Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.     

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.     

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 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.     

Effects of infection time and moisture on development of ear blight and deoxynivalenol production by Fusarium spp. in wheat

Wheat ears were inoculated with conidia of Fusarium spp. at different growth stages between ear emergence and harvest and moist conditions were maintained for up to 7 days subsequently by mist irrigation. Of the fungi tested (Fusarium culmorum, F. avenaceum, F. tricinctum, F. sporotrichioides and Microdochium nivale), only F. culmorum produced ear blight symptoms and grain samples were found subsequently to contain deoxynivalenol. Most ear infection and deoxynivalenol formation occurred following inoculation at about mid-anthesis. Small amounts of deoxynivalenol were formed and some F. culmorum was isolated even in the absence of ear blight symptoms. An overnight wet period was sufficient to initiate infection and deoxynivalenol formation but both were increased by extending the wet period up to at least 3 days. Recovery of Fusarium spp. from harvested grain was usually possible whether or not symptoms developed. F. culmorum usually persisted and often increased to moderately high levels after storage for 7 wk in a range of moisture conditions.     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.     

Degeneration of Streptomyces niveus with Repeated Transfers

Novobiocin production by Streptomyces niveus decreased drastically as the culture was transferred at regular intervals under both sporulating and nonsporulating conditions. Addition of degenerated live mycelium as second inoculum to shake flask fermentations already inoculated with a high-producing strain resulted in sharply depressed novobiocin formation. Fractionated medium of low-producing strain containing either no cells or dead cells had no adverse effect on the antibiotic yield of the high-producing mycelium. It appears that the low-producing mycelium was outgrowing the high-producing mycelium. A study of the growth rates of the two types of mycelium in a clear broth medium indicated no differences in generation time. However, the low-producing strain proved to have a higher efficiency of carbohydrate utilization, thus overgrowing the high-producing strain. It was speculated that culture instability of S. niveus is due to heterocaryosis.     

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.     

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 zearalenone,nivalenol, moniliformin,and wortmannin from toxigenic cultures ofFusarium obtained from pasture soil samples collected in New Zealand

One culture ofF avenaceum, 4 cultures ofF oxysporum, and 11 cultures of Fsambucinum were isolated from soil samples of pasture in New Zealand in 1987. All cultures, when grown on rice media and fed to rats caused a weight loss in rats as well as toxic signs including hemorrhaging and congestion, uterine enlargement, and hematuria. 6 out of 16 cultures caused death in rat feeding tests.F oxysporum #1 killed rats (feeding test) within 5-12hrs. 10 cultures produced zearalenone (19 to 8,849 ppm), 8 cultures produced nivalenol (32 to 117 ppm), 1 culture,F sambucinum #8, produced wortmannin (40 ppm), and 5 cultures produced moniliformin (19 to 9,000ppm). We report for the first time the co-occurrence of zearalenone, nivalenol, and moniliformin produced byF sambucinum #3 in culture.F avenaceum #1 andF oxysporum cultures (nos 1, 2, and 3) produced moniliformin alone.F oxysporum #4 produced zearalenone alone as well.F sambucinum #5 caused erythema in the small intestine of rats and 100% mortality and did not produce any known toxin(s). Nivalenol when administered to the stomach of rats orally at levels 10, 20, and 40mg/kg body weight caused inflammation in the intestines, coma, and death. The mycotoxins T-2 toxin, HT-2 toxin, T-2 tetraol, diacetoxyscirpenol (DAS), monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyl-and 15-acetyldeoxynivalenol, depoxynivalenol, fusarenon-X, alpha-and beta-zearalenone, and fusarochromanone (TDP-1) were not detected in the extracts of these cultures.     

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.     

Mycotoxigenic Fusarium and Deoxynivalenol Production Repress Chitinase Gene Expression in the Biocontrol Agent Trichoderma atroviride P1

Mycotoxin contamination associated with head blight of wheat and other grains caused by Fusarium culmorum and F. graminearum is a chronic threat to crop, human, and animal health throughout the world. One of the most important toxins in terms of human exposure is deoxynivalenol (DON) (formerly called vomitoxin), an inhibitor of protein synthesis with a broad spectrum of toxigenicity against animals. Certain Fusarium toxins have additional antimicrobial activity, and the phytotoxin fusaric acid has recently been shown to modulate fungus-bacterium interactions that affect plant health (Duffy and Défago, Phytopathology 87:1250-1257, 1997). The potential impact of DON on Fusarium competition with other microorganisms has not been described previously. Any competitive advantage conferred by DON would complicate efforts to control Fusarium during its saprophytic growth on crop residues that are left after harvest and constitute the primary inoculum reservoir for outbreaks in subsequent plantings. We examined the effect of the DON mycotoxin on ecological interactions between pathogenic Fusarium and Trichoderma atroviride strain P1, a competitor fungus with biocontrol activity against a wide range of plant diseases. Expression of the Trichoderma chitinase genes, ech42 and nag1, which contribute to biocontrol activity, was monitored in vitro and on crop residues of two maize cultivars by using goxA reporter gene fusions. We found that DON-producing F. culmorum and F. graminearum strains repressed expression of nag1-gox. DON-negative wild-type Fusarium strains and a DON-negative mutant with an insertional disruption in the tricothecene biosynthetic gene, tri5, had no effect on antagonist gene expression. The role of DON as the principal repressor above other pathogen factors was confirmed. Exposure of Trichoderma to synthetic DON or to a non-DON-producing Fusarium mutant resulted in the same level of nag1-gox repression as the level observed with DON-producing Fusarium. DON repression was specific for nag1-gox and had no effect, either positive or negative, on expression of another key chitinase gene, ech42. This is the first demonstration that a target pathogen down-regulates genes in a fungal biocontrol agent, and our results provide evidence that mycotoxins have a novel ecological function as factors in Fusarium competitiveness.     

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.     

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.     

Deoxynivalenol and 3-acetyldeoxynivalenol and Fusarium species in winter triticale

Fusarium species infecting heads of Triticale and mycotoxins presence in infected kernels and chaff were studied during two seasons. The most important species observed on infected heads were in 1986F. avenaceum (39%),F. nivale (21%),F. culmorum (20%),F. graminearum (14%), and others (6%). In 1987 after long and snowy winterF. nivale dominated (64%), followed byF. avenaceum (24%),F. culmorum (6%), andF. graminearum (5%). The mycotoxins deoxynivalenol (DON) and 3-acetyl DON were present in all 11 subsamples of kernels from heads infected byF. culmorum and/orF. Graminearum (1.6–16.4 mg and 0.7–2.4mg/kg, respectively). Chaff from the same subsamples contained 9.9–33.2mg/kg of DON and 5.2–16.0mg/kg of 3-AcDON. Kernels with visibleFusarium-damage contained 2.4–31.2 mg/kg of DON and 1.2–6.0 mg/kg of 3-AcDON. Remaining part of kernels without symptoms of visibleFusarium-damage contained only DON in an amount of 0.9–5.9 mg/kg.     

Mycotoxins produced by Discolor section Fusarium species

An improved TLC method ofFusaria metabolites detection and quantitation has been elaborated. A total 92 isolates of Discolor sectionFusaria from cereals and potato have been examined from the point of view of cultures morphology and ability to produce characteristic mycotoxins. Low nutrient media (CLA, SNA) were found as suitable for production of uniform and typical macroconidia in studied cultures. All 26 isolates ofF. sambucinum Fuckel (=F.sulphureum, Schlecht) formed diacetoxyscirpenol in amount 20-1000 mg/kg and all 17.F. crookwellense Burgess N. & T. produced zearalenone (16-602 mg/kg).F. graminearum Schwabe produced: zearalenone 14/14 isolates, deoxynivalenol 11/14 isolates, both up to 77 mg/kg. Out of 26F. culmorum cultures originating from Poland 22 produced zearalenone up to 675 mg/kg, 17/26 3 acetyldeoxynivalenol up to 280 mg/kg and 16/26 deoxynivalenol up to 220 mg/kg. The difference in metabolism agrees with the difference in morphology of those species.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Comparative Analysis of Genetic Chemotyping Methods for Fusarium: Tri13 Polymorphism Does not Discriminate between 3‐ and 15‐acetylated Deoxynivalenol Chemotypes in Fusarium graminearum

Genetic chemotyping is an essential tool for characterizing Fusarium populations causing head blight on wheat and other cereals. Three PCR methods, based on tri cluster polymorphism, were optimized and compared on 94 single‐spore isolates obtained from three continents belonging to F. gramineaurm, F. culmorum, F. poae, F. avenaceum and Microdochium nivale. While the methods based on the tri3, tri7 and tri12 polymorphism correctly identified all the tested strains, the method based on tri13 polymorphism was unable to discriminate between the 3‐ and 15‐acetylated DON forms in F. graminearum. It is advised to avoid the use of tri13 polymorphism for genetic chemotyping of the two acetylated chemotypes.     

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.     

Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum.

The inhibitory effect of L-lysine on penicillin biosynthesis by Penicillium chrysogenum has been compared in a low-producing strain (Wis. 54-1255) and a high-producing strain (ASP-78). Lysine inhibited total penicillin synthesis to a similar extent in both strains. However, in the high-producing strain the onset of penicillin synthesis occurred even at a high lysine concentration, whereas in the low-producing strain lysine had to be depleted before penicillin production commenced.