ECOTOXICOLOGY Effect of Transgenic Plants Expressing High Levels of a Tobacco Anionic Peroxidase on the Toxicity of falcifera Nucleopolyhedrovirus to Helicoverpa zea (: )

1 2 3 4 R. W. BEHLE, P. F. DOWD, P. TAMEZ-GUERRA, AND L. M. LAGRIMINI

J. Econ. Entomol. 95(1): 81Ð88 (2002) ABSTRACT Wild type and corresponding transgenic tomato (Lycopersicon esculentum Miller) and two tobacco (Nicotiana spp.) plants that express high levels of a tobacco anionic peroxidase were used to determine what type of interactions occurred between peroxidase altered plant chemistry and the baculovirus nucleopolyhedrovirus (AfMNPV) for control of neonate corn ear- worms, Helicoverpa zea (Boddie). Transgenic plants expressed approximately Þve to 400 times higher peroxidase activity than corresponding tissues of wild type plants. The H. zea larvae typically fed 1.5 times less on transgenic compared with wild type leaf disks. There was only one experiment (of three with tomato leaves) where the larvae that fed on transgenic leaves were less susceptible to the virus based on nonoverlapping 95% conÞdence intervals for LC50 values. When the exposure dose was corrected for reduced feeding on the transgenic leaf disks, the insecticidal activity of the virus was not signiÞcantly different for larvae fed on transgenic versus wild type plants. Eight other experiments (with tomato and two species of tobacco) indicated either no signiÞcant effect or enhanced suscep- tibility (when corrected for feeding rates) to the virus of larvae fed on the transgenic leaves. These results indicate enhanced resistance in plants expressing high levels of a speciÞc anionic peroxidase may be compatible with applications of AfMNPV. Potential reasons for this compatibility are discussed.

KEY WORDS Helicoverpa zea, Anagrapha falcifera nucleopolyhedrovirus, peroxidase, transgenic plants

MANY INDIVIDUAL INSECT management strategies, such are two distinct insect management strategies that are as insecticides, host plant resistance, and biological considered relatively benign to the agroecosystem. control, can be effective. However, in developing in- However, applying insect pathogens to plants that tegrated management programs, combining different have allelochemical-based resistance to may management strategies may result in the identiÞcation result in synergistic, antagonistic, or additive interac- of incompatible combinations. This type of association tions. Compatible and incompatible interactions be- is most commonly recognized when, for example, in- tween insect pathogens and host plants or their sec- secticides used against pest insects also result in un- ondary chemistry have been summarized (Duffey et desirable kill of beneÞcial insect predators or parasi- al. 1995). Relevant studies on these interactions that toids, which may result in pest resurgence (e.g., Price involve baculoviruses such as nucleopolyhedroviruses 1976). (NPVs), indicate that the efÞcacy of the baculoviruses Host plant resistance and augmentative biological is often antagonized. Some types of incompatible re- control (such as the application of insect pathogens) actions for baculoviruses in Solanaceous plants have been attributed to the interaction of polyphenoloxi- dases or peroxidases with allelochemical substrates This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recom- such as chlorogenic acid or rutin (review, Duffey et al. mendation by the USDA for its use. 1995; Felton et al. 1987; Felton and Duffey 1990; 1 CropBioprotectionResearch Unit, National Center for Agricul- Hoover et al. 1998a, 1998b), although virus efÞcacy has tural Utilization Research, USDA, Agricultural Research Service, 1815 been enhanced in some cases (Ali et al. 1998). When N. University Street, Peoria, IL 61604. 2 To whom reprint requests should be addressed: Crop Bioprotec- plant tissue is damaged during feeding, these biochem- tion Research Unit, National Center for Agricultural Utilization Re- ical combinations presumably produce reactive com- search, USDA, Agricultural Research Service, 1815 N. University pounds such as semiquinones, quinones or active ox- Street, Peoria, IL 61604 (e-mail: [email protected]). 3 ygen species that derivatize or destroy the integrity of Dep. de Microbiologõ´a e Inmunologõ´a, Fac. de Ciencias Biolo´gcias, viral proteins before successful infection of the target UANL, AP. 46-F, San Nicola´s de los Garza, N.L. Mexico 66451. 4 Current address: Syngenta Biotechnology, 3054 Cornwallis Drive, insect occurs (Duffey et al. 1995), although sloughing Research Triangle Park, NC 27709. of gut cells may also be involved in reducing viral 82 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 95, no. 1 infectivity when insects feed on cotton (Hoover et al. information on plant ages and portions of leaves used 2000). Reduced insecticidal activity of NPVs would for assays in the current study are described in the not be surprising, because peroxidase-allelochemical Results. There were three experiments run with L. interactions that produce reactive compounds are also esculentum, one with N. sylvestris, and Þve with N. thought to be involved in plant disease resistance (e.g., tabacum. Different plant leaf portions and different Bell 1981). aged plants were used to obtain an indication of po- Transgenic plants that express high levels of tobacco tential variation in effects on insecticidal activity of anionic peroxidase have often been more resistant to the baculovirus. different insect species compared with wild type Enzyme Assays. A 5-mm-diameter leaf disk from counterparts (Dowd and Lagrimini 1997a, 1997b; exactly the same leaf position (extreme tip or base) Dowd et al. 1998, 1999a, 1999b; Privalle et al. 1998). corresponding to the same portion of the leaf used in However, the representatives of these transgenic the respective bioassay was removed with a cork plants that have been tested have not shown enhanced borer. When disks for bioassays were taken from the resistance to plant pathogens such as tobacco mosaic entire leaf in younger plants, the leaf disk for perox- virus (Lagrimini et al. 1993), in contrast to transgenic idase determinations was taken from the extreme tip. plants that express high levels of cationic peroxidases Each leaf disk was homogenized in 1 ml of pH 7.4, 0.1 (Rasmussen and Kristensen 1999). This information M phosphate buffer, and centrifuged at 10,000 ϫ g for suggests that anionic peroxidase isozyme transgeni- 15 min (Dowd and Lagrimini 1997a). The supernatant cally expressed in the appropriate plant context may was diluted as necessary and used in duplicate spec- be compatible with use of insect pathogens for insect trophotometric peroxidase assays with guaiacol as a control. Because this enhanced peroxidase activity is substrate at pH 6.0 (the isozyme optimum, Sheen due to a single gene alteration (Lagrimini et al. 1987), 1974). The enzyme reaction was monitored at 470 nm this system also makes it possible to directly evaluate over the linear reaction time according to previously the potential for elevated levels of a speciÞc peroxi- described procedures for optimum enzyme activity dase isozyme to adversely affect insect pathogens. We using a Lambda 4B extended range spectrophotome- now report on studies that generally suggest these ter (Perkin-Elmer, Oakbrook, IL) (Lagrimini et al. transgenic plants are compatible with a potentially 1987, Dowd and Lagrimini 1997b). Linear run times commercial strain of Anagrapha falcifera (Kirby) mul- were 10 min for wild type leaf disk homogenates, and tiply imbedded nucleopolyhedrosis virus (AfMNPV), 2Ð5 min for transgenic leaf disk homogenates. Results originally isolated from A. falcifera whose target spe- are reported as mean change in absorbance over a cies includes the corn earworm, Helicoverpa zea (Bod- 10-min period per leaf disk (which weighed Ϸ15 mg). die). Virus Preparation. For earlier bioassays with to- mato leaves, stock virus of AfMNPV was provided by biosys (now Certis USA, Columbia, MD) and propa- Materials and Methods gated in vivo in our laboratory using Trichoplusia ni Insects. Helicoverpa zea were reared on pinto bean (Hu¨ bner) as described previously (Tamez-Guerra et based diet at 27 Ϯ 1ЊC, 40 Ϯ 10% RH, and a photo- al. 2000). For later bioassays with tobacco, sufÞcient period of 14:10 (L:D) h (Dowd 1988). First instars AfMNPV was supplied by Certis USA so that it could were used for bioassays. be used without further propagation. Stock material Plants. Plants were grown in growth chambers at contained 4.3 ϫ 109 polyhedra occlusion bodies (OBs) 27 Ϯ 1ЊC, 50 Ϯ 5% RH, and a photoperiod of 14:10 per gram. Preliminary bioassays with several OB dos- (L:D) h (Dowd and Lagrimini 1997b). Seed from ages differing by an order of magnitude were used to several selÞngs of wild type tomato, Lycopersicon es- establish an appropriate series of concentrations, de- culentum Miller, ÔOH 7814Õ, and transgenic construct signed to yield well distributed mortality values at, and Õ7BÕ (Lagrimini et al. 1992), wild type ornamental on either side of, the LC50. Ultimately, Þve concen- tobacco, Nicotiana sylvestris L., and transgenic con- trations (1.0 ϫ 107, 3.3 ϫ 106, 1.1 ϫ 106, 3.7 ϫ 105, and struct Õ507-C1Õ (Lagrimini et al. 1987, 1990), and wild 1.2 ϫ 105 OB/ml) were used. These concentrations type tobacco Nicotiana tabacum L. ÔCoker 176Õ and were prepared by mixing serial dilutions of unformu- transgenic construct Ô507-C16Õ (Lagrimini et al. 1987, lated puriÞed OBs with water. 1990) were used. All plants contained the tobacco An additional series of studies involved an experi- anionic peroxidase gene driven by a CaMV promoter mental virus formulation made with lignin, corn ßour, and a NOP terminator (Lagrimini et al. 1987 1990, and titanium dioxide (TiO2) that has been used to 1992). Recently matured (full sized) leaves 1 wk after increase virus Þeld stability to washoff and UV light initiation from all of these plants have often shown (Tamez-Guerra et al. 2000). This formulation may also Ϸ1.5 times to 2 times leaf feeding resistance to Þrst- stabilize the virus in the insect gut through protection instar H. zea (Dowd and Lagrimini 1997b, Dowd et al. from reactive radicals, similar to those thought to 1999b; P.F.D. and L.M.L., unpublished data). Thus, cause deleterious effects when produced by UV light 1-wk-old leaves (third leaf from initiation point) were (Ignoffo and Garcia 1994). Experiments including this used in assays with virus, because these leaves have formulation thus may provide information by com- shown more consistent insect resistance differences in parison with unformulated virus on the nature of neg- past studies (Dowd and Lagrimini 1997a, Dowd et al. ative or positive interactions caused when insects feed 1999b; P.F.D. and L.M.L., unpublished data). Further on plant tissue with higher peroxidase activity due to February 2002 BEHLE ET AL.: NPV AND PEROXIDASE 83

Table 1. Mean ؎ SE peroxidase activity and feeding rates of H. zea on virus treated leaves

Peroxidase activity (10 min Feeding rating Plant type/age/experiment n Correlation coefÞcient (r) ⌬ absorbance at 470 nm) (No. 0.25 mm2 holes) L. esculentum Wild type/2 wk/1 30 0.0056 Ϯ 0.0001a ND ND Transgenic/2 wk/1 30 2.3 Ϯ 0.1b ND ND Wild type/2 wk/2 30 0.0056 Ϯ 0.0001a 46.0 Ϯ 1.9a Transgenic/2 wk/2 30 2.3 Ϯ 0.1b 28.9 Ϯ 1.4b Ϫ0.64* N. sylvestris Wild type/3 wk 30 0.09 Ϯ 0.06a 25.4 Ϯ 1.8a Transgenic/3 wk 30 1.5 Ϯ 0.1b 19.7 Ϯ 1.8b Ϫ0.30* N. tabacum Wild type/3 wk/1 30 0.068 Ϯ 0.005a 29.2 Ϯ 2.1a Transgenic/3 wk/1 30 3.4 Ϯ 0.15b 25.7 Ϯ 1.8a Ϫ0.16 Wild type/5 wk/tip/1 30 1.3 Ϯ 0.2a 46.5 Ϯ 2.9a Transgenic/5 wk/tip/1 30 7.1 Ϯ 0.1b 26.5 Ϯ 1.6b Ϫ0.63* Wild type/5 wk/base/1 30 0.23 Ϯ 0.05a 15.0 Ϯ 1.6a Transgenic/5 wk/base/1 30 2.4 Ϯ 0.04b 9.0 Ϯ 1.0b Ϫ0.38* Wild type/3 wk/2 30 0.018 Ϯ 0.03a 44.9 Ϯ 2.1a Transgenic/3 wk/2 30 1.8 Ϯ 0.1b 26.3 Ϯ 1.3b Ϫ0.69* Wild type/5 wk/tip/2 30 0.48 Ϯ 0.11a 26.7 Ϯ 1.5a Transgenic/5wk/tip/2 30 6.1 Ϯ 0.5b 14.2 Ϯ 0.8b Ϫ0.61* Wild type-combined 150 NA 32.4 Ϯ 1.3a Transgenic-combined 150 NA 20.3 Ϯ 0.8b NA

Mean peroxidase activity and feeding rating values in columns for like experiments followed by different letters are signiÞcantly different by ANOVA at P Ͻ 0.05. Correlation coefÞcients followed by * are signiÞcant at P Ͻ 0.05. ND, not determined. NA, not applicable. potential protective effects of the formulation. The spreading the virus material over the surface of each PC-1307 lignin was obtained from Westvaco (Charles- leaf disk with a glass rod, disks were air dried for Ϸ1 ton, SC) and the corn ßour 965 was obtained from h under laboratory conditions to evaporate the water Illinois Cereal Mills (Paris, IL). The formulation was carrier from the virus application. After drying, 100 ␮l prepared and spray-dried as previously described us- of water was added to the Þlter paper in each petri dish ing a Niro Atomizer spray dryer (Niro Atomizer, Co- to prevent the leaves from drying out during the sub- lumbia, MD) (Tamez-Guerra et al. 2000). The formu- sequent larval feeding period. 9 lated virus contained 2.2 ϫ 10 OBs/g and was mixed Ten Þrst-instar H. zea were transferred into each with water to provide the same OB concentrations as petri dish, the lids were put on, and the dishes were previously described for unformulated virus. Formu- placed in a dark incubator at 28 Ϯ 1ЊCanda40Ϯ 10% lated virus was applied to leaf disks from the same RH for a 24-h feeding period (total of 50 larvae per respective leaves used in the Þrst four experiments treatment dosage per experiment). After feeding, six with ÔCokerÕ tobacco (see Table 1). larvae (from the original 10 per dish) that had obvi- Bioassays. For each experiment, the third leaf from ously fed and were of as uniform size as possible were the initiation point leaf from 2- or 4-wk-old tomato and transferred to individual diet cups containing wheat 3- or 5-wk-old tobacco plants was used. Leaf disks from germ based diet (Behle et al. 2000) and returned to the the same Þve transgenic and Þve wild type plants were incubator (30 larvae per dosage per experiment). Lar- used for every treatment of an individual experiment. vae were initially examined after2dtodetermine Six leaf disks (17 mm diameter) were cut from each mortality caused by handling, and these dead individ- leaf with a #10 cork borer. When the formulated virus uals were excluded from statistical analysis. Thus, dos- was included in the assay, 11 leaf disks were cut from age response analysis was based on a target number of each leaf. Leaf disks were cut from the same position ϫ and portion (tip or base) of each leaf when leaves 150 larvae per plant type (5 dosages 30 larvae per were large enough (older plants only) to examine dosage) for each experiment. Final mortality was eval- position effects. Leaf disks were placed individually on uated after 7 d for bioassays with N. tabacum and after 42.5-mm-diameter Whatman #1 Þlter paper (What- 10 d for bioassays with the other plant species. Dif- man, Maidstone, England) in a 50 by 9-mm petri dish ferent intervals were used because larvae did not die with a tight Þtting lid (Falcon 1006, Becton Dickinson, at the same rate, which affected plateau mortality. Lincoln Park, NJ). For each virus concentration, 25 ␮l After larvae were transferred to diet cups, leaf disks of treatment solution was applied individually to Þve were visually rated for feeding by counting the num- 2 disks from each plant type, one disk from each leaf of ber of 0.25-mm -diameter (head capsule sized) holes each plant. Applying the previously described virus made by feeding larvae, and estimating total equiva- concentrations over the leaf disks provided 0, 14, 41, lent area when holes were larger (Dowd and Lagri- 122, 363, and 1,102 OB/mm2. One leaf disk from each mini 1997b; Dowd et al. 1998, 1999b). Dosages were leaf was left untreated to serve as a control (a leaf disk also corrected for the amount of leaf (and hence virus) from each of the Þve different wild type and Þve consumed by multiplying the leaf area consumed in transgenic plant leaves used per experiment). After mm2 by the OBs/mm2 on the leaf disk. 84 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 95, no. 1

Statistical Analysis. Data were analyzed using SAS with increasing virus concentration (F ϭ 2.46; df ϭ 5, for Windows, version 6.12 (SAS Institute 1990). Per- 54; P ϭ 0.044); whereas for the Þrst N. tabacum ex- oxidase activity and feeding data were analyzed using periment with 3-wk-old plants the feeding rating de- analysis of variance (ANOVA) with PROC GLM and creased with increasing virus concentration (F ϭ 2.94; respective means for wild type and transgenic plants df ϭ 5, 54; P ϭ 0.020). Combining the data for all were separated using the LSMEAN option. Correla- experiments with feeding ratings (one L. esculentum, tions among peroxidase activity and feeding were de- one N. sylvestris, and Þve N. tabacum) gave average termined using PROC REG, option CORR with the feeding ratings of 26.7, 27.4, 26.9, 29.7 26.2, and 27.2 for model: feeding ϭ peroxidase activity. Mortality values treatments of 0, 14, 41, 122, 262, and 1102 OB/mm2, for untreated leaf disks were tested for signiÞcance respectively (F ϭ 0.47; df ϭ 5, 414; P ϭ 0.796). Mor- using PROC FREQ and Chi-square analysis. DoseÐ tality values for insects fed on both types of untreated response mortality data were analyzed using PROC leaf disks ranged from 31.4 to 0.0%, but were predom- PROBIT LOG10 to estimate LC50 values, 95% conÞ- inantly Ͻ20%. There were no signiÞcant differences dence intervals, and probit slopes; as well as to com- between mortality of insects that fed on untreated pare parameters for doseÐresponse curves between wild type versus transgenic leaf disks for any experi- wild type and transgenic plants for each experiment, ment and there was no signiÞcant correlation between and combined data for each plant species as an overall peroxidase activity and mortality for any experiment effect. Additional probit analyses were run on dosages at P Ͻ 0.05. that were corrected for the amount of leaf (and hence In general, estimated LC50 values were not signif- virus) consumed. icantly different (based on overlapping 95% conÞ- dence intervals) for larvae fed on wild type versus transgenic leaf disks in individual experiments (Table Results 2). However, there were some exceptions. In the sec- Transgenic leaf disks of all assay pairings had sig- ond experiment with leaves from 2-wk-old L. esculen- niÞcantly (P Ͻ 0.05) greater peroxidase activity than tum plants (one of three experiments with L. esculen- wild type leaf disks, with transgenic leaf disks ranging tum), the LC50 value for larvae fed on virus-treated from 5.5 times (Þrst experiment with 5-wk-old N. taba- transgenic leaf disks was signiÞcantly higher (95% cum plant using leaf disks from the leaf tip; F ϭ 326.235; conÞdence intervals did not overlap) than the LC50 df ϭ 1, 8; P Ͻ 0.0001) to Ͼ400 times (2-wk-old L. value for larvae fed on virus-treated leaf disks from esculentum plants; F ϭ 210.236; df ϭ 1, 8; P Ͻ 0.0001) wild type plants. When dosages were corrected for relative activity (Table 1). Although peroxidase ac- feeding, probit analysis indicated no signiÞcant dif- tivity was variable depending on the leaf age and ference in LC50 values to virus between cohorts fed position for the same plant species, peroxidase activity wild type versus transgenic tomato leaf disks, based on in the transgenic leaf disks was always signiÞcantly overlapping 95% conÞdence intervals (Table 3). For higher than corresponding wild type leaf disks in a the combined L. esculentum data, doseÐresponse pa- given experiment. rameters (LC50 value estimates and slope of gener- Feeding on transgenic leaf disks was generally Ϸ1.5 ated line) were not signiÞcantly different (␹2 ϭ 3.83). times less compared with wild type leaf disks in cor- In the case of the single experiment with N. sylves- responding assays, with signiÞcant differences ranging tris, although the LC50 value was lower for larvae fed from P ϭ 0.024 (F ϭ 5.243; df ϭ 1, 58; for N. sylvestris on the transgenic compared with wild type leaf disks, experiment) to P Ͻ 0.0001 (F ϭ 55.5815; df ϭ 1, 58; for these values were not signiÞcantly different. For in- the second experiment with 3-wk-old plants of N. dividual analysis of the Þve experiments with the N. tabacum) (Table 1). Based on mean values for wild tabacum Coker leaf disks involving different aged type versus transgenic plants, feeding was generally plants and disks from different positions on leaves, inversely correlated to peroxidase activity within in- LC50 values were often two to four times lower for dividual experiments, which was signiÞcant for the larvae fed on transgenic compared with wild type leaf second experiment with 2-wk-old L. esculentum plants disks, but 95% conÞdence intervals overlapped. (only one experiment of three could be rated for leaf Greater differences in LC50 values for larvae fed on feeding) (F ϭ 40.025, mean square error [MSE] ϭ wild type versus transgenic leaf disks were observed 94.9638, P ϭ 0.0001), N. sylvestris (F ϭ 5.824, MSE ϭ for larvae fed on leaf disks from 3-wk-old versus 5-wk- 92.1064, P ϭ 0.02) and in all but one case for N. tabacum old plants. For the combined N. tabacum Coker ex- (Þrst experiment with 5-wk-old plants and disks from periments, probit analysis indicated lower LC50 val- leaf tips, F ϭ 41.455, MSE ϭ 160.4299, P ϭ 0.0001; Þrst ues for larvae fed on transgenic versus wild type leaf experiment with 5-wk-old plants and disks from leaf disks as expected, but 95% conÞdence intervals again bases; F ϭ 9.552, MSE ϭ 52.9370, P ϭ 0.0031; second overlapped. experiment with 3-wk-old plants, F ϭ 52.697, MSE ϭ In general AfMNPV had greater insecticidal activity 95.4548, P ϭ 0.0001; and second experiment with 5-wk- when applied to transgenic plants compared with wild old plants and disks from leaf tips, F ϭ 34.603, MSE ϭ type plants after dosage exposures were corrected for 52.4058, P ϭ 0.0001) (Table 1). Generally, the pres- feeding rates (Table 3). However, LC50 values on ence of virus had no signiÞcant effect on feeding rates, virus-treated transgenic plants were not signiÞcantly although there were signiÞcant effects in two exper- different from the LC50 values for virus-treated wild iments. For L. esculentum, the feeding rate increased type plants, except in one instance with N. tabacum February 2002 BEHLE ET AL.: NPV AND PEROXIDASE 85

Table 2. Dose-response probit analysis statistics for H. zea larvae exposed to Anagrapha falcifera nucleopolyhedrovirus on wild type and transgenic plants expressing high levels of tobacco anionic peroxidase

Plant LC (occlusion 95% CI n Slope Ϯ SE 50 ␹2 type/age/experiment bodies/ml) Lower Upper L. esculentum Wild type/2 wk/1 150 1.41 Ϯ 0.40 8.07 ϫ 105 5.36 ϫ 105 1.19 ϫ 106 4.30 Transgenic/2 wk/1 149 0.78 Ϯ 0.32 1.07 ϫ 106 5.34 ϫ 105 2.12 ϫ 106 4.91 Wild type/2 wk/2 150 0.87 Ϯ 0.34 3.56 ϫ 105 1.45 ϫ 105 6.42 ϫ 105 2.80 Transgenic/2 wk/2 149 0.99 Ϯ 0.34 1.60 ϫ 106 9.55 ϫ 105 2.88 ϫ 106 0.11 Wild type/4 wk 149 1.30 Ϯ 0.61 1.11 ϫ 106 2.34 ϫ 105 6.31 ϫ 106 7.80 Transgenic/4 wk 150 1.25 Ϯ 0.37 7.80 ϫ 105 4.92 ϫ 105 1.19 ϫ 106 2.86 Wild type-combined 449 1.15 Ϯ 0.41 7.23 ϫ 105 2.25 ϫ 105 1.85 ϫ 106 12.01 Transgenic-combined 448 1.08 Ϯ 0.20 1.16 ϫ 106 8.60 ϫ 105 1.49 ϫ 106 1.24 N. sylvestris Wild type/3 wk 134 1.01 Ϯ 0.37 1.93 ϫ 106 1.14 ϫ 106 3.72 ϫ 106 1.94 Transgenic/3 wk 134 0.94 Ϯ 0.55 1.31 ϫ 106 3.98 ϫ 104 3.16 ϫ 108 6.80 N. tabacum Wild type/3 wk/1 149 0.74 Ϯ 0.55 7.44 ϫ 105 NC NC 8.98 Transgenic/3 wk/1 149 0.56 Ϯ 0.71 2.18 ϫ 105 NC NC 14.13 Wild type/5 wk/tip/1 149 0.87 Ϯ 0.34 2.22 ϫ 106 1.23 ϫ 106 4.91 ϫ 106 1.30 Transgenic/5 wk/tip/1 149 0.48 Ϯ 0.33 1.25 ϫ 106 3.13 ϫ 105 5.85 ϫ 106 3.58 Wild type/5 wk/base/1 149 0.94 Ϯ 0.34 1.80 ϫ 106 1.05 ϫ 106 3.43 ϫ 106 2.25 Transgenic/5 wk/base/1 149 0.90 Ϯ 0.33 7.55 ϫ 105 3.97 ϫ 105 1.33 ϫ 106 4.50 Wild type/3 wk/2 149 0.66 Ϯ 0.17 3.70 ϫ 105 9.23 ϫ 104 7.94 ϫ 105 0.11 Transgenic/3 wk/2 149 0.15 Ϯ 0.16 1.70 ϫ 105 NC NC 0.84 Wild type/5 wk/tip/2 149 0.76 Ϯ 0.41 5.82 ϫ 104 3.00 ϫ 103 1.64 ϫ 105 2.57 Transgenic/5 wk/tip/2 149 0.90 Ϯ 0.44 5.47 ϫ 104 5.24 ϫ 103 1.39 ϫ 105 0.68 Wild type-combined 717 0.76 Ϯ 0.13 7.54 ϫ 105 5.51 ϫ 105 1.01 ϫ 106 1.90 Transgenic-combined 713 0.58 Ϯ 0.15 4.46 ϫ 105 2.68 ϫ 105 6.62 ϫ 105 3.61

NC, not calculated by the SAS program (SAS Institute 1990). when basal leaf disks from 5-wk-old plants were used. ferences in probit line slopes were noted for larvae fed In this case, the 95% conÞdence intervals did not any transgenic versus wild type Nicotiana spp. plants overlap, indicating the H. zea larvae that fed on the for any experiment or combination. transgenic leaf disks were signiÞcantly more suscep- Our experiments using formulated virus in the same tible to the AfMNPV than larvae fed on wild type leaf transgenic system showed no reduction in insecticidal disks. As stated previously, only the second experi- activity when applied to transgenic N. tabacum plants. ment (of three) with leaf disks from 2-wk-old L. es- Overall, the formulated virus showed lower activity culentum plants resulted in a lower LC50 value for than unformulated virus, indicating a general loss of virus-treated wild type disks compared with the trans- activity. Because of the low activity, probit analysis of genic disks, which was not signiÞcant based on over- the individual experiments gave poor results. The lapping 95% conÞdence intervals. No signiÞcant dif- combined data of the four experiments Þt the probit

Table 3. Dose-response probit analysis statistics corrected for feeding for H. zea larvae exposed to Anagrapha falcifera nucleopoly- hedrovirus on wild type and transgenic leaves expressing high levels of tobacco anionic peroxidase

Plant LC (occlusion 95% CI n Slope Ϯ SE 50 ␹2 type/age/experiment bodies per larva) Lower Upper L. esculentum Wild type/2 wk/2 152 0.91 Ϯ 0.36 46.8 20.0 82.3 2.52 Transgenic/2 wk/2 149 1.05 Ϯ 0.37 125.2 77.2 217.1 0.04 N. sylvestris Wild type/3 wk 134 0.91 Ϯ 0.34 136.5 75.9 283.6 2.83 Transgenic/3 wk 134 0.84 Ϯ 0.51 67.7 34.0 138.0 7.20 N. tabacum Wild type/3 wk/1 149 0.88 Ϯ 0.59 75.2 NC NC 7.58 Transgenic/3 wk/1 149 0.64 Ϯ 0.76 15.2 NC NC 13.5 Wild type/5 wk/tip/1 142 0.87 Ϯ 0.35 280.6 157.0 624.9 1.99 Transgenic/5 wk/tip/1 125 0.51 Ϯ 0.35 94.5 25.6 396.7 3.30 Wild type/5 wk/base/1 140 0.96 Ϯ 0.35 81.1 47.7 152.9 2.48 Transgenic/5 wk/base/1 150 0.88 Ϯ 0.33 20.0 11.9 35.7* 5.63 Wild type/3 wk/2 136 0.66 Ϯ 0.32 48.1 11.9 103.9 0.12 Transgenic/3 wk/2 140 0.13 Ϯ 0.29 10.3 NC NC 0.91 Wild type/5 wk/tip/2 150 0.74 Ϯ 0.41 2.8 0.08 9.13 3.36 Transgenic/5 wk/tip/2 149 0.92 Ϯ 0.45 2.5 0.24 6.17 1.33

* Indicates that LC50 values within the same plant type, age and experiment are signiÞcantly different based on non-overlapping 95% CI levels. NC, not calculated by SAS program (SAS Institute 1990). 86 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 95, no. 1 model for both plant types (wild type ␹2 ϭ 2.72, trans- such as tobacco mosaic virus, due to the production of genic ␹2 ϭ 2.81). When formulated virus was applied the tobacco anionic peroxidase in transgenic plants to wild type N. tabacum, probit analysis indicated no (Lagrimini et al. 1993), it is not surprising that we did signiÞcant difference in LC50 values (wild type LC50 not observe detrimental effects for the insect NPV and lower and upper 95% CIs were 3.1 ϫ 107, 1.6 ϫ 107, tested in the current study. and 3.5 ϫ 108 OBs/ml, respectively; transgenic LC50 Our studies using formulated virus in the same and lower and upper 95% CIs were 4.2 ϫ 107, 1.3 ϫ 107, transgenic system support our observations with un- and 7.2 ϫ 108 OBs/ml, respectively) or line slopes for formulated virus; i.e., the insecticidal activity of the insects fed on wild type versus transgenic plants. virus was not negatively affected by the higher levels of peroxidase in the transgenic plants. The potential protective effects of the lignin against reactive radicals Discussion were apparently irrelevant in the assay system. Lack of Negative Interactions. Negative interac- Positive Interactions. There were indications in the tions between baculoviruses, such as NPVs, and So- current study that the efÞcacy of the NPV may have lanaceous plant chemicals that are mediated by adding been enhanced when H. zea were fed on the trans- or inducing oxidizing enzymes such as peroxidase or genic plants expressing high levels of a tobacco anionic polyphenoloxidase, have been reported for H. zea in peroxidase, especially when the dosage was corrected the past (Felton et al. 1987; Felton and Duffey 1990; based on quantity of foliage consumed. Positive (ad- Forschler et al. 1992; Hoover et al. 1998a, 1998b), and ditive/synergistic) interactions between peroxidase appear to be mainly due to generation of hydrogen and NPV have been reported previously when per- peroxide (Hoover et al. 1998b). Other studies with H. oxidase was used to counteract the effects of UV gen- zea comparing tomato foliage versus artiÞcial diet in- erated reactive radicals on NPV baculovirus (Ignoffo dicated no differences in susceptibility to HzNPV and Garcia 1994). SigniÞcantly enhanced mortality of when the two types of diet were fed to H. zea (For- H. zea fed virus on wounded (where peroxidase would schler et al. 1992). In longer duration studies with presumably be induced) versus nonwounded tomato several wound-induced plant species (under condi- foliage was noted in long-term studies (Ali et al. 1998). tions where oxidases would likely be induced [Stout Prior theory indicates that the type of relationships et al. 1996]), signiÞcant reductions in baculovirus ef- between the plant environment, the insect, and the Þcacy were seldom noted (Ali et al. 1998). We also entomopathogenic virus is the net effect of several typically noted no negative effect when leaf disks from different positive and negative individual factors transgenic plants expressing high levels of tobacco (Duffey et al. 1995). The presence or increased levels anionic peroxidase, compared with wild type plant of oxidative enzymes, such as polyphenoloxidase or disks, were fed to H. zea. peroxidase, may affect many factors that enhance in- Plant peroxidases induced by mechanical damage secticidal activity of virus treatments. The peroxidase/ and caterpillar feeding on tobacco and tomato leaves polyphenoloxidase mediated factors that may en- are cationic (Lagrimini and Rothstein 1987, Dowd and hance virus efÞcacy include reduced proteolytic Lagrimini 1997b, Dowd et al. 1999b). Typically, cat- digestion, increased nutritional limitations, increased ionic peroxidases are thought to be relatively more tissue toughness, reduced rates of feeding, and in- effective at generating hydrogen peroxide from creased general stress (Duffey et al. 1995). All of these NADH, whereas anionic ones are less effective factors may have occurred in one form or another with (Campa 1991). These tobacco anionic peroxidases the transgenic plants examined in the current study. (that include the one enhanced transgenically in the The potential role of these factors in the transgenic current study) are much poorer generators of hydro- plants we studied is examined in the following discus- gen peroxide than are cationic peroxidases from to- sion. bacco (Ma¨der et al. 1980). However, the tobacco an- Semiquinones/quinones, which may be produced ionic peroxidase that we studied is known to actively by peroxidases from allelochemicals, have been noted consume hydrogen peroxide when the peroxidase ox- as protease inhibitors for some time (Hoffman-Osten- idizes phenolics (e.g., Gazaryan et al. 1996). Thus, the hof 1947). Gut proteases in H. zea appear to be mainly transgenic plants we studied are theoretically capable serine proteases (Purcell et al. 1992). The serine hy- of consuming hydrogen peroxide at a higher rate per droxyl group is a moiety that is potentially susceptible unit area/volume due to higher levels of peroxidase in to attack by quinones, as are other amino acids con- the plants. This information may explain why some taining -SH or -NH(2) groups (Felton et al. 1989, earlier studies, where peroxidase isozyme mixtures 1992). We have not yet determined if protease activity (combined with plant secondary metabolites) or in- is inhibited in larvae fed transgenic versus wild type duced tissues (involving enhanced levels of cationic plants, but consider it a likely occurrence based on the peroxidases) were used, have noted negative interac- types of products produced by peroxidase and the tions between peroxidase products and NPVs, susceptibility of different protease amino acids to whereas ours have not. In the past, parallels have been these products. Conceivably, this type of reaction may drawn between negative interactions of enzymatically serve to protect the viral protein from digestion. The oxidized plant chemicals and both plant viruses and alkaline pH of the gut, which is also important in insect viruses (Felton et al. 1987). Because we have releasing virions (Evans and Entwistle 1989), may be not seen any detrimental effect on plant pathogens, sufÞcient in and of itself to promote viral infection. February 2002 BEHLE ET AL.: NPV AND PEROXIDASE 87

Thus, proteolysis would mainly be harming the virus zea (in contrast with most other insect species tested) and thus reducing viral infection, and therefore inhi- (Dowd et al. 1998), indicating that the results of the bition of proteolysis by peroxidase products would current study may not necessarily be applicable to all serve to enhance virus efÞcacy. plant systems. However, this study does provide in- Nutritional limitations can stress the insect and limit formation that suggests it is possible to enhance plant the general host defense mechanisms (Duffey et al. resistance through increased expression of appropri- 1995). Reduction in digestibility (a form of nutritional ate isozymes of oxidative enzymes such as peroxidase, limitation) has been previously reported in different and that this resistance can be compatible with appli- experimental systems using H. zea where polyphe- cation of insect pathogens for insect control. noloxidase or peroxidase activity was increased in combination with allelochemicals, apparently ulti- Acknowledgments mately producing quinones that bind to protein and prevent its digestion (Felton et al. 1989, 1992). We We thank E. Bailey, M. Giovanini, K. Girsch, H. Goebel, have had evidence that tissue from transgenic plants D. A. Lee, J. Petersen, and R. Rodriguez for technical assis- expressing high levels of peroxidase is less digestible in tance, D. Palmquist for suggestions on statistical analysis, and studies with larger insects (Dowd et al. 1999a, 1999b, C. A. Mullin and F. E. Vega for comments on prior drafts of 2000). the manuscript. We also thank Certis USA for providing the In some cases, leaf resistance to European unformulated virus. corn borer, Ostrinia nubilalis (Hu¨ bner), is related to leaf toughness, which can be mediated by peroxidase References Cited activity (Bergvinson et al. 1995). We have also noted Ali, M. I., G. W. Felton, T. Meade, and S. Y. Young. 1998. that the peroxidase enhanced transgenic tissues are Inßuence of interspeciÞc and intraspeciÞc host plant vari- physically tougher (Dowd and Lagrimini 1997a, ation on the susceptibility of Heliothines to a baculovirus. 1997b; Dowd et al. 1998, 1999a, 1999b, 2000), and that Biol. Control 12: 42Ð49. somewhat higher levels of lignin are present in leaves Behle, R. W., M. R. McGuire, and P. Tamez-Guerra. 2000. due to larger numbers of smaller cells (and as a result, Effects of light energy on alkali released virions from ligniÞed cell walls) in the same sized leaves (Lagrimini Anagrapha falcifera nucleopolyhedrosis virus. J. Inver- 1992, Lagrimini et al. 1993). Reduced rates of feeding tebr. Pathol. 79: 120Ð126. and smaller larval size (apparently due to increased Bell, A. A. 1981. Biochemical mechanisms of disease resis- toxicity) are a frequent result when Þrst-instar H. zea tance. Annu. Rev. Plant Physiol. 32: 21Ð81. Bergvinson, D. J., R. I. Hamilton, and J. T. Arnason. 1995. are fed transgenic versus wild type leaves from to- Leaf proÞle of maize resistance factors to European corn bacco and tomato (Dowd and Lagrimini 1997a, 1997b; borer, Ostrinia nubilalis (Hu¨ bner). J. Chem. Ecol. 21: Dowd et al. 1998, 1999a, 1999b, 2000). This reduced 343Ð354. feeding rate (perhaps also contributed to by higher Campa, A. 1991. Biological roles of plant peroxidases: lignin levels) coupled with the stress imposed by the known and potential functions, pp. 25Ð50. In J. Everse, apparently more toxic transgenic materials (Dowd K. E. Everse, and M. B. Grisham [eds.], Peroxidases in and Lagrimini 1997a, 1997b; Dowd et al. 1998, 1999 a, chemistry and biology. CRC, Boca Raton, FL. 1999b, 2000) appear to be the most consistent resis- Dowd, P. F. 1988. Toxicological and biochemical interac- tance mechanisms noted with the transgenic plants tions of the fungal metabolites fusaric acid and kojic acid with xenobiotics in Heliothis zea and Spodoptera frugi- tested. These reasons, in addition to increased rates of perda. Pestic. Biochem. Physiol. 32: 123Ð134. hydrogen peroxide consumption previously men- Dowd, P. F., and L. M. Lagrimini. 1997a. The role of per- tioned, appear to be the most likely explanation for oxidase in host insect defenses, pp. 195Ð223. In N. Carozzi increasing virus efÞcacy in transgenic versus wild type and M. Koziel [eds.], Advances in insect control: the role plants. of transgenic plants. Taylor and Francis, London. In conclusion, this study suggests that the particular Dowd, P. F., and L. M. Lagrimini. 1997b. Examination of tobacco anionic peroxidase transgenically expressed different tobacco (Nicotiana spp.) types under- and over- at high levels in tobacco and tomato, and which con- producing tobacco anionic peroxidase for their leaf re- fers H. zea resistance, is likely to be compatible with sistance to Helicoverpa zea. J. Chem. Ecol. 23: 2357Ð2370. Dowd, P. F., L. M. Lagrimini, and D. A. Herms. 1998. Dif- the use of the AfMNPV virus tested in the current ferential leaf resistance to insects of transgenic sweetgum study, and perhaps other baculoviruses. Further test- (Liquidambar styraciflua) expressing tobacco anionic ing under Þeld conditions will be needed to verify the peroxidase. Cell. Mol. Life Sci. 54: 712Ð720. compatibility of the plants expressing high levels of Dowd, P. F., L. M. Lagrimini, and D. A. Herms. 1999a. anionic peroxidase and AfMNPV noted in this labo- Tobacco anionic peroxidase often increases resistance to ratory study. This study also suggests that interpreting insects in different dicotyledonous species. Pestic. Sci. 55: or predicting interactions with oxidative enzymes is 633Ð634. more complex than simply measuring a total type of Dowd, P. F., L. M. Lagrimini, and T. C. Nelsen. 1999b. oxidative enzyme activity. The speciÞcity of the indi- Relative resistance of transgenic tomato tissues express- ing high levels of tobacco anionic peroxidase to different vidual isozyme and the plant context of the isozyme insect species. Nat. Toxins 6: 241Ð249. are also important in determining the end result, as Dowd, P. F., D. A. Herms, M. A. Berhow, and L. M. Lagri- suggested previously by Dowd and Lagrimini (1997a). mini. 2000. Mechanisms of insect resistance in trans- Producing the tobacco anionic peroxidase in sweet- genic plants (over)expressing a tobacco anionic peroxi- gum, Liquidambar styraciflua, enhanced damage by H. dase. Proceedings of the 1998 International Plant 88 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 95, no. 1

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