Postharvest Pathology and Mycotoxins Pathogenesis in Aspergillus Ear Rot of Maize: Aflatoxin B1 Levels in Grains Around Wound-Inoculation Sites

Smart, M. G., Shotwell, O. L., and Caldwell, R. W. 1990. Pathogenesis in aspergillus ear rot of maize: Aflatoxin B1 levels in grains around woundinoculation sites. Phytopathology 80: 1283-1286. Aflatoxin contamination of preharvest maize intermittently presents serious problems in grain storage and in animal health. To determine whether this mycotoxin can be translocated through the ear in the absence of hyphae, we harvested 21 grains from around each of 21 wound-inoculation sites of maize ears matured at 34/30 C (day/night). Individual grains were analyzed for aflatoxin by enzyme-linked immunosorbent assay. Maize spikelets are borne in pairs and, if aflatoxin is transported in the rachis, grains in a pair should have similar toxin levels, but nonpaired grains mayor may not. Statistical treatment showed that no two grains Additional keywords: Aspergillusflavus, ELISA, mycotoxins, Zea mays. Accumulated aflatoxin in stored grain and seeds is concentrated in a small number of individuals (2,7,12,13). Lee et al (8) reported a pattern of highly variable toxin levels in single maize grains taken from the Aspergillus flavus Link:Fr. infested portion of three insect-damaged ears. They also found highly contaminated grains (up to 8 X 104 ng aflatoxin g-I) adjacent to grains lacking detectable aflatoxin. The pattern of aflatoxin accumulation in individual grains around an inoculation site has not been investigated directly. However, clues to toxin variability are found in the data of Wicklow and coworkers (18,19), who grouped intact grains surrounding sites of inoculation with A. flavus according to their proximity to the wound. The authors found that, on average, aflatoxin levels decreased with increasing distance from the wound, but, within groups, levels were variable. Very little is known of the mechanics of toxin accumulation in seeds on developing maize ears. In the most general sense, the variability evident in work published previously could have arisen in two ways: either the toxin alone (in the absence of hyphae) was able to move by diffusion through tissues of the rachis to contaminate other grains, or the grains first were infected by hyphae before aflatoxin could accumulate. The data of Lee et al (8) are inconclusive on this point but did identify grains which had aflatoxin but no visible evidence of fungal colonization. In this regard, Mertz et al (10) injected aflatoxin into an internode of the ear shank and recovered aflatoxin from the grains 1 mo later, indicating toxin movement. We undertook this study to further document the variability in toxin levels and to investigate the mechanism of toxin accumulation in grain. We harvested 21 grains from around each of 21 wound-inoculation sites and determined aflatoxin levels in the individual grains. We also noted the incidence of fungal signs This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1990. chosen at random had different average toxin levels: there was no pattern discernible in toxin accumulation. Highly contaminated individual grains rarely had highly contaminated neighbors. Finally, of the 413 grains assayed, almost 80% either were aflatoxin-positive and showed signs of the fungus or were not aflatoxin-positive and had no signs of the fungus. Only 58 grains (14%) had detectable toxin levels without fungal signs. We conclude that long-distance transport of aflatoxin does not occur in infected ears independently of the hyphae. in the grains. The data provide clues to the mechanics of pathogenesis in this disease and form the basis of a histological study of the in situ growth of A. flavus (14). MATERIALS AND METHODS Suscept and pathogen material. Maize (Zea mays L. DeKalb hybrid XLl2) was grown under controlled conditions at the University of Wisconsin Biotron, Madison (I). Briefly, the plants were grown under a l2-hr / 12-hr day/night photoperiod with a daytime flux of 600 j.LEm-sphotosynthetically active radiation measuring 1.5 m above the topmost leaf). The temperature was 30 C during the day and 20 C during the night until pollination was complete. After pollination, the temperature was raised to 34 C during the day and 30 C during the night. Relative humidity was approximately 85%, and the plants were watered liberally to prevent stress. The inoculum used was a mixture of conidia from 10 isolates of A. flavus obtained from the collection of the Northern Regional Research Center. Their numbers were as follows: NRRL 6536, NRRL 6537, NRRL 6539, NRRL 6540, NRRL 6576, NRRL 6577, NRRL 6578, NRRL 6579, NRRL 6580, and NRRL 658 I. All isolates were collected originally from maize (17). A 10-isolate mixture was used because a single isolate did not spread from the wound sites under a 30/20 day/night temperature regime (see the accompanying paper (14) for a full discussion of this point). Preparation of inoculum and inoculation. The isolates were grown separately on malt extract agar for 7 days at 22 C in the dark. Conidial suspensions were prepared by flooding the plates with sterile, distilled water containing 0.01% Triton X100. Spore concentration was adjusted to I X 10 ml, and equal volumes of the 10 suspensions were combined. Maize ears were wounded at 21 days after silk emergence by inserting a sterile toothpick through the husks into a grain and the underlying rachis. The toothpick was withdrawn, dipped in the spore suspension, Vol. 80, No. 12, 199