, Jena, Germany). macroconidia formation in both F. graminearum chemo- Each treatment had three replicates and the experiment types; (2) to assess the germination rate, a good indicator of was repeated twice. macroconidia viability or infection capacity (Harris 2005); and (3) to investigate the effect of liquid media on the Macroconidia germination profile of mycotoxins. Germination assays on F. graminearum macroconidia were performed in U-bottom microplates (96-well) using potato Materials and methods dextrose broth (PDB) (Difco, Becton Dickinson). On day 7, macroconidia suspensions from BM amended with 1 g/L Fungal isolates and growth CMC and 1% sucrose medium for all six F. graminearum isolates were mixed with PDB in microplates at a ratio of Selected F. graminearum 3 and 15 (3- and 15-ADON) 1:1 (1 part macroconidia suspension: 1 part PDB). isolates were obtained from various sources as summarized Inoculated microplates were incubated at 23°C in darkness in Table 1. Two chemotypes were identified according to for an additional day prior to examination of spore the procedure outlined in Von der Ohe et al. (2010). F. germination. The percentage of germinated Fusarium graminearum isolates were grown and maintained on PDA macroconidia was obtained by scoring the spores in the (Difco, Becton Dickinson, Sparks, MD) at 4°C in darkness macroconidia suspension from microplates using 200× and for 1 week prior to the study. 400× objectives of Carl Zeiss Axioskop2 microscope and systematically choosing 50 macroconidia, starting at the top Macroconidia production right corner and continuing to count until 50. There were three replicates per treatment, and experiments were Mycelial plugs (~0.5 cm2) from the actively growing repeated twice. A macroconidium was considered germi- zone were cut and transferred into six different liquid nated only when the germ tubes exceeded half the length of media: (1) basal medium (BM) with 1 g/L CMC (Sigma, the macroconidium (approximately 10 μm). Germinated macroconidia were counted and recorded as a percentage of the total macroconidia number. Table 1 Fusarium graminearum 3- and 15-ADON (acetyldeoxynivalenol- producer) isolates used in this study Trichothecene mycotoxin extraction

F. graminearum isolate Accession no. Sourcea For mycotoxin analyses, only treatments with macroconidia 3-ADON chemotypes SMCD 2243 SMCD Saskatoon were selected for further studies. DON, ZEA, 3-ADON and wrs 2070 AAFC Winnipeg 15-ADON mycotoxins from treatments with macroconidia SIA-06-3 CCFC Guelph were extracted from the different liquid media on day 14 15-ADON chemotypes wrs 2085 AAFC Winnipeg using three 10 mL volumes of ethyl acetate (Vasavada and wrs 2073 AAFC Winnipeg Hsieh 1987). Samples were shaken vigorously, sonicated on M2-06-2 CCFC Guelph ice, and allowed to stand for 5 min for separation of phases. The organic phase was siphoned off and passed through a AAFC Agriculture and Agri-Food Canada, Winnipeg, MN, Canada sodium sulfate to remove water. The solvent was allowed to (Dr. A. Tekauz). CCFC University of Guelph Collection, Guelph, ON, evaporate at room temperature (23°C) for 3 days. The Canada (Dr. Lily Tamburic-Ilincic); SMCD Saskatchewan Microbial Collection and Database, University of Saskatchewan, Saskatoon, SK, residue was then re-dissolved in 2 mL acetonitrile for Canada (Dr. V. Vujanovic). HPLC analyses. Ann Microbiol (2012) 62:965–971 967

HPLC analyses and 10, with the exception of the strain wrs 2085 on day 3 (Table 2). Basal media supplemented with CMC triggered Standard trichothecene mycotoxins of DON, ZEA, 3- macroconidia production in some but not all F. graminea- ADON and 15-ADON were purchased from Sigma (HPLC rum isolates (Table 2). A high variation in abundance of grade; Oakville, ON). Mycotoxins extracted from different macroconidia was observed in all isolates tested and at all treatments were analyzed with a Water’s 2695 HPLC CMC concentrations. In contrast, 1% sucrose-water medi- system with: 250×4.60 mm, Luna 5 μ C18 (2) 100A um induced a relatively stable frequency, whereas the column (Phenomenex, Torrance, CA) and a photodiode- number of macroconidia (105/mL) ranged from 74 to 92 array (PDA) detector was used with an isocratic solvent in 3-ADON, and 41 to 64 in 15-ADON isolates by day 7 system [methanol: water-methanol containing 5% (v/v) (Table 2). After 7 days of incubation, macroconidia (90:10) ratio]. The PDA detector measured the UV produced by most F. graminearum isolates in 1% sucrose spectrum (190–500 nm). Samples were dissolved in medium began to germinate (Fig. 1). A subsequent macro- acetonitrile and 10 μL loaded onto the column using an conidia formation, statistically insignificant in terms of automatic injector. Mycotoxins were eluted with solvent or macroconidia abundance (P<0.05), was found on day 7 and mobile phase at a rate of 0.75 mL min−1 for 25 min. 14. No germination of macroconidia was detected in other Standard curves for respective mycotoxins were generated media at the incubation times tested in this study. based on five different concentrations of pure toxins, and Solid sucrose medium containing 15% agar allowed a absorbances obtained from HPLC analyses. reduced, but stable, macroconidia production in comparison with 1% sucrose-water broth, which was retained for Statistical analyses further assays.

The number of macroconidia produced by different F. Macroconidia germination graminearum isolates in six different media for four separate incubation times was not distributed normally. Differences in BM amended with 1 g/L CMC, or with 1% sucrose-water the number of macroconidia produced at each incubation medium, was found to induce macroconidia formation in all time for six F. graminearum isolates in the six different F. graminearum isolates, whereas no conidia germination media were analyzed using Kruskal-Wallis test (SPSS, was observed in the tested media on days 3 and 7 (Fig. 1). Chicago, IL). The quantities of different mycotoxins gener- Therefore, macroconidia from both treatments on day 7 ated by each individual F. graminearum isolate in four were transferred to microplates (96 well) with PDB to different media (basal media supplemented with 1, 7 and assess the ability of asexual spores to germinate. Macro- 15 g/L CMC), and one F. graminearum 3-ADON chemotype conidia of each F. graminearum isolate retrieved from both SMCD 2243 and one 15-ADON chemotype wrs 2085 in 1% BM-1 g/L CMC and 1% sucrose-water media germinated in sucrose medium were analyzed with ANOVA-LSD test. equal numbers (P<0.05, with t-test) (data not shown). However, quantities of different mycotoxins produced by individual F. graminearum isolates (other than SMCD 2243 Mycotoxin production and wrs 2085) for two media (BM with 1 g/L CMC and 1% sucrose medium) were assessed using a t-test (Norusis 1990). Mycotoxins were extracted from F. graminearum chemotype cultures showing macroconidia production. The different toxins were analyzed using HPLC to determine the quantity Results of toxins produced in each medium. The quantity of mycotoxins produced by F. graminearum chemotype 3 (3- Macroconidia production ADON-producer) and F. graminearum chemotype 15 (15- ADON-producer) isolates are summarized in Fig. 2.In Amounts of macroconidia produced by the six different F. general, the F. graminearum chemotypes tested produced graminearum isolates, grown in six different media and at trichothecene mycotoxins, DON, 3-ADON or 15-ADON, four separate incubation times, are summarized in Table 2. and ZEA in different concentrations depending on the type In general, 1% sucrose medium induced the highest amount of liquid medium (Fig. 2). In all treatments, a lower quantity of macroconidia formation in all six F. graminearum of ZEA than trichothecene mycotoxins was produced. isolates during the first 7 days of incubation (Table 2). Although there was no correlation between the number of Macroconidia were detected from day 2 of incubation in macroconidia produced in the different liquid media and the 1% sucrose-water medium, which was faster compared to quantity of different F. graminearum mycotoxins, 3-ADON other media (data not shown). The number of macroconidia SMCD 2243 (Fig. 2a1) and 15-ADON wrs 2085 (Fig. 2b1) produced by days 7 and 14 were higher compared to days 3 isolates accumulated the most mycotoxins. 968 Ann Microbiol (2012) 62:965–971

Table 2 Number of macroconidia produced by six different F. ± standard deviation. Means within each row of incubation time for graminearum isolates (Fgra3—chemotype 3 and Fgra15—chemotype each F. graminearum isolate in six different treatments followed by the 15) in six different media for four separate incubation periods. same letter are not significantly different at P<0.05 according to the Numbers in each column represented mean of macroconidia produced Kruskall-Wallis test. BM Basal medium

Fusarium graminearum Incubation time‡ Number of macroconidia (105/mL) isolate (days) BM+1 g/L BM+7 g/L BM+15 g/L BM+7 g/L CMC Sucrose1% Sterilized distilled CMC CMC CMC without yeast extract water

Fgra3 SMCD 2243 3 2.5±0.6 c 11.7±3.1 b 0 d 0 d 34.5±6.6 a 0 d 7 12.7±3.2 c 24.3±1.2 b 1±0.5 d 1±0.5 d 74±7.6 a 0 d 10 12.7±2.4 c 111±6.8 a 1±0.3 d 1±0.3 d 34±2.1 b 0 d 14 18±2 c 224.7±9.8 a 1±0.2 d 0 d 92±4.0 b 0 d wrs 2070 3 11.5±3.8 b 0 d 0 d 2±1.5 c 21±5.6 a 0 d 7 30±3.7 b 0 d 0 d 9±4.5 c 92.3±5.1 a 0 d 10 74.5±8.2 a 0 d 0 d 2.3±1.5 c 59±5.8 b 0 d 14 111+12 a 0 c 0 c 0 c 61.3±5.1 b 0 c SIA-06-03 3 0 b 0 b 0 b 0 b 30±8.3 a 0 b 7 6.7±1.7 c 11.3±2.1 b 0 d 0 d 74±4.8 a 0 d 10 21±1 b 33.5±1.6 a 0 c 0 c 37±2.5 a 0 c 14 15.7±1.3 c 30±3.5 b 0 d 0 d 67.2±6.0 a 0 d Fgra15 wrs 2085 3 2.5±1.3 b 2.5±0.8 b 3.17±1.2 b 0 c 50.5±1.6 a 0 c 7 8±0.8 bc 5±2 c 12±5.2 b 0 d 41±11.2 a 0 d 10 42±7.3 a 21.3±3.8 b 28±5.3 b 0 d 36.7±3.8 a 0 d 14 82.7±8.6 a 58±3.2 bc 41±6.2 c 0 d 67.2±4.7 b 0 d wrs 2073 3 10±2 b 0 d 2±1 c 0 d 26.1±3.4 a 0 d 7 22±5.8 b 0 d 3.17±0.6 c 0 d 64±3.3 a 0 d 10 43±3.3 a 0 c 6±1.3 b 0 c 42±5.4 a 0 c 14 73±4.1 a 0 d 8±2.5 c 0 d 59.3±3.8 b 0 d M2-06-2 3 1±0.9 b 0 b 0 b 0 b 34±8.7 a 0 b 7 14±2.6 b 0 c 0 c 0 c 56±8.5 a 0 c 10 30.5±4.3 a 0 b 0 b 0 b 28.5±1.6 a 0 b 14 51.7±5.2 a 0 c 0 c 0 c 36.7±4.3 b 0 c

Discussion chemotypes of F. graminearum using a very simple medium. Fusarium macroconidia, which are required for experimental The present work deals with the enhancement of macroconidia inoculations, are difficult to obtain even on complex media: production in both the 3- and the 15-acetyldeoxynivanenol therefore the sucrose-based medium would be very useful for both plant pathologists and cereal or maize breeders. Previous cultural methods for large-scale macroconidia production involve more complex steps in media preparation and expensive chemicals (Harris 2005). These include carnation leaf agar (CLA) and wheat bran, CMC and V8 medium (Eudes et al. 2001;Fisheretal.1982; Hassan and Bullerman 2009), and Bilay’s low-dextrose liquid medium proposed for field pathogenicity tests (Reid et al. 1992;ReidandZhu 2005). Hence, the search for an alternative medium was warranted. Another advantage of this medium is its capability of inducing macroconidiation in all the strains tested, which in contrast seemed erratic on the other media. In this study, a Fig. 1 Fusarium graminearum macroconidia produced in 1% sucrose 1% sucrose-water medium showed the earliest formation medium begin germination on day 8 at 23°C. Bar: 20 μm and highest number of macroconidia in all 3- and 15- Ann Microbiol (2012) 62:965–971 969 a1 b1

a2 b2

a3 b3

Fig. 2 Quantity of mycotoxins produced by F. graminearum chemo- inoculated in 1 g/L CMC (■), 7 g/L CMC (□), 15 g/L CMC ( ), and type 3 (a) and 15 (b) isolates in different treatments; F. graminearum 1% sucrose ( ). ZEA Zearalenone, DON deoxynivalenol, 3ADON 3- isolates used were (a1) SMCD 2243, (a2) wrs 2070, (a3) SIA-06-3, acetyldeoxynivalenol, 15ADON 15-acetyldeoxynivalenol (b1) wrs 2085, (b2) wrs 2073, (b3) M-06-02. These isolates were

ADON isolates tested. Maximal conidia production was Beside the enhanced and uniform macroconidia produc- achieved after 7 days and 14 days, which corresponds to the tion, the saccharose-based medium induced higher myco- ’s microcycle of sporogenesis. The weekly micro- toxin accumulation—an important advantage when testing cycle interval consists of sporulation–conidia germination– isolate virulence or aggressiveness. Variation in aggressive- sporulation steps. In agreement with a previous report by ness and mycotoxin production has been observed in Ward et al. (2008), the average number of conidia produced isolates of F. graminearum in Europe (Miedaner et al. was higher in 3- than in 15-ADON chemotypes. 2010) and North America (Von der Ohe et al. 2010). Since 970 Ann Microbiol (2012) 62:965–971 some mycotoxins, such as DON, affect isolate aggressive- in most isolates. A better understanding of DON biosyn- ness (Gardiner et al. 2009), using toxigenic fungal isolates thesis in F. graminearum induced by sucrose would have as a source of inoculum is advisable (Presello et al. 2011). implications in research seeking to develop toxin manage- Fusarium graminearum DON-non-producing isolates can- ment strategies. This medium is also highly concurrent with not spread within the wheat head (Bai et al. 2001) and natural (CLA and WB) media plates (Hassan and Bullerman cannot produce symptoms, suggesting that DON is a 2009), the handling of which is time-consuming; UV light virulence factor enhancing the ability of the fungal is a known mutagenic agent whereas potato-dextrose agar pathogen to colonize the host and cause disease (Nicholson leads to the development of irregular conidia (Leslie and 2009; Gale et al. 2011). In this study, higher mycotoxin Summerell 2006)ofF. graminearum isolates. Future concentrations are linked to the presence of a simple carbon greenhouse and field experiments would be useful in (sucrose) in the culture medium—an easily accessible validating the advantages and disadvantages of this medium energy source for the biosynthesis of Fusarium secondary for pathological studies using different crops or host species metabolites (Jiao et al. 2008; Ueno et al. 1975). Reverberi of F. graminearum across different environments. et al. (2010) reported that mycotoxin biosynthesis can be enhanced through frequent non-mycelial morphological and Acknowledgments This research was supported financially by a metabolic transition stages, and that it could be related with Natural Sciences and Engineering Research Council of Canada a microcycle of sporogenesis in F. graminearum observed Discovery Grant to V.V. The authors are thankful to Dr. A. Tekauz in the sucrose medium. Gardiner et al. (2009) demonstrated and Dr. L. Tamburic-Ilincic for providing fungal cultures. that low H+ ion concentration promotes DON production in F. graminearum. From that perspective, sucrose added to References water does not release H+ ion and the pH of the water remains unchanged. Bai G, Shaner G (1994) Scab of wheat: prospects for control. Plant Microbiologists and environmental scientists may profit Dis 78:760–766 equally from using this medium to distinguish suppressive Bai G, Shaner G (2004) Management and resistance in wheat and versus conductive conditions associated with Fusarium to Fusarium head blight. Annu Rev Phytopathol 42:135– toxicity and food contamination, especially with respect to 161 Bai G, Desjardins A, Plattner R (2001) Deoxynivalenol non- mycotoxin accumulation (Desjardins 2006). The 1% producing Fusarium graminearum causes initial infection but sucrose-water medium can be used for trichothecene and does not cause disease spread in wheat spikes. Mycopathologia zearalenone mycotoxin production in most F. graminearum 153:91–98 isolates in vitro. The sucrose-water medium induced high- Desjardins AE (2006) Fusarium mycotoxins. Chemistry, genetics, and biology, 424. American Phytopathological Society Press, St. est mycotoxin production in the 3-ADON population, Paul, MN noting ZEA, DON, and 3-acetyl-DON at ranges of 300- Desjardins AE, Proctor RH (2007) Molecular biology of Fusarium 500 μg/L, 600–1,000 μg/L, and 600–1,100 μg/L, respec- mycotoxins. Int J Food Microbiol 119:47–50 tively (Fig. 2). Environmental toxicology and plant pathol- Desjardins AE, Proctor RH (2011) Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from ogy especially could benefit from such a method, because it maize in Nepal and identification of a putative new lineage. can be employed for monitoring of mycotoxin formation in Fungal Biol 115:38–48 toxigenic isolates, alone or in combination with techniques Eudes F, Comeau A, Rioux S, Collin J (2001) Impact of trichothe- like mung bean agar (MBA) medium, on which F. cenes on Fusarium head blight (Fusarium graminearum) development in spring wheat (Triticum aestivum). Can J Plant graminearum does not produce DON mycotoxin (Evans Pathol 23:318–322 et al. 2000). Disruption of the genes encoding DON and/or Evans CK, Xie W, Dill-Macky R, Mirocha CJ (2000) Biosynthesis of zearelanone synthesis in 3- and 15-ADON chemotypes deoxynivalenol in spikelets of barley inoculated with macro- should allow generation of mutants (Maier et al. 2006)in conidia of Fusarium graminearum. Plant Dis 84:654–660 Fisher NL, Burgess LW, Toussoun TA, Nelson PE (1982) Carnation which the effects of the sucrose-water medium on F. leaves as a substrate and for preserving cultures of Fusarium graminearum can be further evaluated. species. Phytopathology 72:151–153 In conclusion, this study demonstrates the possibility of Foroud NA, Eudes F (2009) Trichothecenes in cereal grains. Int J Mol replacing basal CMC (CMC 1 g/L; CMC 7 g/L; CMC 15 Sci 10:147–173 Gale LR, Harrison SA, Ward TJ, O’Donnell K, Milus EA, Gale SW, g/L; and CMC 7 g/L without yeast extract) media, or time- Kistler HC (2011) Nivalenol-type populations of Fusarium consuming media containing natural substrates such as graminearum and F. asiaticum are prevalent on wheat in southern carnation leaves or wheat bran, with an affordable and Louisiana. Phytopathology 101:124–134 simple sucrose-water medium that does not affect the Gardiner DM, Osborne S, Kazan K, Manners JM (2009) Low pH regulates the production of deoxynivalenol by Fusarium grami- morphological/growth characteristics of the Fusarium che- nearum. Microbiology 155:3149–3156 motypes, and which promotes macroconidia formation, as Gilbert J, Abramson D, McCallum B, Clear R (2001) Comparison of well as high trichothecene and zearalenone accumulation, Canadian Fusarium graminearum isolates for aggressiveness, Ann Microbiol (2012) 62:965–971 971

vegetative compatibility, and producti on of ergosterol and ination caused by Fusarium spp. in maize. Euphytica 178:23–29. mycotoxins. Mycopathologia 153:209–215 doi:10.1007/s10681-010-0255-3 Harris SD (2005) Morphogenesis in germinating Fusarium graminea- Reid LM, Zhu X (2005) Screening corn for resistance to common rum macroconidia. Mycologia 97:880–887 diseases in Canada. Tech Bull Publications A42-103-2005E, p 27 Harris LJ, DesjardinsAE PRD, Nicholson P, Butler G, Young JC, Reid LM, Mather DE, Hamilton RI, Bolton AT (1992) Genotypic Weston G, Proctor RH, Hohn TM (1999) Possible role of differences in the resistance of maize silk to Fusarium grami- trichothecene mycotoxins in virulence of Fusarium graminearum nearum. Can J Plant Pathol 14:211–214 on maize. Plant Dis 83:954–960 Reverberi M, Ricelli A, Zjalic S, Fabbri AA, Fanelli C (2010) Natural Hassan YI, Bullerman LB (2009) Wheat bran as an alternative functions of mycotoxins and control of their biosynthesis in substrate for macroconidia formation by some Fusarium species. fungi. Appl Microbiol Biotechnol 87:899–911 J Microbiol Methods 77:134–136 Seifert K (2006) Fuskey: Fusarium interactive Key. Agriculture and Jiao F, Kawakami A, Nakajima T (2008) Effects of different carbon Agri-Food Canada, Ottawa. A42-66/1996E-IN. pp.17 sources on trichothecene production and Tri gene expression by Sutton JC (1982) Epidemiology of wheat head blight and maize ear Fusarium graminearum in liquid culture. FEMS Microbiol Letts rot caused by Fusarium graminearum. Can J Plant Pathol 4:195– 285:212–219 209 Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Ueno Y, Sawano M, Ishii K (1975) Production of trichothecene Blackwell, New York mycotoxins by Fusarium species in shake culture. Appl Micro- Maier FJ, Miedaner T, Hadeler B, Felk A, Saloman S, Lemmens M, biol 30:4–9 Kassner H, Schafer W (2006) Involvement of trichothecenes in Vasavada AB, Hsieh PH (1987) Production of 3-acetyldeoxynivalenol fusarioses of wheat, barley and maize evaluated by gene by Fusarium graminearum R2118 in submerged cultures. Appl disruption of the trichodiene synthase (Tri5) gene in three field Microbiol Biotechnol 26:517–521 isolates of different chemotype and virulence. Mol Plant Pathol Von der Ohe C, Gauthier V, Tamburic-Ilincic L, Brule-Babel A, 7:449–461 FerJnando WGD, Clear R, Ward TJ, Miedaner T (2010) A Miedaner T, Bolduan C, Melchinger AE (2010) Aggressiveness and comparison of aggressiveness and deoxynivalenol production mycotoxin production of eight isolates each of Fusarium between Canadian Fusarium graminearum isolates with 3-acetyl graminearum and Fusarium verticillioides for ear rot on and 15-acetyldeoxynivalenol chemotypes in field-grown spring susceptible and resistant early maize inbred lines. Eur J Plant wheat. Eur J Plant Pathol 127:407–417 Pathol 127:113–123 Ward TJ, Bielawski JP, Kistler HC, Sullivan E, O'Donnell K (2002) Nicholson P (2009) Fusarium and Fusarium–cereal interactions. In: Ancestral polymorphism and adaptive evolution in the trichothe- Encyclopedia of Life Sciences (ELS). Wiley, Chichester. cene mycotoxin gene cluster of phytopathogenic Fusarium. Proc doi:10.1002/9780470015902.a0021266 Natl Acad Sci USA 99:9278–9283 Norusis MJ (1990) SPSS/PC+4.0 Advanced Statistics Manual. SPSS, Ward TJ, Clear RM, Rooney AP, O’Donnell K, Gaba D, Patrick S, Chicago, IL Starkey DE, Gilbert J, Geiser DM, Nowicki TW (2008) An Ohara T, Inoue L, Namiki F, Kunoh H, Tsuge T (2004) REN1 is adaptive evolutionary shift in Fusarium head blight pathogen required for development of microconidia and macroconidia, but populations is driving the rapid spread of more toxigenic not of chlamydospores, in the plant pathogenic fungus Fusarium Fusarium graminearum in North America. Fungal Genet Biol oxysporum. Genetics 166:113–124 45:473–484 Presello DA, Pereyra AO, Iglesias J, Fauguel CM, Sampietro DA, Wing N, Burgess LW, Bryden WL (1995) Cultural degeneration in two Eyhérabide GH (2011) Responses to selection of S5 inbreds for Fusarium species and its effects on toxigenicity and cultural broad-based resistance to ear rots and grain mycotoxin contam- morphology. Mycol Res 99:615–620