J. Genet. & Breed. 56: 165-169 (2002)

Southwestern corn borer damage and aflatoxin accumulation in a diallel cross of

W.P. Williams, F.M. Davis, -G.L. Windham and P.M. Buckley USDA-ARS Corn Horst Plant Resistance Research Unit, Box 9555, • Mississippi State, MS 39762, USA. Fax: (662) 325-8441. • Received September 18, 2001

ABSTRACT Southwestern corn borer, grandiosella Dyar, is a serious pest of maize, mays L., in the southern USA. When plants are infested during and after anthesis, larvae feed on the husks and developing cars before tunneling into the stalk. Larval feeding also provides potential sites for fungi to enter developing ears. Aflatoxin, produced by the fungus Aspergillus flavus Link: Fr, is a potent carcinogen, and its presence at levels exceeding 20 ng g restricts maize from interstate commerce. Aflatoxin contamination is a chronic problem in maize produced in the southern USA. Little is cur- rently known about the value of resistance to southwestern corn borer in reducing aflatoxin accu- mulation. This investigation was undertaken to compare aflatoxin accumulation in crosses among in- bred crosses with different levels of southwestern corn borer resistance and to study the importance of general and specific combining ability in the inheritance of resistance to southwestern corn bor- er and aflatoxin accumulation in an eight-parent diallel cross. Our results indicated that general com- bining ability was a highly significant source of variation in the inheritance of resistance to stalk tun- neling and ear damage by southwestern corn borer and resistance to aflatoxin accumulation. Stalk tunneling, ear damage, and aflatoxin accumulation were lowest in hybrids with the inbred line MP496 as a parent. Key words: Aspergillus flavus, Diatraea grandiosella, Zea mays. Abbreviations: AF, Aspergillus flavus; GCA, general combining ability, SCA, specific combining ability, SWCB, southwestern corn borer.

INTRODUCTION Drought and high temperatures have fre- quently been linked with high levels of aflatox- Aflatoxin, a naturally occurring toxin pro- in accumulation in corn (DIENER, 1989; PAYNE, düced by the fungus Aspergillus flavus Link: Fr., 1992). damage to developing maize ears is a potent carcinogen (CASTEGNARO and Mc- and kernels has also been associated with high GREGOR; PITrET, 1998). Aflatoxin contamination aflatoxin levels (GUTHRIE et al., 1981; MCMILLIAN, of maize, Zea mays L., is a sporadic problem in 1983; MCMILLIAN et al., 1985; WINDHAM et al., the Midwest, but a frequent problem in the 1999). Increased aflatoxin contamination of har- Southeast (PAYNE, 1992; WIDSTROM, 1996). Afla- vested grain has been attributed to several in- toxin B 1 is the most commonly found form of sects including corn earworm, Helicoverpa zea aflatoxin in maize. The U.S. Food and Drug Ad- (Boddie); European corn borer, Ostrinia nubi- ministration has a tolerance of 20 ng g for B1 lalis (Hubner), fall armyworm, Spodoptera in maize grain; grain with higher levels is re- frugiperda Q.E. Smith); and southwestern corn stricted from interstate commerce (GoUltiviA and borer, Diatraea grandiosella Dyar. BULLERMAN, 1995). Many countries have set even Both insect damage and aflatoxin accumula- lower tolerances for aflatoxin in maize. tion vary widely from year to year, and the re- \," Corresponding author. E-mail: [email protected] Joint contribution of USDA-ARS and the Mississippi Agricultural and Forestry Experiment Station, Mississippi Agric. And Forestry Exp. Stn. Journal No. J-9759. Purchase BN of Agriculture Use Only 166 lationship between the two is difficult to define. morillonitic, nonacid, thermic vertic Haplaquept) soil. WINDHAM et al. (1999) reported that aflatoxin lev- The single-row plots were approximately Sm long, els were significantly higher in when com- spaced I m apart, and thinned to 20 plants. 1995 In 1998, 7 d after 50% of the plants in a plot had mercial hybrids were both infested with south- silked, plants were infested with 60 southwestern corn western corn borer larvae and inoculated with borer larvae. Fifteen neonates were placed in the leaf A. flavus spores than when only inoculated with axil above the top ear and 45 in the axil of the top- A. flavus spores. In 1996, however, aflatoxin con- ear leaf (DAvis and WILLIAMS, 1994) using a portable plastic dispensing device (MIHM, 1983). On the fol- tamination was generally lower and was not in- lowing day, the top ear of each plant was inoculated creased significantly by infesting developing ears with a 3.4-ml suspension containing 3 x 108 A. flavus with southwestern corn borer larvae. Substantial conidia in distilled water using a treemarking gun effort has been invested in developing technique (ZUMMO and Scorr, 1989). Inoculum was prepared as for identifying maize germpiasm with resistance described by WINDHAM and WILLIAMS (1999) with A. flavus isolate NRRL 3357, which is known to produce to southwestern corn borer by USDA-ARS sci- aflatoxin (Scorr and ZUMMO, 1988). The experiment, entists at Mississippi State (DAvIs et al., 1989; was repeated in 1999. In 1999, three additional repli-, DAvis and WILLIAMS, 1997). Several germplasm cations were grown and inoculated with A. flavus. , but lines with resistance to leaf feeding by south- not artificially infested with southwestern corn borer. western corn borer when plants are in the whorl No insecticides were applied to prevent natural insect infestation. stage of growth have been developed and re- Mature ears in all experiments were hand har- leased (WILLIAMS and DAVTS, 1997). Although vested approximatel y 56 d after inoculation with A. germplasm with resistance to southwestern corn flavus and dried for 7 d at 38° C. In 1999, ears were borer after anthesis has not yet been released, visually rated for feeding damage by southwestern inbred lines Mp305, Mp496, and SC213 have cx- corn borer and other before shelling. Ten ears from each plot were scored using the following hibitedreduced damage when infested 7 to 21d scale: (1) no Lepidoptera damage to any ears, (2) ear after anthesis (unpublished data). tip damage only to three or fewer ears, (3) ear tip This research investigates the association be- damage to four to six ears and no damage below tips, tween resistance to southwestern corn borer (4) ear tip damage to most ears and light additional damage and aflatoxin accumulation in maize. damage (2 or 3 kernels at I or 2 sites destroyed) to three or fewer ears, (5) ear tip damage to most ears Specific objectives were to (i) evaluate, in hy- and light damage to kernels below tips of four to six brids, inbred lines of corn with different levels cars, (6) ear tip damage and light damage below tips of resistance to southwestern corn borer dam- of most ears or moderate damage (4 to 6 kernels de- age and aflatOxin accumulation, (ii) obtain in- stroyèd at I to 3 sites) below tips of three or fewer formation on the relative importance of general ears, (7) ear tip damage and moderate to heavy dam- age (kernels destroyed at more than four sites or more and specific combining ability in the inheritance than six kernels destroyed at fewer sites) to four to of resistance to southwestern corn borer dam- six ears, (8) car tip damage and moderate to heavy1 age and aflatoxin accumulation, and (iii) com- damage below tips of seven or eight ears, and (9) ear pare ear damage and aflatoxin accumulation in tip damage and moderate to heavy damage below tips a diallel cross when infested and not infested of nine or ten ears. Ears from each plot were shelled, and the grain c with southwestern corn borer. was mixed by pouring through a sample-splitter twice. Grain was ground using a Romer mill (Union, MO). Afiatoxin contamination in 50-g subsamples from MATERIALS AND METHODS each plot was determined by the Vicam Aflatest (Wa- tertown, MA). This procedure detects aflatoxin (B1, Eight inbred lines of maize, Ab24E, GA209, 132 , G 1 , G2) at concentrations as low as 2 ng g. GE333, Mp305, Mp488, Mp496, SC213, and T202, In both 1998 and 1999, stalks in plots that had - were selected as parents of a diallel cross. Previous been infested with southwestern corn borer were dis- evaluations indicated that among a group of inbred sected after harvest. Tunneling from the top ear node lines infested with southwestern corn borer larvae af- to the base of the plant was measured. ter anthesis, Mp305, Mp496, and SC213 sustained less Plot means for all traits were calculated and used damage than other lines evaluated; GE333, Mp488, in an analysis of variance. To equalize variances and and, Ab24E were heavily damaged (unpublished da- because some values were zero, the data for aflatox- ta). The 28 single cross hybrids were planted in three in contamination were transformed by adding I and replications on 4 May 1998 and six replications on 3 taking the logarithm of each number [log (y+l)] pri- May 1999 in a randomized complete block design at or to analysis. Data on tunneling and aflatoxin for the Starkville, MS in a Leeper silty clay loam (fine mont- two years were combined for analyses over years. A. 167

combined analysis of data on ear damage and afla- TABLE 2 toxin accumulation with and without southwestern Estimates of general combining ability effects for corn borer infestation in 1999 was performed..Varia- stalk tunneling and aflatoxin accumulation following tion among hybrids was partitioned into"general infestation with sotuhwestern corn borer and (GCA) and specific (SCA) combining ability using inoculation with A. flavus in 1998 and 1999 GRIFFINGS Method 4, Model 1 (1956). Hybrids, years, and treatments (with and without southwest- Inbred Tunneling Aflatoxin ern corn borer infestation) were considered as fixed cm Log(ng g- + 1) effects.

Mp305 4.56 -0.07 Ah24E 3.92 0.18 T202 2.14 0.32 RESULTS AND DISCUSSION Mp488 1.41 -0.19 GE333 0.55 0.00 Stalk tunneling following southwestern corn GA209 -1.20 0.38

borer infestation was twice as extensive in 1999 SC2 13 _4.68 -0.01

- as in 1998. Mean tunneling was 17cm per plant Mp496 _0.61 in 1998, and ranged from 6 (T202 x Mp496, Significantly different from 0 at P = 0.01. SC21.3 x Mp496) to 38cm (T202 x Mp305). in 1999, tunneling ranged from 20 (Mp496 x SC213) to 52cm (Mp305 x Mp333). In the com- bined analysis of the two years data, years and er evaluations. The resistance expressed previ- year x hybrid interactions were highly signifi oustybtIihbred not line Mp305 appar- was cant sources of variation. Both general (GCA) ent ?in this investigation; the GCA effect for and specific combining ability (SCA) were high- Mp305 was highly significant and positive..: ly significant in the inheritance of resistance to Ear damage was rated in 1999 in both those stalk tunneling (Table 1). As indicated by GCA plots infested with southwestern corn borer and effects (Table 2), Mp496 contributed most to- in plots not infested with southwestern corn bor- ward reduced stalk tunneling in its hybrids. The er. The mean rating for the infested plots was GCA effect for SC213 was also negative and 6.1, the mean rating for the noninfested plots, highly significant. In earlier field evaluations, 4.2. When infested with southwestern corn bor- Mp496 and SC2 13 exhibited resistance to south- er larvae, T202 x Ab24E was most heavily dam- western corn borer when plants were infested aged (8.0), and Mp496 x GA209, least damaged after anthesis (unpublished data). in the earlier (3.7). The same two hybrids also exhibited the evaluations, GE333, Mp488, and Ab24E were extremes in ear damage (T202 x Ab24E, 5.3; susceptible. Their performance in crosses in Mp496 x GA209, 3.0) in the plots that were not these evaluations was consistent with the earli- infested with southwestern corn borer. Differ- ences in ear damage between hybrids infested TABLE 1 with southwestern corn borer and those not in- fested were highly significant (Table 3). When Analysis of variance of tunneling within stalks and aflatoxin in grain following infestation with not artificially infested with southwestern corn southwestern corn borer and inoculation with A. flavus borer, many ears exhibited damage only to the of a diallel cross in 1998 and 1999 tips, or damage to the ear tips with slight addi- Mean Square tional damage below the tips on a few ears. This Source dl Tunneling Aflatoxin damage could have resulted from natural infes- tation by southwestern corn borer, corn ear- Years I 11255 383.7 worm, or fall armyworm. GCA was a highly sig- Rcsps (years) 4 254 2.4 nificant source of variation in the inheritance of Hybrids 27 255 GCA 7 . 571 resistance to ear damage (Table 3). Mp496 con- scA 20 144 1.1 tributed most to reduced car damage (Table 4). Years x hibrids 27 116 1.7 Conversely, T202, Ab24E, and Mp305 con- En-or 108 56 0.6 tributed to significantly greater ear damage. Significant at P = 0.05% and P = 0.01% levels of probability, Aflatoxin levels in maize grain were high in respectively. 1998 when high temperatures and drought

168

TABLE 3 TABLE 4 Analysis of variance of ear damage ratings Estimates of general combining ability effects and aflatoxinaccumulation in grain following for ear damage and aflatoxin accumulation infestation with southwestern corn borer following A. fluzvus inoculation with and and inoculation with A flavus or inoculation without southwestern corn borer infestation of a with A. flavus without southwestern corn borer diallel cross in 1999 infestation of a diallel cross in 1999 Inbred Ear damage Aflatoxin Mean Square Log(ng g-+ 1) Source df Ear damage Aflatoxin Mp305 0.26 0.01 Treatments . 1 141.5 51.6 Ab24E 0.38 -0.04 Resps (treatments) 4 0.7, 5.7 T202 0.51 0.49 Hybrids 27 2.7,2.2 Mp488 -0.07 -0.09 GCA 7 8.4 2.9 GE333 -0.15 4.22 SCA 20 0.8 1.9 GA209 -0.01 0.31 07 Treatments x hibrids 27 1.1 SC213 0.05 -0.07 Error 108 0.4 0.6 Mp496 _0.96

Significant at P = 0.05 and P = 0.01 levels of probability, re- Significantly different from 0 at P = 0.01. spectively.

plagued Mississippi. Among the 28 single cross tive and highly significant. Hybrids with these hybrids evaluated, aflatoxin contamination two lines exhibited the highest levels of aflatox- ranged from 236 ng g for Mp496 x Mp488 to in contamination. 6491 ng g for T202 x Ab24E following infesta- Aflatoxin accumulation was 200% greater tion with southwestern corn borer and inocula- with the addition of southwestern corn borer in tion with A. flavus. The overall mean was 2160 1999, indicating that damage from this insect ng g, 1998. In contrast, the mean aflatoxin lev- can be instrumental in increasing aflatoxin ac- el for hybrids infested with southwestern corn cumulation in maize. The correlation between borer and inoculated with A. flavus in 1999 was ear damage and aflatoxin accumulation was sig- only 86 ng g. Aflatoxin levels for the hybrids nificant (r = 0.40, P < 0.05). On the other hand, ranged from 22 (Mp488 x Ab24E) to 385 ng g mean stalk tunneling was 17 cm in 1998 and 34 (T202 x GA209). For hybrids inoculated with A. cm in 1999, and overall aflatoxin Ievels in the flavus but not infested with southwestern corn same experiments were 2160 and 86 ng g, re- borer, in 1999, aflatoxin levels ranged from 5 for spectively. Unfortunately, ear damage was not Mp496 x Mp488 and GE333 x SC213 to 124 ng rated in 1998. It would be informative to have g for T202 x Mp305. The mean was 28 ng g. data on ear damage and its correlation with afla- Years and years x hybrids were highly signifi- toxin contamination under the high tempera- cant sources of variation in the inheritance of tures and drought experienced in 1998. resistance to aflatoxin contamination (Table 1). Although it is difficult to quantify the effects Both GCA and SCA were significant sources of of southwestern corn borer damage on aflatox- variation in the inheritance of resistance to afla- in accumulation, it is interesting to note that toxin contamination following southwestern among the inbred parents included in this dial- corn borer infestation (Table 1). When data from lel cross, Mp496 contributed toward reduced 1999 for aflatoxin accumulation with and with- stalk tunneling, reduced ear damage, and re- out southwestern corn borer infestation were an- duced aflatoxin accumulation in hybrid combi- alyzed, both GCA and SCA were highly signifi- nations with the other parental inbred lines. This cant sources of variation (Table 3). line, which was originally released as a source Estimates of GCA effects indicate the Mp496 of resistance to leaf feeding by southwestern was the only parental inbred that made a sig- corn borer and fall armyworm (Scorr and DAVIS) nificant contribution toward reduced aflatoxin 1981) could be useful in developing corn hybrids contamination when used in hybrids (Tables 2, with resistance to ear damage by Lepidoptera 4). GCA effects for GA209 and T202 were posi- and resistance to aflatoxin accumulation. It 169

could be especially important if used in combi- GUTHRIE W.D., LILLEHOJ E.B., McMILLIAM WW., BARRY D., nation with other lines released as sources of re- KWOLEK WE, Fiwz A.O., CATALANO E.A., RUSSEL W.A. and WIDSTROM NW., 1981. Effect of hybrids with dif- sistance to A. flavus kernel infection and afla- ferent levels of susceptibility to second-generation Eu- toxin accumulation in maize grain. ropean corn borers on aflatoxin contamination in corn. J. Agric. Food Chem., 29: 1170-1172. MCMILLIAN W.W., 1983. Role of in field contam- ACKNOWLEDGMENTS ination. In: U.L. Deiner, R.L. Asquith and J.W. Dickens ed. Aflatoxin and Aspergillus flavus in corn., South. Coop. The authors thank Gerald A. Matthews and Diane Ser. Bull. 279. Alabama Agric. Exp. Stn., Auburn, AL, p. Keller for assistance in preparing the manuscript. Ap- 20-22. preciation is also extended to Michael Alpe, Summer MCMILLIAN W.W., WILSON D.M. and WIDSTROM NW., 1985. Gingrich, Gerald Matthews, Lee Scruggs, Patrick Aflatoxin contamination of preharvest corn in Georgia: Tranum, and Susan Wolf for technical assistance. a six-year study of insect damage and visible Aspergillus flavus. J. Enviion. Qual., 14: 200-202. MIHM J.A., 1983. Efficient mass rearing and infestation tech- REFERENCES niques to screen for host plant resistance to fall army- / worm, Spodoptera frugiperda, International Maize and CASTEGNARO M., MCGREGOR D., 1998. Carcinogenic risk as- Wheat Improvement Center (CIMMYT). Mexico City, sessment of mycotoxins. Revue Med. Vet., 149: 671-678. Mexico. DAVIS F.M. and WILLIAMS W.P., 1994. Evaluation of repro- PAYNE GA., 1992. Aflatoxin in maize. Crit. Rev. Plant Sci., ductive stage maize for resistance to southwestern corn 10: 423-440. borer (Lepidoptera: Pyralidae) using visual rating scores PIrFET A., 1998. Natural occurrence of mycotoxin in foods of leaf sheath and husk damage. J. Econ. Entomol., 87: and feeds-an updated review. Revue Med. Vet., 149: 479- 1105-1112. 492. DAVIS F.M. and WILLIAMS W.P., 1997. A new technique for Scorr G.E. and DAVIS F.M., 1981. Registration of Mp496 in- evaluating southwestern corn borer damage to post-an- bred of maize. Crop Sci., 21: 353. thesis maize. P. 189-194. In:J.A. Mihrn ed. Insect Re- Scorr G.E. and ZUMMO N., 1988. Sources of resistance in sistant Maize: Recent Advances and Utilization. Proc. maize to kernel infection by Aspergillus flavus in the Symp. International Maize and Wheat Improvement field. Crop Sci., 28: 504-507. Center (CIMMYT), 27 Nov.-3 Dec. 1994. CIMMYT, Mex- WIDSTROM N.W., 1996. The aflatoxin problem with corn ico City, Mexico. grain. Advances in Agronomy, 56: 219-280. DAvIs EM., WILLIAMS W.P. and WISEMAN B.R., 1989. Methods WILLIAMS W.P. and DAVIS EM., 1997. Maize germplasrn with used to screen maize for, and to determine mechanisms resistance to southwestern corn borer and fall army- of resistance to the southwestern corn borer and fall worm. In: J.A. Mihm J.A. ed., Insect Resistance Maize: armyworm. In: Toward Insect Resistant Maize for the Recent Advances and Utilization. Proc. Symp. Interna- Third World, Proceedings of mt. Symp. On Methodolo- tional Maize and Wheat ImprOvement Center (CIM- gies for Developing Host Plant Resistance to maize In- MYT), 27 Nov.-3 Dec. 1994. CIMMYT, Mexico City, Mex- sects. International Maize and Wheat Improvement Cen- ico, p. 226-225. ter (CIMMYT), 9-14 mar. 1987, CIMMYT, Mexico City, WINDHAM G.L. and WILLIAMS W.P., 1999. Aflatoxin accumu- Mexico, p. 101-108. lation in commercial corn hybrids in 1998. Miss. Agric. DIENER U.L., 1989. Preharvest aflatoxin contamination of and Forestry Exp. Stn. Res. Rep., Vol. 8, No. 8. peanuts, corn and cottonseed. Biodeterioration. Res., 2: WINDHAM G.L., WILLIAMS W.P. and DAVIS EM., 1999. Effects 217-244. of the southwestern corn borer on Aspergillus flavus ker- GOURMA H. and BULLERMAN L.B., 1995. Aspergillus flavus and nel infection and aflatoxin accumulation in maize hy- Aspergillus parasiticus: aflatoxigenic fungi of concern in brids. Plant. Dis., 83: 535-540. foods and feeds. J. Food Prof., 58: 1395-1404. ZUMMO N. and Scorr G.E., 1989. Evaluation of field inocu- ,GRJFFIN B., 1956. Concept of general and specific combining lation techniques for screening maize genotypes against ability in relation to diallel crossing systems. Aust. J. Bi- kernel infection by Aspergillus flavus in Mississippi. ol. Sci., 9: 463-495. Plant. Dis., 73: 313-316.