84 Journal of Food Protection, Vol. 60, No.1, 1997, Pages 84~7 Copyright
©, International
Association
of Milk, Food and Environmental
Sanitarians
Research Note
Growth of an Aspergillus flavus Transformant Expressing Escherichia coli p-Glucuronidase in Maize Kernels Resistant to Aflatoxin Production ROBERT L. BROWN,I* THOMAS E. CLEVELAND,' GARY A. PAYNE,z CHARLES P. WOLOSHUK,3 and DONALD G. WHITE4 ISouthern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, New Orleans, Louisiana 70179; 2Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27965; 3Department of Botany and Plant Pathology, Purdue University, West Lofayette, Indiana 47907; and 4Department of Crop Science, University of Illinois, Urbana, Illinois 61801, USA (MS# 96-55: Received 1 March 1996/Accepted
ABSTRACT Kernels of a maize inbred that demonstrated resistance to aflatoxin production in previous studies were inoculated with an Aspergillus flavus strain containing the Escherichia coli f3-Dglucuronidase reporter gene linked to a f3-tubulin gene promoter and assessed for both fungal growth and aflatoxin accumulation. Prior to inoculation, kernels were pin-wounded through the pericarp to the endosperm, pin-wounded in the embryo region, or left unwounded. After 7 days incubation with the fungus, f3-glucuronidase activity (fungal growth) in the kernels was quantified using a fluorogenic assay and aflatoxin B, content of the same kernels was analyzed. Kernels of a susceptible inbred, similarly treated, served as controls. Results indicate a positive relationship between aflatoxin levels and the amount of fungal growth. However, resistant kernels wounded through the peri carp to the endosperm before inoculation supported an increase in aflatoxin B lover levels observed in nonwounded kernels, without an increase in fungal growth. Wounding kernels of the resistant inbred through the embryo resulted in both the greatest fungal growth and the highest levels of aflatoxin B, for this genotype. Maintenance of resistance to aflatoxin B1 in endosperm-wounded kernels may be due to the action of a mechanism which limits fungal access to the kernel embryo. Key words: Aflatoxin; maize; resistance; f3-glucuronidase; Aspergillus flavus
Contamination of cottonseed, peanuts, tree nuts or maize with aflatoxins, the toxigenic and carcinogenic secondary metabolites of the fungi Aspergillus fiavus Link:Fr and A. parasiticus Speare, poses serious health hazards to humans and to domestic animals (5, 7). Aflatoxin contamination of maize is both a preharvest and a postharvest problem;
* Author
for correspondence. Tel: 504-286-4359; E-mail:
[email protected]
Fax: 504-286-4419;
27 May 1996)
A. fiavus can infect the crop before harvest and remain with the grain throughout harvest, storage, and use (11). Recent progress has been made in identifying maize genotypes that resist aflatoxin production (2, 3, 4, 18). A significant level of resistance also was found in some genotypes after kernel pericarps were breached (2, 3, 8). These studies have also demonstrated a relationship between lower aflatoxin levels and either limited external sporulation on kernels or limited fungal spread (2, 3, 8). However, the actual relationship between fungal growth and aflatoxin contamination in resistant kernels has not been determined. Previous studies have shown varied results when quantitatively comparing aflatoxin production to fungal growth or infection (13). Also, methods for quantifying fungal growth in natural substrates often have been unsatisfactory (13). In the present study, the ~-glucuronidase (GUS) gene fusion system was employed to quantify fungal infection in inoculated resistant and susceptible maize genotypes. A brief description of this technique is included in a previous study (2). The usefulness of GUS gene reporter constructs for quantifying fungal growth in plant tissues has been demonstrated (6,12,14,17). The present authors thoughtthat using this approach would generate more information about the response of nonwounded and wounded resistant kernels to infection by an aflatoxin-producing fungus. We also sought to demonstrate whether the mode of action of the resistant mechanisms being studied was antifungal or directed at aflatoxin biosynthesis. MATERIALS AND METHODS Maize entries Inbred MI82 and 33-16 kernels were obtained from the Department of Plant Pathology of the University of Illinois. MI82 was identified as potentially resistant to aflatoxin production in field studies and in a kernel-screening laboratory assay (2, 4).
FUNGAL GROWTH
IN RESISTANT
Inbred 33-16 is susceptible to the elaboration of aflatoxins (2, 4). All kernels were dried in a forced-air oven at 45°C for 2 days and kept in sealed plastic containers containing silica-gel desiccant until used. Fungal strains and growth conditions The strain of A. flavus (GAP 2-4) used in this study is a transformant containing the E. coli J3-glucuronidase (GUS) gene linked to an A. flavus J3-tubulin gene promoter. It has been fully described in other studies (2, 19). Cultures were grown at 37°C in the dark on potato-dextrose agar. Conidia from 4- to 7-day-old cultures suspended in deionized water served as inocula. Inoculations with GAP 2-4 Kernels of the genotypes being investigated were treated in one of three ways: by being (i) wounded through the pericarp to the endosperm to a depth of 1 mm (3), (ii) wounded in the embryo region (1 mm from the pedicel) to a depth of 1.5 mm (3), or (iii) left unwounded. Wounding was performed with a 26-gauge, 13-mm hypodermic needle (Becton Dickinson & Co., Rutherford, NJ) (3). All kernels tested were then surface-sterilized with a 4-min wash in 70% ethanol (with stirring) followed by a quick rinse with deionized water and then three 3-min deionized water washes (with stirring) (16). Kernels were inoculated by dipping them into a spore suspension of GAP 2-4 (4.0 X 106 conidia per ml). They were then incubated at 37°C for 1 day and at 31°C for 6 days using a kernel-screening assay (KSA) as described in previous studies (2, 3). In the KSA, kernels are maintained in an atmosphere of high humidity (3). Each treatment was replicated seven times with each replicate containing 3 kernels. The tests were performed twice. Quantitation of GUS activity After incubation, the kernels were subjected to a protocol for fluorogenic GUS quantification (9). Kernels were first ground to a powder in an analytical mill (Tekmar A-lO, Janke and Kunkel GmbH & Co., Staufen, Germany) and then homogenized in a GUS extraction buffer (2, 9) with a mortar and pestle. The volume of extraction buffer used equaled twice the weight of the sample. The homogenate was then centrifuged for 2 min at 14,000 rpm and the supernatant removed for use in enzyme assays and protein determinations. The remaining pellet was dried at 60°C for 1 day and the dry weight determined, prior to analysis for aflatoxins (3). For each enzyme reaction, 10 to 50 fll of crude extract was added to the assay buffer containing the substrate and reactions
85
MAIZE KERNELS
were stopped after 10 or 20 min. Preliminary J3-glucuronidase kinetic experiments were performed to identify the amount of crude extract to use in each test and to identify a time that fell within the linear portion of the enzyme reaction curve. GUS activity in samples was determined by using a Gilford Auoro IV spectrofluorimeter (Coming Laboratory Sciences Co., Oberlin, OH); excitation was at 360 nm and emission at 455 nm. GUS activity was normalized through protein determinations in crude extracts by the method of Sedmak and Grossberg (15), and expressed in nanomoles of MU (methyl-umbelliferone) produced per minute per milligram of protein. Aflatoxin analyses The aflatoxin BI content of replicates from all tests was determined by a modification of the procedure employed in an earlier study (3). In the present investigation, methylene chloride was added to the dried pellet that remained after the supernatant (used for GUS and protein determinations) was separated from the kernel homogenate. Statistical analyses Analyses of aflatoxin and GUS activity data were performed with the Statistical Analysis Software System (SAS Institute, Inc., Cary, NC). Treatment replicates from each test were first subjected to analysis of variance followed by mean comparisons of either toxin or GUS values, log transformations of these values, or square-root transformations of these values. Transformations were performed to equalize treatment variances. Differences among treatment means were determined by the least significant difference test.
RESULTS AND DISCUSSION There was a significant interaction (P = 0.05) between test and treatment variables for both aflatoxin and GUS data. The magnitude of A. fiavus growth and aflatoxin production varied significantly between tests 1 and 2 (Table 1). However, genotype-treatment responses to GUS activity and aflatoxin production and their respective rankings were identical for the two tests. The aflatoxin BI content of resistant MI82 nonwounded kernels was the lowest detected among all treatments in both tests. Wounding MI82 kernels through the pericarp to the endosperm increased the amount of aflatoxin supported; however, wounding the embryo prior to inoculation resulted in an even greater increase. Suscep-
TABLE 1. J3-Glucuronidase (GUS) activity and aflatoxin production in inoculated kernels of maize inbreds M182 and 33-16 Test I (n = 7)
Maize entry
MI82
33-16
Treatment
Nonwounded Endosperm-wounded Embryo- wounded Nonwounded Endosperm-wounded Embryo-wounded
GUS activity" (nmol MU/minlmg)
6.7c 7.4c 45.4B 136.6A 208.3A 191.6A C
Test 2 (n = 7)
Aflatoxin
BIb
(ng/g)
56D 313c 5,439B 19,797A 47,7l8A 27,221A
GUS activity" (nmol MU/minlmg)
0.9c l.lc 6.3B l8.6A 24.4A 28.8A
Aflatoxin
BIb
(ng/g)
590D 1,959c l2,575B 50,372A 68,295A 2l,8l2A
" GUS (J3-glucuronidase) activity is expressed in nmol of MU (methylumbelliferone) produced per min per mg of kernel protein. b Aflatoxin BI measured in ng per g of extracted kernel dry weight. c Values in a column followed by the same letter are not significantly different by the least significant difference test. Data were log transformed prior to analyses to equalize variances.
86
BROWN, CLEVELAND,
PAYNE, WOLOSHUK,
tible genotype 33-16 supported the highest levels of aflatoxin B 1, and wounding had no significant effect on aflatoxin accumulation. GUS activity in nonwounded kernels of inbred MI82 was the same as that in endosperm-wounded MI92 kernels, and was lower than that in embryo-wounded kernels. Levels of GUS activity in nonwounded, endosperm-wounded, and embryo-wounded kernels of inbred 33-16 were highest among respective treatments in each test, and wounding did not significantly influence the levels of GUS activity. Inbred M182, as in previous studies (2, 4), demonstrated resistance to aflatoxin production in non wounded kernels. In an investigation of the resistant maize population, GT-MAS: gk, nonwounded kernel resistance was partially attributed to the pericarp wax and cutin layers (2). Further investigation is needed to determine if this mechanism is also operable in MI82 kernels. When MI82 kernels were wounded through the pericarp to the endosperm, higher aflatoxin B J levels were seen than in nonwounded kernels; however, resistance was still maintained. The growth of A. flavus, as detected through GUS activity, did not increase, despite wounding. In a previous study which used a histochemical assay for detection of GUS activity (2), low levels of fungal colonization and aflatoxin production in MI82 nonwounded and endospermwounded kernels were also found. However, distinctions between fungal spread and the elaboration of aflatoxins in response to endosperm wounding were not as clear-cut as they are in the present study. This may highlight a distinct advantage of the quantitative technique employed here over visual assessment. The previous histochemical study (2) also demonstrated fungal colonization of the MI82 kernel aleurone layer and production of aflatoxins in kernels that otherwise had a "clean" appearance. Utilization of the aleurone as a preferred substrate for aflatoxin production also has been observed by other investigators (10). In the present study, increased fungal access to the MI82 kernel aleurone tissue, due to wounding, may have stimulated aflatoxin production without significantly stimulating fungal growth. The approximately fivefold increase in aflatoxin BJ produced per unit of fungal growth (determined as methylumbelliferone [MU] produced) in endosperm-wounded kernels over nonwounded kernels (e.g., Table 1: from 56 ng of aflatoxin BJ per gram per 6.7 nmol of MU to 313 ng/g per 7.4 nmol of MU) may be a demonstration of direct stimulation of the aflatoxin biosynthetic pathway by this kernel tissue. The ability of MI82 kernels to support high levels of both fungal infection and aflatoxins after embryos have been invaded was demonstrated in this investigation. Embryo vulnerability to high aflatoxin levels was previously demonstrated in maize population GT-MAS:gk (3). Embryo viability was also shown to be necessary for the expression of resistance to aflatoxin production in this same genotype (3). MI82 embryos and endosperm tissue were observed through histochemical detection to be relatively fungus-free in appearance, even after endosperm wounding (2). The ability of MI82 kernels to limit A. flavus colonization to a small area after wounding may provide kernels with a two-pronged
AND WHITE
defense: (i) it may prevent interruption of whole-kernel expression of an embryo-based resistance mechanism; and (ii) it may deny the fungus easy access to the oil-rich embryonic substrate (1). The GUS gene fusion system has been shown to be a useful tool for quantifying fungal growth in maize kernels and for facilitating comparisons with aflatoxin production in the same seed. This technique may prove useful in evaluating resistance to aflatoxin contamination and in determining whether A. flavus or aflatoxin biosynthesis is the primary target of kernel resistance mechanisms. ACKNOWLEDGMENTS We thank Rodney Reddix, Daniel Whittington, and Herbert Holen for technical assistance, and Dr. Brian Vinyard for statistical assistance.
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