Tracking Aspergillus flavus Infection in Cottonseed to Prevent Preharvest Aflatoxin Contamination

The saprophytic soil-borne fungus Aspergillus flavus produces the carcinogenic aflatoxins in lipid-rich seeds of corn, cotton, peanuts and tree nuts.   In order to develop an effective control strategy, it is important to study and evaluate the fungal interaction with the susceptible plant.   We have developed a GFP-expressing A. flavus strain to study fungal invasion and colonization process in cottonseed in real time without the need for destructive sampling.  Using fluorescence microscopy of open bolls and seeds inoculated with the GFP-expressing A. flavus strain we have demonstrated the following:  1) Fungal invasion is rapid within the boll; infection of the distal seeds was not significantly different from the apical seeds within a locule. 2) Seed infection proceeds from the chalazal end to the micropylar end in intact seeds.  3) The seed coat is colonized rapidly (48-72 h), with penetration into the seed apparently occurring through the “chalazal plug” within 24-48 h of infection.  4) Once the fungus has penetrated, hyphal spread appears to progress along the seed coat-cotyledon interface and within about 48-72 h the cotyledon shows signs of fungal infection.  5) GFP in the cotyledon could be detected at about 48 h followed closely by aflatoxin production beginning at about 72 h and reaching high levels (200 ppm) by 168 h.   6) In addition, we were able to evaluate resistance to A. flavus due to expression of antifungal proteins and peptides by using the GFP strain.  We conclude that this rapid method of evaluation in situ or in planta is very useful in studies on preharvest aflatoxin contamination due to Aspergillus, an opportunistic saprophyte that does not follow the classical host-pathogen relation postulates.