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United States Department of Agriculture

Agricultural Research Service

Research Project: Improvement of Biological Control Fungi for Reduction of Aflatoxin Contamination

Location: Food and Feed Safety Research

2013 Annual Report

1a. Objectives (from AD-416):
1. Understand the phylogenetics of aflatoxin production through an examination of the population dynamics of aflatoxigenic and atoxigenic Aspergillus (A.) species in mixed cultures. Determine the potential for restoration of toxigenicity in toxigenic strains of A. flavus and development of toxicity in other species or genera which contain partial aflatoxin gene clusters. 2. Identify A. flavus pathogenicity factors required during invasion of oilseed crops and link these factors to aflatoxin contamination. Identify the critical enzymes that will allow the formulation of viable biological control strategies for a given crop situation.

1b. Approach (from AD-416):
Aflatoxins (AFs) are polyketide-derived, toxic and carcinogenic secondary metabolites produced mainly by Aspergillus (A.) flavus on cotton, corn, peanuts, and tree nuts. One of the main strategies for prevention of AF contamination in these crops involves introducing a non-aflatoxigenic competitor Aspergillus to the soil of the plants during the growing season. Recent work revealed that A. flavus has a complicated evolutionary history that includes a history of recombination. It is the purpose of this proposal to better understand A. flavus population dynamics in agricultural environments in order to more safely use biocontrol as a strategy to reduce crop contamination with AFs. To do this we will develop new competitor strains with improved properties for tracking their dispersal after introduction onto crops and with improved ability to over-winter in order to decrease the need for annual reapplication. Optimal candidates should be unable to produce the neurotoxin, cyclopiazonic acid, without altering their competitive ability. The potential for restoration of both AF- and CPA-producing ability of the atoxigenic strain in the laboratory or the field will be assessed. We will determine if the ability to produce hydrolases by the biocontrol strain is important for its competitive ability. With these studies, we expect to be able to either develop new biocontrol strains or improve the design of currently used biocontrol strains to reduce preharvest AF contamination. We also expect to provide additional insight into the evolution of diversity within A. flavus.

3. Progress Report:
Production of green fluorescent protein (eGFP) by the fungus had no effect upon its growth or ability to serve as a competitor. Strain aggressiveness appeared to be enhanced by introduction of the fluorescent marker. The eGFP-transformed strains (with all mating-type MAT1-2 i.e. equivalent of one type sex in higher organisms) were paired with a toxigenic Aspergillus (A.) flavus isolate having the MAT1-1 mating type (i.e. the equivalent of the other type of sex). The results provide evidence of mating as early as two months after inoculation when the fungi were grown under conditions similar to those expected in nature. Two eGFP-transformed biocontrol strains, AF36 and Afla-guard strain, could also produce offsprings. Some transformed strains (in which genetic material has been added) showed no difference in growth habit and colonization potential, while others seemed to be inhibited by their eGFP transformation. Some transformed isolates maintain fluorescence at the colony margin with non-melanized tissue. Two transformed atoxigenic isolates show improved fitness and aggressiveness, and exhibit fluorescence throughout their colonies, so they are promising candidate strains for future greenhouse or field studies. Field studies of competitor potential in corn in Texas have just been initiated using the fluorescent-tagged mutants. Invasiveness of aflatoxin strains depends on production of extracellular hydrolases. Cotton carpel tissue (35-45 days after flowering) was treated with a mixture of xylanolytic hydrolases (enzymes that break down tissues) derived from A. flavus. Immunocytochemical analysis (a method of analysis of the content of a cell by using antibodies) revealed severe degradation of the secondary wall structure. Treatment with an A. flavus 14-kD endoxylanase (another enyzme) also resulted degraded secondary wall xylans (a complex polysaccharide made up of a type of sugar and found as a component of a cell wall). Arabinogalactan proteins (a type of protein found in cell wall) were not as severely affected by the xylanolytic hydrolases. Pectins (complex sugar found in cell walls) could only be detected in the samples treated with xylanolytic enzymes, indicating that the pectins were being masked by xylans. These results are consistent with the conclusion that the xylanolytic hydrolase complex of A. flavus is a critical factor for host cell wall maceration and may represent another important fungal virulence factor. Atoxigenic A. flavus biocontrol agents produced extracellular xylanolytic and pectinolytic hydrolases when grown on a medium with larch (wood) xylan; with AflaGuard displaying lower levels pectinase P2c. The tested A. flavus isolates produced endoxylanases when grown on a xylan medium. Both soil and maize kernel isolates produced extracellular xylanolytic and pectinolytic hydrolases and expressed high levels of pectinolytic activity when grown on a pectin medium due to pectinase P2c. The kernel isolates expressed an additional catabolite repressed pectinase (type of enyzmes) (P1/P3).

4. Accomplishments
1. Recombination among Aspergillus (A.) flavus isolates. Assessing the ability of A. flavus to outcross (introducing unrelated genetic material) is necessary to accurately determine the fate of introduced non-aflatoxigenic biocontrol strains to mediate aflatoxin production. Work was done by scientists at North Carolina State University, Department of Plant Pathology, Raleigh, NC and in the Food and Feed Safety Research Unit at the Southern Regional Research Center, New Orleans, LA. Out-crossing among A. flavus mutants and wild-type strains has been proven to occur under laboratory conditions and under conditions closely mimicking field conditions. Out-crossing can occur between strains from different vegetative compatibility groups (VCGs) proving that VCG is not a barrier to recombination. Mating type populations dictate both the isolate’s ability to out-cross as well as its ability to infect the seed. Formation of sexual structures allowing such out-crossing can occur under optimal conditions in as little as two months. This research provides strong evidence that outcrossing among biocontrol and natural populations in the field is likely to occur within a single growing season and refutes prior studies that suggested that such outcrossing could not occur.

2. Extracellular hydrolase (enzyme that breaks down chemical bonds) activity is different in some strains of Aspergillus (A.) flavus used for biocontrol. This research is necessary to determine if introduced non-aflatoxigenic biocontrol A. flavus would be less or more likely to cause damage to the crop. The research was done by ARS scientists in the Food and Feed Safety Research Unit at Southern Regional Research Center, New Orleans, LA. Afla-guard, an atoxigenic strain used for biocontrol of aflatoxin in corn, produced lower pectinolytic activity (ability to break down pectin) when pectin (a complex sugar found in cell walls) was used for enzyme induction than either AF36, a strain used predominantly for biocontrol of aflatoxin in cottonseed, or the wild-type aflatoxin-producing A. flavus used for comparison. Generally, xylanolytic (ability to break down xylan, another complex sugar) and pectanolytic activities were similar for all strains when the fungi were grown on a natural substrate, corn. These studies show that there is no more likelihood for crop damage from the introduced biocontrol strain than from the natural populations in the agricultural fields.

Review Publications
Chettri, P., Ehrlich, K., Cary, J.W., Collemare, J., Cox, M.P., Griffiths, S.A., Olson, M.A., De Wit, P.J., Bradshaw, R.E. 2013. Dothistromin genes at multiple separate loci are regulated by AflR. Fungal Genetics and Biology. 51:12-20.

Ehrlich, K., Mack, B.M., Wei, Q., Li, P., Roze, L.V., Dazzo, F., Cary, J.W., Bhatnagar, D., Linz, J.E. 2012. Association with AflR in endosomes reveals new functions for AflJ in aflatoxin biosynthesis. Toxins. 4:1582-1600.

Chang, P.-K., Scharfenstein, L.L., Ehrlich, K., Wei, Q., Bhatnagar, D., Ingber, B.F. 2012. Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production. Fungal Biology. 116:298-307.

Chang, P-K., Scharfenstein, L.L., Mack, B.M., Ehrlich, K. 2012. Deletion of the Aspergillus flavus orthologue of A. nidulans fluG reduces conidiation and promotes production of sclerotia but does not abolish aflatoxin biosynthesis. Applied and Environmental Microbiology. 78(21):7557-7563.

Bradshaw, R.E., Slot, J.C., Moore, G.G., Chettri, P., De Wit, P., Ehrlich, K., Ganley, A., Olson, M.A., Rokas, A., Carbone, I., Cox, M.P. 2013. Fragmentation of an aflatoxin-like gene cluster in a forest pathogen. New Phytologist. 198:525-535.

Chang, P.-K., Ehrlich, K. 2013. Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus. Applied Microbiology and Biotechnology. 97(10):4289-4300.

Mellon, J.E., Zelaya, C.A., Dowd, M.K., Beltz, S.B., Klich, M.A. 2012. Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi. Journal of Agricultural and Food Chemistry. 60:2740-2745.

Mellon, J.E., Dowd, M.K., Beltz, S.B. 2013. Effects of temperature and medium composition on inhibitory activities of gossypol-related compounds against aflatoxigenic fungi. Journal of Applied Microbiology. 115:179-186.

Last Modified: 05/28/2017
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