Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/27/2004
Publication Date: 10/21/2004
Citation: Molyneux, R.J., Mahoney, N.E., Kim, J.H., Campbell, B.C. 2004. Inhibition of aflatoxin biosynthesis in aspergillus flavus by phenolic natural products. Meeting Abstract.
Technical Abstract: Agricultural crops, especially corn, cotton, peanuts and treenuts, may be infected by Aspergillus flavus and A. parasiticus, resulting in contamination by aflatoxins which can present a risk of hepatocarcinogenicity to humans. All tree nuts are subject to contamination but walnuts are exceptionally resistant to aflatoxigenesis. We have investigated the compounds naturally present in the walnuts which are responsible for the inhibition of aflatoxin formation. The resistance factor(s) are restricted to the seed coat (pellicle) and are not present in the kernel. Chemical analysis of the seed coat has established that the inhibitory activity resides in a complex of hydrolysable tannins common to all walnut cultivars. In vitro experiments showed that tannin from the cultivar 'Tulare' completely suppressed growth of A. flavus at a concentration of 0.5% in the media, with no aflatoxin formed. The extracellular tannase present in A. flavus produces gallic acid and ellagic acid by hydrolysis of the tannin. In vitro time-course experiments with Vogel's medium N (VMN) showed that gallic acid completely inhibited aflatoxin production whereas ellagic acid was essentially inactive. Treatment of walnut seed coat tissue with anhydrous methanolic HCl yields methyl gallate and ellagic acid, the levels of which can be measured by reverse phase HPLC. Gallic acid levels in seed coat of 'Tulare' and the variety 'Chico', which is susceptible to aflatoxin formation, were determined on a biweekly basis throughout the growing season during 2002 and 2003. Levels in 'Tulare' were significantly higher, and were maintained throughout the growing season, whereas those in 'Chico' declined steadily as the nuts matured. At maturity, 'Tulare' had a gallic acid content of 3.3% (dry weight basis) while the level in 'Chico' was only 1.4%. Pistachio seed coat had 0.5% gallic acid, but almond varieties only had trace levels (<0.1%). Within the group of tree nut seed coats tested so far, inhibition of aflatoxin production correlated with the amount of gallic acid present. The evidence indicates that hydrolysable tannins are capable of inhibiting growth of A. flavus and that aflatoxigenicity is phytochemically inhibited by biosynthesis and maintenance of high levels of tannins throughout the growing season. Gallic acid, produced in situ by a fungal tannase, is the specific tannin component responsible for suppression of aflatoxin biosynthesis by the fungus and should be amenable to enhancement by conventional breeding or genetic manipulation. The mechanism whereby gallic acid suppresses aflatoxin biosynthesis is currently under active investigation. Neither norsolorinic acid, an early precursor in the aflatoxin biosynthetic pathway, nor any of the subsequent anthraquinone metabolites, appear to be formed. This suggests that gallic acid could be affecting the genes controlling fatty acid synthase (aflA and aflB) and/or polyketide synthase (aflC) involved in the synthesis of the polyketide precursor of norsolorinic acid. However, the pathway regulator gene aflR is still expressed in the presence of gallic acid. The efficiency of gallic acid as an antioxidant suggests that it may act by down-regulating a transcriptional activation factor through a signaling pathway involved in the oxidative stress response. This hypothesis is being tested by using complementation analysis of deletion mutants of the yeast Saccharomyces cerevisiae as a model system. The approach allows for high throughput screening of genes involved in oxidative stress response induction of aflatoxin biosynthesis.