A field of cotton

2005 Aflotoxin Program Objectives

Atoxigenic Strain Technology for Aflatoxin Management in Cotton

Principal Investigators: Larry Antilla, Director, Az. Cotton Research & Protection Council Peter J. Cotty, Ph.D., Research Plant Pathologist, USDA Ag Research Service


Aflatoxin contamination of cottonseed has, for decades, created a major negative impact on the cotton production systems of Arizona and other southwestern desert areas. In order to ensure a healthy food supply in the United States, harvested seed containing more than 20 ppb of aflatoxin cannot enter the market as feed for dairy cattle. Widespread aflatoxin contamination results in sizable annual revenue losses to cotton producers, often in excess of $50 per acre.


For the past several years, technology, utilizing atoxigenic strains of the common fungus Aspergillus flavus, has produced highly encouraging results in reducing the aflatoxin producing potential of fungal communities in both pilot scale field tests and area wide commercial program settings. As a precursor to program development, the construction of a commercial scale grower owned manufacturing facility has enabled the production of atoxigenic strains to keep pace with expanding demand. Due to the complexities of any newly developing biological control system, this proposal requests continued funding for 2005 to assist in the extensive array of analyses necessary to evaluate both the development and implementation of the program as well as its potential for expansion to other crops.

Objectives include:

  1. Evaluation of crop microenvironment and ultraviolet light effects on atoxigenic strain AF36 in various stages of crop canopy development.
  2. Analysis of soil type in relation to application timing, moisture retention and atoxigenic strain development.
  3. Evaluation of the effects of nutrient enriched substrate medium with respect to fungal growth acceleration.
  4. Field evaluation of various additives for bird, insect and rodent repellence with possible effects on soil and crop carryover.
  5. Field test analysis of various substrates as potentially improved commercial carriers (i.e. sorghum vs. wheat).
  6. Complete stability studies and multi-batch aflatoxin analysis as required by EPA.
  7. Continue to evaluate product packaging and storage containers relative to product stability and/or contamination potential.
  8. Conduct preliminary field studies on selected seed coatings or encapsulates to provide UV and heat stress relief from atoxigenic strain wheat seed media.
  9. Begin screening tests on the effects of mixtures of atoxigenic strains in commercial treatment settings.
  10. Initiate efforts to develop predictive heat unit models for fungal strain developmental curves.
  11. Collect and analyze geographically diverse soil and crop fungal data as a basis for geostatistical analysis of area-wide aflatoxin distribution in relation to the make-up of fungal communities.

Technical Review

Aflatoxins are a group of toxic, carcinogenic fungal metabolites produced by specific isolates of Aspergillus flavus. Concern for human and animal health has led to the establishment of regulatory limitations on the quantity of aflatoxins permitted in foods and feeds throughout the world. Aflatoxin contamination creates serious problems for the United States cottonseed industry. This is due to the ability of dairy cattle to readily transmit aflatoxins from feed to milk in slightly modified form. In order to ensure a healthy food supply in the United States, laws prohibit the aflatoxin content of milk from exceeding 0.5 ppb. To ensure this level, cottonseed fed to dairy cattle is mandated to contain less than 20ppb. Overseas, aflatoxin controls are frequently more stringent than in the U.S. Thus, aflatoxin content can limit export of commodities and aflatoxin regulations can serve as non-tariff barriers to trade. The desert southwest and particularly Arizona is at a serious disadvantage in this area due to aflatoxin.


The fungal isolate AF36 is an atoxigenic member of the species Aspergillus flavus that has been shown to have the ability to competitively exclude aflatoxin-producing fungi and thereby reduce aflatoxin contamination of cottonseed. In greenhouse, field-plot, and commercial field tests, the efficacy of Aspergillus flavus AF36 has repeatedly been demonstrated. Results to date suggest that applications of AF36 reduce the average aflatoxin producing potential of fungal communities within both the treated and nearby fields. Treatments also have been shown to cause long-term reductions in the average aflatoxin producing potential of fungi in treated fields and in so doing provide the opportunity to reduce contamination throughout entire treatment areas over the long run. Evaluating the extent to which such reductions can be economically achieved is a major emphasis of the current work.


Data covering more than seven years of intensive field studies exists which supports the use of atoxigenic strains of Aspergillus flavus to reduce aflatoxin contamination. The application of this bio-control management strategy in both full field and area wide management settings strongly suggests that this technology will be capable of producing long term reductions in the quantity of aflatoxins that the fungi in an area are capable of producing. However, as stated above, this needs to be tested and agronomic procedures to optimize are-wide programs are needed. Efforts to develop area-wide management programs based on atoxigenic strain technology have been undertaken since 1999 with total cumulative statewide acres treated exceeding 105,000. Despite this level of use, many questions still demand resolution for atoxigenic strain technology to meet its potential of being a reliable tool for area-wide aflatoxin management programs.


Based on analysis of the data, the assumption is that utilization of atoxigenic strain technology in a large scale or area wide setting would significantly increase the level of program effectiveness over time. This is true for two principal reasons: 1) tests have shown that AF36 applications have influences beyond treated fields and 2) increased levels of AF36 in field soils tend to persist from one season or cropping cycle to the next, thereby resulting in a cumulative increase in the atoxigenic strain. These phenomena may very well result in a cost benefit ratio, which would be economically attractive to Arizona cotton producers.

Research Approach

Evaluation of the effects of the geographically diverse Arizona production areas on atoxigenic strain performance in aflatoxin reduction will be continued in 2005. Preliminary studies conducted in 2004 strongly indicated that crop microenvironment and ultraviolet light effects are linked to AF36 (atoxigenic strain) development and sporulation. Following commercial application in 2005, seed collections will be made from replicated fields representative of varying degrees of crop maturity and canopy closure and analyzed for AF36 development. Post harvest cottonseed samples and 2006 soil analysis will provide additional data relative to the evaluation process. A similar approach will be utilized to analyze the benefits of nutrient additives and various non-wheat substrates for AF36 development. Replicated tests will also be run to analyze the effectiveness of various bird, insect and rodent repellants as well as seed coatings with UV protectants to maximize AF36 field efficacy and durability. Efforts will also be initiated to screen mixtures of atoxigenic strains for potential improvements in field efficacy of the technology. Pre and one year post treatment soil samples in addition to post application seed analyses will be collected from replicated test areas and utilized for evaluation. Similar techniques will be employed to collect and analyze soil and crop fungal data which along with aflatoxin test results from cooperating gins, will be supplied to Dr. Cotty’s ARS group at the University of Arizona, Tucson, for geostatistical analysis.

Relevance Statement

The sale of cottonseed for dairy and feedlot cattle represents a key element in the agricultural economy of Arizona. The closing of the State’s last cottonseed oil mill in 1998 resulted in the elimination of a critical outlet for seed with high aflatoxin levels. The annual value of Arizona cottonseed exceeds $38 million. Losses due to high aflatoxin levels often are calculated at more than $9 million. This represents a significant loss to the State’s cotton producers. Prior to the introduction of atoxigenic strain technology no effective aflatoxin management tools existed. Data collected and analyzed from the past five years of are-wide commercial evaluation of this unique biological control system have validated the benefits of the technology. From a manufacturing standpoint, refinements in processing technique coupled with equipment modifications continue to improve and standardize the AF36 program.

Tentative Schedule of Events 2005

January – March: Stability studies and multi-batch aflatoxin analysis as required by EPA.


March – April: Collect soil samples throughout Arizona including treated and control fields.


May – July: Coordinate treatment of fields in Arizona with AF36. Monitor treatments for calibration and efficacy. Monitor cultural and agronomic practices for representative growers throughout the state. Gather test data for studies relevant to crop microenvironment, product nutrient and repellant additives. Perform soil type analyses and test seed coat encapsulates.


April – September: Utilize the Arizona Cotton Research & Protection Council assessment lab resources to analyze fungal communities in soils. Collect data for future use in geostatistical analysis. Coordinate operations with Dr. Cotty’s labs in Tucson and Southern Regional Research Center, New Orleans.


September – March: Extensive sampling and lab analysis of seed from 2005 crop. Samples to be taken from treated and non-treated fields in all program sites in Arizona. Acquire toxin data from gins and supply to Cotty’s group for geostatistical analysis.

Facilities and Equipment

The Arizona Cotton Research & Protection Council has two fully equipped laboratories; one for manufacturing and quality control of atoxigenic strain inoculum and one for conducting soil, crop and air sample fungal evaluation.


Field sampling activities necessary to generate soil, crop and air samples for fungal analyses are organized through six district supervisors working out of five field offices geographically located throughout Arizona’s cotton grower regions.


Investigators Qualifications


Larry Antilla, Staff Director

Arizona Cotton Research & Protection Council

3721 E. Wier Avenue, Phoenix, AZ 85040

B.S. 1966 Entomology, Arizona State University

Graduate Studies in Entomology (PhD Candidacy) 1966-1971


The investigator has developed and implemented area-wide pest management programs for more than thirty years. Currently administers field and research activities ($1.2 million annual budget) of the Arizona Cotton Research & Protection Council. Duties include: program design and implementation and cooperative interaction with local, state and federal organizations.


Peter J. Cotty, Research Plant Pathologist

Southern Regional Research Center

Agricultural Research Service, USDA

P.O. Box 1987, New Orleans, LA

PhD 1984, Plant Pathology, University of Arizona

M.S. 1980, Plant Pathology, Rutgers University

B.A. 1976, Biology, Boston University


The investigator has performed research on aflatoxin contamination of cottonseed since 1986. Throughout this period, his work has included field experiments performed in western Arizona. The investigator has been the principle developer of the use of atoxigenic strains to prevent aflatoxin contamination of cottonseed.