Joe W. Dorner

Biological Control of Aflatoxin Contamination of Crops

Aflatoxin contamination of crops compromises the safety of food and feed supplies and causes significant economic losses each year. Of the many research approaches being studied to reduce and, ultimately, eliminate aflatoxin contamination, biological control is one of the more promising, particularly for the near‐term. Numerous organisms have been tested for biological control of aflatoxin contamination including bacteria, yeasts, and nontoxigenic strains of the causal organisms, Aspergillus flavus and A. parasiticus. Most of the field successes to date have been achieved by applying certain nontoxigenic strains of A. flavus and A. parasiticus to soil of susceptible crops, such as peanuts, cotton, and corn. The applied strains occupy the same niche as the naturally occurring toxigenic strains and competitively exclude them when crops are susceptible to infection. Various formulations have been used to apply the nontoxigenic strains to soil, but the most effective methods have been to combine the desired strain with a carrier/substrate, such as a small grain. This was done either by minimally growing the desired strain on sterilized grain or by coating the surface of the grain with conidia of the strain. After application to the field and uptake of moisture, the fungus completely colonizes the grain, and abundant sporulation provides inoculum levels sufficient to achieve a competitive advantage for the nontoxigenic strain. In several years of field studies, particularly with peanuts and cotton, significant reductions in aflatoxin contamination in the range of 70–90% have been achieved consistently. Two separate products have recently received EPA registration as biopesticides to control aflatoxin contamination in cotton (AF36) and peanuts (afla‐guard®).

The relationship of Aspergillus flavus and Aspergillus parasiticus with reference to production of aflatoxins and cyclopiazonic acid

Forty-seven isolates of Aspergillus parasiticus were analyzed for production of aflatoxins B1, B2, G1, G2, and cyclopiazonic acid. None produced cyclopiazonic acid, whereas 46 of 47 produced aflatoxins B1 and G1. These data are related to previous studies pertaining to A. flavus and illustrate species validity from a biochemical standpoint.

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus.

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.

Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis

Aspergillus flavus is the major producer of carcinogenic aflatoxins (AFs) in crops worldwide. Natural populations of A. flavus show tremendous variation in AF production, some of which can be attributed to environmental conditions, differential regulation of the AF biosynthetic pathway and deletions or loss‐of‐function mutations in the AF gene cluster. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative differences in aflatoxigenicity. Several population studies using multilocus genealogical approaches provide indirect evidence of recombination in the genome and specifically in the AF gene cluster. More recently, A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses using toxin phenotype assays, DNA sequence‐based markers and array comparative genome hybridization. We show high AF heritability linked to genetic variation in the AF gene cluster, as well as recombination through the independent assortment of chromosomes and through crossing over within the AF cluster that coincides with inferred recombination blocks and hotspots in natural populations. Moreover, the vertical transmission of cryptic alleles indicates that while an A. flavus deletion strain is predominantly homokaryotic, it may harbour AF cluster genes at a low copy number. Results from experimental matings indicate that sexual recombination is driving genetic and functional hyperdiversity in A. flavus. The results of this study have significant implications for managing AF contamination of crops and for improving biocontrol strategies using nonaflatoxigenic strains of A. flavus.

Effect of competition and adverse culture conditions on aflatoxin production by Aspergillus flavus through successive generations

Strains of Aspergillus flavus often degenerate with serial transfers on culture media, resulting in morphological changes and loss of aflatoxin production. However, degeneration does not readily occur in nature as indicated by the wild-type morphological characters of newly isolated strains and the high percentage of aflatoxigenic A. flavus from soil and crops in some geographic regions. In this study, three aflatoxin-producing strains of A. flavus were serially transferred using conidia for 20 generations (three independent generation lines per strain) on potato dextrose agar at 30 C. The rate of degeneration was compared to that of cultures grown in the presence of competing fungi (A. terreus, Penicillium funiculosum, and the yeast, Pichia guilliermondii) and under adverse conditions of elevated temperature, reduced water activity, low pH, and nutrient deprivation. Formation of morphological variants and the associated loss of aflatoxin production over generations varied considerably according to strain and the generation line within each strain. In the strain most sensitive to degeneration on potato dextrose agar, aflatoxin-producing ability was maintained to varying degrees under adverse culture conditions, but not when A. flavus was competing with other fungi.

Conidial movement of nontoxigenic Aspergillus flavus and A. parasiticus in peanut fields following application to soil.

The use of nontoxigenic strains of Aspergillus flavus and A. parasiticus in biological control effectively reduces aflatoxin in peanuts when conidium-producing inoculum is applied to the soil surface. In this study, the movement of conidia in soil was examined following natural rainfall and controlled precipitation from a sprinkler irrigation system. Conidia of nontoxigenic A. flavus and A. parasiticus remained near the soil surface despite repeated rainfall and varying amounts of applied water from irrigation. In addition, rainfall washed the conidia along the peanut furrows for up to 100 meters downstream from the experimental plot boundary. The dispersal gradient was otherwise very steep upstream along the furrows and in directions perpendicular to the peanut rows. The retention of biocontrol conidia in the upper soil layers is likely important in reducing aflatoxin contamination of peanuts and aerial crops such as corn and cottonseed.

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Abstract Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.

Isolation and identification of two new biologically active norditerpene dilactones from Aspergillus wentii

Abstract Two new biologically-active norditerpenoid dilactones were purified from culture extracts of Aspergillus wentii and assigned the trivial names wentilactone A and wentilactone B. The absolute chemical structure of wentilactone A was determined by single crystal X-ray diffraction and circular dichroism. The structure of wentilactone B was determined by 1 H and 13 C NMR analyses. Wentilactone A had an ld 50 of 7.0 mg/kg when administered orally to 1-day-old chickens. Both metabolites inhibited growth in wheat coleoptile bioassays.

Combined effects of the mycotoxins aflatoxin B1 and cyclopiazonic acid on Sprague-Dawley rats

This study was conducted to determine whether exposure to cyclopiazonic acid (CPA) and aflatoxin B1 (AFB1) would alter the toxicity associated with exposure to either toxin individually. Groups of male rats were administered 0, 0.1 or 4.0 mg CPA/kg body weight/day intragastrically (three groups per dose level) for three consecutive days and 30 min after each of these CPA doses the rats were dosed by gavage with 0, 0.1 or 2.0 mg AFB1/kg body weight/day. Six of the 12 rats given each of these nine treatments were killed on day 4 after the initial dosing, and the rest were allowed a recovery period of 4 days prior to being killed. Weight loss in the three groups receiving 2.0 mg AFB1/kg/day occurred within 24 hr of the first doses. Feed consumption by these rats was about 60% of that in the other groups. By the end of the recovery period, rats in these three groups had lost an average of 31-38 g. Feed consumption throughout the recovery period by rats in the 2.0-mg AFB1 groups was about 50% of the control value, except in the group that also received the high dose of CPA, in which it was 75%. Gross pathological findings were primarily limited to rats in the high AFB1 group, and included icterus, shrunken liver and lesions in the kidney at the cortico-medullary junction. Microscopic changes were characteristic of aflatoxicosis in rats. Glycocholic acid assays indicated liver damage only in those groups that received the high AFB1 dose. We conclude that neither toxin potentiates the action of the other at the dose levels used in this study.

Increased accumulation of the lipophilic cation tetraphenylphosphonium+ by cyclopiazonic acid‐treated renal epithelial cells

Pig kidney renal epithelial cells (LLC-PK1) in culture were used to determine the effects of cyclopiazonic acid (CPA) on the uptake of the transmembrane potential probe, [3H]tetraphenylphosphonium bromide (TPP+). CPA had a significant stimulatory effect on TPP+ accumulation, which occurred in a dose-related manner. TPP+ accumulation in the presence of CPA was significantly reduced by high-potassium media (HK) and carbonylcyanide m-chlorophenylhydrazone (CCCP), but neither HK nor the protonophore CCCP, could completely abolish the stimulatory effect of CPA. The apparent transmembrane potential difference (delta psi), calculated based on the difference in accumulation of TPP+ in low-potassium and HK media, ranged from -55.9 to -85.7 mV for control cells and -89.4 to -109.0 mV for CPA-treated cells (20 mg CPA/I). The mechanism of CPA stimulation of TPP+ accumulation was not known. However, it was hypothesized that the effect could be a result of alterations in ion pumps or altered membrane permeability. The fact that the stimulatory effect could not be completely abolished by high potassium or CCCP suggested that there was some interaction between CPA and TPP+ or there were sites of TPP+ accumulation that were insensitive to K+ and H+ permeability.