Multiplex Real-Time PCR Detection of Fumonisin-Producing and Trichothecene-Producing Groups of Fusarium Species

Bluhm, Burt H.; Cousin, M. A.; Woloshuk, Charles P.

doi:10.4315/0362-028x-67.3.536

Cited by 82 publications

(49 citation statements)

References 32 publications

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“…PCR assays developed for Fusarium species were reviewed by Nicholson et al (2003). More recently, real-time quantitative PCR assays with fluorogenic sequence-specific probes for fusaria have been developed (Bluhm et al, 2004;Reischer et al, 2004;Waalwijk et al, 2004;Klemsdal et al, 2006;Yli-Mattila et al, 2006). Real-time PCR provides a means for detection and quantification of DNA targets by monitoring PCR product accumulation during the thermal cycling as indicated by increased fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

Sarlin

Yli-Mattila

Jestoi³

et al. 2006

Eur J Plant Pathol

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A real-time PCR technique was applied for the quantification of trichothecene-producing Fusarium species (TMTRI assay) as well as the highly toxigenic Fusarium graminearum (TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCyclerÒ system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium DNA and F. graminearum DNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4-5 log units when pure Fusarium DNAs were tested. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearum gave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearum DNA were found to be naturally low in Finnish barleys.

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Section: Introductionmentioning

confidence: 99%

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

Sarlin

Yli-Mattila

Jestoi³

et al. 2006

Eur J Plant Pathol

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“…A common method for detect-ing and quantifying Fusarium in field samples is real-time PCR using species-specific primers (28). Multiplex real-time PCR has been used for the simultaneous detection of multiple Fusarium species (29,30), but detection is still limited to a few species. Description of Fusarium diversity without targeting specific species is traditionally performed by isolation on selective media, and identification is based on morphological and/or molecular characteristics, but this is a time-consuming procedure (27,31).…”

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confidence: 99%

Genus-Specific Primers for Study of Fusarium Communities in Field Samples

Karlsson

Edel-Hermann

Gautheron

et al. 2016

Appl Environ Microbiol

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Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genusspecific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa؉7/Ra؉6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa؉7/Ra؉6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

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“…The primer and probes used to detect the members of the genus Fusarium (ITS assay) as well as the trichothecene producing group of Fusarium (TRI 6 assay) have been reported previously (Bluhm et al 2004). Using the TRI 6 assay, the concentration of DNA encoding trichothecenes (TRI-DNA) was determined and expressed in ng TRI-DNA per g dry matter of wheat heads.…”

Section: Dna Extraction and Real Time Pcrmentioning

confidence: 99%

Impact of aggressiveness of Fusarium graminearum and F. culmorum isolates on yield parameters and mycotoxin production in wheat

et al. 2011

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Plant-associated isolates from Fusarium graminearum and F. culmorum were inoculated on wheat in field experiments in 2007 and 2008 to ascertain their influence on fungal colonization of the ears, as well as mycotoxin contamination (deoxynivalenol, DON; nivalenol, NIV; zearalenone, ZEA) and yield parameters in the mature crop after inoculation with or without irrigation. The isolates were assigned to four different groups of aggressiveness on the basis of pathogenic symptom development and mycotoxin production in vitro. Increased levels of trichothecene-producing Fusarium DNA in the ears indicated a successful inoculation of the plants, which resulted in increased DON content in the wheat kernels in 2007. Dry conditions at anthesis markedly suppressed fungal colonization as well as mycotoxin accumulation. However, due to precipitation during the ripening period, yield and thousand-kernel weight were similar whether or not irrigation was applied at the time of inoculation. The level of aggressiveness among the isolates as determined in vitro was not reflected in the field experiment. The activity of the extracellular invertase in developing ears increased as a plant response to pathogen infection, especially when the plants were irrigated at the time of inoculation. In 2008, the Fusarium inoculation of wheat heads did not cause fungal growth and mycotoxin contamination in the grain, because of the dry weather conditions that occurred over the entire period of anthesis and ripening. The risk of future mycotoxin contamination in grains was discussed based on climate change prognosis.

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Multiplex Real-Time PCR Detection of Fumonisin-Producing and Trichothecene-Producing Groups of Fusarium Species

Cited by 82 publications

References 32 publications

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

Genus-Specific Primers for Study of Fusarium Communities in Field Samples

Impact of aggressiveness of Fusarium graminearum and F. culmorum isolates on yield parameters and mycotoxin production in wheat

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