FNS  Vol.4 No.4 , April 2013
Testing the Efficacy of eGFP-Transformed Aspergillus flavus as Biocontrol Strains
Abstract: Current biological control methods to prevent pre-harvest aflatoxin contamination of corn, cottonseed, and ground and tree nuts involve field inoculation of non-aflatoxigenic Aspergillus flavus. To date, the efficacy of this approach requires annual reapplication of the biocontrol agent. The reason for this requirement is uncertain. To track the dispersal and test the longevity of these strains, we prepared fluorescent biocontrol strains by incorporating into them the gene expressing the enhanced green fluorescent protein (eGFP). We first investigated the effects of eGFP transformation on the ability of the fluorescent fungus to compete with its non-fluorescent homolog, and then with other heterologous non-aflatoxigenic strains as well as with aflatoxigenic isolates. Our findings indicate that, in these studies, detection of fluorescence was variable, with some fluorescent strains exhibiting enhanced growth and sporulation post-transformation. In our tests, not all transformed strains proved to be good candidates for tracking because their fluorescence was reduced over the course of our study. Most of the transformed strains retained fluorescence and showed robust colony growth in an artificial competitor environment; therefore, they should be suited for further trial under more natural settings. Our ultimate objective is to determine if out-crossing between biocontrol strains and native field populations is occurring in a natural setting.
Cite this paper: G. Moore, B. Mack and S. Beltz, "Testing the Efficacy of eGFP-Transformed Aspergillus flavus as Biocontrol Strains," Food and Nutrition Sciences, Vol. 4 No. 4, 2013, pp. 469-479. doi: 10.4236/fns.2013.44060.

[1]   K. C. Ehrlich and P. J. Cotty, “An Isolate of Aspergillus flavus Used to Reduce Aflatoxin Contamination in Cottonseed Has a Defective Polyketide Synthase Gene,” Applied Microbiology and Biotechnology, Vol. 65, No. 4, 2004, pp. 473-478. doi:10.1007/s00253-004-1670-y

[2]   P.-K. Chang, B. W. Horn and J. W. Dorner, “Sequence Breakpoints in the Aflatoxin Biosynthesis Gene Cluster and Flanking Regions in Nonaflatoxigenic Aspergillus flavus Isolates,” Fungal Genetics and Biology, Vol. 42, No. 11, 2005, pp. 914-923. doi:10.1016/j.fgb.2005.07.004

[3]   G. G. Moore, R. Singh, B. W. Horn and I. Carbone, “Re combination and Lineage-Specific Gene Loss in the Aflatoxin Gene Cluster of Aspergillus flavus,” Molecular Ecology, Vol. 18, No. 23, 2009, pp. 4870-4887. doi:10.1111/j.1365-294X.2009.04414.x

[4]   J. W. Dorner, “Biological Control of Aflatoxin Contamination in Corn Using a Nontoxigenic Strain of Aspergillus flavus,” Journal of Food Protection, Vol. 72, No. 4, 2009, pp. 801-804.

[5]   P. J. Cotty, “Influence of Field Application of an Atoxigenic Strain of Aspergillus flavus on the Populations of A. flavus Infecting Cotton Bolls and on the Aflatoxin Con tent of Cottonseed,” Phytopathology, Vol. 84, No. 11, 1994, pp. 1270-1277. doi:10.1094/Phyto-84-1270

[6]   J. W. Dorner, “Development of Biocontrol Technology to Manage Aflatoxin Contamination in Peanuts,” Peanut Science, Vol. 36, No. 1, 2009, pp. 60-67. doi:10.3146/AT07-002.1

[7]   C. Huang, A. Jha, R. Sweany, C. DeRobertis, K. E. Damann Jr., “Intraspecific Aflatoxin Inhibition in Aspergillus flavus Is Thigmoregulated, Independent of Vegetative Compatibility Group and Is Strain Dependent,” PLoS One, Vol. 6, No. 8, 2011, Article ID: e23470. doi:10.1371/journal.pone.0023470

[8]   P.-K. Chang, H. K. Abbas, M. A. Weaver, et al., “Identification of Genetic Defects in the Atoxigenic Biocontrol Strain Aspergillus flavus K49 Reveals the Presence of a Competitive Recombinant Group in Field Populations,” International Journal of Food Microbiology, Vol. 154, No. 3, 2012, pp. 192-196.

[9]   F. Wu, Y. Liu and D. Bhatnagar, “Cost-Effectiveness of Aflatoxin Control Methods: Economic Incentives,” Toxin Reviews, Vol. 27, No. 3-4, 2008, pp. 203-225. doi:10.1080/15569540802393690

[10]   P. J. Cotty, “Aflatoxin Control in Pistachios, Almonds and Figs: Biocontrol Using Atoxigenic Strains,” 2011. = 412279

[11]   J. W. Dorner and B. W. Horn, “Separate and Combined Applications of Nontoxigenic Aspergillus flavus and A. parasiticus for Biocontrol of Aflatoxin in Peanuts,” My copathologia, Vol. 163, No. 4, 2007, pp. 215-223. doi:10.1007/s11046-007-9004-0

[12]   H. K. Abbas, R. M. Zablotowicz, B. W. Horn, et al., “Comparison of Major Biocontrol Strains of Non-Aflato xigenic Aspergillus flavus for the Reduction of Aflatoxins and Cyclopiazonic Acid in Maize,” Food Additives and Contaminants, Vol. 28, No. 2, 2011, pp. 198-208. doi:10.1080/19440049.2010.544680

[13]   C. Accinelli, M. L. Saccà, H. K. Abbas, et al., “Use of a Bioplastic Formulation for Carrying Conidia of a Non Aflatoxigenic Strain of Aspergillus flavus,” Bioresource Technologies, Vol. 100, No. 17, 2009, pp. 3997-4004. doi:10.1016/j.biortech.2009.03.010

[14]   B. W. Horn, J. H. Ramirez-Prado and I. Carbone, “The Sexual State of Aspergillus parasiticus,” Mycologia, Vol. 101, No. 2, 2009, pp. 275-280. doi:10.3852/08-205

[15]   B. W. Horn, G. G. Moore and I. Carbone, “Sexual Production in Aspergillus flavus,” Mycologia, Vol. 101, No. 3, 2009, pp. 423-429. doi:10.3852/09-011

[16]   B. W. Horn, G. G. Moore and I. Carbone, “Sexual Re production in Aflatoxin-Producing Aspergillusnomius,” Mycologia, Vol. 103, No. 1, 2011, pp. 174-183. doi:10.3852/10-115

[17]   C. J. Worthington, B. W. Horn, G. G. Moore, et al., “Hybridization between Aspergillus flavus and Aspergillus parasiticus,” The 26th Fungal Genetics Conference, Pacific Grove, 15-20 March 2011.

[18]   R. A. Olarte, B. W. Horn, J. W. Dorner, et al., “Effect of Sexual Recombination on Population Diversity in Aflatoxin Production by Aspergillus flavus and Evidence for Cryptic Heterokaryosis,” Molecular Ecology, Vol. 21, No. 6, 2012, pp. 1453-1476. doi:10.1111/j.1365-294X.2011.05398.x

[19]   K. C. Ehrlich, P.-K. Chang, J. Yu and P. J. Cotty, “Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation,” Applied and Environmental Microbiology, Vol. 70, No. 11, 2004, pp. 6518-6524. doi:10.1128/AEM.70.11.6518-6524.2004

[20]   P.-K. Chang, K. C. Ehrlich and S.-S. T. Hua, “Cladal Relatedness among Aspergillus oryzae Isolates and Aspergillus flavus S and L Morphotype Isolates,” International Journal of Food Microbiology, Vol. 108, No. 2, 2006, pp. 172-177. doi:10.1016/j.ijfoodmicro.2005.11.008

[21]   J. H. Ramirez-Prado, G. G. Moore, B. W. Horn and I. Carbone, “Characterization and Population Analysis of the Mating-Type Genes in Aspergillus flavus and Aspergillus parasiticus,” Fungal Genetics and Biology, Vol. 45, No. 9, 2008, pp. 1292-1299. doi:10.1016/j.fgb.2008.06.007

[22]   K. Rajasekaran, J. W. Cary, P. J. Cotty and T. E. Cleve land, “Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed,” Mycopathologia, Vol. 165, No. 2, 2008, pp. 89-97. doi:10.1007/s11046-007-9085-9

[23]   J. W. Cary, P. Y. Harris-Coward, K. C. Ehrlich, et al., “NsdC and NsdD Affect Aspergillus flavus Morphogene sis and Aflatoxin Production,” Eukaryotic Cell, Vol. 11, No. 9, 2012, pp. 1104-1111. doi:10.1128/EC.00069-12

[24]   R. K. Garber and P. J. Cotty, “Formation of Sclerotia and Aflatoxins in Developing Cotton Bolls Infected by the S Strain of Aspergillus flavus and Potential for Biocontrol with an Atoxigenic Strain,” Phytopathology, Vol. 87, No. 9, 1997, pp. 940-945. doi:10.1094/PHYTO.1997.87.9.940

[25]   R. Jamie-Garcia and P. J. Cotty, “Aspergillus flavus in Soils and Corncobs in South Texas: Implications for Management of Aflatoxins in Corn-Cotton Rotations,” Plant Disease, Vol. 88, No. 12, 2004, pp. 1366-1371. doi:10.1094/PDIS.2004.88.12.1366

[26]   B. W. Horn and J. W. Dorner, “Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States,” Applied and Environmental Microbiology, Vol. 65, No. 4, 1999, pp. 1444-1449.