JOURNAL
OF INVERTEBRATE
Specificity
PATHOLOGY
56, 258-266 (1990)
and Efficacy of Purified Bacillus thuringiensis against Agronomically Important insects
Proteins
SUSAN C. MACINTOSH,’ TERRY B. STONE, STEVE R. SIMS, PENNY L. HUNST, JOHN T. GREENPLATE, PAMELA G. MARRONE, FREDERICK J. PERLAK, DAVID A. FISCHHOFF, AND ROY L. FUCHS Plant
Science
Technology,
Monsanto
Agriculrural Company, St. Louis, Missouri 63198
700 Cheste$eld
Village
Parkway,
Received October 30, 1989; accepted February 7, 1990 The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. rhuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kursiaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. renebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (L&J varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. ihuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC,). This extremely sensitive assay enabled detection of B. rhuringiensis ssp. kurstaki HD-73 levels as low as 1 rig/ml. 8 1990academic press, IIIC. KEY WORDS:Bacillus thuringiensis ssp. lenebrionis; Bacillus rhuringiensis ssp. kursraki; insecticidal spectrum; purified 8-endotoxin.
INTRODUCTION
Bacillus thuringiensis represents the major class of microbes used for insect biocontrol (Klausner, 1984). B. thuringiensis is a gram-positive, spore-forming bacterium that characteristically produces a parasporal crystal protein. Crystal and spore preparation of B. thuringiensis ssp. kurstaki such as Dipel (Abbott Laboratories, North Chicago, Illinois) and Thuricide (Sandoz, Basei, Switzerland) are effective against some 55 lepidopteran pest species (Wilcox et al., 1986) and have been used as commercial insecticides for many years. At least 11 distinct genes encoding lepidopteran-specific proteins have been cloned (Wilcox et al., 1986; Whiteley and Schnepf, 1986) and 4 have been expressed at insecticidal levels in transgenic plants (Adang et al., 1987; Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). Recently, another class of B. thuringien’ To whom correspondence should be addressed. 258 0022-201 l/90 $1.50 Copyright 6 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
sis proteins active against coleopterans has been isolated from B. thuringiensis ssp. tenebrionis (Krieg et al., 1983, 1987a) and B. thuringiensis ssp. san diego (Herrnstadt et al., 1986). These two strains have been shown to be indistinguishable (Krieg et al., 1987a) and are designated CryIIIA (H6fte and Whiteley, 1989). A commercial formulation of B. thuringiensis ssp. salt diego effective against specific coleopteran pests (e.g., Colorado potato beetle, cottonwood leaf beetle) was introduced in 1988 as MOne (Mycogen, San Diego, California). The genes encoding the B. thuringiensis ssp. tenebrionis and B. thuringiensis ssp. san diego proteins have been cloned and sequenced and shown to be identical (Herrnstadt et al., 1987; HGfte et al., 1987; Jan et al., 1987; McPherson et al., 1988; Sekar et al., 1987). The cryZZZA gene has also been expressed at insecticidal levels in plants (D. A. Fischhoff et al., unpubl.). Determining the host range and relative efficacy for these individual B. thuringiensis proteins are important steps in the as-
EFFICACY
sessment of their commercial potential. Little quantitative data are available on the insecticidal spectrum of single purified B. thuringiensis proteins against a wide range of agronomically important pests. Most previous host range studies focused on examining a wide variety of B. thuringiensis strains against a limited group of insects (Hiifte et al., 1988; Jaquet et al., 1987; Krieg and Langenbruch, 1981; Lecadet and Martouret, 1987). The B. thuringiensis strains in these reports were typically tested as whole cultures with spores and crystals that contained multiple B. thuringiensis proteins. The combined potencies of the various B. thuringiensis proteins in these B. thuringiensis strains accounted for the observed insecticidal spectra and efficacies. The insecticidal activity of individual B. thuringiensis proteins was impossible to determine. In reports where purified B. thuringiensis proteins were used, only a few agronomically important insects were assayed (Hofte et al., 1988; Jaquet et al., 1987). The present study established the host range of individually purified B. thuringiensis ssp. tenebriunis (CryIIIA), B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)), and HD-73 (Cry IA(c)) proteins by screening 18 agronomically important insects. Bioassays were used to determine the level of insecticidal activity (LC,,) for each sensitive insect species and larval growth inhibitory activity (EC,,,) for a selected subset of these insects. MATERIALS
259
OF B. rhunizgiensis PROTEINS
scribed (McPherson et al., 1988). Washed protein inclusions were solubilized for 2 hr at room temperature in 100 rnkr sodium carbonate buffer, pH 10, and the insoluble material was removed by centrifugation (15,000g). The solubilized CryIIIA protein was adjusted to a concentration of 2 to 5 mg/ml and extensively dialyzed against 10 mM sodium phosphate buffer, pH 6.0. The fine white precipitate was collected by centrifugation and dissolved in 100 mM sodium carbonate buffer, pH 10. This novel pHdependent, preferential precipitation method produced CryIIIA protein of greater than 95% purity (Fig. 1). CryIA(c) protein crystals which contain a single insecticidal protein were isolated from strain B. thuringiensis ssp. kurstaki HD-73 using a modification of the procedure of Yamamoto and Odoni (1983). Washed crystals were solubilized in 100 mM sodium carbonate buffer, pH 10, with 10 mM dithiothreitol (D’IT). The solubilized protein was separated from spores and other particulates by centrifugation and fil12
34
5
92kDa 66kDa -
AND METHODS
All reagents, unless otherwise indicated, were reagent grade and obtained from Sigma Chemical Co., St. Louis, Missouri. Protein purification. CryIIIA protein was purified from Escherichia coli JMlOl that contains a plasmid (pMON5456) encoding an N-terminal truncation of the CryIIIA protein (McPherson et al., 1988). This CryIIIA protein begins at amino acid 48 of the deduced full-length sequence. Cultures were grown, induced, and harvested and protein inclusions were isolated as de-
43kDa -
21 kDa FIG. 1. SDS-PAGE analysis of purified Bacillus thuringiensis proteins on a 9% silver-stained polyacrylamide gel. Lanes 1 and 5, molecular weight standards; lane 2, CryIIIA; lanes 3 and 4, CryIA(b) and CryIA(c), respectively.
260
MACINTOSH
tration through a 0.2~pm filter. Excision grade trypsin (Calbiochem, LaJolla, California) was substituted for gut juice to cleave the full-length CryIA(c) protein to the trypsin resistant fragment. Trypsin was mixed with CryIA(c) protein in a I:5 (w:w) ratio and incubated at 18°C for 6 hr. The solution was stored at 4°C and the extent of cleavage, to a 63-kDa protein, was determined by SDS-PAGE analysis (Laemmli, 1970). CryIA(b) protein was isolated from an E. coli strain containing a plasmid that encodes the full-length CryIA(b) protein comparable to pMAP 4 (Fischhoff et al., 1987) but engineered for expression in E. coli. This cryZA(b) gene was isolated from the B. thuringiensis ssp. kurstaki HD-1 strain which contains at least three different B. thuringiensis ssp. kurstaki genes (Wilcox et al., 1986). Cultures were grown, induced, and harvested and protein inclusions were isolated as described for CryIIIA. The in-
TABLE
ET AL.
clusion bodies were extensively washed and the CryIA(b) protein was solubilized in 100 mM sodium carbonate, 10 mM DTT buffer, pH 10. The clear supematant was precipitated with 40% ammonium sulfate, dissolved in 100 mM sodium carbonate, 10 mM DTT buffer, pH 10, and subjected to ion-exchange chromatography on a MonoQ (HRlO/lO) column (Pharmacia, Uppsala, Sweden) utilizing a 0 to 1 M potassium thiocyanate gradient. The purified full-length CryIA(b) protein was trypsinized as described above for the CryIA(c) protein. Protein determination. Protein concentrations were determined by the method of Bradford (1976), using bovine serum albumin as the standard. Insect bioassays. Seventeen agronomitally important insects and one species of mite were selected which represented a wide range of insect species. These insects were from colonies established at Monsanto or obtained from outside sources.
1
Bacillus thuringiensis HOST RANGE
Common name
Abbreviation
Alfalfa weevil Cotton bollweevil* Horseradish flea beetle Southern corn rootwormb White grub (Japanese beetle) Colorado potato beetle Beet armywormb Black cutwormb Cabbage loopeP Corn earwormb European corn bore? Tobacco budwormb Tobacco hornworm’ Mosquito Cockroach Green peach aphid Termite Spider mite
ALW CBW FB SCRW WG CPB BAW BCW CL CEW ECB TBW THW MOS CR GPA TER SM
Scientific name Hypera postica Antonomus grandes Phyllotreta armoraciae Diabrotica undecimpunctata Popilla japonica Leptinotarsa decemlineata Spodoptera exigua Agrotis ipsilon Trichoplusia ni Heliothis zea Ostrinia nubilialis Heliothis virescens Manduca sexta Aedes aegypti Blatella germanica Myzus persicae Reticulitermes jlavipes Tetranychus urticae
howardii
Sensitivity to B. thuringiensis proteins -0 +c + + + + + + + -
a Significant insect mortality (>25%) for any of the three B. thuringiensis proteins. b Insects were tested in a diet incorporation assay as described under Materials and Methods. c NO significant insect mortality (80 3.6 34
0.02-O. 12 O.l(M.39 1.02-3.19 15.4-l 17.8 ND 1.76-6.89 26.34.3
a LC, denotes the concentration of CryIA protein causing 50% mortality. EC, denotes the concentration of CryIA protein causing 50% larval growth reduction. All LC, and EC,, values expressed in t&ml. b Not determined.
l&ml with 95% fiducial limits of 3.4-11.5 kg/ml. Table 2 lists the sensitive lepidopteran insects in order of decreasing LCsO values to CryIA(c) protein. The relative sensitivity of these insects to CryIA(b) protein typically coincided with that of CryIA(c) protein, but the ratio of LC,, values of these 7 insects were different for the two proteins. The LC,, for five of the lepidopterans, cabbage looper (CL), tobacco budworm (TBW), corn earworm (CEW), and black cutworm (BCW) was 2- to 5-fold lower for CryIA(c) than for CryIA(b) protein. European corn borer (ECB) was more sensitive to CryIA(b) than CryIA(c) protein by lo-fold. Beet armyworm (BAW) and tobacco hornworm (THW) showed similar LCso values for both CryIA(b) and CryIA(c) protein. An approximately 450fold range in LCsa values for CryIA(c) protein was observed from the least sensitive insect, BAW, to the most sensitive insects CL and THW. The overall range in activity of CryIA(b) protein was approximately 2000-fold across the 7 sensitive insects. Detailed analysis of the mortality data for a subset of these insects with CryIA(c) protein showed very different dose-response curves (Fig. 2). Both CL and THW had steep slopes, with a narrow effective concentration range of about one log, i.e., a dose of 0.05 l.&nl of CryIA(c) protein produced only slight mortality whereas 0.5 &ml killed all insects. In contrast, the
curves for TBW and CEW covered about 2 to 3 logs of CryIA(c) protein concentrations. Limited mortality of TBW was observed at 0.2 pg/ml and 100% mortality at levels above 20 l&ml CryIAfc) protein. CEW had a very flat dose-response curve with insufficient data at the highest CryIA(c) protein concentrations to fully evaluate mortality curves. Limited mortality was observed at 1 to 2 pg/ml and about 80% mortality found at the highest level tested, 100 &nl CryIA(c) protein. Sublethal doses of CryIA(c) protein caused dramatic reduction in larval growth at surprisingly low protein concentrations, particularly for TBW and CEW (Fig. 3). The EC,, values for THW and CL were loo-
> 80
0.01
0.1 HD-73
1 10 CONCENTRATION (,,&nl)
1 3
FIG. 2. Mortality dose-response curves for four lepidopteran insects. The artificial diet incorporation bioassay described under Materials and Methods was used to determine the percentage corrected mortality as a function of CryIA(c) protein concentration.
EFFICACY
OF B. thuringiensis
h ,1
0.01 0.1 HD-73 CONCENTRATION (ug/ml)
1
FIG. 3. Average larval weight dose-response curves for four lepidopteran insects. The artificial diet incorporation bioassay described under Materials and Methods was used to determine the average insect weight as a function of CryIA(c) protein concentration.
approximately IO-fold below their LC,, values. However, for TBW and CEW, the ECSo values were approximately 500- and lOOO-fold below the respective LCsO values (Table 2). The differences in relative EC,d L&s values accounted for the fact that although THW and CL were the most sensitive lepidopterans assayed in terms of mortality (i.e., L&J, TBW was approximately lo-fold more sensitive than THW and CL in terms of growth inhibition (i.e., EC&. The corresponding dose-response curves for CL, THW, TBW, and CEW illustrated these conclusions; ie., the growth inhibition dose-response curves for TBW and CEW were shallow, especially in comparison to the steep curve for the CL (Fig. 3). DISCUSSION
The host range of three B. thuringiensis insect control proteins and the relative sensitivities of the 18 different insects to these proteins were determined. Ten insects were insensitive to all of the B. thuringiensis proteins tested, 7 were sensitive to both CryIA proteins, and 1 insect was sensitive to CryIIIA protein. Only a limited number of coleopteran insects were included in the present study. Those insects selected were chosen for
PROTEINS
263
their agronomic importance in the United States. Krieg et al. (1987b) reported activity of B. thuringiensis ssp. tenebrionis spores and crystals against seven different coleopterans. CPB was the only insect of that group to be included in the present study. Herrnstadt et al. (1986, 1987) reported that corn rootworm (CRW), cotton bollweevil (CBW), and CPB were also sensitive to crude B. thuringiensis ssp. sun diego cultures. Although all three of these insects were included in this screen, only CPB was found to be sensitive to the puritied CryIIIA protein. No mortality was observed against CRW, CBW, or ALW (an additional coleopteran) with purified CryIIIA protein even at very high concentrations, 2 to 5 mg/ml (R. Fuchs and S. Macintosh, unpubl.). Hermstadt et al. (1987) reported strong insecticidal activity of B. thuringiensis ssp. sun diego spores and crystals for CPB and CBW and only weak activity for CRW. This lack of activity observed against SCRW and CBW with purified CryIIIA protein suggests that some other protein(s), metabolite(s), or synergism between spores and crystals may be responsible for the activity observed by Herrnstadt et al. (1986, 1987) Data on the insecticidal spectrum and relative efficacy of individual proteins are crucial to identify the preferred gene(s) to use to produce B. thuringiensis microbial biocontrol agents and insect tolerant transgenic plants. This is especially true for lepidopteran-specific CryIA proteins because of the numerous distinct genes from various multigene B. thuringiensis strains that have been isolated and characterized (Wilcox et al., 1986; Whiteley and Schnepf, 1986). The present study compared the relative activity of the single protein CryIA(c), produced by the B. thuringiensis ssp. kurstaki HD-73 strain, and a purified gene product, CryIA(b), from the B. thuringiensis ssp. kurstaki HD-1 strain. The ratio of differential activities of the CryIA(b) and CryIA(c) proteins varied among the seven sensitive
264
MACINTOSH
insects from 2- to lo-fold. This difference is of particular significance in selecting a gene or gene product targeted at the agronomitally important, less sensitive insects (e.g., BCW, CEW), where CryIA(c) was approximately 4-fold more active than the CryIA(b) protein. In addition the enhanced sensitivity to CryIA(b) of ECB may be important in areas where this insect pest is prevalent. These data confirm a recent report (Jaquet et al., 1987) that showed that purified, activated CryIA(c) protein was more potent against TBW than the purified activated proteins from the B. thuringiensis ssp. kurstaki HD-1 strain. Jaquet et al. (1987) used the combined purified proteins from the HD-1 strain, which contains three different B. thuringiensis ssp. kurstaki proteins having differential insect activities (Wilcox et al., 1986). Hofte et al. (1988) reported that the CryIA(c) protein showed quantitative differences in insecticidal activity compared to the CryIA(b) protein against two of the five insects tested (TBW and Mamestra brassicae). They found that TBW was 5- to 6-fold more sensitive to the CryIA(c) protein but that M. brassicae was lo- to 15fold more sensitive to the CryIA(b) protein. The other three insects showed no quantitatively significant differences in sensitivity to the CryIA(b) and CryIA(c) proteins. Compilation of these data suggest that selection of the gene for engineering into microbes or plants is dependent on the insect(s) targeted for control. The present study shows that many of the agronomically important lepidopteran pests in the United States are more sensitive to the CryIA(c) protein rather than the CryIA(b) protein. The increased efficacy of purified CryIA(c) protein relative to CryIA(b) protein was confirmed with E. coli cultures that expressed either fulllength or genetically truncated genes that encode these individual B. thuringiensis ssp. kurstaki proteins. Cultures expressing the CryIA(c) protein showed 3- to 5-fold higher specific activity, based on quantita-
ET
AL.
tion by Western analysis (Towbin et al., 1979), than the corresponding cultures expressing CryIA(b) protein for TBW and CEW (R. Fuchs and F. Perlak, unpubl.). In addition to selecting a highly efficacious B. thuringiensis ssp. kurstaki gene for introduction into microbes and plants, the relative sensitivity of a specific target insect(s) dictates the expression level required for effective control. For example, based on LC,, values, control of the beet armyworm requires about lOOO-fold more CryIA(c) protein than control of the tobacco hornworm. Therefore, correspondingly higher expression levels in transgenic plants are required to control the beet armyworm. Prior to this report, the magnitude of sensitivity differences (LC,,) for single B. thuringiensis proteins was not determined for many agronomically important insects. Most previous reports of the host range for the lepidopteran-active B. thuringiensis ssp. kurstaki proteins (Dulmage 1981; Krieg and Langenbruch, 1981; Trottier et al., 1988) used crude B. thuringiensis ssp. kurstaki cultures, many of which contained multiple proteins and spores (i.e., B. thuringiensis ssp. kurstaki HD-1 strain). These relative rankings of insect sensitivities previously reported typically agree with those described in this study. However, sensitivities were reported in relative terms; LC5a values were not determined. In the studies where LC5a values were reported (Jaquet et al., 1987; Lacadet and Martouret, 1987; Li et al., 1987), purified crystals solubilized from B. thuringiensis ssp. kurstaki strains were used. Only a limited number of agronomically important pests were included in these reports and in the most recent study that used purified proteins of single cloned gene products (Hofte et al., 1988). The present study provides specific L& and ECso values of single purified B. thuringiensis proteins for a broad collection of agronomically important pests. These insects, with known LCsO and
EFFICACY
OF
B. thuringiensis
ECSo values, provide a valuable resource for the detection and quantitation of B. thuringiensis ssp. kurstuki expression levels. The reported expression levels of B. thuringiensis proteins in transgenic plants to date have been extremely low (Adang et al., 1987; Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). Therefore, a very sensitive bioassay is essential to detect bioactivity. The most sensitive insects assayed in this study in terms of mortality, THW and CL, showed insecticidal sensitivity (LC,,) as low as 0.04 kg/ml. The level of detection of CryIA(c) protein was extended 50-fold to 1 rig/ml for the most sensitive insect, TBW, by using the growth inhibition assay (EC,,) (Table 2). This growth inhibition or stunting effect was previously used to assess insect control in transgenic plants (Barton et al., 1987), but was used only qualitatively and not quantitatively. As increased expression levels in transgenic plants are achieved, the mortality and growth inhibition assays, which enable B. thuringiensis ssp. kurstaki quantitation over a 6 log range, will be extremely useful to monitor expression levels. ACKNOWLEDGMENTS We thank Jamie Wibbenmeyer and Mary Taylor for their technical assistance in purifying the CryIA(b) and CryIA(c) protein. We also thank Paul Gahr and Christine Olsen for assistance with the insect bioassays.
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1970-1980” (H.D. Burges, Ed.), pp. 8388%. Academic Press, New York. KRIEG, A., HUGER, A. M., AND SCHNETTER, W. 1987a. “Bacillus thuringiensis var. sun diego” Stamm M-7 ist identisch mit dem zuvor in Deutschland isolierten kaferwirksamen B. thuringiensis subsp. tenebrionis Stamm BI 256-82. .I. Appl. Entomol., 104, 417-424. KRIEG, A., SCHNETTER, W., HUGER, A. M., AND LANGENBRUCH, G. A. 1987b. Bacillus thuringiensis subsp. tenebrionis strain BI 256-82: A third pathotype within the H-serotype 8a8b. Syst. Appl. Microbiol., 9, 138-141. LAEMMELI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 681-685. LECADET, M. M., AND MARTOURET, D. 1987. Host specificity of the Bacillus thuringiensis &endotoxin toward lepidopteran species: Spodoptera littoralis Bdv. and Pieris brassicae. J. Invertebr. Pathol., 49, 37-48. LI, R. S., JARRE’IT, P., AND BURGES, H. D. 1987. Importance of spores, crystals, and &endotoxins in the patbogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae. J. Invertebr. Pathol., 50, 277-284. MCCLANAHAN, R. J., AND FOUNK, J. 1983. Toxicity of insecticides to the green peach aphid (Homoptera: Aphididae) in laboratory and field tests, 1971-1982. J. Econ. Entomol., 76, 899-905. MCLAUGHLIN, R. E., DULMAGE, H. T., ALLS, R., COUCH, T. L., DAME, D. A., HALL, I. M., ROSE, R. I., AND VERSOI, P. L. 1981. U.S. standard bioassay for the potency assessment of Bacillus thuringiensis serotype H-14 against mosquito larvae. Bull. Entomol. Sot. Amer., 30, 26-29. MARRONE, P. G., FERRI, F. D., MOSLEY, T. R., AND MEINKE, L. J. 1985. Improvements in laboratory rearing of the southern corn rootworm, Diubrotica undecimpunctn howardi Barber (Coleoptera: Chrysomelidae), on and artificial diet and corn. J. Econ. Entomol., 78, 29&293. MCPHERSON, S. A., PERLAK, F. J., FUCHS, R. L., MARRONE, P. G., LAVRIK, P. B., AND FISCHHOFF, D. A. 1988. Characterization of the coleopteran-
ET AL. specific protein gene of Bacillus thuringiensis var. tenebrionis. BiolTechnol., 6, 61-66. RUST, M. K., AND REIERSON, D. A. 1981. Attraction and performance of insecticidal baits for German cockroach control. Znt. Pest Control., 23, 106-109. SEKAR, V., THOMPSON, Q. V., MARONEY, M. J., BOOKLAND, R. G., AND ADANG, M. J. 1987. Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc. Natl. Acad. Sci. USA, 84, 70367040. STONE, T. B., SIMS, S. R., AND MARRONE, P. G. 1989. Selection of tobacco budworm for resistance to a genetically engineered Pseudomonas jluorescens containing the &endotoxin of Bacillus thuringiensis subsp. kurstaki. J. Znvertebr. Pathol., 53, 228-232. TOWBIN, H. T., STAELHELIN, T., AND GORDON, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA, 76, 4350-4354. TROTTIER, M. R., MORRIS, 0. N., AND DULMAGE, H. 1988. Susceptibility of the Bertha armyworm, Mamestra configuratu (Lepidoptera, Noctuidae), to sixty-six strains from ten varieties of Bacillus thuringiensis. J. Invertebr. Pathol., 51, 242-249. VAECK, M., REYNAERTS, A., H~FTE, H., JANSENS, S., DEBEUCKELEER, M., DEAN, C., ZABEAU, M., VAN MONTAGU, M., AND LEEMANS, J. 1987. Transgenie plants protected from insect attack. Nature (London), 328, 33-37. WHITELEY, H. R., AND SCHNEPF, H. E. 1986. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Annu. Rev. Microbiol., 40, 549-576. WILCOX, E. R., SHIRAKUMA, A. G., MELIN, B. E., MILLER, M. F., BENSON, T. A., SCHOPP, C. W., CASUTO, D., GUNDLING, G. J., BOLLONG, T. J., SPEAR, B. B., AND Fox, J. L. 1986. Genetic engineering of bioinsecticides. In “Protein Engineering: Applications in Science, Medicine and Industry” (M. Inouye and R. Sarma, Eds.), pp. 395-413. Academic Press, New York. YAMAMOTO, T., AND ODONI, T. 1983. Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis var. kurstaki. Arch. Biochem. Biophys., 227, 233-241.
OF INVERTEBRATE
Specificity
PATHOLOGY
56, 258-266 (1990)
and Efficacy of Purified Bacillus thuringiensis against Agronomically Important insects
Proteins
SUSAN C. MACINTOSH,’ TERRY B. STONE, STEVE R. SIMS, PENNY L. HUNST, JOHN T. GREENPLATE, PAMELA G. MARRONE, FREDERICK J. PERLAK, DAVID A. FISCHHOFF, AND ROY L. FUCHS Plant
Science
Technology,
Monsanto
Agriculrural Company, St. Louis, Missouri 63198
700 Cheste$eld
Village
Parkway,
Received October 30, 1989; accepted February 7, 1990 The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. rhuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kursiaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. renebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (L&J varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. ihuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC,). This extremely sensitive assay enabled detection of B. rhuringiensis ssp. kurstaki HD-73 levels as low as 1 rig/ml. 8 1990academic press, IIIC. KEY WORDS:Bacillus thuringiensis ssp. lenebrionis; Bacillus rhuringiensis ssp. kursraki; insecticidal spectrum; purified 8-endotoxin.
INTRODUCTION
Bacillus thuringiensis represents the major class of microbes used for insect biocontrol (Klausner, 1984). B. thuringiensis is a gram-positive, spore-forming bacterium that characteristically produces a parasporal crystal protein. Crystal and spore preparation of B. thuringiensis ssp. kurstaki such as Dipel (Abbott Laboratories, North Chicago, Illinois) and Thuricide (Sandoz, Basei, Switzerland) are effective against some 55 lepidopteran pest species (Wilcox et al., 1986) and have been used as commercial insecticides for many years. At least 11 distinct genes encoding lepidopteran-specific proteins have been cloned (Wilcox et al., 1986; Whiteley and Schnepf, 1986) and 4 have been expressed at insecticidal levels in transgenic plants (Adang et al., 1987; Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). Recently, another class of B. thuringien’ To whom correspondence should be addressed. 258 0022-201 l/90 $1.50 Copyright 6 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
sis proteins active against coleopterans has been isolated from B. thuringiensis ssp. tenebrionis (Krieg et al., 1983, 1987a) and B. thuringiensis ssp. san diego (Herrnstadt et al., 1986). These two strains have been shown to be indistinguishable (Krieg et al., 1987a) and are designated CryIIIA (H6fte and Whiteley, 1989). A commercial formulation of B. thuringiensis ssp. salt diego effective against specific coleopteran pests (e.g., Colorado potato beetle, cottonwood leaf beetle) was introduced in 1988 as MOne (Mycogen, San Diego, California). The genes encoding the B. thuringiensis ssp. tenebrionis and B. thuringiensis ssp. san diego proteins have been cloned and sequenced and shown to be identical (Herrnstadt et al., 1987; HGfte et al., 1987; Jan et al., 1987; McPherson et al., 1988; Sekar et al., 1987). The cryZZZA gene has also been expressed at insecticidal levels in plants (D. A. Fischhoff et al., unpubl.). Determining the host range and relative efficacy for these individual B. thuringiensis proteins are important steps in the as-
EFFICACY
sessment of their commercial potential. Little quantitative data are available on the insecticidal spectrum of single purified B. thuringiensis proteins against a wide range of agronomically important pests. Most previous host range studies focused on examining a wide variety of B. thuringiensis strains against a limited group of insects (Hiifte et al., 1988; Jaquet et al., 1987; Krieg and Langenbruch, 1981; Lecadet and Martouret, 1987). The B. thuringiensis strains in these reports were typically tested as whole cultures with spores and crystals that contained multiple B. thuringiensis proteins. The combined potencies of the various B. thuringiensis proteins in these B. thuringiensis strains accounted for the observed insecticidal spectra and efficacies. The insecticidal activity of individual B. thuringiensis proteins was impossible to determine. In reports where purified B. thuringiensis proteins were used, only a few agronomically important insects were assayed (Hofte et al., 1988; Jaquet et al., 1987). The present study established the host range of individually purified B. thuringiensis ssp. tenebriunis (CryIIIA), B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)), and HD-73 (Cry IA(c)) proteins by screening 18 agronomically important insects. Bioassays were used to determine the level of insecticidal activity (LC,,) for each sensitive insect species and larval growth inhibitory activity (EC,,,) for a selected subset of these insects. MATERIALS
259
OF B. rhunizgiensis PROTEINS
scribed (McPherson et al., 1988). Washed protein inclusions were solubilized for 2 hr at room temperature in 100 rnkr sodium carbonate buffer, pH 10, and the insoluble material was removed by centrifugation (15,000g). The solubilized CryIIIA protein was adjusted to a concentration of 2 to 5 mg/ml and extensively dialyzed against 10 mM sodium phosphate buffer, pH 6.0. The fine white precipitate was collected by centrifugation and dissolved in 100 mM sodium carbonate buffer, pH 10. This novel pHdependent, preferential precipitation method produced CryIIIA protein of greater than 95% purity (Fig. 1). CryIA(c) protein crystals which contain a single insecticidal protein were isolated from strain B. thuringiensis ssp. kurstaki HD-73 using a modification of the procedure of Yamamoto and Odoni (1983). Washed crystals were solubilized in 100 mM sodium carbonate buffer, pH 10, with 10 mM dithiothreitol (D’IT). The solubilized protein was separated from spores and other particulates by centrifugation and fil12
34
5
92kDa 66kDa -
AND METHODS
All reagents, unless otherwise indicated, were reagent grade and obtained from Sigma Chemical Co., St. Louis, Missouri. Protein purification. CryIIIA protein was purified from Escherichia coli JMlOl that contains a plasmid (pMON5456) encoding an N-terminal truncation of the CryIIIA protein (McPherson et al., 1988). This CryIIIA protein begins at amino acid 48 of the deduced full-length sequence. Cultures were grown, induced, and harvested and protein inclusions were isolated as de-
43kDa -
21 kDa FIG. 1. SDS-PAGE analysis of purified Bacillus thuringiensis proteins on a 9% silver-stained polyacrylamide gel. Lanes 1 and 5, molecular weight standards; lane 2, CryIIIA; lanes 3 and 4, CryIA(b) and CryIA(c), respectively.
260
MACINTOSH
tration through a 0.2~pm filter. Excision grade trypsin (Calbiochem, LaJolla, California) was substituted for gut juice to cleave the full-length CryIA(c) protein to the trypsin resistant fragment. Trypsin was mixed with CryIA(c) protein in a I:5 (w:w) ratio and incubated at 18°C for 6 hr. The solution was stored at 4°C and the extent of cleavage, to a 63-kDa protein, was determined by SDS-PAGE analysis (Laemmli, 1970). CryIA(b) protein was isolated from an E. coli strain containing a plasmid that encodes the full-length CryIA(b) protein comparable to pMAP 4 (Fischhoff et al., 1987) but engineered for expression in E. coli. This cryZA(b) gene was isolated from the B. thuringiensis ssp. kurstaki HD-1 strain which contains at least three different B. thuringiensis ssp. kurstaki genes (Wilcox et al., 1986). Cultures were grown, induced, and harvested and protein inclusions were isolated as described for CryIIIA. The in-
TABLE
ET AL.
clusion bodies were extensively washed and the CryIA(b) protein was solubilized in 100 mM sodium carbonate, 10 mM DTT buffer, pH 10. The clear supematant was precipitated with 40% ammonium sulfate, dissolved in 100 mM sodium carbonate, 10 mM DTT buffer, pH 10, and subjected to ion-exchange chromatography on a MonoQ (HRlO/lO) column (Pharmacia, Uppsala, Sweden) utilizing a 0 to 1 M potassium thiocyanate gradient. The purified full-length CryIA(b) protein was trypsinized as described above for the CryIA(c) protein. Protein determination. Protein concentrations were determined by the method of Bradford (1976), using bovine serum albumin as the standard. Insect bioassays. Seventeen agronomitally important insects and one species of mite were selected which represented a wide range of insect species. These insects were from colonies established at Monsanto or obtained from outside sources.
1
Bacillus thuringiensis HOST RANGE
Common name
Abbreviation
Alfalfa weevil Cotton bollweevil* Horseradish flea beetle Southern corn rootwormb White grub (Japanese beetle) Colorado potato beetle Beet armywormb Black cutwormb Cabbage loopeP Corn earwormb European corn bore? Tobacco budwormb Tobacco hornworm’ Mosquito Cockroach Green peach aphid Termite Spider mite
ALW CBW FB SCRW WG CPB BAW BCW CL CEW ECB TBW THW MOS CR GPA TER SM
Scientific name Hypera postica Antonomus grandes Phyllotreta armoraciae Diabrotica undecimpunctata Popilla japonica Leptinotarsa decemlineata Spodoptera exigua Agrotis ipsilon Trichoplusia ni Heliothis zea Ostrinia nubilialis Heliothis virescens Manduca sexta Aedes aegypti Blatella germanica Myzus persicae Reticulitermes jlavipes Tetranychus urticae
howardii
Sensitivity to B. thuringiensis proteins -0 +c + + + + + + + -
a Significant insect mortality (>25%) for any of the three B. thuringiensis proteins. b Insects were tested in a diet incorporation assay as described under Materials and Methods. c NO significant insect mortality (80 3.6 34
0.02-O. 12 O.l(M.39 1.02-3.19 15.4-l 17.8 ND 1.76-6.89 26.34.3
a LC, denotes the concentration of CryIA protein causing 50% mortality. EC, denotes the concentration of CryIA protein causing 50% larval growth reduction. All LC, and EC,, values expressed in t&ml. b Not determined.
l&ml with 95% fiducial limits of 3.4-11.5 kg/ml. Table 2 lists the sensitive lepidopteran insects in order of decreasing LCsO values to CryIA(c) protein. The relative sensitivity of these insects to CryIA(b) protein typically coincided with that of CryIA(c) protein, but the ratio of LC,, values of these 7 insects were different for the two proteins. The LC,, for five of the lepidopterans, cabbage looper (CL), tobacco budworm (TBW), corn earworm (CEW), and black cutworm (BCW) was 2- to 5-fold lower for CryIA(c) than for CryIA(b) protein. European corn borer (ECB) was more sensitive to CryIA(b) than CryIA(c) protein by lo-fold. Beet armyworm (BAW) and tobacco hornworm (THW) showed similar LCso values for both CryIA(b) and CryIA(c) protein. An approximately 450fold range in LCsa values for CryIA(c) protein was observed from the least sensitive insect, BAW, to the most sensitive insects CL and THW. The overall range in activity of CryIA(b) protein was approximately 2000-fold across the 7 sensitive insects. Detailed analysis of the mortality data for a subset of these insects with CryIA(c) protein showed very different dose-response curves (Fig. 2). Both CL and THW had steep slopes, with a narrow effective concentration range of about one log, i.e., a dose of 0.05 l.&nl of CryIA(c) protein produced only slight mortality whereas 0.5 &ml killed all insects. In contrast, the
curves for TBW and CEW covered about 2 to 3 logs of CryIA(c) protein concentrations. Limited mortality of TBW was observed at 0.2 pg/ml and 100% mortality at levels above 20 l&ml CryIAfc) protein. CEW had a very flat dose-response curve with insufficient data at the highest CryIA(c) protein concentrations to fully evaluate mortality curves. Limited mortality was observed at 1 to 2 pg/ml and about 80% mortality found at the highest level tested, 100 &nl CryIA(c) protein. Sublethal doses of CryIA(c) protein caused dramatic reduction in larval growth at surprisingly low protein concentrations, particularly for TBW and CEW (Fig. 3). The EC,, values for THW and CL were loo-
> 80
0.01
0.1 HD-73
1 10 CONCENTRATION (,,&nl)
1 3
FIG. 2. Mortality dose-response curves for four lepidopteran insects. The artificial diet incorporation bioassay described under Materials and Methods was used to determine the percentage corrected mortality as a function of CryIA(c) protein concentration.
EFFICACY
OF B. thuringiensis
h ,1
0.01 0.1 HD-73 CONCENTRATION (ug/ml)
1
FIG. 3. Average larval weight dose-response curves for four lepidopteran insects. The artificial diet incorporation bioassay described under Materials and Methods was used to determine the average insect weight as a function of CryIA(c) protein concentration.
approximately IO-fold below their LC,, values. However, for TBW and CEW, the ECSo values were approximately 500- and lOOO-fold below the respective LCsO values (Table 2). The differences in relative EC,d L&s values accounted for the fact that although THW and CL were the most sensitive lepidopterans assayed in terms of mortality (i.e., L&J, TBW was approximately lo-fold more sensitive than THW and CL in terms of growth inhibition (i.e., EC&. The corresponding dose-response curves for CL, THW, TBW, and CEW illustrated these conclusions; ie., the growth inhibition dose-response curves for TBW and CEW were shallow, especially in comparison to the steep curve for the CL (Fig. 3). DISCUSSION
The host range of three B. thuringiensis insect control proteins and the relative sensitivities of the 18 different insects to these proteins were determined. Ten insects were insensitive to all of the B. thuringiensis proteins tested, 7 were sensitive to both CryIA proteins, and 1 insect was sensitive to CryIIIA protein. Only a limited number of coleopteran insects were included in the present study. Those insects selected were chosen for
PROTEINS
263
their agronomic importance in the United States. Krieg et al. (1987b) reported activity of B. thuringiensis ssp. tenebrionis spores and crystals against seven different coleopterans. CPB was the only insect of that group to be included in the present study. Herrnstadt et al. (1986, 1987) reported that corn rootworm (CRW), cotton bollweevil (CBW), and CPB were also sensitive to crude B. thuringiensis ssp. sun diego cultures. Although all three of these insects were included in this screen, only CPB was found to be sensitive to the puritied CryIIIA protein. No mortality was observed against CRW, CBW, or ALW (an additional coleopteran) with purified CryIIIA protein even at very high concentrations, 2 to 5 mg/ml (R. Fuchs and S. Macintosh, unpubl.). Hermstadt et al. (1987) reported strong insecticidal activity of B. thuringiensis ssp. sun diego spores and crystals for CPB and CBW and only weak activity for CRW. This lack of activity observed against SCRW and CBW with purified CryIIIA protein suggests that some other protein(s), metabolite(s), or synergism between spores and crystals may be responsible for the activity observed by Herrnstadt et al. (1986, 1987) Data on the insecticidal spectrum and relative efficacy of individual proteins are crucial to identify the preferred gene(s) to use to produce B. thuringiensis microbial biocontrol agents and insect tolerant transgenic plants. This is especially true for lepidopteran-specific CryIA proteins because of the numerous distinct genes from various multigene B. thuringiensis strains that have been isolated and characterized (Wilcox et al., 1986; Whiteley and Schnepf, 1986). The present study compared the relative activity of the single protein CryIA(c), produced by the B. thuringiensis ssp. kurstaki HD-73 strain, and a purified gene product, CryIA(b), from the B. thuringiensis ssp. kurstaki HD-1 strain. The ratio of differential activities of the CryIA(b) and CryIA(c) proteins varied among the seven sensitive
264
MACINTOSH
insects from 2- to lo-fold. This difference is of particular significance in selecting a gene or gene product targeted at the agronomitally important, less sensitive insects (e.g., BCW, CEW), where CryIA(c) was approximately 4-fold more active than the CryIA(b) protein. In addition the enhanced sensitivity to CryIA(b) of ECB may be important in areas where this insect pest is prevalent. These data confirm a recent report (Jaquet et al., 1987) that showed that purified, activated CryIA(c) protein was more potent against TBW than the purified activated proteins from the B. thuringiensis ssp. kurstaki HD-1 strain. Jaquet et al. (1987) used the combined purified proteins from the HD-1 strain, which contains three different B. thuringiensis ssp. kurstaki proteins having differential insect activities (Wilcox et al., 1986). Hofte et al. (1988) reported that the CryIA(c) protein showed quantitative differences in insecticidal activity compared to the CryIA(b) protein against two of the five insects tested (TBW and Mamestra brassicae). They found that TBW was 5- to 6-fold more sensitive to the CryIA(c) protein but that M. brassicae was lo- to 15fold more sensitive to the CryIA(b) protein. The other three insects showed no quantitatively significant differences in sensitivity to the CryIA(b) and CryIA(c) proteins. Compilation of these data suggest that selection of the gene for engineering into microbes or plants is dependent on the insect(s) targeted for control. The present study shows that many of the agronomically important lepidopteran pests in the United States are more sensitive to the CryIA(c) protein rather than the CryIA(b) protein. The increased efficacy of purified CryIA(c) protein relative to CryIA(b) protein was confirmed with E. coli cultures that expressed either fulllength or genetically truncated genes that encode these individual B. thuringiensis ssp. kurstaki proteins. Cultures expressing the CryIA(c) protein showed 3- to 5-fold higher specific activity, based on quantita-
ET
AL.
tion by Western analysis (Towbin et al., 1979), than the corresponding cultures expressing CryIA(b) protein for TBW and CEW (R. Fuchs and F. Perlak, unpubl.). In addition to selecting a highly efficacious B. thuringiensis ssp. kurstaki gene for introduction into microbes and plants, the relative sensitivity of a specific target insect(s) dictates the expression level required for effective control. For example, based on LC,, values, control of the beet armyworm requires about lOOO-fold more CryIA(c) protein than control of the tobacco hornworm. Therefore, correspondingly higher expression levels in transgenic plants are required to control the beet armyworm. Prior to this report, the magnitude of sensitivity differences (LC,,) for single B. thuringiensis proteins was not determined for many agronomically important insects. Most previous reports of the host range for the lepidopteran-active B. thuringiensis ssp. kurstaki proteins (Dulmage 1981; Krieg and Langenbruch, 1981; Trottier et al., 1988) used crude B. thuringiensis ssp. kurstaki cultures, many of which contained multiple proteins and spores (i.e., B. thuringiensis ssp. kurstaki HD-1 strain). These relative rankings of insect sensitivities previously reported typically agree with those described in this study. However, sensitivities were reported in relative terms; LC5a values were not determined. In the studies where LC5a values were reported (Jaquet et al., 1987; Lacadet and Martouret, 1987; Li et al., 1987), purified crystals solubilized from B. thuringiensis ssp. kurstaki strains were used. Only a limited number of agronomically important pests were included in these reports and in the most recent study that used purified proteins of single cloned gene products (Hofte et al., 1988). The present study provides specific L& and ECso values of single purified B. thuringiensis proteins for a broad collection of agronomically important pests. These insects, with known LCsO and
EFFICACY
OF
B. thuringiensis
ECSo values, provide a valuable resource for the detection and quantitation of B. thuringiensis ssp. kurstuki expression levels. The reported expression levels of B. thuringiensis proteins in transgenic plants to date have been extremely low (Adang et al., 1987; Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). Therefore, a very sensitive bioassay is essential to detect bioactivity. The most sensitive insects assayed in this study in terms of mortality, THW and CL, showed insecticidal sensitivity (LC,,) as low as 0.04 kg/ml. The level of detection of CryIA(c) protein was extended 50-fold to 1 rig/ml for the most sensitive insect, TBW, by using the growth inhibition assay (EC,,) (Table 2). This growth inhibition or stunting effect was previously used to assess insect control in transgenic plants (Barton et al., 1987), but was used only qualitatively and not quantitatively. As increased expression levels in transgenic plants are achieved, the mortality and growth inhibition assays, which enable B. thuringiensis ssp. kurstaki quantitation over a 6 log range, will be extremely useful to monitor expression levels. ACKNOWLEDGMENTS We thank Jamie Wibbenmeyer and Mary Taylor for their technical assistance in purifying the CryIA(b) and CryIA(c) protein. We also thank Paul Gahr and Christine Olsen for assistance with the insect bioassays.
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crystal protein gene in tobacco plants. In “Molecular Strategies for Crop Protection” (C. J. Amtzen and C. Ryan, Eds.), pp. 345-353. A. R. Liss, New York. BARTON, K. A., WHITELEY, H. R., AND YANG, K. S. 1987. Bacillus thuringiensis 8-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects. Plant Physiol., 85,
1103-1109. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.
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C. C., AND PEREGRINE, D. J. 1986. Methods for the routine screening of acaracides against the citrus rust mite Phyllocoptruta oleivora (Acari:Eriophyidae). Brit. Crop Prot. Conf. Pest
CHILDERS,
Dis., 3C-17. DULMAGE, H. T. 1981. of Bacillus thuringiensis
Insecticidal activity of isolates and their potential for pest control. In “Microbial Control of Pests and Plant Diseases 1970-1980” (H. D. Burges, Ed.), pp. 193222. Academic Press, New York. FISCHHOFF, D. A., BOWDISH, K. S., PERLAK, F. J., MARRONE, P. G., MCCORMICK, S. M., NIEDERMEYER, J. G., DEAN, D. A., KUSANO-KRETZMER, K., MAYER, E. J., ROCHESTER, D. E., ROGERS, S. G., AND FRALEY, R. T. 1987. Insect tolerant transgenic tomato plants. BiolTechnol., 5, 807-813. HERRNSTADT, C., SOARES, G. G., WILCOX, E. R., AND EDWARDS, D. L. 1986. A new strain of Bacillus thuringiensis with activity against coleopteran insects. BiolTechnol., 4, 305-308. HERRNSTADT, C., GILROY, T. E., SOBIESKI, D. A., BENNETT, B. D., AND GAERTNER, F. H. 1987. Nu-
cleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego. Gene, 51, 3746. H~FTE, H., SEURINCK, J., VAN HOUTVEN, A., AND VAECK, M. 1987. Nucleotide sequence of a gene encoding an insecticidal protein B. thuringiensis var. tenebrionis toxic against coleoptera. Nucleic Acids Res., 15, 7183. H~FTE, H., VAN RIE, J., JANSEN, S., VAN HOUTVEN, A., VANDERLIRUGGEN, H., AND VAECK, M. 1988.
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Cloning of an insecticidal toxin gene of Bacillus thuringiensis subsp. tenebrionis and its expression in Escherichia coli cells. FEMS Microbial. Lett., 48, 311-315. JAQUET,
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R., AND LUTHY, P. 1987. Specthuringiensis delta-endotoxin. Microbial., 53, XW504. 1984. Microbial insect control. Biol
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