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).     

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.     

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.     

Production of culmorin compounds and other secondary metabolites by Fusarium culmorum and F. graminearum strains isolated from Norwegian cereals

Twenty-three Fusarium culmorum and 21 F. graminearumisolates were studied for their ability to produce mycotoxins and other secondary metabolites. The strains were cultivated on rice, and the extracts analysed by gas chromatography mass spectrometry (GC-MS) after derivatization with pentafluoropropionic (PFP) reagent. Two F. culmorum strains formed nivalenol and its acetylated derivatives (chemotype II), while all F. graminearum and the otherF. culmorum isolates produced deoxynivalenol (DON) via 3-acetyldeoxynivalenol (3-acetyl-DON) (chemotype IA). 15-hydroxy-culmorin, followed by 5-hydroxy-culmorin were the main other metabolites produced F. culmorum, while 5-, 12- and an unidentified hydroxy-culmorin, suggested to be 14-hydroxy-culmorin, were the main metabolites of F. graminearum. The hydroxy-culmorin profile was found to be significantly different for the two Fusarium species. Minor amounts of about ten other hydroxy-culmorins, four hydroxy-culmorones and 3,13-dihydroxy-epiapotrichothecene were also detected in most cultures. Traces of sambucinol seemed to be present in some of the isolates, but were not detected in any significant amounts. The precursors in the biosynthetic sequence to 3-acetyldeoxynivalenol,7,8-dihydroxycalonectrin and 15-deacetyl-7,8-dihydroxycalonectrin,were detected in most cultures. We also report the assignment of both the ¹H and¹³C NMR data of 15-deacetyl-7,8-dihydroxycalonectrin, which has only been reported incorrectly before. This revised version was published online in August 2006 with corrections to the Cover Date.     

Toxigenic Fusarium species infecting wheat heads in Poland

Toxigenic Fusarium species are common pathogens of wheat and other cereals worldwide. In total, 449 wheat heads from six localities in Poland, heavily infected with Fusarium during 2009 season, were examined for Fusarium species identification. F. culmorum was the most common species (72.1% on average) with F. graminearum and F. avenaceum the next most commonly observed, but much less frequent (13.4 and 12.5% respectively). F. cerealis was found in 1.8% of all samples, and F. tricinctum was found only in one sample (0.2%). Subsequent quantification of the three major mycotoxins (deoxynivalenol, zearalenone and moniliformin) in grain and chaff fractions with respect to associated prevailing pathogen species uncovered the following patterns. Moniliformin (MON) was found in low amounts in all samples with F. avenaceum present. In contrast, deoxynivalenol (DON) and zearalenone (ZEA) were the contaminants of F. culmorum- and F. graminearum-infected heads. The highest concentration of DON was recorded in grain sample collected in Radzików (77 µg g^?1). High temperatures in Central Poland during July and August accompanied with high rainfall in July were responsible for this high DON accumulation. Trichothecene, zearalenone, enniatin and beauvericin chemotypes were identified among 21 purified isolates using gene-specific PCR markers.     

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.     

Trichothecene mycotoxins in kernels and head fusariosis susceptibility in winter triticale

A single isolates ofFusarium graminearum Schwabe KF 366 andFusarium culmorum (W.G.Sm.) Sacc. KF 365 were used to infect 10 genotypes (9 lines and one cultivar) of winter triticale, 1 rye cultivar and 1 wheat cultivar, and amounts of mycotoxins in kernels were analysed at the same stage of development. One genotype of triticale CHD 353/79 and rye “Chodan” were found to be most resistant towards both species infection with lowest amount of mycotoxins (deoxynivalenol) content in kernels and also the lowest yield reduction. The most susceptible line CZR 142 cumulated in kernels about ten times higher amount of mycotoxins (up 53 mg DON/kg and 16 mg 3AcDON/kg, and 5 mg zearalenone/kg). GenerallyF, culmorum formed higher level of mycotoxins in kernels of infected heads thanF. graminearum. In kernels of more susceptible genotypes except deoxynivalenol, 3 acetyldeoxynivalenol and zearalenone also were present.     

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.     

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.     

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.     

Zearalenone formation by<Emphasis Type="Underline">Fusarium crookwellense</Emphasis> /Burgess,Nelson and Toussoun/ isolates from Poland and their pathogenicity towards cereals

Fusarium crookwellense /B.N. and T./ isolated from affected cereals in Poland formed zearalenone on wheat grain up to 602 mg/kg. Tested isolates have been found strong to severe pathogens of wheat, rye, triticale and barley seedlings and corn ears with pathogenicity similar to that ofF. culmorum andF. graminearum.     

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.     

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.     

Fusarium species and their mycotoxins in infected corn in Italy

Surveys of corn (infected plants and commercial kernels) forFusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries.Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). TheFusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were:F. moniliforme;F. culmorum (ZON, ZOL, DON, 3AcDON);F. equiseti (ZON, ZOL); andF. proliferatum (MF). Along with these species,F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON);F. chlamydosporum;F. acuminatum (type-A trichothecene derivatives); andF. semitectum were often found to be associated.F. heterosporum (ZON, ZOL);F. solani;F. crookwellense (ZON, ZOL, FUS, NIV);F. oxysporum (MF);F. avenaceum (MF);F. sporotrichioides (T-2 toxin and derivatives); andF. poae (DAS, MAS) were occasionally isolated.     

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).     

Occurrence of toxigenic strains of Fusarium in maize and barley in Germany

Summary A survey was made of maize and barley in Germany for the occurrence of toxigenic strains of Fusarium and of the mycotoxins produced in culture by these strains.The following 6 species of Fusarium were found: F. avenaceum, F. culmorum, F. equiseti, F.oxysporum, F. poae, and F. tricinctum. The species most commonly isolated from bird-damaged maize ears was F. avenaceum while F. culmorum was consistently isolated from maize stem rot. The predominant species in barley grain was F. poae while F. avenaceum, F. culmorum, and F. tricinctum were also isolated frequently.Cultures on autoclaved maize of all the Fusarium strains were assayed for toxicity by feeding to 1-day-old chickens for 14 days. Some strains of F. avenaceum, F. culmorum, F. equiseti, and F. oxysporum proved to be acutely toxic to chickens and caused mortality as well as marked reductions in weight gain and feed consumption. All the strains of F. poae and F. tricinctum had a low degree of toxicity.Culture material of all the strains were analyzed for the presence of 11 known Fusarium mycotoxins. The following 4 mycotoxins were detected in the strains examined: moniliformin in 9 out of 9 F. avenaceum strains (2 to 760 ppm) and in the single strain of F. oxysporum (1150 ppm); zearalenone in 4 out of 5 F. culmorum strains (320 to 1400 ppm); deoxynivalenol in 3 out of 5 F. culmorum strains.(1 to 15 ppm); and acetyldeoxynivalenol (1 to 2 ppm) in 3 out of 5 F. culmorum strains. This is the first report of moniliformin production by F. avenaceum and F. oxysporum and also the first report of the occurrence of moniliformin-producing Fusarium strains in Europe.     

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.     

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.     

Zearalenone-producing species of Fusarium on barley seed

Isolates of Fusarium were obtained from samples of barley grain at harvest in 1971, 1973 and 1974. The ability of these isolates to produce the myco-toxin zearalenone was examined, the grain also being tested for its presence in 1974. The most common isolate was F. culmorum, 60% of which in 1974 produced zearalenone. Other species isolated which produced this myco-toxin were F. monilijorme, F. graminearum, F. avenaceum, F. nivale and F. sambucinum var coeruleum, the last three species have not previously been recorded as producing zearalenone.     

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.