Effect of Transgenic Plants Expressing High Levels of a Tobacco Anionic Peroxidase on the Toxicity of Anagrapha Falcifera Nucleo
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ECOTOXICOLOGY Effect of Transgenic Plants Expressing High Levels of a Tobacco Anionic Peroxidase on the Toxicity of Anagrapha falcifera Nucleopolyhedrovirus to Helicoverpa zea (Lepidoptera: Noctuidae) 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 Anagrapha falcifera 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 insect 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 insects 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.