Tri5 Gene Research Articles

PCR Analysis of the Tri13 gene to Determine the Genetic Potential of Fusarium graminearum Isolates from Iran to Produce Nivalenol and Deoxynivalenol

Fusarium graminearum trichothecene producing isolates can be broadly divided into two chemotypes based on the production of the 8- ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). Functional Tri13 gene required for the production of NIV and 4- acetyl NIV, whereas in the isolates producing DON and its acetylated derivates, this gene is nonfunctional. In this study, a total of 57 isolates from different fields of Mazandaran province, Iran were identified as F. graminearum using classical methods and species specific primers. In order to assess the potential of isolates to produce NIV or DON, we used PCR to determine whether isolates carried a functional or nonfunctional Tri13 gene. Out of the 57 tested F. graminearum isolates with Tri13 PCR assays, 46 yielded an amplicon similar to the size predicted for nivalenol production, while 11 yielded an amplicon similar to the size predicted for deoxynivalenol production. From regions where more than one F. graminearum isolate was obtained, isolates were not exclusively of a single chemotype. It seems that genetic diversity among the isolates has relation with geographical region and wheat cultivar. The assay can provide information about the distribution of Tri13 haplotype that can be used in tracing of trichothecene contaminated samples.

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue.

Transcriptome Analysis of Trichothecene-Induced Gene Expression in Barley

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

Magnesium represses trichothecene biosynthesis and modulates Tri5, Tri6, and Tri12 genes expression in Fusarium graminearum

Growth and production of type-B trichothecenes mycotoxins by the Fusarium graminearum strain CBS 185.32 were compared in GYEP medium supplemented with Mg(2+) at different concentrations (0-4 mM). Mg(2+) led to a strong decrease in toxin accumulation without affecting the mycelial growth, suggesting a specific Mg(2+) effect on fungal secondary metabolism. Expression of Tri5, Tri6, and Tri12 genes was followed throughout the time courses of type-B trichothecenes (TCTB) yield in standard and 2 mM Mg(2+)-supplemented GYEP media. Mg(2+) addition significantly decreased Tri5, Tri6, and Tri12 expression. The inhibition of toxin production by Mg(2+ )was shown to be highly correlated with inhibition of Tri5 transcription and, to a lesser extend, of Tri6 and Tri12. This is the first report of a transcriptional control of TCTB production by Mg(2+).

Detection of toxigenic fungi and quantification of type A trichothecene levels in some food and feed materials from India

In this study, the direct detection of toxigenic fungi and the quantification of trichothecene levels in food and feed materials from India were investigated. A total of 40 different food and feed materials were subjected to PCR for the detection of toxigenic Fusarium spp. PCR indicated the presence of tri5 gene in seven samples (six sorghum samples and one poultry feed). Results indicated the presence of T-2 toxin and diacetoxyscirpenol (DAS) in all the six sorghum samples whereas only T-2 content was noticed in poultry feed. Levels of T-2 and DAS varied from 0.012 (±0.004) to 0.13 (±0.03) mg/kg and 0.014 (±0.004) to 0.084 (±0.004) mg/kg, respectively. The levels of these toxins were falling within the range detected previously in India and in other parts of the world.

Molecular and Genetic Studies ofFusariumTrichothecene Biosynthesis: Pathways, Genes, and Evolution

Trichothecenes are a large family of sesquiterpenoid secondary metabolites of Fusarium species (e.g., F. graminearum) and other molds. They are major mycotoxins that can cause serious problems when consumed via contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene Tri5 encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes (Tri genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the Tri5 gene cluster. At least three pathway genes, Tri101 (separated alone), and Tri1 and Tri16 (located in the Tri1-Tri16 two-gene cluster), were found outside of the Tri5 gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned Tri genes, and the evolution of Tri genes, focusing on Fusarium species.

Applying Real-Time Quantitative PCR to Fusarium Crown Rot of Wheat.

Fusarium crown rot (FCR) of wheat is a persistent problem that causes significant losses worldwide. In Montana, FCR is caused primarily by Fusarium culmorum and F. pseudograminearum. Recently, a real-time quantitative PCR (QPCR) assay was developed for FCR using primers and probes specific for a segment of the trichodiene synthase (tri5) gene. The purpose of this study was to determine the utility of QPCR for accessing FCR severity on wheat in field experiments. In 2004 and 2005, plots of spring and durum wheat were inoculated with varying levels of F. pseudograminearum oat inoculum and grown under rain-fed conditions. Two weeks prior to harvest, plants were collected from the plots and assessed for FCR severity and analyzed by QPCR for Fusarium DNA quantities. Disease severity scores (DSS) and Fusarium DNA quantities were positively correlated with each other for all three cultivars in 2004 but for only the durum cultivar in 2005 (P < 0.05). In 2004, grain yields for both spring wheat cultivars were negatively correlated with Fusarium DNA quantities (P > 0.05). When DSS and Fusarium DNA quantities negatively correlated with yield, both measurements were comparable in predicting yield reduction (R = -0.64 and -0.77, respectively). Results indicate that this QPCR assay is effective in measuring FCR severity in wheat.

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.

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.

Contamination Evaluation of Lyophilizated Processed-Foods

P-142 Introduction: The new technological acquisitions in alimentary field have encouraged researchers to study those transformation processes useful to the conservation of foodstuffs without losing their nourishing value and their microbiological safety. One of the conservation techniques is lyophilizing, which offers the advantage of remarkably reducing food volume while increasing its lifespan without altering its structure. However, lyophilized foodstuffs are not free from polluting agents, since conservation processes are not always effective on the latter if they are present in the primary products. Objective of the study is to estimate the presence of the tri-5 gene, and concentration of Zearalenon (ZEA), Nivalenol (NIV) and Deossinivalenol (DON), metals (Pb, Cd, As and Hg) and chlorinated pesticides, both, in the primary products used for the preparation of lyophilized foodstuffs and in the final products. Methods: The tri-5 gene search has been carried out through PCR. Mycotoxines ZEA, DON and NIV have been extracted and purified by using specific immunoaffinity columns and quantified by means of high performance liquid chromatography with fluorescence and UV detector. Metals (Pb, Cd, As) have been detected through atomic absorption spectrophotometry with grafite furnace while the mercury has been detected through cold vapors technique, after acid digestion. Chlorinated pesticides have been extracted through FAST-PSE, purified through GPC, identified and quantified through GC with ECD. Results: The Tri-5 gene is always present in prontocrepes and always absent in prontocone. With the air treatment a reduction of DON and NIV in both products has been obtained. The nitrogen treatment has had a single infuence on the DON and in both products, in which has had a reduction to 50%. While, the NIV with the same treatment has reached the lowest percentage of reduction in both products. Cadmium has been found to inferior concentration, regarding the ingredients, in both products. Lead has been found to greater concentration in both products and with both treatments. Arsenic results inferior to the sensibility limit of method in all the samples. Mercury reach inferior concentration in the air treatment and more elevated concentration in the nitrogen treatment. Chlorurated pesticides has been found to elevated concentrations regarding the ingredients. Discussion and Conclusions: The treatments influence relatively the contamination of processed-foods that, therefore, sure it depends on the first quality of the primary products.

Enhanced mycotoxin production of a lipase-deficient Fusarium graminearum mutant correlates to toxin-related gene expression

Fusarium graminearum causes important diseases of small-grain cereals and maize and produces several mycotoxins. Among them, deoxynivalenol (DON) and zearalenone (ZEA) can accumulate in feedstuffs and foods to health-threatening levels. Although DON is important for fungal virulence in wheat, disease severity in the field does not correlate with mycotoxin concentrations. We compared gene expression and mycotoxin production of lipase-deficient mutants (Dfgl1), strongly reduced in virulence, and the respective wild-type isolate. Dfgl1 mutants exhibited up-regulated DON production during wheat head infection. On isolated wheat kernels, DON was only produced in low quantities, but higher in wild-type than in Dfgl1 mutants. In contrast, neither wild-type nor Dfgl1 mutants produced ZEA during wheat head infection. However, ZEA was clearly detectable on wheat kernels. Here, Dfgl1 mutants revealed a dramatically enhanced ZEA production. We could correlate the altered amounts of DON and ZEA directly with the expression of the toxin-related genes Tri5 for DON and PKS4 and PKS13 for ZEA. Based on Tri5 expression and the infection pattern of the wild-type and Dfgl1 mutants, we suggest that the transition zone of rachilla and rachis is important in the induction of DON synthesis. Gene expression studies indicate an involvement of the lipase FGL1 in regulation of 8 PKS genes and ZEA production. Abbreviations: FHB – Fusarium head blight; DON – deoxynivalenol; ZEA – zearalenone; PKS – polyketide synthase; Dpi – days post-inoculation

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

SUMMARY Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.

A role for the mycotoxin deoxynivalenol in stem colonisation during crown rot disease of wheat caused by Fusarium graminearum and Fusarium pseudograminearum

Fusarium graminearum ( Fg) can cause head blight and crown rot (CR) diseases of wheat but fungal colonisation and mycotoxin production by Fg during CR are little understood. Studies of an Australian strain of Fg demonstrated that expression of the Tri5 gene of Fg and deoxynivalenol (DON) production were induced during infection of the stem base and to levels equivalent to those observed in inoculated heads. To study fungal colonisation and DON production in CR disease, we inoculated stem bases, 14 days after sowing, with macroconidia of Australian and USA strains of Fg and of an Australian strain of Fusarium pseudograminearum, a related pathogen frequently associated with CR. At maturity, the fungal pathogen was subsequently detected by isolation on growth media in high percentages of plants at the stem base and the lowest node adjacent to the inoculation site (50–100%), in the non-inoculated flag-leaf node (FLN) (35–95%) and in asymptomatic non-inoculated mature heads (50–60%) and kernels (20%). Microscopic analysis suggested that colonisation of upper nodes occurred primarily via the pith parenchyma and lumen. Significant concentrations of DON (up to 35 ppm) were detected in the FLN and the head/rachis following inoculation of the stem base. To test the role of DON in CR a transgenic strain of Fg from the USA with the Tri5 gene deleted was compared with its original wild-type and these produced similar levels of CR lesion development and necrosis indicating that DON production was not necessary to cause CR disease symptoms. However, the DON-minus mutant was less frequently recovered from the FLN than the wild type suggesting that DON has a role in stem colonisation by the fungus.

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes inFusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.

Trichothecene and zearalenone production, in culture, by isolates of Fusarium pseudograminearum from western Canada

One hundred and twenty-seven isolates of Fusarium pseudograminearum were obtained from seeds and vegetative parts of cereals and other gramineae grown in western Canada. Culture of the isolates on agar selective for Fusarium graminearum allowed a successful differentiation from F. graminearum. Sterilized rice (40% moisture content) was inoculated with a single germinated spore of isolates identified as F. pseudograminearum and was incubated at 23 °C. After 14 days of incubation, mycelium was taken from each culture, and the DNA was extracted to detect the tri5 gene by polymerase chain reaction analysis and confirm the species identity. After 21 days of incubation, mycotoxins were quantified in dried rice. All isolates contained the tri5 gene. Deoxynivalenol (DON) was produced in 125 of the 127 isolates, 3-acetyldeoxynivalenol (3-ADON) in 122 isolates, 15-acetyldeoxynivalenol (15-ADON) in 2 isolates, diacetoxyscirpenol (DAS) in 17 isolates, and zearalenone in 100 isolates; both nivalenol (NIV) and fusarenon X (FX) were detected in 1 isolate. There appears to be three chemotypes: DON–3-ADON, DON–15-ADON, and NIV. Neither of the two 15-ADON producers or the NIV producer formed DAS. This is the first time that the production of DAS and FX is associated with F. pseudograminearum. None of the isolates produced HT-2 toxin or T-2 toxin at detectable levels.

Pathogenicity and real-time PCR detection of Fusarium spp. in wheat and barley roots

The pathogenicity of five Fusarium spp. frequently isolated from wheat and barley roots in southern Idaho was investigated during four growth-chamber experiments and two field studies. A real-time PCR assay for quantifying the presence of F. culmorum from infected root tissue was also developed based on nucleotide sequence for the tri5 gene. Fusarium culmorum, followed by F. acuminatum and F. reticulatum, resulted in the largest root lesions and percent infected root area. However, F. semitectum, followed by F. acuminatum and F. equiseti, had the greatest impact on total root length. The TaqMan-based real-time assay was able to quantify F. culmorum in root tissue from both growth-chamber and field studies down to 61 pg. The assay also detected F. pseudograminearum and F. graminearum but could not distinguish among these three Fusarium spp.

Assessment of trichothecene chemotypes of Fusarium culmorum occurring in Europe

Fusarium trichothecenes are a group of fungal toxic metabolites whose synthesis requires the action of gene products from three different genetic loci. We evaluated, both chemically and by PCR assays, 55 isolates of Fusarium culmorum from eight European countries and different host plants for their ability to produce trichothecenes. Specific sequences in the Tri6–Tri5 intergenic region were associated with deoxynivalenol production. Sequences in the Tri3 gene were also associated with deoxynivalenol production and specific primer sets were selected from these sequences to identify 3–acetyl–deoxynivalenol or 15–acetyl–deoxynivalenol chemotypes. Specific sequences in the Tri5 and Tri7 genes were associated with the nivalenol chemotype but not with the deoxynivalenol chemotype. Two chemotypes were identified by chemical analysis and confirmed by PCR. Strains of the nivalenol chemotype produced nivalenol (up to 260 µg g−1) and 4–acetyl–nivalenol (up to 60 µg g−1), strains with the 3–acetyl–deoxynivalenol chemotype produced deoxynivalenol (up to 1700 µg g−1) and 3–acetyl–deoxynivalenol (up to 600 µg g−1). Three strains of F. culmorum from France, previously reported as 15–acetyl–deoxynivalenol producers, had the 3–acetyl–deoxynivalenol chemotype. The results are consistent with data from other European countries on the occurrence of the nivalenol and 3–acetyl–deoxynivalenol chemotypes and provide support for the hypothesis that European isolates of F. culmorum producing deoxynivalenol belong only to the 3–acetyl–deoxynivalenol chemotype. The production of trichothecenes from F. culmorum isolates from walnut (3–acetyl–deoxynivalenol chemotype) and leek (nivalenol chemotype) is reported for the first time.

A simple multiplex polymerase chain reaction assay for the identification of four environmentally relevant fungal contaminants

Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time-consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. The polymerase chain reaction (PCR) has shown great promise in its ability to identify and quantify individual organisms from a mixed culture environment; however, the cost effectiveness of single organism PCR reactions is quickly becoming an issue. Our laboratory has developed a simple method to identify multiple fungal species, Stachybotrys chartarum, Aspergillus versicolor, Penicillium purpurogenum, and Cladosporium spp. by performing multiplex PCR and distinguishing the different reaction products by their mobility during agarose gel electrophoresis. The amplified genes include the β-Tubulin gene from A. versicolor, the Tri5 gene from S. chartarum, and ribosomal sequences from both P. purpurogenum and Cladosporium spp. This method was found to be both rapid and easy to perform, while maintaining high sensitivity and specificity for characterizing isolates, even from a mixed culture.

Isolation and characterisation of a trichodiene synthase homologous gene in Trichoderma harzianum

Trichoderma species have long been known as effective biocontrol agents of major plant diseases, and some strains have also been reported to produce toxic metabolites, such as trichothecenes. In order to clarify the molecular aspect of trichothecene biosynthesis the strain ATCC 90237 of Trichoderma harzianum, a recognised producer of the trichothecene harzianum A, was investigated. An amplification fragment of 600 bp obtained by using a degenerate PCR approach showed several degrees of identity with trichodiene synthase genes previously reported from other fungal species. Flanking genomic regions were identified through genomic walking technique. The analysis of resultant sequence allowed the identification of an open reading frame of 1226 nucleotides. The expression of the gene was confirmed by the determination of the corresponding cDNA. The comparison with the genomic sequence revealed the presence of a unique 59 bp intron that presents consensus border and internal sequence typical of filamentous fungi. The deduced putative protein of 388 amino acids displayed an identity of about 60% to the enzyme trichodiene synthase which catalyses the first step in the trichothecene pathway of other mycotoxins producing fungi. The isolation of a putative trichodiene synthase ( tri5) gene required for trichothecene biosynthesis was described for the first time in Trichoderma and enabled the design of specific tri5 primers for a PCR assay that demonstrated the presence of the trichodiene synthase gene in the genomes of different Trichoderma species.

Mycotoxin Production and Molecular Variability of European and American Isolates of Fusarium Culmorum

The main causative agents of Fusarium head blight are Fusarium graminearum and Fusarium culmorum. We examined the mycotoxin-producing abilities and molecular variability of 37 Fusarium culmorum isolates collected from the Pan-Northern Hemisphere, together with isolates representing related species. Mycotoxin-producing abilities of the isolates were tested by thin layer chromatography and by PCR using primer pairs specific for the Tri7 and Tri13 genes. Thirty isolates belonged to chemotype I (producing deoxynivalenol and 3-acetyl-deoxynivalenol), while seven represented chemotype II (producing nivalenol and/or fusarenone X). The presence of a functional Tri7 gene correlated well with nivalenol production. Isolates belonging to chemotype I were in general more pathogenic in in vitro tests than those belonging to chemotype II. Phylogenetic analysis of the random amplified polymorphic DNA profiles (RAPD) of the isolates enabled the isolates to be clustered into different groups. Most isolates from Hungary exhibited identical RAPD profiles. A similar clustering was found on the tree based on restriction analysis of the intergenic spacer region data. Sequence analysis of a putative reductase gene fragment of the isolates was also carried out. A correlation was detected between the geographic origin of the isolates and their position on the cladogram produced based on sequence data. The presence of mating type gene homologues was also tested with primer pairs specific for MAT1-1 and MAT1-2. The isolates carried either MAT1-1 or MAT1-2 homologues. No correlation was observed between clustering of the isolates based on RAPD, restriction analysis of the intergenic spacer region or sequence data and the distribution of MAT idiomorphs. Similarly, no correlation was detected between mycotoxin-producing abilities or aggressiveness and molecular characteristics of the isolates. Statistical analysis of RAPD data and lack of strict correlation between trees based on different data sets supported the view that Fusarium culmorum has a recombining population structure. The presence of mating type gene homologues in the isolates indicates that the recombining population structure is caused by ongoing or past meiotic exchanges.