Evaluation of the Atoxigenic Aspergillus flavus Strain AF36 in Pistachio Orchards

Doster, M. A., Cotty, P. J., and Michailides, T. J. 2014. Evaluation of the atoxigenic Aspergillus flavus strain AF36 in pistachio orchards. Plant Dis. 98:948-956. The atoxigenic strain Aspergillus flavus AF36, which has been extensively used as a biocontrol agent in commercial corn and cotton fields to reduce aflatoxin contamination, was applied in research pistachio orchards from 2002 to 2005 and in commercial pistachio orchards from 2008 to 2011. AF36 was applied as hyphae-colonized steamsterilized wheat seed (the same product and same application rate as used in cotton fields). In all orchards, applying the wheat-AF36 product substantially increased the proportion of vegetative compatibility group (VCG) YV36, the VCG to which AF36 belongs, within A. flavus soil communities. Application of the AF36 product in additional years further increased YV36 in the soil until it composed 93% of the A. flavus isolates in treated commercial orchards. Nonetheless, application of the AF36 product did not result in increased incidence of kernel decay of the nuts. For nuts harvested from commercial orchards, reductions in percentages of samples contaminated with aflatoxin from treated orchards (relative to that for untreated orchards) ranged from 20 to 45%, depending on the year. Because of the high value of pistachio nuts and the costs associated with rejection of shipments due to aflatoxin contamination, these reductions are significant and valuable to the pistachio industry. Aflatoxins, which are potent toxins and carcinogens, are widely regulated by governments, with the tolerances set very low in food (43). Aflatoxins are mainly produced by two closely related fungi, Aspergillus flavus Link and A. parasiticus Speare, and they naturally grow in many crops, including corn, peanut, and cottonseed. These aflatoxin-producing fungi also grow at low levels in pistachio nuts (Pistacia vera L.) in commercial orchards in California, occasionally resulting in aflatoxin contamination (27). The incidence of aflatoxin-contaminated pistachio nuts in California is somewhat low. For example, it has been estimated at 1 aflatoxincontaminated nut per 25,000 nuts (41). Nonetheless, due to very low tolerances of regulatory agencies and the problems associated with the rejection of shipments of pistachio nuts, these levels of aflatoxin contamination result in significant financial problems for the pistachio industry in California. The most common Aspergillus sp. found decaying pistachio nuts in California is A. niger, which is much more common than the aflatoxin-producing A. flavus and A. parasiticus (27). The aflatoxin-producing fungi typically grow in plant litter on the orchard floor of pistachio orchards (28) and, in late summer, can infect nuts on the tree, especially nuts with both hull and shell split known as early split nuts (27,29). Naturally occurring isolates of A. flavus that are not able to produce aflatoxin (“atoxigenic” strains) have been used successfully to reduce aflatoxin contamination in cottonseed (3,5,14,19), peanut (24,26), and corn (7,8,12,25). Atoxigenic strains reduce aflatoxin contamination by displacing or excluding aflatoxin-producing fungi (17). The atoxigenic A. flavus AF36 has been developed as a biocontrol agent for preventing aflatoxin contamination of cottonseed (3,5). Currently, AF36 is used in commercial cotton production in Arizona, California, and Texas and in corn production in Texas and Arizona. Steam-sterilized wheat seed colonized by A. flavus isolate AF36 is applied in fields to the surface of the soil once each year in the late spring or early summer at the rate of 11.2 kg/ha. After exposure to moisture through irrigation, dew, rain, or high humidity, the wheat becomes rehydrated, allowing the fungal biocontrol agent to grow on the biomass of the dead wheat seed and produce spores for dispersal. The effectiveness of AF36 applications in cotton was determined by quantifying the effect on fungal populations and by measuring the reduction in aflatoxin contamination of the crop (3,5,14). The standard method used for identifying the AF36 fungus within A. flavus populations has been to determine whether A. flavus isolates belong to the same vegetative compatibility group (VCG) as the AF36 isolate, VCG YV36 (3,14,21). The major objectives of this research were to (i) quantify the occurrence of VCG YV36 (the VCG of the AF36 isolate) in commercial pistachio orchards in California prior to the application of the AF36 product in any commercial orchard, (ii) measure the effectiveness of applying the AF36 product in increasing the frequency of VCG YV36 in the fungal population in commercial pistachio orchards, (iii) evaluate whether application of the AF36 product results in increases in the incidence of decayed nuts, and (iv) determine the influences of application of the AF36 product on the aflatoxin contamination of the nuts. Materials and Methods Natural occurrence in commercial orchards. Incidences of VCG YV36, the VCG to which AF36 belongs, in commercial pistachio orchards was determined with a collection of 1,255 A. flavus isolates from pistachio orchards (38 different commercial orchards in eight counties) already present in our culture collection at the Kearney Agricultural Center. The A. flavus isolates had been obtained during our various studies of aflatoxin contamination in commercial pistachio orchards in California between 1990 and 2007, prior to application of the AF36 product in commercial orchards. Although most of the isolates were obtained from pistachio orchard soil, many isolates were also obtained from inflorescences, Corresponding authors: M. A. Doster, E-mail: [email protected]; and T. J. Michailides, E-mail: [email protected] This research was supported by the United States Department of Agriculture Cooperative Research Agreement Number 58-5325-1-194 and 585325-6-844 and the California Pistachio Research Board. Accepted for publication 12 February 2014. http://dx.doi.org/10.1094/PDIS-10-13-1053-RE © 2014 The American Phytopathological Society