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A new PCR approach for the identification of Fusarium graminearum.

ABSTRACT: The main objective of this work was to develop a PCR protocol for the identification of Fusarium graminearum, based on a pair of primers targeted to a segment of the 3´coding region of the gaoA gene that codes for the enzyme galactose oxidase (GO). This region has low homology with the same region of GO genes from other fungi. Genomic DNA from 17 strains of Fusarium spp. isolated from diseased cereals, from several other Fusarium species, and from other fungi genera was analyzed in a PCR assay using this primer set. The 17 strains of Fusarium spp. were also analyzed for the GO enzyme production in submerse fermentation in a new formulated liquid medium. All strains that were morphologically and molecularly identified as F. graminearum were able to secrete the enzyme and had a positive result in the used PCR protocol. No DNA fragment was amplified using genomic DNA from other Fusarium species and species of other fungi genera. The results suggest that the proposed PCR protocol is specific and can be considered as a new molecular tool for the identification of F. graminearum. In addition, the new formulated medium is a cheap alternative for screening for GO screening production by F. graminearum.

SUBMITTER: de Biazio GR

PROVIDER: S-EPMC3768439 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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A new PCR approach for the identification of Fusarium graminearum.

de Biazio Gleison Ricardo GR Leite Gabriela Guimarães Sousa GG Tessmann Dauri José DJ Barbosa-Tessmann Ione Parra IP

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20080701 3

The main objective of this work was to develop a PCR protocol for the identification of Fusarium graminearum, based on a pair of primers targeted to a segment of the 3´coding region of the gaoA gene that codes for the enzyme galactose oxidase (GO). This region has low homology with the same region of GO genes from other fungi. Genomic DNA from 17 strains of Fusarium spp. isolated from diseased cereals, from several other Fusarium species, and from other fungi genera was analyzed in a PCR assay u ...[more]

PMID: 24031265

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Project description:Fusarium graminearum is the pathogenic agent of Fusarium head blight (FHB), which is a destructive disease on wheat and barley, thereby causing huge economic loss and health problems to human by contaminating foods. Identifying pathogenic genes can shed light on pathogenesis underlying the interaction between F. graminearum and its plant host. However, it is difficult to detect pathogenic genes for this destructive pathogen by time-consuming and expensive molecular biological experiments in lab. On the other hand, computational methods provide an alternative way to solve this problem. Since pathogenesis is a complicated procedure that involves complex regulations and interactions, the molecular interaction network of F. graminearum can give clues to potential pathogenic genes. Furthermore, the gene expression data of F. graminearum before and after its invasion into plant host can also provide useful information. In this paper, a novel systems biology approach is presented to predict pathogenic genes of F. graminearum based on molecular interaction network and gene expression data. With a small number of known pathogenic genes as seed genes, a subnetwork that consists of potential pathogenic genes is identified from the protein-protein interaction network (PPIN) of F. graminearum, where the genes in the subnetwork are further required to be differentially expressed before and after the invasion of the pathogenic fungus. Therefore, the candidate genes in the subnetwork are expected to be involved in the same biological processes as seed genes, which imply that they are potential pathogenic genes. The prediction results show that most of the pathogenic genes of F. graminearum are enriched in two important signal transduction pathways, including G protein coupled receptor pathway and MAPK signaling pathway, which are known related to pathogenesis in other fungi. In addition, several pathogenic genes predicted by our method are verified in other pathogenic fungi, which demonstrate the effectiveness of the proposed method. The results presented in this paper not only can provide guidelines for future experimental verification, but also shed light on the pathogenesis of the destructive fungus F. graminearum.

Project description:BackgroundFusarium head blight (FHB) of wheat caused by Fusarium graminearum species complex (FGSC) is a devastating disease worldwide. The pathogens not only reduce the yield of wheat, but also impact the quality of wheat by contamination with trichothecene mycotoxins. A systematic investigation on the pathogens of FHB in Taiwan is lacking. Here, molecular and morphological approaches were used to identify species of the Taiwanese FGSC isolates and determine their trichothecene genotypes.ResultsIn this study, a total of 195 isolates of FGSC from diseased wheat were collected from 8 areas of northern and central Taiwan. All isolates were subjected to seedling inoculation for verification of pathogenicity. The pathogenic isolates were genetically characterized by sequence characterized amplified region (SCAR), PCR- restriction fragment length polymorphism (RFLP), phylogenetic analysis and fixed nucleotides to clarify their phylogenetic species, and by PCR assays of TRI genes to determine trichothecene genotypes. They were identified as F. asiaticum, F. graminearum sensu stricto, F. meridionale and an unknown species. Isolates of F. asiaticum were the major causal agents (98%) in this investigated population and were comprised of SCAR type 5 (75%), SCAR type 4 (21%) and SCAR type 3 (2%). Their trichothecene genotypes were either 15-acetyl-deoxynivalenol (15-ADON) (83%) or nivalenol (NIV) genotype (17%). These genetic characterizations indicated that F. asiaticum (15-ADON SCAR type 5) accounts for 60% of this Taiwanese population. Virulence assay on wheat heads indicated virulence of F. asiaticum isolates in subpopulations divided by SCAR types or trichothecene genotypes were comparable, suggesting other factors influence the unequal subpopulation sizes.ConclusionsThis is the first study that FGSC isolates in Taiwan were systematically collected and characterized. In addition to F. graminearum sensu stricto and F. meridionale, F. asiaticum with 15-ADON genotype was identified as the predominate species in Taiwan. In contrast to Chinese and Japanese populations that F. asiaticum isolates were typically of 3-ADON or NIV genotype, the predominate 15-ADON genotype in Taiwanese population was unique among F. asiaticum populations and represented the southernmost 15-ADON genotype population in East Asia.

Project description:BackgroundFusarium graminearum virus 1 strain-DK21 (FgV1-DK21) is a mycovirus that confers hypovirulence to F. graminearum, which is the primary phytopathogenic fungus that causes Fusarium head blight (FHB) disease in many cereals. Understanding the interaction between mycoviruses and plant pathogenic fungi is necessary for preventing damage caused by F. graminearum. Therefore, we investigated important cellular regulatory processes in a host containing FgV1-DK21 as compared to an uninfected parent using a transcriptional approach.ResultsUsing a 3'-tiling microarray covering all known F. graminearum genes, we carried out genome-wide expression analyses of F. graminearum at two different time points. At the early point of growth of an infected strain as compared to an uninfected strain, genes associated with protein synthesis, including ribosome assembly, nucleolus, and ribosomal RNA processing, were significantly up-regulated. In addition, genes required for transcription and signal transduction, including fungal-specific transcription factors and cAMP signaling, respectively, were actively up-regulated. In contrast, genes involved in various metabolic pathways, particularly in producing carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines, showed dramatic down-regulation at the early time point. Moreover, genes associated with transport systems localizing to transmembranes were down-regulated at both time points.ConclusionThis is the first report of global change in the prominent cellular pathways in the Fusarium host containing FgV1-DK21. The significant increase in transcripts for transcription and translation machinery in fungal host cells seems to be related to virus replication. In addition, significant down-regulation of genes required for metabolism and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence. Taken together, our data aid in the understanding of how FgV1-DK21 regulates the transcriptional reprogramming of F. graminearum.

Project description:BackgroundFusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration.ResultsHere, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1.ConclusionThe TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation.

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A new PCR approach for the identification of Fusarium graminearum.

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A new PCR approach for the identification of Fusarium graminearum.

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