LAB93937 - Studies on Aspergillus Flavus and Biological Control of Aflatoxin Contamination of Louisiana Corn

Robert Carver, Damann, Jr., Kenneth E.

START: 01 OCT 2008 TERM: 30 SEP 2013 FY: 2010

INVESTIGATOR: Damann, K. E.; DeRobertis, C.; Huang, C.; Sweany, R.



KA Subject Science Pct
215 1510 1160 10
215 4020 1030 10
215 4020 1150 10
215 4020 1160 10
712 1510 1030 10
712 1510 1150 10
712 1510 1160 10
712 4020 1030 10
712 4020 1150 10
712 4020 1160 10

CLASSIFICATION HEADINGS: R215 . Biological Control of Pests Affecting Plants; S1510 . Corn; F1160 . Pathology; S4020 . Fungi; F1030 . Cellular biology; F1150 . Toxicology; R712 . Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins


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 specificity involved can lead to better approaches to deploying atoxigenic inoculum in a form which is maximally effective.

OBJECTIVES: Determine the population biology or spectrum of diversity of Aspergillus flavus in the Louisiana corn agro-ecosystem. Determine the specificity profiles for intraspecific 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.

APPROACH: 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. dsRNAs 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 noninhibitory atoxigenic isolates and also by comparing toxigenic isolates which are and are not inhibited by a particular nontoxigenic 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.

KEYWORDS: aflatoxins; intra-specific inhibition; vcg; touch inhibition; ssr fingerprinting; specificity profiles; root infection; dsrna; cyclopaizonic acid; biological control; biocontrol; aspergillus flavus

PROGRESS: 2010/01 TO 2010/12
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 Engineering 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.

IMPACT: 2010/01 TO 2010/12
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 successfully 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.

PUBLICATIONS (not previously reported): 2010/01 TO 2010/12
1. 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.
2. 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.
3. Damann, K. E., DeRobertis, C., and Sweany, R. 2010. Mating between Aspergillus flavus cryptic species I and II. Phytopathology 100(6):S28.
4. 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.
5. 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: 2009/01/01 TO 2009/12/31
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.

IMPACT: 2009/01/01 TO 2009/12/31
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 nontoxigenic 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 potential 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 nontoxigenic isolates which inhibit toxin production by those toxigenic isolates/VCG's which end up colonizing the corn niche and the nontoxigenic 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.

PUBLICATIONS: 2009/01/01 TO 2009/12/31
1. Damann, K, Huang, C, Jha, A, Sweany, R, DeRobertis, C, Danmek, K, and Punnapayk, H. 2009. Biological control of aflatoxin contamination using nontoxigenic Aspergillus flavus.(abstract) The 2nd Thai-American Symposium on Plant Biomass, Biotechnology and Agriculture,S2. April 2,Bangkok, Thailand.
2. Damann, K, Huang, C, Jha, A, Sweany, R, DeRobertis, C, Danmek, K, and Punnapayk, H. 2009. Biological control of aflatoxin contamination using nontoxigenic Aspergillus flavus.(abstract)RGJ-PhD Congress X, April 3-5,p116.Pattaya, Thailand.
3. 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.
4. Damann, K, Huang, C, Jha, A, Sweany, R, DeRobertis, C. 2009. Biological control of aflatoxin contamination using nontoxigenic Aspergillus flavus.(abstract) Phytopathology 99(6):S27.
5. Sweany, R. and Damann, K. 2009. Comparison of aflatoxigenicity of corn kernel and soil populations of Aspergillus flavus. (abstract) Phytopathology 99(6):S126.


Name: Damann, K. E.
Phone: 225-578-1401
Fax: 225-578-1415
7/20/2011 9:21:30 PM
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