Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil
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Aflatoxin B1 is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28 °C for 2 weeks and 1 month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.
KeywordsBiocontrol Soil population composition Gene deletion in atoxigenic strain ddPCR assay
This work is supported by in-house research program funds from the US Department of Agriculture, Agricultural Research Services (Project Number: 5325-42000-039-00). We thank Oscar Castillo and Nicholas Hayter for technical assistance.
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Conflict of interest
- Abbas HK, Zablotowicz RM, Horn BW, Phillips NA, Johnson BJ, Jin X, Abel CA (2011a) Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 28:198–208. https://doi.org/10.1080/19440049.2010.544680 CrossRefPubMedGoogle Scholar
- Atehnkeng J, Ojiambo PS, Ikotun T, Sikora RA, Cotty PJ, Bandyopadhyay R (2008) Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 25:1264–1271. https://doi.org/10.1080/02652030802112635 CrossRefPubMedGoogle Scholar
- Atehnkeng J, Ojiambo PS, Cotty PJ, Bandyopadhyay R (2014) Field efficacy of a mixture of atoxigenic Aspergillus flavus link: FR vegetative compatibility groups in preventing aflatoxin contamination in maize (Zea mays L.) Biol Control 72:62–70. https://doi.org/10.1016/j.biocontrol.2014.02.009 CrossRefGoogle Scholar
- Bandyopadhyay R, Ortega-Beltran A, Akande A, Mutegi C, Atehnkeng J, Kaptoge L, Senghor AL, Adhikari BN, Cotty PJ (2016) Biological control of aflatoxins in Africa: current status and potential challenges in the face of climate change. World Mycotoxin J 9:771–789. https://doi.org/10.3920/WMJ2016.2130 CrossRefGoogle Scholar
- Chang PK, Scharfenstein LL, Solorzano CD, Abbas HK, Hua SST, Jones WA, Zablotowicz RM (2015) High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae. Int J Food Microbiol 200:66–71. https://doi.org/10.1016/j.ijfoodmicro.2015.01.021 CrossRefPubMedGoogle Scholar
- Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–8610. https://doi.org/10.1021/ac202028g CrossRefPubMedPubMedCentralGoogle Scholar
- Horn BW, Dorner JW (2009) Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations. Biocontrol Sci Tech 19:249–262. https://doi.org/10.1080/09583150802696541 CrossRefGoogle Scholar
- Hruska Z, Rajasekaran K, Yao H, Kincaid R, Darlington D, Brown RL, Bhatnagar D, Cleveland TE (2014) Co-inoculation of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels. Front Microbiol 5:122. https://doi.org/10.3389/fmicb.2014.00122 CrossRefPubMedPubMedCentralGoogle Scholar
- Hua SST, Chang PK, Palumbo JD (2017) Mycotoxins. In: Witczak A SZ (ed) Toxins and other harmful compounds in foods. CRC Press, Taylor & Francis Group, Boca Raton, Fl, pp 153–168Google Scholar