Plant Pathology and Microbiology, Texas A&M University
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Christensen, S., Borrego, E., Shim, W. B., Isakeit, T., Kolomiets, M. Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays. J. Vis. Exp. (62), e3727, doi:10.3791/3727 (2012).
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.
1. Maize Kernel Bioassay
Note: For seeds or fungi other than those described in these methods, the amount of inoculum required may be different and should be derived experimentally.
2. Conidia Enumeration
3. Aflatoxin Quantification
Note: When using the Aflatest FGIS protocol, the measurement from the fluorometer is calculated based upon an initial 50 grams of sample extracted in 100 ml. If you consider the dilution of the sample with water, the 1 ml applied to a column represents 0.166 grams of sample. So, when modifying the protocol, you need to take into account the differences in sample size and the initial extraction solution. For example, 2 grams of kernels are extracted in 20 ml 80% methanol. This is 0.1 grams/ml. When 1 ml of this sample is mixed with 2 ml water, the proportion of sample in the liquid is now 0.033 grams/ml. If this sample gives a fluorometer reading of 100 ppb, the actual concentration is based on the proportion of sample to 0.166, that is, ppb = (0.166 gram X 100 ppb)/ 0.1 gram), or 166 ppb. For alternative aflatoxin quantification methods, see reference 7.
4. Fumonisin B1 (FB1) Analysis
5. Ergosterol Analysis
6. Representative Results
Following inoculation and incubation in a humidity chamber for two to three days, fungal growth should begin to appear on the kernels. Seven days post treatment, vegetative growth on treated plants should be clearly visible, while mock controls should be uninfected (Figure 1B, top). Periods of longer incubation are conducive to more copious vegetative growth (Figure 1B, bottom). Under the conditions described herein (Figure 2G), A. flavus wild-type NRRL 3357 displayed maximum values of colonization, aflatoxin accumulation, and conidia production at 8, 6, and 8 days, respectively (Figure 2, A-C). However, when mycotoxin contamination and conidiation were compared per unit of ergosterol, the greatest levels were observed at 4 and 6 days, respectively (Figure 2, D-E). Figure 2F summarizes these fungal biomass-dependent maximum-value observations. Interestingly, between 4-6 days post-inoculation, the kernels undergo nucleic acid degradation, as seen by total RNA from samples on the time-course (Figure 2H, bottom).
Several studies, including our own, have examined F. verticilliodies infection on maize kernels2,8,9,10,11,12 and successfully used 7-13 days as time-points for observations. Using the methods described herein, fumonisin levels range from 3,500-8,000 ng/g kernel4,13 and ergosterol levels range from 5,000-10,000 ng/g kernel14,13. Figure 3 shows representative peaks for fumonisin (top) and ergosterol (bottom) from HPLC chromatograms. Measurement of ergosterol can also be carried out via absorbance spectra 15 .
Figure 1. Flow chart for the quantification of fungal biomass, sporogenesis, and mycotoxin production and representative results for vegetative growth from kernel bioassays. A) Flow chart outlining the described method for quantification of fundamental biological parameters used to assess fungal pathogenesis on maize kernels. B) Representative results for kernel bioassays. Top, A. flavus (NRRL3357) vegetative growth on maize kernels in the B73 genetic background. Seeds were inoculated with 200 μl of 106 spores/mL and pictures were taken 7 days post inoculation. Bottom, F. verticillioides inoculated kernels in B73 background 13 days post infection. Kernels were inoculated with 200 μl of 106 spores/mL.
Figure 2. Aspergillus flavus kernel bioassay time-course. B73 kernels were inoculated with 200 μl of 106 spores/ml Aspergillus flavus conidial suspension and incubated for 2-8 days (G). All values were determined from the dry weight average of 4 kernels (n = 3-4; mean ± SE). A) Colonization (based on ergosterol), (B) aflatoxin, and (C) conidiation were quantified using methods described above. E) Aflatoxin and (F) conidiation are displayed as a function of fungal biomass as measured through h ergosterol. F) Maximum values for ergosterol, aflatoxin accumulation, and conidiation as a function of fungal biomass - maximum values for each quantity observed was set to 100%. (H) 1 μg per lane of total RNA from time-course.
Figure 3. Representative High Performance Liquid Chromatography (HPLC) chromatograms for fumonisin B1 (top) and ergosterol (bottom) isolated from kernels infected with Fusarium verticillioides (M3125).
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 kernel facilitates infection while maintaining the viability of the seed.
Effectively run kernel assays have been shown to parallel results from the field. In a previous study 4, kernel bioassays showed that lox3-4 mutant lines were more susceptible to aflatoxin contamination than wild-type plants. This outcome was directly in line with results seen from four independent field trials 4. This suggests that kernel bioassays may be effective tools for determining and/or predicting the outcome of plant-pathogen interactions in large-scale settings.
I have nothing to disclose.
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.
|Potato Dextrose Agar||Fisher Scientific||S71659A|
|Plastic incubation container||Sterilite||1713LAB06|
|24 cm Fluted Filter Papers||Vicam||31240|
|1.5 Ám glass microfibre||Vicam||31955|
|Afla Test column||Vicam||G1024|
|Afrla Test Developer||Vicam||32010|
|C-18 solid phase extraction column (Prep SEP SPE C18 Column)||Fisher Scientific||60108-304|
|Boric acid||Fisher Scientific||BP168-500|
|Sodium borate||Fisher Scientific||RDCS0330500|
|Shimadzu HPLC LC-20AT (Pump)||Shimadzu Corporation||LC-20AT|
|Zorbax ODS column (4.6x150mm)||Agilent Technologies||443905-902|
|Shimatzu RF-10Axl fluorescence detector||Shimadzu Corporation||RF-10AXL|
|Sodium phosphate||Fisher Scientific||AC38987-0010|
|13 mm syringe filter with 0.45 um nylon membrane (HPLC)||Pall Corporation||4426|
|Scintillation vials||VWR international||66021-602|