The occurrence of culmorin and hydroxy-culmorins in cereals

Forty-five samples from 1988–1995 of naturally contaminated grain, barley, wheat and oats, three samples of mixed feed, and 16 samples of grain artificially inoculated with Fusarium culmorum during the flowering stage were analysed for deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-acetyl-DON), culmorin and hydroxy-culmorins. These compounds are secondary metabolites produced by the fungal species F. culmorum and F. graminearum. Acetonitrile-water extract of the samples was purified on a Mycosep^TM#225 column, derivetized using pentafluoropropionic anhydride (PFPA) and analysed by gas chromatography-mass spectrometry (GC-MS). The amount of each of culmorin, 5-, 12-, 14 and 15-hydroxy-culmorin and one unknown hydroxy-culmorin were determined relative to the amount of DON plus 3-acetyl DON for each sample. The ratio between the total amount of culmorin compounds and the DON compounds ranged from 0.14 to 1.07 in the samples. This study shows that there is a strong correlation between the amount of DON present in the grain and the amount of culmorin and hydroxy-culmorins present. The ratio of each of the culmorin compounds relative to the amount of DON compounds were in the same range in the grain artificially inoculated by F. culmorum as found in an earlier study for F. culmorum strains cultivated on rice, while the hydroxy-culmorin profile in the naturally contaminated grain was more similar to what was found for the F. graminearum cultures in the same study [1]. These results indicate that F. graminearum may be a relatively important source for DON in grain also in relatively cold areas. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chemotyping of Fusarium graminearum and F. culmorum Isolates from Turkey by PCR Assay

Fusarium graminearum and F. culmorum are the major causal agents of Fusarium head blight in Turkey. They produce trichothecenes such as deoxynivalenol (DON), nivalenol (NIV) and their several acetylated derivatives, 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON). In this study, a total of thirty-three isolates of F. graminearum and F. culmorum were collected from various regions and three different hosts. They were identified by amplification of tri5 gene cluster. Totally 32 isolates, 21 of F. culmorum and 11 of F. graminearum, were determined as DON chemotype, while only one F. graminearum isolate (1F) was detected as a NIV. A 282 base pair (bp) band for tri13 gene and also ranging from 458 to 535 bp bands for tri7 gene were amplified in all DON producers’ genomes. Further analysis of DON chemotype based on tri3 gene amplification showed that all isolates of F. graminearum displayed 15-ADON sub-chemotype. They yielded a 863 bp amplicon. Similarly, 3-ADON sub-chemotype was identified in F. culmorum’ isolates except F13. As a result of tri3 gene assay, it was produced a 583 bp fragment in these twenty isolates. It is the first report that a F. graminearum isolate depicts NIV chemotype in agricultural regions of Turkey. According to our findings, DON chemotype is predominating in our country. Also, it is presented that most of the F. graminearum isolates have 15-ADON sub-chemotype, while all F. culmorum’s belong to 3-ADON which possess full length amplicon of tri7 gene.     

Trichothecene production by Fusarium species isolated from grain and pasture throughout New Zealand

Liquid cultures of 200 Fusarium isolates selected to represent the most common species found in autumn pasture (70 isolates) and in grain (130 isolates) grown in New Zealand were analysed for trichothecenes and related compounds. Production of butenolide, cyclonerodiol derivatives and culmorins was also measured. The principal trichothecenes produced were derivatives of either nivalenol (NIV), deoxynivalenol (DON) or scirpentriol (Sctol), in order of frequency. The principal trichothecene producing species were F. crookwellense, F. culmorum and F. graminearum. Isolates of the first two species were predominantly NIV-chemotypes with one or two isolates respectively as Sctol-chemotypes. F. graminearum showed equal quantities of NIV- and DON-chemotypes, with the DON-chemotypes producing primarily 15-acetyldeoxynivalenol (15-ADON).     

Production of trichothecene mycotoxins byFusarium graminearum andFusarium culmorum on barley and wheat

Wheat cultivars (Stoa, MN87150, SuMai-3, YMI-6, Wheaton) and barley cultivars (Robust, Excel, Chevron, M69) were inoculated in the field with isolates ofFusarium graminearum andF. culmorum. The diseased (Fusarium head blight) kernels were analyzed for deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV).F. culmorum produced all three trichothecenes on all cultivars tested whereasF. graminearum only produced DON and 15-ADON. There was no well defined correlation between DON production in the host and resistance although the data tended to favor SuMai-3 as having definitive resistance to bothF. graminearum andF. culmorum.Minnesota Agricultural Experiment Station, Paper No. 20 279.     

Wheat-infectingFusarium species in Poland — their chemotypes and frequencies revealed by PCR assay

ThreeFusarium species:F. graminearum, F. culmorum andF. cerealis were identified in laboratory cultures and in sporodochia from spikelets of scabby wheat. SCAR (sequence characterized amplified region) primers were used to identifyFusarium species and nivalenol (NIV) and deoxynivalenol (DON) chemotypes within species in laboratory cultures and field collected heads harvested in 2006. Results from PCR analyses confirmed preliminary identifications of species on the basis of examination of macroconidia under a light microscope and identification of cultures on agar media. NIV and DON (3Ac-DON and 15Ac-DON) chemotypes were identified using PCR assay. Among samples and isolates ofF. graminearum, the 15Ac-DON chemotype dominated, and among those whereF. culmorum was identified, the 3Ac-DON chemotype prevailed. Only 5 of the 41 isolates ofF. graminearum tested, displayed the NIV chemotype. An increase in the frequency ofF. graminearum and a decrease in the frequency ofF. culmorum were found during 1998 to 2006.     

Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China

A large number of isolates from the Fusarium graminearum clade representing all regions in China with a known history of Fusarium head blight (FHB) epidemics in wheat were assayed using PCR to ascertain their trichothecene mycotoxin chemotypes and associated phylogenetic species and geographical distribution. Of the 299 isolates assayed, 231 are from F. asiaticum species lineage 6, which produce deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON); deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON); and nivalenol and 4-acetylnivalenol (NIV) mycotoxins, with 3-AcDON being the predominant chemotype. Ninety-five percent of this species originated from the warmer regions where the annual average temperatures were above 15 °C, based on the climate data of 30 y during 1970–1999. However, 68 isolates within F. graminearum species lineage 7 consisted only of 15-AcDON producers, 59 % of which were from the cooler regions where the annual average temperatures were 15 °C or lower. Identification of a new subpopulation of 15-AcDON producers revealed a molecular distinction between F. graminearum and F. asiaticum that produce 15-AcDON. An 11-bp repeat is present in F. graminearum within their Tri7 gene sequences but is absent in F. asiaticum, which could be directly used for differentiating the two phylogenetic species of the F. graminearum clade.     

Variation in 8-ketotrichothecenes and zearalenone production by Fusarium graminearum isolates from corn and barley in Korea

A total of 214 Fusarium graminearum isolates were obtained from corn and barley which were collected from Kangwon province and the southern part of Korea, respectively, and were tested for 8-ketotrichothecenes and zearalenone (ZEA) production on rice grains. The incidences of trichothecene production by 105 isolates of F. graminearum from corn were 59.0% for deoxynivalenol (DON), 37.1% for 15-acetyldeoxynivalenol(15-ADON), 13.3% for 3-acetyldeoxynivalenol (3-ADON), 7.6% for 3,15-diacetyldeoxynivalenol (3,15-DADON), 20.0% for nivalenol (NIV), 6.7% for 4-acetylnivalenol (4-ANIV), and 1.0% for 4,15-diacetylnivalenol (4,15-DANIV). DON chemotypes frequently produced 15-ADON as the major isomer rather than 3-ADON and 9 of the 61 DON chemotypes produced low levels of NIV. On the other hand, the incidences of trichothecene production of 109 isolates by F. graminearum from barley were 24.8% for DON, 72.5% for NIV, 62.4% for 4-ANIV, and 10.1% for 4,15-DANIV. Of these isolates, 78 were NIV chemotypes and only one isolate produced DON and 3-ADON as major toxins. In addition, 26 of the 78 NIV chemotypes produced low levels of DON. ZEA was frequently produced by the trichothecene-producing isolates and the incidences of ZEA were 51.4% and 31.2% for the isolates from corn and barley, respectively. There was a great regional difference in trichothecene production by F. graminearum isolates between corn- and barley-producing areas in Korea.     

Analysis of Pectic Enzyme Zymograms of Fusarium Species.

Zymograms of the extracellular polygalacturonase (PG), produced by isolates of F. culmorum and F. graminearum originating from different geographic locations and different sources, were compared. PG patterns were prepared by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) of untreated fluid from liquid pectin salts cultures. There was no intraspecific variability between isolates of both Fusarium species. Electrophoretic and isofocusing PG patterns were species specific. On the basis of IEF patterns, F. culmorum (4 isozymes, estimated pI's 6.4, 6.6, 6.9, 7.1) and F. graminearum (5 isozymes, estimated pI's 6.4, 6.6, 6.9, 7.1, 7.5) could be separated from one another by the pH 7.5 PG isozyme.     

Susceptibility of Barley Cultivars and Lines to Fusarium Infection and Mycotoxin Accumulation in Kernels

Heads of 12 barley genotypes (8 cultivars and 4 lines) were inoculated with conidial suspension of the following single isolates: F. culmorum no. 3, F. graminearum no. 122 and F. sporotrichioides no. ATCC 62 360. The number of kernels per head. 1000 Kernel weight and yield have been calculated for each genotype. Seed samples collected at harvest were analysed for each genotype. Seed samples collected at harvest were analysed for several trichothecene mycotoxins and zearalenone.The mycotoxin concentrations (mg/kg) in barley kernels inoculated with F. graminearum were as follows. deoxynivalenol (DON) 0.1 to 5.4 (av. 2.3). 3-acetyldeoxy-nivalenol (3-AcDON) 0.0–0.2 (av. 0.1), 15-acetyldeoxynivalenol (15-AcDON) 0.0–0.7 (av.0.2), nivalenol (NIV) 0.0–0.8 (av. 0.3). zearalenone (ZEA) 0.0–0.1 (av. 0.0); F. culmorum: DON 0.6 to 12.0 (av. 5.3), 3-AcDON 0.1 to 1.0 (av. 0.6). 15-AcDON nd. NIV 0.1–0.7 (av. 0.3). ZEA 0.1–0.5 (av. 0.2). F. sporotrichioides T-2 toxin 2.4–13.9 (av. 6.0), HT-2-toxin 0.1–0.8 (av.0.3) and neosolaniol 0.2–1.5 (av.0.7).     

Identification of Toxigenic Fusarium Species using PCR Assays

Isolates of the toxigenic cereal pathogens Fusarium culmorum, Fusarium graminearum, Fusarium crookwellense and Fusarium avenaceum, from Poland (48 isolates) and 12 from England, New Zealand, Italy and Canada, were examined using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), sequence-characterized amplified regions (SCARs), morphology and mycotoxin production under laboratory conditions. Their DNA products were compared by RAPD-PCR, which showed species-specific bands and the greatest diversity among isolates of F. avenaceum. PCR using three 20-mer-primer-pairs that are reported to be useful for identification of F. culmorum and F. graminearum group 2 confirmed their species-specificity. The same species-specific PCR product was observed in isolates of both nivalenol and deoxynivalenol chemotypes of F. culmorum or F. graminearum. A clear relationship was found between morphological and species-specific PCR identification of F. culmorum and F. graminearum isolates. However, F. avenaceum can be confused when using primers FA-ITS F/R (SCAR 2-14) with Fusarium tricinctum because the same band 272 bp appears in the gel, in both species probes.     

Competitive Aggressiveness in Binary Mixtures of Fusarium graminearum and F. culmorum Isolates Inoculated on Spring Wheat with Highly Effective Resistance QTL

Fusarium head blight (FHB) caused by Fusarium graminearum and F. culmorum is a devastating disease with high effects on grain yield and quality. We developed spring wheat lines incorporating the highly effective FHB resistance quantitative trait loci (QTL) Fhb1 and Qfhs.ifa‐5A. Whether these QTL lead to competition within Fusarium populations in the field resulting in isolates with higher aggressiveness has not been analysed. The aims of this study were to determine (i) the aggressiveness potential of F. graminearum and F. culmorum isolates, (ii) competition effects of these isolates in binary mixtures and (iii) the stability of resistant hosts. Six F. graminearum, two F. culmorum isolates and seven binary mixtures containing these isolates were tested for their aggressiveness and mycotoxin production at two locations in South Germany in 2007 and 2008. Host lines were four spring wheat lines containing the resistance QTL Fhb1 and/or Qfhs.ifa‐5A or none of them and one standard variety. Re‐isolates were sampled from plots inoculated with the binary mixtures to identify the percentage of each isolate in the mixture by simple sequence repeat markers. Resistant host lines reacted as expected and had a high stability to all isolates and mixtures. Only less important host × mixture interactions were detected. Aggressiveness among isolates and mixtures was significantly different. Type and amount of mycotoxin and high single isolate aggressiveness were not necessarily advantageous in the mixture. However, both F. culmorum isolates outcompeted F. graminearum isolates. Significant deviations from the inoculated 1 : 1 proportions occurred in 34 of 49 cases, illustrating that competition effects appeared in the mixtures. These differences depended mainly on the year and not on the level of host resistance. We conclude that resistance should not be affected by the Fusarium isolates and mixtures.     

Comparison of Canadian Fusarium graminearum isolates for aggressiveness,vegetative compatibility,and production of ergosterol and mycotoxins

Fusarium graminearum is the predominant pathogen causing fusarium head blight of cereals in North America. Fifteen Canadian isolates of Fusarium graminearum were highly diverse in terms of vegetative compatibility grouping (VCG) and varied for production of ergosterol and mycotoxin production in rice culture. Aggressiveness was assessed by scoring the disease severity incited in wheat spikes by each isolate. Two inoculation methods, single-floret injection and spray of entire spikes, were used to screen 4 wheat varieties for reaction to the F. graminearum isolates. All isolates were of broadly similar aggressiveness, with disease severity ranging from 17.2 to 39.1 for single floret injection, and 39.1 to 69.0 for spray inoculation. Disease severity, ergosterol production, and mycotoxin development were not correlated. Using nitrate non-utilizing mutants the 15 isolates were grouped into 14 VCGs. Deoxynivalenol (DON) was produced by all isolates in rice culture, at levels between 0.2 and 249 ppm. 15-acetyldeoxynivalenol was produced by 14 of the 15 isolates at levels between 0.4 and 44.6 ppm. These results reveal a high level of diversity for several characteristics among F. graminearum isolates from Canada. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pathogenicity of Fusarium Isolates from Wheat, Rye and Triticale Towards Seedlings and their Ability to Produce Trichothecenes and Zearalenone

37 Fusarium isolates (F. culmorum 20, F. graminearum 4, F. avenaceum 3, F. solani 4, and F. equiseti 6 from wheat, rye and triticale) were examined for pathogenicity and tested for zearalenone (F-2) and trichothecenes production in vitro. Strong pathogens (F. culmorum and F. graminearum) produced deoxynivalenol and 3-acetyl-deoxynivalenol and zearalenone in considerable quantities.     

Patterns of mycotoxin production by Fusarium gmminearum isolated from Argentine wheat

Fusarium graminearum was isolated from several wheat samples of the 1985/86 Argentine crop, taken from lots that had suffered extensive invasion by this fungus. Previous chemical analysis of the cereal, had revealed contamination with deoxynivalenol (DON), but the presence of the other mycotoxins could not be excluded with certainty, due to the low sensitivity of the analytical methodology employed.Twenty four F. graminearum isolates were grown on white corn with 50% water, for 21 days at 28 °C, or in liquid medium (Sucrose 3%, Peptone 0,1% and Yeast Extract 0,1%) for 7 days at 28 °C without shaking, and tested for the production of mycotoxins. Eight isolates (33% of the total) were found to produce toxins in one or both media. Toxins detected were: DON (6 isolates), 15-AcetylDON (5), 3-AcetylDON (2) and Zearalenone (3). No traces of Nivalenol, Fusarenon-X or other trichothecenes were found.These results suggest that strains of F. graminearum, prevailing in Argentine wheat-growing regions, might belong to the DON/AcetylDON chemotype, since no organisms of the Nivalenol/Fusarenon-X chemotype were detected in this study.     

Mycotoxins produced by <Emphasis Type="Italic">Fusarium</Emphasis> spp. associated with Fusarium head blight of wheat in Western Australia

An isolated occurrence of Fusarium head blight (FHB) of wheat was detected in the south-west region of Western Australia during the 2003 harvest season. The molecular identity of 23 isolates of Fusarium spp. collected from this region during the FHB outbreak confirmed the associated pathogens to be F. graminearum, F. acuminatum or F. tricinctum. Moreover, the toxicity of their crude extracts from Czapek-Dox liquid broth and millet seed cultures to brine shrimp (Artemia franciscana) was associated with high mortality levels. The main mycotoxins detected were type B trichothecenes (deoxynivalenol and 3-acetyldeoxynivalenol), enniatins, chlamydosporol and zearalenone. This study is the first report on the mycotoxin profiles of Fusarium spp. associated with FHB of wheat in Western Australia. This study highlights the need for monitoring not just for the presence of the specific Fusarium spp. present in any affected grain but also for their potential mycotoxin and other toxic secondary metabolites.     

Molecular identification and determination of the trichothecene chemotypes of fungi causing crown and root in different growth stages of wheat in north of Iran

In order to determine the crown and root agents and their mycotoxins produced in different growth stages of wheat including seedling, tillering and heading, sampling was done in north of Iran, during 2011–2012. From 160 isolates of Fusarium, eight species were obtained including F. graminearum, F. culmorum, F. equiseti, F. nygamai, F. semitectum, F. solani, F. acuminatum and F. oxysporum. Sampling at different growth stages showed that F. graminearum was the predominant causal agent of crown and root at the heading stage, whereas other species of Fusarium were mostly observed at the seedling and tillering stages. Moreover, identification of pathogenic species was confirmed using species-specific primers pairs. In F. graminearum isolates, presence of Tri13 gene, responsible for nivalenol (NIV) and deoxynivalenol (DON) mycotoxins biosynthesis, was detected using specific PCR primers. Finally, the ability of trichothecene production of five F. graminearum isolates was confirmed with high-performance liquid chromatography.     

Preservation of fungi in water (Castellani): 20 years

Sixty-two isolates of Fusarium were obtained from pasture grass and soil from various areas of New Zealand and identified as F. anthophilum [2], F. avenaceum [17], F. crookwellense [8], F. culmorum [4], F. graminearum [1], F. nivale [3], F. oxysporum [3], F. sambucinum [17], F. semitectum [1], F. tricinctum [1] and an unidentified Fusarium spp. [5]. These isolates were grown on autoclaved rice and tested for toxicity to rats in feeding tests. Eighty two percent of the isolates were toxic, of which twenty-four percent were severely toxic and caused hemorrhages of stomach and intestine, hematuria, and finally death. Cultures of the most toxic isolates contained 0.1 to 104 ppm of deoxynivalenol, 0.7 and 7 ppm of 15- and 3-acetyldeoxynivalenol respectively, 0.2 to 4 ppm of fusarenon- X, 11 to 1021 ppm zearalenone, 40 to 272 ppm of the hemorrhagic factor (wortmannin), 2,100 to 7,200 ppm of moniliformin, 565 ppm of the cytotoxic factor (HM-8) and enniatin in substantial concentrations. F. sambucinum is reported as a moniliformin producer for the first time.     

Effects of Fusarium graminearum Metabolites on Wheat Tissue in Relation to Fusarium Head Blight Resistance

Fusarium head blight (FHB) of wheat is caused by Fusarium graminearum which produces many secondary metabolites including the trichothecene mycotoxins deoxynivalenol (vomitoxin) and 3-acetyldeoxynivalenol. Coleoptile tissue segements from 14 spring wheat cultivars were exposed to the F. graminearum metabolites deoxynivalenol, 3-acetyldeoxynivalenol, butenolide (all known mycotoxins), sambucinol, culmorin and dihydroxycalonectrin in a bioassay. The tissue of most cultivars was inhibited, at a concentration of 10^?6M by the trichothecenes tested and up to 10^?3M for the other compounds. Deoxynivalenol and 3-acetyldeoxynivalenol, which affect protein synthesis at the ribosome, are therefore potent phytotoxins in addition to being mycotoxins. The resistance or susceptibility of each cultivar to FHB was established in a field experiment. A comparison of the two sets of data indicated that resistant cultivars could tolerate much higher concentrations of the metabolites tested than susceptible cultivars. Some resistant material can tolerate 10 to 1000 times the concentration of the trichothecenes, compared with susceptible cultivars, with no effect on growth. The data suggest that it may be possible to screen germplasm rapidly for FHB resistance in vitro and a new type of resistance in wheat to this disease is proposed based on the apparent insensitivity to trichothecenes by resistant cultivars, additional to the three types of resistance described in the literature.     

Production of trichothecene and non-trichothecene mycotoxins by Fusarium species isolated from maize in Minnesota

Eighty-two cultures of Fusarium species isolated in 1986 from moldy maize in Minnesota were each cultured on rice for 4 weeks and found to produce the following mycotoxins: F. graminearum isolates, deoxynivalenol (DON, 4–225 g/g), 3-acetyldeoxynivalenol (3-ADON, 2–4g/g), 15-acetyldeoxynivalenol (15-ADON, 1–35 g/g) and zearalenone (ZEA, 5–4350 g/g); F. moniliforme, fusarin C (detectable amounts to 1000 g/g); F. mòniliforme, F. oxysporum, F. proliferatum and F. subglutinans isolates, moniliformin (15–6775 g/g); F. moniliforme, F. proliferatum, and F. subglutinans isolates, fusaric acid (detectable amounts). Other mycotoxins screened for in each rice sample and not detected were T-2 toxin, HT-2 toxin, neosolaniol, T-2 tetraol, nivalenol, fusarenon-X, scirpenols, alpha and beta trans-zearalenols, wortmannin, and fusarochromanone. The rat feeding bioassay indicated that other, unidentified toxins may be present.     

Variability and characterization of mycotoxin-producing Fusarium spp isolates by PCR-RFLP analysis of the IGS-rDNA region

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB₁, FB₂, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be␣divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA␣and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.