Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Oct 1, 2008
Project End Date
Sep 30, 2013
Grant Year
Project Director
Damann, K.
Recipient Organization
BATON ROUGE,LA 70803-0100
Performing Department
Plant Pathology & Crop Physiol
Non Technical Summary
Aflatoxin contamination of corn is a chronic problem in the Gulf south and an economic concern to Louisiana growers. Biological control can help ameliorate this problem. Our previous work has helped elucidate the probable mechanism of biological control which is touch inhibition of aflatoxin synthesis. Further understanding of this mechanism and the specificitiy involved can lead to better approaches to deploying atoxigenic inoculum in a form which is maximally effective.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
Determine the population biology or spectrum of diversity of Aspergillus flavus in the Louisiana corn agro-ecosystem. Determine the specificity profiles for intra-specific aflatoxin inhibition, i.e., which atoxigenic isolates are capable of inhibiting which toxigenic isolates by the touch inhibition mechanism. Determine the efficacy of selected atoxigenic isolates to inhibit aflatoxin contamination when deployed in field trials. Investigate the mechanism of touch inhibition of aflatoxin biosynthesis. Investigate the potential of Aspergillus flavus to infect roots of corn and become systemic, thereby gaining access to the developing corn kernels.
Project Methods
Fungal isolates from soil and kernels will be grown on rice and extracts checked for aflatoxins and cyclopiazonic acid by HPLC and TLC, respectively. VCG's will determined by the nit mutant method as well as attempts to develop PCR amplified microsatellite loci fingerprint patterns specific to VCG's. dsRNA's will be purified from a cellulose column and characterized on PAGE. VCG profiles from soil/saprophytic and kernels/parasitic will be compared to discern if there is specificity. Specificity profiles of intra-specific aflatoxin inhibition among paired toxigenic and atoxigenic isolates will be developed by growing conidial suspensions in Eppendorf tubes for 5 days prior to aflatoxin analysis. Patterns of inhibition will be compared among all possible combinations. Broad spectrum inhibitory isolates will be deployed in corn field trials, and challenged with toxigenic isolates to determine efficacy in minimizing aflatoxin contamination. Mechanism of touch inhibition will be investigated by comparing inhibitory and non-inhibitory atoxigenic isolates and also by comparing toxigenic isolates which are and are not inhibited by a particular non-toxigenic isolate. Ability to root infect corn will be done in the growth chamber with selected isolates. Systemic spread and ability to reach developing kernels will also be addressed by homogenizing corn tissues and selective plating. Toxin content will also be determined.

Progress 10/01/08 to 09/30/13

Target Audience: National Corn Growers Association, Louisiana, Arkansas, Texas and Mississippicorn producers and consultants, scientists worldwide interested in Aspergillus flavus and aflatoxin contamination of commodities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A cooperative agreement between USDA-ARS-SRRC scientists in New Orleans has been executed and provides stimulation in both directions.A California scientist has indicated that they have investigated the touch inhibition of aflatoxin by growing toxigenic and atoxigenic strains and are finding the same results using their strains as reported in our 2011 PLoSONE paper. Rebecca Sweany completed an MS degree in Plant Pathology at LSU ona population biology study is now working on a PhD to extend findings from her previous work. How have the results been disseminated to communities of interest? The list of talks given to scientists, consultants, growers, and other interested parties (See Other Products Section) has disseminated results. What do you plan to do during the next reporting period to accomplish the goals? N/A

What was accomplished under these goals? Analysis of Aspergillus flavus isolates from corn kernels fromLouisiana corn fields indicated a skewed distribution (>90%)toward Mat1-2 strains. Soil isolates from the same fields approximated a 1:1 distribution of Mat1-1:Mat1-2 strains. Sampling of airborne conidia from Louisiana corn fields indicated a similar skewed distribution of Mat1-2 strains. Strains with Mat1-2, which is a transcriptional activator, have an inherent greater ability to infect corn than Mat1-1 strains in Louisiana. However, Aspergillus isolates isolated from kernels grown in S. Illinois gavea less skewed distribution. Isolates (90 of 574)were characterizedin a 39:49:2 Mat1-1:Mat1-2:no amplification ratio. The predominant strain (80% of kernel isolates) in corn kernels from a 2007 survey produced less than 5 ppb aflatoxin when grown on a sterile rice medium while the second most predominant strain (10% of kernel isolates)produced over 25,000 ppb of aflatoxin. The predominate low toxin producing strain is acting as an endemic natural biocontrol strain to protect against the high toxin producing strains. The infectivity of two Mat1-2 strains isolated from kernels was compared with two Mat1-1 strains from soil and not found in kernels. The isolate from the predominant strain infected about half of the kernels when sprayed on the silks while the others infected only ~5-7%. The predominant strain genotype referred to as VCG1 was more infectious than the other 3 strain isolates.


  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Damann, KE, & DeRobertis, C. 2013. Mating of Aspergillus flavus x Aspergillus minisclerotigenes hybrids: Are they functionally mules? Phytopathology 103(Supplement 2)6:32.

Progress 01/01/12 to 12/31/12

OUTPUTS: A collection of 12 parents and 120 ascospore progeny of sexual crosses between A. flavus (cryptic species 1) and A. minisclerotigenes (cryptic species 2) were deposited in the Fungal Genetics Stock Center, Kansas City, MO. Information generated by this work was orally disseminated to corn growers at the National Corn Growers Association Corn Utilization and Technology Conference, to faculty and students at departmental seminar, to the Louisiana Soybean and Grain Research and Promotion Board at their annual meeting, to members of the American Phytopathological Society at the annual meeting. One peer-reviewed publication was produced. PARTICIPANTS: K.E. Damann (PI), Catherine DeRobertis, Nipur Patel, LSU AgCenter; Robert Bellm, Univ. of Illinois, Kenneth Ehrlich, Jay Mellon, ARS, USDA, SRRC, New Orleans; Dean Malvik, Univ. of Minnesota; Burton Bluhm, University of Arkansas; and Tom Allen, Mississippi State University. TARGET AUDIENCES: The Louisiana corn growers as well as corn growers across the nation whose corn fields are subject to aflatoxin contamination. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Field experiments were conducted to investigate various parameters affecting atoxigenic biological control of aflatoxin contamination of corn. Both simultaneous and delayed treatments were efficacious giving comparable results, thus there was no clear indication that pre-establishment of a biocontrol strain was better than co-establishment. The high rate of biocontrol strain application at the V10 stage of development was far superior to other treatments. This was explained by the fact that the ear emerges from the V10 leaf position and thus the whorl at the V10 stage contains inoculum which" bathes" the emerging ear facilitating infection and thereby biological control. A synergistic effect of Afla-Guard+LA17 and Afla-Guard+LA51 demonstrated superior efficacy to all biocontrol strains applied singly. Analysis of spore trap isolates indicated a predominance (95%) of Mat1-2 strains. This is consistent with the predominance of Mat1-2 strains in isolates from infected kernels. Lab experiments (intraspecific aflatoxin inhibition) identified potential biological control strains from an atoxigenic highly infective vegetative compatibility group. Four VCG 1 isolates S3, S10, S17, and S19, were selected for further field testing based on their inability to produce cyclopiazonic acid and their intraspecific aflatoxin inhibition ability. The collection of parents and their sexual progeny submitted to the FGSC confirms that there are no intrinsic impediments to mating between these two "species" and strongly suggests that the current species designations are artificial.


  • Abbas, HK, Mascagni, HJ, Bruns, A, Shier, WT, Damann, KE. 2012. Effect of planting density, irrigation regimes, and maize hybrids with varing ear size on yield, and aflatoxin and fumonisin contamination levels. American Journal of Plant Sciences doi:10.4236/ajps.2012.
  • Damann, KE. 2012. A role for mating type in Aspergillus flavus infection of corn and in biological control Phytopathology 102(7):S4.29.

Progress 01/01/11 to 12/31/11

OUTPUTS: The Syngenta Afla-guard tests alone and in combination with Louisiana Aspergillus strains improved protection against aflatoxin contamination of grain. There appear to be three A. flavus strains (potential products) from Louisiana which when individually combined with Afla-guard increase non-toxigenic biological control of aflatoxin contamination of corn. One of them also performed well alone. Further analysis of the collection of isolates of this vcg revealed ~20 of the 483 isolates which failed to produce aflatoxin and ~5 of these failed to produce another mycotoxin, cyclopiazonic acid. These five are candidates for further testing as putative biocontrol strains. All these results were reported to the Louisiana Soybean and Grain Research and Promotion Board. Research demonstrated a 100-fold improvement in efficacy of itraconazole treatment to inhibit A. flavus when the antifungal was encapsidated in nanoparticles. This information was shared commercial industries. PARTICIPANTS: K.E. Damann (PI), N. Patel, LSU AgCenter; J. Cary, ARS USDA SRRL, Khanchai Danmek, Chulalongkorn University, Bangkok, Thailand, Thai Research Fund; Hunsa Punnapayak of Chulalongkorn; Texas Corn Growers; Burt Fluhm, University of Arkansas; Tom Allen, Mississippi State University. TARGET AUDIENCES: Louisiana corn industry and National Corn Growers. PROJECT MODIFICATIONS: Expansion of the field testing of biocontrol isolates to a broader audience (Arkansas, Mississippi),through support of the Texas Corn Growers AMCOE grant program.

These results are supported by the work of Huang et al 2011, which documents the specificity of intraspecific aflatoxin inhibition among various strains of A. flavus, and provides the mechanistic basis of the inhibition as being mediated by physical interaction resulting in a thigmo-down-regulation of aflatoxin synthesis. The improvement in efficacy is most likely related to the broader specificity of intraspecific aflatoxin inhibition achieved by the differing complementary spectra of inhibition by the various isolates. Experiments to follow up on the previous results suggested the importance of mating type in corn infection. They indicate a strong bias toward corn infection by A. flavus strains of mating type 1-2 rather than 1-1. Further analysis of these results revealed that the predominant(~80%) vcg infecting corn in 2007 was a very low toxin producer while the next most frequent (~10%) was a very high toxin producer when cultured on sterile rice. This result suggested the possibility that the predominant vcg was an example of a natural "native" biocontrol strain. The extremely high frequency(>95%)of corn kernel isolates of the Mat1-2 mating type strongly suggests that Mat1-2 which is a transcriptional activator of genes is turning on genes which enhance corn infection. All of these biocontrol isolates are Mat1-2 which further supports the importance of kernel infection by the biocontrol strains in mediating control. The occurrence of the putative "native" biocontrol strain provides an explanation of why aflatoxin contamination outbreaks are absent when environmental conditions are conducive. Nanoparticles encapsidating or covalently labeled with a fluorescent probe were almost immediately internalized into the fungus hyphae where the fluorescence was observed. Nanoparticles were endocytosed by the fungus thus providing a "Trojan horse" explanation of the increased antifungal efficacy.


  • Huang, CW, Jha, A, Sweany, R, DeRobertis, C, Damann, KE. 2011. Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent. PLoS ONE 6(8):e23470. doi:10.1371/journal.pone.0023470.
  • Sweany, RR, Damann, KE Jr, Kaller, MD. 2011. Comparison of soil and corn kernel Aspergillus flavus populations: evidence for niche specialization. Phytopathology 101:952-959.
  • Patel, NR, Damann, K, Leonardi, C, Sabliov, CM. 2011. Size dependency of PLGA-nanoparticle uptake and antifungal activity against Aspergillus flavus. Nanomedicine 6(8):1381-1395.
  • Danmek, K, Prasongsuk, S, Lotrakuk, P, Damann, K E, Eveleigh, D E, Punnapayak, H. 2011. Effect of Avid on the synnema-like formation of Aspergillus flavus grown on Czapek medium. African Journal of Microbiology Research 5(18):2812-2815.

Progress 01/01/10 to 12/31/10

OUTPUTS: A significant output during the year was the application of technology that allowed the sexual crossing or mating of the fungus Aspergillus flavus which was previously thought to be only asexually reproducing. The results were disseminated at the 2010 meeting of the American Phytopathological Society. Another significant output was the culmination of a population biology study of Aspergillus flavus from corn field soil and kernels from Louisiana fields. This established that there are subsets in the soil population that do infect corn and subsets that do not infect corn. These results were reported at the 2010 meeting of the American Phytopathological Society and as an MS thesis. A third significant output involved the use of nanoparticles encapsidating a fungicide which showed 100-fold greater efficacy than the fungicide alone. The results were submitted and published. This work resulted from mentoring and collaboration with an MS student in another department. Finally biocontrol experiments pairing each one of our 3 selected biocontrol isolates with Syngenta's Aflaguard resulted in significantly better protection from aflatoxin contamination than any treatment alone. This work was presented to the Louisiana Soybean and Grain Research and Promotion Board. PARTICIPANTS: The nanoparticle work was done by an MS student of major professor Christina Sabliov in Biological and Agricultural Enginering at LSU, Nipur Patel, on whose graduate committee I served. Donation of several antifungal natural products from the lab of Dr. David Wedge, ARS, USDA natural products chemist from Oxford, MS. The biocontrol work was in cooperation with Dr. Bobby Bassi of Syngenta Corp. Other individuals contributing to the project were Catherine DeRobertis, research associate in my lab, and Rebecca Sweany, MS student in my lab. TARGET AUDIENCES: The scientific community, aspergillusologists and the corn growers of Louisiana. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

The results of the sexual crossing/mating work between what was known as A. flavus cryptic species I and II revealed positive matings between the two species. Cryptic species II was transferred by taxonomists to A. minisclerotigenes. This means that there are no intrinsic blocks to cross species mating in these two Aspergillus species and probably that many other Aspergillus species that have Mat1-1 or Mat1-2 mating types can be sucessfully mated. This calls into question the validity of the abundance of Aspergillus species which taxonomists have described. The dogma in aspergillusology has been that one isolate of A. flavus is as good as another when it comes to infecting hosts and producing aflatoxin. The work of Sweany suggests that there is specificity in the population with some vegetative compatibility groups being specialized to exist only saprophytically in the soil and other vegetative compatibility groups that also grow in the soil can be facultatively parasitic/pathogenic on corn. Further implications of this work suggest that biological control can focus on the facultative parasites and "ignore" the soil saprophyte only subset of the population. The finding that nanoparticle encapsidation of antifungals significantly enhances efficacy has broad potential impact both medically and agriculturally.


  • Patel, N. R., Damann, K., Leonardi, C. and Sabliov, C. M. 2010. Itraconazole-loaded poly(lactic-co-glycolic) acid nanoparticles for improved antifungal activity. Nanomedicine 5(7):1037-1050.
  • Chen, Z. Y., Brown, R. L., Damann, K. E., and Cleveland, T. E. 2010. PR10 expression in maize and its effect on host resistance against Aspergillus flavus infection and aflatoxin production. Molecular Plant Pathology 11(1):69-81.
  • Damann, K. E., DeRobertis, C., and Sweany, R. 2010. Mating between Aspergillus flavus cryptic species I and II. Phytopathology 100(6):S28.
  • Sweany, R., and Damann, K. E. 2010. A comparison of soil and corn kernel Aspergillus flavus populations: evidence for niche specialization. Phytopathology 100(6):S28.
  • Sweany, Rebecca Ruth, 2010. A comparison of soil and corn kernel Aspergillus flavus populations: evidence for niche specialization. LSU electronic thesis and dissertation collection-Sweany Thesis.pdf-URN:etd-03172010-103211.

Progress 01/01/09 to 12/31/09

OUTPUTS: Field experiments were conducted at Ben Hur, Macon Ridge and the St. Joseph experiment stations which examined the efficacy of 4 of our identified Aspergillus flavus biocontrol isolates in comparison with several commercially available or soon to be available isolates from others. In addition the corn hybrid test at Macon Ridge was pinbar inoculated with toxigenic A. flavus, harvested and hybrids analyzed for aflatoxin content (this was in cooperation with Dr. Mascagni who conducts the hybrid tests and who will post the aflatoxin results along with the yield data on the web). This should aid farmers in selecting hybrids with lower aflatoxin contamination potential. The hybrid rankings and amounts of contamination were reported at the Soybean and Grain Research and Promotion Board meeting November 20, 2009, as were the results of the biocontrol experiments. The biocontrol research was also reported to the assembled masses at the St. Joseph Field day conducted on June 19, 2009. I gave an oral presentation of my biocontrol work and co-chaired a session at the annual meeting of the American Phytopathological Society in Portland, OR, August 1-5, 2009. In addition my student presented her MS work there as well as receiving a travel award from the APS to attend. Invited to speak on biocontrol in corn and present a poster on aflatoxin analysis at the "Reducing Aflatoxin Contamination in Corn" a USDA meeting Oct. 27-28, 2009, at MSU in Starkville, MS . The oral presentations were posted online at . Instigated an informal three way project dealing with drug delivery to inhibit A. flavus / fumigatis between Dr. Cristina Sabliov and her student Nipur Patel, Biol. & Ag Engineering, LSU AgCenter, and Dr. David Wedge, ARS, USDA, National Center for Natural Products Research, University, MS, and myself. Sabliov and Patel make nanoparticles which encapsidate potential fungicidal products provided by Wedge, and assay them for efficacy against A. flavus and GFP-A. flavus isolates in assays which I provide. Participated as co-major professor for Khanchai Danmek, a PhD student at Chulalongkorn Univ, Bangkok, Thailand. Attended his dissertation defense April 6, 2009, in Bangkok. Presented an invited lecture April 2, 2009 at the 2nd Thai-American Symposium , Bangkok, and the 10th RGJ PhD Congress, Pattaya, Thailand, April 3-5, 2009. Met with representative of Syngenta which markets Aflaguard a biocontrol formulation of A. flavus, to discuss possible collaborations. Initiated work involving the determination of mating type in our A. flavus isolates and pairing opposite mating types to produce the sexual stage of A. flavus. The ability to perform crosses of isolates and determine segregation of phenotypic traits will allow elucidation of the genetic control and inheritance of traits such as aflatoxin production, CPA production, VCG determination, and biocontrol efficacy. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

The discovery of the "touch inhibition" requirement for intra-specific aflatoxin inhibition has led to the proposal of a role for "touch inhibition" in biological control. The previous competitive exclusion model focused on soil competition and minimizing inoculum potential of indigenous toxigenic strains in the soil. The "touch inhibition" model changes the focus to the infection court, silk, kernel, location and predicts that the biological control results from "touch inhibition" occurring in the infection court. We have used touch inhibition assays to screen non-toxigenic isolates for broadness of efficacy against multiple toxigenic isolates. The demonstration of specificity in the "touch inhibition" phenomenon allows further testing of the role of "touch inhibition" in biocontrol. There appear to be two phases where biocontrol can be exhibited. The saprophytic phase which occurs in the soil which is/should be required for long term sustainable control and the parasitic phase which occurs in the infection court, alluded to previously. This means that both the biocontrol and the "touch inhibited" toxigenic isolate should be present in the infection court. Work is ongoing to determine the mechanism of "touch inhibition". The 4 potenital biocontrol isolates selected by screening for "touch inhibition" all performed as well as if not better than the commercially available or soon to be available strains in the tests conducted at the 3 research stations. Work from her MS reported by R. Sweany at the APS meetings in Portland, OR, supported the idea that the A.flavus VCG populations found in corn kernels differed from the VCG populations present in the soil. There appear to be VCG's which have a preferred niche in corn kernels and are not highly represented in the soil, and conversely VCG's which have a preferred niche in the soil and are not highly represented in corn kernels. This means that biological control needs to utilize non-toxigenic isolates which inhibit toxin production by those toxigenic isolates/VCG's which end up colonizing the corn niche and the non-toxigenic isolates themselves must be capable of colonizing the corn kernel niche if our parasitic "touch inhibition" concept is correct. Further work indicated that the Mat 1-2 mating type isolates seemed to predominate in the corn kernel niche, while soil isolates were more evenly distributed between Mat 1-1 and Mat 1-2. Work looking at nanoparticle encapsidated vs non-encapsidated drug/antibiotic (sporanox) efficacy indicated that the encapsidated form of drug delivery was much more efficacious. This will be continued by looking at several other potential fungitoxic natural products. We successfully made crosses between the two A. flavus mating types, produced ascospores and are looking at segregation of various traits.


  • Damann, K, Huang, C, Jha, A, Sweany, R, DeRobertis, C, Danmek, K, and Punnapayk, H. 2009. Biological control of aflatoxin contamination using non-toxigenic Aspergillus flavus.(abstract) The 2nd Thai-American Symposiium on Plant Biomass, Biotechnology and Agriculture,S2. April 2,Bangkok, Thailand.
  • Damann, K, Huang, C, Jha, A, Sweany, R, DeRobertis, C, Danmek, K, and Punnapayk, H. 2009. Biological control of aflatoxin contamination using non-toxigenic Aspergillus flavus.(abstract)RGJ-PhD Congress X, April 3-5,p116.Pattaya, Thailand.
  • DeRobertis, C, Sweany, R, Damann, K. 2009. Atoxigenic biocontrol in corn:the Louisiana experience. Reducing Aflatoxin in Corn,p 12 (abstract) Oct 27-28,2009, Mississippi State University.
  • Damann, K, Huang, C, Jha, A, Sweany, R, DeRobertis, C. 2009. Biological control of aflatoxin contamination using non-toxigenic Aspergillus flavus.(abstract) Phytopathology 99(6):S27.
  • Sweany, R. and Damann, K. 2009. Comparison of aflatoxigenicity of corn kernel and soil populations of Aspergillus flavus. (abstract) Phytopathology 99(6):S126.