Susceptibility of Cry1F-susceptible and -resistant fall armyworm to transgenic corn plants containing single or pyramided Bt genes Y. Niu1, R. Meagher2, F. Yang1, V. Dangal1 and F. Huang1, 1Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA. 2Behavioral and Biological Control Unit, USDA - ARS, Gainesville, FL Abstract The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a major target of transgenic corn expressing Bacillus thuringiensis (Bt) proteins. A highly Cry1F-resistant FAW strain was established from a field collection in Puerto Rico in 2011. In this study, three greenhouse trials were conducted to evaluate larval survival and plant injury of Cry1F-susceptible, -resistant, and -heterozygous genotypes of FAW on five non-Bt and eight Bt corn hybrids. The Bt corn products included two single-gene Bt corn hybrids containing Herculex®I (Cry1F) and YieldGard® (Cry1Ab) traits, respectively, and six pyramided Bt corn hybrids representing four traits: Genuity® VT Double ProTM, VT Triple ProTM, SmartStaxTM, and Agrisure® VipteraTM 3111. In each trial, 3-5 neonates of an insect genotype were placed into the whorl of a plant. Larvae of the three insect genotypes on non-Bt corn hybrids survived well and caused serious plant injury. Cry1Ab corn was ineffective against all three insect genotypes. After 12-15 d, live FAW larvae were also recovered from 72.9% Cry1F corn plants that were infested with resistant neonates and the survival resulted in an average of leaf injury rating (Davis’ 1 to 9 scales) of 7.6. In contrast, no live larvae with little or no leaf injury were observed on the Cry1F corn plants that were infested with susceptible or heterozygous genotypes, suggesting that the Cry1F-resistant FAW was highly resistant to whole plants of Cry1F corn and the resistance was recessive. There was no survival with little or no leaf injury on pyramided Bt corn plants infested with the three insect genotypes. The results showed that the pyramided Bt corn products can overcome the Cry1F resistance and thus should provide a means for managing Cry1F resistance in FAW. Introduction Results Leaf injury ratings : All three insect genotypes caused heavy injuries to non-Bt corn plants with an average leaf injury rating of 7.6 after 12-15 d (Fig.1 A). Both Cry1F-SS and -RS caused only little damage to HX1 (Cry1F). However, leaf injury rating of HX1 by the Cry1F-RR was high, averaged 7.6. Leaf injury rating of YG plants was also high with an average of 6.1. In contrast, no or little leaf damage was observed on the four pyramided Bt corn traits infested with the three insect genotypes across all three trials. Larval survival: Live larvae were observed on 85.1% of the non-Bt plants after 12-15 d (Fig.1 B). No live larvae were recovered from HX1 plants infested with either SS or RS, while 72.9% HX1 plants that were infested with RR contained live larvae, which was not significantly different from that recorded on non-Bt corn plants. YG was ineffective against any of the three insect genotypes. An average of 93.4% YG plants contained live larvae, which were also not significantly different from the larval survivorship rates observed on the non-Bt corn plants. In contrast, no live larvae were found from plants of the four pyramided Bt corn traits infested with any of the three insect genotypes across all three trials. The fall armyworm (FAW), Spodoptera frugiperda, is a common pest of field corn. Except for Cry1F corn, the first generation Bt corn expressing a single Bt gene is usually not very effective against FAW. With the recent availability of the more effective pyramided Bt corn products, FAW becomes a target species of Bt corn in both North and South America (Matten et al. 2012). During 2011, a FAW population was collected from Puerto Rico. Prior studies showed that this Puerto Rico population was highly resistant to both Cry1F protein and Cry1F corn leaf tissue (Niu et al. 2013) and thus it provides a good opportunity for analyzing if any cross-resistance exists to the second generation pyramided Bt corn products. Objective To evaluate susceptibility of Cry1F-susceptible, -resistant, and -heterozygous genotypes of FAW on transgenic corn containing single or pyramided Bt genes. Leaf damage rating (Davis’ 1 to 9 scales) Corn Hybrid % Plants with live larvae A: Leaf injury ratings B: Larval survival Materials and Methods Insect sources: Three FAW genotypes were tested in this study, which included a Cry1F-susceptible (Cry1F-SS), a Cry1F-resistant (Cry1F-RR), and an F1 hybrid (Cry1F-RS) that was developed by crossing the Cry1F-SS with Cry1F-RR. Cry1F-RR was developed from larvae collected from a corn field in Puerto Rico during 2011 and it has shown high resistance to both purified Cry1F protein and Cry1F corn leaf tissue (Niu et al. 2013). Cry1F-SS was collected from two locations in Louisiana and Florida and was documented to be susceptible to Cry1F (Niu et al. 2013). Corn hybrids and traits: Five non-Bt and eight Bt corn hybrids representing six Bt corn traits were evaluated in this study (Table 1). In each trial, two corn seeds of a hybrid were planted in 18.9-liter pots containing 5 kg of a standard potting soil mixture. Pots were placed in a greenhouse in Baton Rouge, LA. Table 1. Corn hybrids evaluated in the study Traits Corn hybrid Used in Abbreviation in the figures Bt genes Targets Non-Bt DKC 61-22 Trial 1 closely related to DKC 61-21 -- DKC 67-86 closely related to DKC 67-88 DKC 63-45 Trial 2&3 closely related to DKC 63-97 N78N-GT closely related to N78N-3111 Pioneer 31P40 closely related to Pioneer 31D59 Herculex®I Pioneer 31D59 Trial 1,2&3 HX1 Cry1F lepidoptera YieldGard® DKC 69-70 YG Cry1Ab Genuity®VT Double ProTM   DKC 64-04 VT-2P Cry1A.105, Cry2Ab2 DKC 63-87 Genuity®VT Triple ProTM DKC 67-88 VT-3P Cry1A.105, Cry2Ab2, Cry3Bb1 lepidoptera & rootworms DKC 62-97 Genuity® SmartStax® DKC 61-21 SMT Cry1A.105, Cry2Ab, Cry1F, Cry3Bb1, Cry34/35Ab Agrisure® VipteraTM 3111 N78N-3111 VIP Vip3A,Cry1Ab,mCry3A  Fig.1. Leaf injury rating (A) and larval survival (B) of Cry1F-susceptible (Cry1F-SS), -resistant (Cry1F-RR, and -heterozygous (Cry1F-RS) genotypes of Spodoptera frugiperda on non-Bt and Bt corn plants containing single or multiple Bt traits. Mean values followed by a same letter are not significantly different (P >0.05; Tukey’s test). Conclusions The FAW population collected from Puerto Rico was highly resistant to Cry1F corn plants and the resistance was recessive. Corn hybrids containing anyone of the four pyramided Bt traits were effective in controlling the Cry1F-resistant FAW and thus should provide a means for managing Cry1F resistance in FAW. However, these pyramided Bt corn traits may not be considered as “pyramided” anymore if resistance of a target species to one Bt protein (e.g. Cry1F) in the plant occurs and the corresponding resistance management strategies for such conditions still need to be investigated. Acknowledgments This study was sponsored by Louisiana Soybean and Feed Grain Promotion Board and NC-205. Greenhouse study: A total of three trials were conducted in 2011-2013. In each trial, three (trial 1) or five (trials 2 and 3) neonates of an insect genotype were manually placed into the whorl of a plant at V6-V10 plant stages. Treatment in each trial was replicated four times in a randomized complete block design with 4 plants (or 2 pots) in each replication. Larval survival and corn leaf damage ratings (Davis’ 1 (no damage) to 9 (most leaves with long lesions) scales) (Davis et al. 1992) were checked 12th -15th d after larval inoculation when majority of the larvae were at 4th-5th instars on non-Bt corn plants. References Davis, F.M., Ng, S.S., Williams, W.P., 1992. Visual rating scales for screening whorl-stage corn for resistance to fall armyworm. Miss. Agric. Forestry Exp. Stn. Tech. Bull. 186. Matten, S.R., Frederick, R.J., Reynolds, A.H., 2012. United States Environmental Protection Agency insect resistance management programs for plant-incorporated protectants and use of simulation modeling. pp 175-267. In: C.A. Wozniak, A. McHughen (eds), “Regulation of agricultural biotechnology: The United States and Canada”. Springer. Niu, Y., Meagher, Jr, R.L., Yang, F., Huang, F., 2013. Susceptibility of field populations of the fall armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to purified Cry1F and corn leaf tissue containing single and pyramided Bt genes. Fla. Entomol. 96, 701-713. Data analysis: The overall results were very consistent across the three trials and thus data for each variable were combined for each corn trait across the three trials. The combined data were then analyzed using mixed models with trial as a random factor. Tukey’s tests were used to determine treatment differences at the α= 0.05 level.