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

Summary

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.

Abstract

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 contamination^1,2,3,4,5,6. 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.

Protocol

1. Maize Kernel Bioassay

1. Two weeks prior, culture the fungal pathogens on Potato Dextrose Agar (PDA) at 28 °C.
2. 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.
3. Surface sterilize kernels by shaking tubes at room temperature for 5 minutes with 70% ethanol, 1 minute with sterile water, and 10 minutes with 6% sodium hypochlorite. Rinse, three times, for 5 minutes each time, with sterile water while continuing shak....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Potato Dextrose Agar	Fisher Scientific	S71659A
Tween-20	Fisher Scientific	BP337-100
Plastic incubation container	Sterilite	1713LAB06
Blender	Vicam	20200
24 cm Fluted Filter Papers	Vicam	31240
1.5 μm glass microfibre	Vicam	31955
Afla Test column	Vicam	G1024
Afrla Test Developer	Vicam	32010
Methanol	Vicam	35016
Acetonitrile	Fisher Scientific	AC14952-0025
Ethanol	Fisher Scientific	AC39769-0025
C-18 solid phase extraction column (Prep SEP SPE C18 Column)	Fisher Scientific	60108-304
O-phthalaldehyde (OPA)	Sigma-Aldrich	79760-5g
Boric acid	Fisher Scientific	BP168-500
Sodium borate	Fisher Scientific	RDCS0330500
Mercapt–thanol	Fisher Scientific	45-000-231
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
FB1 standards	Sigma-Aldrich	F1147-1mg
Chloroform	VWR international	MK444410
13 mm syringe filter with 0.45 um nylon membrane (HPLC)	Pall Corporation	4426
Ergosterol	Sigma-Aldrich	45480-50G-F
Scintillation vials	VWR international	66021-602
Sodium Chloride	Vicam	G1124