Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: Research work developed a multiplex real-time PCR assay to detect and quantify mycotoxigenic fungi, specifically of Fusarium, Penicillium, and Aspergillus. The strategy was to utilize broad-spectrum primers to amplify the targeted genomic DNA and specific fluorescent probes to detect and quantify the different fungal genera. 1. A PCR primers were developed that amplified more than 40 Aspergillus species, 23 Fusarium species, and 32 Penicillium species as well as 64 other fungal genera. 2. Genus-specific Taqman probes were designed from ITS sequences of rDNA to detect mycotoxigenic Fusarium, Penicillium, and Aspergillus. A multiplex assay was developed. 3. The assay was validated by analyzing fungal growth in distiller's grain, an animal feedstock that accumulates as a by-product when ethanol is produced from corn. 4. One graduate student was mentored and received a Master's degree based on this work. 5. Two undergraduates were mentored on molecular techniques and mycotoxin analysis as part of the work. 6. One peer-reviewed journal article was published in 2009. 7. Results were presented in five invited talks. 8. Results were displayed in two poster presentations. Between 2005 and 2008 a yearly survey of the preharvest corn crop was conducted to determine the incidence and severity of ear rots and mycotoxins. Each year, ears were collected from 150 fields, two samples of five ears from each field. Ears were assessed for ear rots caused by Gibberella, Diplodia, Fusarium, Aspergillus, Penicillium and Trichoderma. Mycotoxin analysis was conducted on a subset of the samples. 1. Results were communicated each year to producers and grain industry at extension workshops. 2. Results were published each year in extension newsletters. Ozone is highly reactive molecule widely used for sanitation and odor reduction. A study was conducted to evaluate the efficacy of an auger system to treat grain with ozone. The ozone concentration within the auger was 47,800 ppm and the average retention time of one corn kernel moving through the system was 1.8 min. The potential effectiveness of the auger treatment to reduce mold on the surface of corn kernels was explored. 1. Mold counts, specifically Aspergillus flavus, were reduced by 96 % in a single pass through the auger. 2. Three passes through the auger reduced the mold count by more than 2-log units. 3. The ozone reduced aflatoxin in the grain, however, the reduction was not sufficient enough to be of commercial value. 4. A manuscript describing the work was sent for publication. 5. Two Ph.D graduate students and two undergraduate students were mentored as part to the work. 6. Results were presented at an International Society for Mycotoxicology conference in Austria 2009. PARTICIPANTS: O3Co of Aberdeen, ID provided the ozone auger system Lynntech Inc. of College Station, TX provided an ozone generator TARGET AUDIENCES: Plant pathologists conducting research on maize mycotoxins PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1. The PCR detection assay provides a useful tool for the detection of mycotoxigenic fungi. 2. The PCR assay publication (2009) has been cited 4 times. 3. Numerous calls were received about the results from the ear rot survey data 4. The ozone study results provide valuable information for estimating the parameters needed for effectively treating grain in an auger under commercial settings.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Aspergillus flavus causes a devastating rot on both maize ears and kernels. Environmental stresses, such as drought and insect damage, increase the severity of the disease. A. flavus produces the secondary metabolite aflatoxin, which is a potent liver toxin and carcinogen. The objective of this study was to examine gene expression profiles during colonization of different developmental stages of the maize kernel. The major question is what impact does kernel composition in the various stages have on A. flavus. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Growth analysis (ergosterol content) demonstrated that there were no significant changes during colonization of different kernel development stages. When looking at the change in gene expression as a whole, A. flavus metabolism reflects the change in available nutrients during the development stages. During the maturation of the kernel, free amino acids become unavailable resulting in a higher individual synthesis by A. flavus rather than transporting them in during colonization of the blister kernel. Also, as soluble sugars are converted by the endosperm during kernel development, proteins such as zein increase to create storage vacuoles. A. flavus adapts by changing its metabolism to lipid based. Five lipid metabolism genes become highly expressed as this conversion occurs; whereas, eleven carbohydrate metabolism genes are lost. There are genes uniquely expressed during growth on each kernel type. For example, during dent colonization multiple genes involved in micronutrient transport, such as zinc, are expressed. The expression of zinc transporters is due to localization and binding of zinc by phytate in the germ which causes a zinc limiting environment during dent. Transition from dough to dent leads to a major shift in gene expression in A. flavus. Of the genes that show significant up regulation during the later development stage phases, 58 show a sharp increase at the dent stage. This group of genes show steady expression through the blister, milk, and dough, yet are highly expressed in the dent. This pattern incorporates 83% of all the genes over-expressed in dent. The drastic change in gene expression reflects the uniqueness of the composition of the dent kernel.
Publications
- Suanthie, Y., Cousin, M.A. and Woloshuk, C. P. 2009. Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium, and Fusarium. J. Stored Prod. Res. 45:139-145.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The purpose of this research was to develop a multiplex real-time PCR assay to detect and quantify mycotoxigenic fungi. Our strategy was to utilize broad-spectrum primers to amplify the targeted genomic DNA and specific fluorescent probes to detect and quantify the different fungal genera. An analysis of PCR primer indicated PCR products from more than 40 Aspergillus species, 23 Fusarium species, and 32 Penicillium species as well as 64 other fungal genera. Genus-specific Taqman probes were designed from ITS sequences of rDNA to detect mycotoxigenic Fusarium, Penicillium, and Aspergillus. The specificity of the probes was established against a wide range of fungal species. To increase the utility of assay, multiplex conditions were developed. The assay was validated by analyzing fungal growth in distiller's grain, an animal feedstock that accumulates as a by-product when ethanol is produced from corn. PARTICIPANTS: Yenny Suanthie, Graduate student and Maribeth A. Cousin, Professor of Food Science, Purdue University TARGET AUDIENCES: Interested parties would include companies that provide analytical service to the food and grain industry, scientist involved in adapting our assay to microarray technology, and regulatory agencies looking for new approaches of assessing food and grain quality. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A comprehensive scheme to analyze grain and grain products for the presence of mycotoxigenic fungi should begin with the detection and quantification of the key mycotoxigenic genera. The information derived from the preliminary analysis would then guide subsequent analyses with mycotoxin-specific or species-specific measures. The assay we have developed can be used as an initial step to evaluate the mycotoxigenic potential of distiller's grain and various other agricultural commodities. The method has an advantage over the traditional plating methods that are time consuming, laborious, and require an expertise for fungal identification. A further advantage is the PCR assay can detect these mycotoxigenic fungi within distiller's grain, which often contain enormous yeast populations that interfere with plate-method quantification of less numerous fungal species. The assay is applicable for the analysis of stored grains and foods and could be used as an initial step in quality assessment in conjunction with other mycotoxin-specific tests such as chemical analysis or PCR.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Mycotoxins are fungal secondary metabolites that cause severe health-related problems when consumed by humans and animals. The most important mycotoxins that contaminate agricultural commodities are produced by several species of Aspergillus, Fusarium, and Penicillium. Aflatoxins, ochratoxins, and sterigmatocystin are produced by Aspergillus species. Fusarium species produce fumonisins, trichothecenes, and zearalenone, and Penicillium species produce ochratoxins, patulins, and citrinins. Cheap and quick methods to detect the presence of mycotoxigenic fungi are needed for surveillance purposes. Conventional detection methods involve plating techniques that are time consuming and identification that requires expertise. Polymerase Chain Reaction (PCR) is a powerful tool that provides a cheap, fast, and reliable detection and identification of fungi. Real-Time PCR methods allow one to monitor DNA amplification by the stoichiometric accumulation of fluorecence, which eliminate
the need for post-PCR gel electrophoresis. SYBR green and Taqman probes are typically fluorescent dye used in Real-Time PCR. Taqman probes are 20-30 bp oligonucleotides that are modified at the 5' with reporter fluorophore and a quencher at the 3' end (Fig. 1). During PCR, the 5' exonuclease activity of the DNA polymerase cleaves the fluorophore from the probe, which is annealed to the target DNA. Fluorescent from the released fluorophore provides a measure of the amplified DNA. As part of a comprehensive approach for monitoring the presence of mycotoxigenic fungi, our goal is to develop Real-Time PCR assays that will detect, identify, and quantify mycotoxigenic fungi. In this study, our objectives were to developed genus-specific Taqman probes for Aspergillus, Fusarium, and Penicillium and use them in a multiplex assay to quantify Aspergillus, Penicillium, and Fusarium species in important agricultural food commodities.
TARGET AUDIENCES: Plant pathologists and ethanol plant managers
Impacts
The non-specific primers we designed amplify all the Aspergillus, Penicillium, Fusarium, and other fungal species such as Botrytis cinerea. The probes we developed for Aspergillus, Penicillium, and Fusarium are genus-specific and they can be multiplexed. For the multiplex assay, we use 5 pmol of each of the probes and the detection limit is 10 pg to 100 ng. Also, we can use the multiplex real-time PCR assay to detect and quantify fungal growth. Samples of distillers' grain (DG) were collected from the concrete pad for wet DG at a ethanol facility in Clymers, IN. A glass vial containing 6 g of DG was autoclaved for 45 minutes, and inoculated with 50,000 spores of Aspergillus flavus, Penicillium aurantiogriseum, and Fusarium verticillioides. The vials were incubated at 28C in a humid chamber, and vials were collected in triplicate after 0 and 7 days. For negative control, we used autoclaved DG without inoculation in duplicate for 0 and 7 days. The moisture contents of
the DG was determined at 0 and 7 days to be 50% by weight. DNA was extracted from 0.2 g of the vial content, which we further analyze by real-time PCR. The results in showed that all three fungi grew in the inoculated DG. This assay will be useful for monitoring DGs to evaluate management methods for preventing spoilage.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
We have identified target genes for developing mutiplex real-time PCR probes and primers to detect Fusarium, Penicillium, and Aspergillus species. We are also testing the application of supercritical fluid carbon dioxide to efficiently disrupt fungal spore that are present in a low concentration.
Impacts
We anticipate the application of these procedures for monitoring air-borne pathogen populations around fields and for detecting mycotoxigenic fungi in food products.
Publications
- No publications reported this period
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