P.J. Cotty, Page NO.1 of 8.

P.J. Cotty, Page NO.1 of 8. POSITION AND AFLATOXIN LEVELS OF TOXIN POSITIVE BOLLS ON COTTON PLANTS. P.J. Cotty and L.S. Lee Research Plant Pathologist and Research Chemist USDA, ARS, Southern Regional Research Center New Orleans, LA Abstract Cotton bolls collected from fields in the Yuma Valley during 1987 and 1988 were assayed for aflatoxin on an individual basis. Distribution of aflatoxin contaminated bolls was scattered but, generally most aflatoxin occurred in the lower half of a plant. A greater proportion of pink bollworm (PBW) damaged bolls produced seed contaminated with aflatoxins than bolls not damaged and over ge% of the total aflatoxin detected was in seed produced in PBW damaged bolls. During 1988, bolls that formed early in ~he season contained more aflatoxin than bolls that formed later. Bolls damaged by PBW early in the season when fungal inoculum was low were infected at equal or greater frequency than bolls damaged late in the season. The results suggest that protection of early bolls from PBW damage should be a component of management programs directed towards preventing aflatoxin contamination of cottonseed. Introduction Aflatoxins are toxic, carcinogenic chemicals that are frequent contaminants of agricultural commodities and pose a threat to the health of humans and domestic animals (7). Economic loss due to aflatoxin contamination of cottonseed still persists more than 25 years after aflatoxins were first detected in cottonseed and cottonseed meal (7, 12). Little information is available on the distribution of aflatoxin contamination in cotton fields. Ashworth observed that most toxin occurs in bolls borne close to the soil (3) and several researchers have associated aflatoxin contamination of cottonseed with insect damage in the field (2,11,8).A. flavus colonization of pink bollworm (PBW), Pectinophora gossypiella (Sanders), exit holes results in seed infection (2). However, a specific and consistent association between PBW damage of bolls and aflatoxin contamination of harvested cottonseed has not been directly established (8,le). The current study was carried out to describe the distribution of aflatoxin contamination among bolls produced in the Yuma Valley. It was

P.J. Cotty, Page NO.2 of 8. hoped that this description development of techniques contamination of cottonseed. might facilitate the to manage aflatoxin Materials and Methods In 1987, cotton bolls were collected from cotton fields in the Yuma Valley in October. In one experiment, at randomly selected locations in each of two fields, fully mature bolls in the lower half of the plants were examined for pink bollworm exit holes in the carpel walls. Each boll with an exit hole was collected along with an apparently undamaged boll at a similar position on the same plant. Bolls were dried and analyzed for aflatoxins as previously described (5) Twenty five pairs of bolls from each field were collected. In a second study, all bolls on a plant were individually harvested, labeled by position, dried and analyzed for aflatoxins (5). Onehundredtwentynine bolls from a total of six plants collected from four fields were examined. In 1988 cotton was planted on 40 inch centers at the Yuma Valley Agricultural Center near Yuma, Arizona. Four rows of Gossypium hirsutum cultivar Oeltapine (OP) 90 were alternated with four rows of cultivar OP 61. Eight replicate blocks were sampled; each replicate consisted of four rows of each cultivar. In mid-july, in each replicate, 60 bolls (20 to 30 days old) and 60 flowers of each cultivar were tagged for identification at harvest. All tagged bolls were harvested on October 25, dried, subsampled and analyzed for toxin content as previously described (5). Pink bollworm control sprays were not initiated until a 30% infestation was reached. This permitted extensive damage to the crop and increased the likelyhood of a high incidence of aflatoxin contaminated cottonseed. Entire plants of OP 90 were also collected from this field and boll positions were noted prior to boll removal as in 1987. After drying, the bolls were examined for pink bollworm exit holes and the lint of bolls with exit holes was examined under an ultraviolet light for bright-green-yellow fluorescence (BGYF) as previously described (3,5). Presence of this fluorescence indicates infection by A. flavus (3). Results During 1987, pairs of bolls collected from the lower half of cotton plants in two fields in the Yuma Valley had different levels of aflatoxins in PBW damaged and non-damaged bolls (Table 1). Pink bollworm damaged bolls contained over 95% of the aflatoxin.

P.J. Cotty, Page NO.3 of 8. The 129 bolls from six plants assayed during the 1987 season bore seed which contained an overall average aflatoxin Bl level of 28~ PPB. Nine percent of the bolls contained aflatoxins (limit of detection 5 PPB). Twenty-two percent of the bolls were pink bollworm damaged and these bolls contained 99.6% of the toxin. However, over 98% of the toxin was contained in a single pink bollworm damaged boll located near the bottom of a plant (Figure 1). In field plots at the Yuma Valley Agricultural Center (1988), where PBW control was not initiated until a 3~% infestation was reached, 3~ to 40 percent of the bolls resulting from flowers produced in late June were damaged by the pink bollworm. Ninety to 100 percent of bolls resulting from flowers formed in mid-july were pink bollworm damaged. The earlier bolls of OP 90 and DP 61 bore seed which contained average aflatoxin Bl concentrations of 8,452 PPB and 7,~72 PPB, respectively. Bolls of these cultivars formed 20 to 30 days later contained significantly less aflatoxin Bl (7~8 PPB and 2040 PPB respectively) even though they were more extensively damaged by PBW. Infection of pink bollworm A. flavus, as indicated by BGYF frequently on early formed bolls late in the season (Table 2). damaged bolls by on lint, occured more than on bolls formed Discussion The results presented here suggest two important components of aflatoxin contamination of cottonseed in the Yuma Valley: 1) A direct relationship between pink bollworm damage of bolls in the lower half of the plant and aflatoxin contamination. 2) Bolls produced early in the season and damaged by PBW tend,to become infected with A. flavus at a high frequency. Results agree with previous observations on association of contamination with insect damage (2,8,11) and bolls formed near the soil (1,3) and early in the season (10). Results from the 1988 field plot studies indicate much more toxin formation in bolls forming from flowers produced in late June than in bolls formed about four weeks later even though the later formed bolls were more extensively damaged by PBW. Furthermore, the rate of infection of PBW damaged bolls (as indicated by BGYF) in the lower portion of plants was greater than in the later formed bolls, even though it has clearly been demonstrated that inoculum of A. flavus increases as the season progresses (9). This suggests that early inoculum has either a vector association or

P.J. Cotty, Page NO.4 of 8. competitive advantage that is lacking in the inoculum occurring later in the season. Damaged bolls formed early in the season or close to the soil may be associated with a vector that other bolls are not. Presence of BGYF on lint does not indicate the level of secondary infection occurring after bolls open because BGYF is typically not produced on lint of most bolls infected after opening (1,3). Inoculum built up during the season may be most significant in inciting secondary infections of fully mature bolls. Results of the current study suggest that although preharvest insecticide treatments have not consistently demonstrated a clear quantitative r~lationship between PBW damage and aflatoxin contamination of cottonseed (8), previous researchers were correct in recommending control of PBW populations to reduce aflatoxin levels (2,11) Failure to statistically relate PBW populations to aflatoxin levels in some fi~ld studies (8), may be attributable to the great variability in both PBW damage and aflatoxin. It may also be attributable in part to the differential importance of pink bollworm damage at different periods during the season. Results of the current study suggest that initiation of insecticide applications for PBW control aft~r a 5 or 10% infestation has been reached may not be adequate to produce seed with acceptable aflatoxin levels in the Yuma Valley. The high frequency of infection and contamination of PBW damaged bolls early in the season suggests that the first bolls formed need to be preferentially protected to optimize management practices. A sound management program for aflatoxin contamination of cottonseed should include prevention of PBW damage to early bolls and timely harvest (4). Acknowledgements This research was conducted in cooperation with the Yuma Valley Agricultural Center and the Deparment of Plant Pathology of the University of Arizona. We thank both these departments for their generous help and cooperation. We thank the National Cottonseed Products Association for assistance with travel expenses. We also thank Paulette Francisco, Lisa G. Fortune, and Lisa A. Williams for technical assistance. References 1. Ashworth, L.J., 1969. Infection of Epidemiology of the 193-202. Jr., J.L. McMeans and C.M. Brown. cotton by Aspergillus flavus: Disease. J. stored Prod. Res. 5,

P.J. Cotty, Page NO.5 of 8. 2. Ashworth, L.J., Jr., R.F. Rice, J.L. McMeans, and C.M. Brown. 1971. The relationship of insects to infection of cotton bolls. Phytopathology 61:488-493. 3. Ashworth, L.J., Jr., and Association of Aspergillus flavus greenish yellow fluorescence Phytopathology 56:1104-1105. McMeans, J.L. 1966. and aflatoxins with a of cotton seed. 4. Cotty, P.J. 1989. Influence of harvest date on aflatoxin contamination of cottonseed. Proc. Beltwide Cotton Prod. Res. Conf. pg. 235. 5. Cotty, P.J. 1989. Effects of cultivar and boll age on aflatoxin in cottonseed after inoculation with Aspergillus flavus at simulated exit holes of the pink bollworm. Plant Disease 73:489-492. 6. Cotty, P.J. contamination of bollworm damaged 29:273-277. and L.S. Lee. 1989.. Aflatoxin cottonseed: comparison of pink and undamaged bolls. Trop. Sci. 7. Goldblatt, L.A. 1970. Chemistry and ~ontrol of Aflatoxin. Pure and Appl. Chem. 21:331-353. 8. Henneberry, T.J., I.A. Bariola, and T.E. Russell 1978. Pink bollworm: Chemical control in Arizona and relationship to infestations, seed damage, and aflatoxin in cottonseed. J. Econ. Entomol. 71:440-448. 9. Lee, L.S., L.V. Lee, Jr. and T.E. Russell. 1986. Aflatoxin in Arizona cottonseed: Field inoculation of bolls by Aspergillus flavus spores in wind-driven soil. J. Am. Oil Chemists Soc. 63:530-532. 10. Russell, T.E., L.S. Lee, and S. Buco. 1987. Seasonal formation of aflatoxins in cottonseed produced in Arizona and California. Plant Disease 71:174-177. 11. Russell, T.E., T.F. Watson and G.F. Ryan. 1976. Field accumulation of aflatoxin in cottonseed as influenced by irrigation termination dates and pink bollworm infestation. Appl. Environ. Microbiol. 31:711-713. 12. Whitten, M.E. 1966. A rapid screening method for detecting aflatoxins in cottonseed products. The Cotton Gin and Oil Mill Press 67:7-8.

P.J. Cotty, Page NO.6 of 8. Table 1- Aflatoxin B, content of cottonseed from bolls damaged and not damaged by the pink bollworm Af latoxin B, (PPB) ---------------------------- Field Boll type Percent Maximum Minimum Average 1 Damaged 68 54,612 0 4,378 Not damaged 8 12 0 2 2 Damaged 54 44,380 8 5,289 Not damaged 0 93 0 5 1/ This table is adapted from data Each value represents data from individual bolls. in 25 reference 6. analyses of

P.J. Cotty, Page NO.7 of 8. Table 2. The relation of boll position to bright-green-yellow-fluorescence (BGYF) on lint of pink. bollworm damaged bolls. Branch Number of Percent with Number Bolls BGYF 1 11 55 2 13 31 3 15 53 4 15 40 5 15 27 6 13 38 7 13 23 8 13 46 9 10 10 10 8 25 11 5 8~ 12 9 56 13 6 17 14 5 0 15 3 0 16 1 0 17 1 0 1/ Values are averages from twelve plants sampled in a single field in which the level of pink bollworm infestation was allowed to reach 90 to 95%. 2/ Only branches with bolls at harvest were numbered. The branch closest to the soil was numbered 1.

P.J. Cotty, Page NO.8 of 8. PLANT AVERAGE - 6504 PPB ARROWS INDICATE 22.240 PPB _ i ' Figure 1. Distribution of aflatoxin contaminated bolls on a cotton plant collected in the Yuma Valley.