We would like to thank Brandon Hassett and Carlos Ortiz for their technical assistance. This work was supported by the NSF grants IOB-0544428, IOS-0951272, and IOS-0925561 to Dr. Michael Kolomiets, and by the USDA National Institute of Food and Agriculture (NIFA), AFRI Plant Breeding and Education Grant #2010-85117-20539 to Drs. Seth Murray, Thomas Isakeit, and Michael Kolomiets.

1. Maize Kernel Bioassay
<ol>
	<li>Two weeks prior, culture the fungal pathogens on Potato Dextrose Agar (PDA) at 28 &deg;C.</li>
	<li>Select kernels with similar size and shape, preferably flattened so they lay level with the bottom of bioassay vials, and place in 50 ml falcon tubes. Kernels selected must have been produced concurrently in same environment to ensure similar seed age and metabolite composition.</li>
	<li>Surface sterilize kernels by shaking tubes at room temperature for 5 minutes with 70% ethanol, 1 mi...

<ol>
	<li>Tsitsigiannis, D. I., Keller, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxylipins+as+developmental+and+host-fungal+communication+signals.">Oxylipins as developmental and host-fungal communication signals.</a> Trends Microbiol. 15, 109-118 (2007).</li><li>Gao, X., Shim, W. -. B., G&#246;bel, C., Kunze, S., Feussner, I., Meeley, R., Balint-Kurti, P., Kolomiets, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+a+maize+9-lipoxygenase+results+in+increased+resistance+to+fungal+pathogens+and+reduced+levels+of+contamination+with+mycotoxin+fumonisin.">Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin.</a> Mol. Plant-Microbe Interact. 20, 922-933 (2007).</li><li>Brodhagen, M., Tsitsigiannis, D. I., Hornung, E., Goebel, C., Feussner, I., Keller, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+oxylipin-mediated+cross-talk+in+the+Aspergillus+-+seed+pathosystem.">Reciprocal oxylipin-mediated cross-talk in the Aspergillus - seed pathosystem.</a> Mol. Microbiol. 67, 378-391 (2008).</li><li>Gao, X., Brodhagen, M., Isakeit, T., Brown, S. H., G&#246;bel, C., Betran, J., Feussner, I., Keller, N. P., Kolomiets, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+lipoxygenase+ZmLOX3+increases+susceptibility+of+maize+to+Aspergillus+spp.">Inactivation of the lipoxygenase ZmLOX3 increases susceptibility of maize to Aspergillus spp.</a> Mol. Plant-Microbe Interact. 22, 222-231 (2009).</li><li>Gao, X. Q., Kolomiets, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+lipids+and+oxylipins+are+crucial+signals+in+modulating+mycotoxin+production+by+fungi.">Host-derived lipids and oxylipins are crucial signals in modulating mycotoxin production by fungi.</a> Toxin Rev. 28, 79-88 (2009).</li><li>Mukherjee, M., Kim, J. -. E., Park, Y. -. S., Kolomiets, M. V., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+G+protein+signaling+in+F.+verticillioides+mediate+differential+host-pathogen+responses+on+non-viable+versus+viable+maize+kernels.">Regulators of G protein signaling in F. verticillioides mediate differential host-pathogen responses on non-viable versus viable maize kernels.</a> Mol. Plant Pathol. 12, 479-491 (2011).</li><li>Zheng, M. Z., Richard, J. L., Binder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+rapid+methods+for+the+analysis+of+mycotoxins.">A review of rapid methods for the analysis of mycotoxins.</a> Mycopathologia. 161, 261-273 (2006).</li><li>Bacon, C. W., Bennett, R. M., Hinton, D. M., Voss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+Fusarium+moniliforme+within+asymptomatic+maize+kernels+and+kernels+associated+with+equine+leukoencephalomalacia.">Scanning electron microscopy of Fusarium moniliforme within asymptomatic maize kernels and kernels associated with equine leukoencephalomalacia.</a> Plant Dis. 76, 144-148 (1992).</li><li>Munkvold, G. P., Hellmich, R. L., Rice, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fumonisin+concentrations+in+kernels+of+transgenic+Bt+maize+hybrids+and+nontransgenic+hybrids.">Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and nontransgenic hybrids.</a> Plant Dis. 83, 130-138 (1999).</li><li>Sagaram, U. S., Shaw, B. D., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusarium+verticillioides+GAP!,+a+gene+encoding+a+putative+glycolipid-anchored+surface+protein,+participates+in+conidiation+and+cell+wall+structure+but+not+virulence.">Fusarium verticillioides GAP!, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence.</a> Microbiol. 153, 2850-2861 (2007).</li><li>Shim, W. -. B., Flaherty, J. E., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Fumonisin+B1+biosynthesis+in+maize+germ+and+degermed+kernels+by+Fusarium+verticillioides.">Comparison of Fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides.</a> J. Food Protect. 66, 2116-2122 (2003).</li><li>Shim, W. -. B., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fumonisin+B1+biosynthesis+and+conidiation+in+Fusarium+verticillioides+by+a+cyclin-like+(C-type)+gene,+FCC1.">Regulation of fumonisin B1 biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1.</a> Appl. Environ. Micrbiol. 67, 1607-1612 (2001).</li><li>Christensen, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conversation with: Won-Bo Shim. , (2011).</li><li>Shin, J. -. H., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PPR1+and+PPR2,+genes+encoding+regulatory+subunits+of+protein+phosphatase+2A+in+Fusarium+verticillioides.">Characterization of PPR1 and PPR2, genes encoding regulatory subunits of protein phosphatase 2A in Fusarium verticillioides.</a> Phytopathol. 99, S119 (2009).</li><li>Breivik, O. N., Owades, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Semimicrodetermination+of+Ergosterol+in+Yeast.">Spectrophotometric Semimicrodetermination of Ergosterol in Yeast.</a> Yeast. Agric. and Food Chem. 5, 360-363 (1957).</li></ol>

The methods described here have been tested extensively and proven to be robust in the generation of quantifiable results for fungal colonization, sporogenesis, and production of mycotoxins. Moreover, these methods should be applicable to seeds from other plant species that are susceptible to contamination with mycotoxigenic fungi (e.g. peanuts, wheat, cotton, pistachios, etc.). For competent plant-pathogen interaction analyses, it is imperative that the seeds be kept alive. A small wound site on the embryo side of the ...

<table border="1">
<tbody>
<tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog #</td>
</tr>
<tr>
 <td>Potato Dextrose Agar</td>
 <td>Fisher Scientifc</td>
 <td>S71659A</td>
</tr>
<tr>
 <td>Tween-20</td>
 <td>Fisher Scientifc</td>
 <td>BP337-100</td>
</tr>
<tr>
 <td>Plastic incubation container</td>
 <td>Sterilite</td>
 <td>1713LAB06</td>
</tr>
<tr>
 <td>Blender</td>
 <td>Vicam</td>
 <td>20200</td>
</tr>
<tr>
 <td>24 cm Fluted Filter Papers</td>
 <td>Vicam</td>
 <td>31240</td>
</tr>
<tr>
 <td>1.5 &mu;m glass microfibre</td>
 <td>Vicam</td>
 <td>31955</td>
</tr>
<tr>
 <td>Afla Test column</td>
 <td>Vicam</td>
 <td>G1024</td>
</tr>
<tr>
 <td>Afrla Test Developer</td>
 <td>Vicam</td>
 <td>32010</td>
</tr>
<tr>
 <td>Methanol</td>
 <td>Vicam</td>
 <td>35016</td>
</tr>
<tr>
 <td>Acetonitrile</td>
 <td>Fisher Scientifc</td>
 <td>AC14952-0025</td>
</tr>
<tr>
 <td>Ethanol</td>
 <td>Fisher Scientifc</td>
 <td>AC39769-0025</td>
</tr>
<tr>
 <td>C-18 solid phase extraction column (Prep SEP SPE C18 Column)</td>
 <td>Fisher Scientifc</td>
 <td>60108-304</td>
</tr>
<tr>
 <td>O-phthalaldehyde (OPA)</td>
 <td>Sigma Chemical Co</td>
 <td>79760-5g</td>
</tr>
<tr>
 <td>Boric acid</td>
 <td>Fisher Scientifc</td>
 <td>BP168-500</td>
</tr>
<tr>
 <td>Sodium borate</td>
 <td>Fisher Scientifc</td>
 <td>RDCS0330500</td>
</tr>
<tr>
 <td>Mercaptoethanol</td>
 <td>Fisher Scientifc</td>
 <td>45-000-231</td>
</tr>
<tr>
 <td>Shimadzu HPLC LC-20AT (Pump)</td>
 <td>Shimadzu Scientific Instruments, Inc.</td>
 <td>LC-20AT</td>
</tr>
<tr>
 <td>Zorbax ODS column (4.6x150mm)</td>
 <td>Agilent Technologies</td>
 <td>443905-902</td>
</tr>
<tr>
 <td>Shimatzu RF-10Axl fluorescence detector</td>
 <td>Shimadzu Scientific Instruments, Inc.</td>
 <td>RF-10AXL</td>
</tr>
<tr>
 <td>Sodium phosphate</td>
 <td>Fisher Scientifc</td>
 <td>AC38987-0010</td>
</tr>
<tr>
 <td>FB1 standards</td>
 <td>Sigma Chemical Co.</td>
 <td>F1147-1mg</td>
</tr>
<tr>
 <td>Chloroform</td>
 <td>VWR</td>
 <td>MK444410</td>
</tr>
<tr>
 <td>13 mm syringe filter with 0.45 um nylon membrane (HPLC)</td>
 <td>Pall Life Science</td>
 <td>4426</td>
</tr>
<tr>
 <td>Ergosterol</td>
 <td>Sigma-Aldrich</td>
 <td>45480-50G-F</td>
</tr>
<tr>
 <td>Scintillation vials</td>
 <td>VWR</td>
 <td>66021-602</td>
</tr>
<tr>
 <td>Sodium Chloride</td>
 <td>Vicam</td>
 <td>G1124</td>
</tr>
</tbody>
</table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Research

JoVE Journal

Immunology and Infection

17.9K Views.  Texas A&M University. The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.