(Bt) for the Control of Insect Pests in Stored Tobacco: a Review* By

Home , Bacillus thuringiensis, Ephestia elutella

Beiträge zur Tabakforschung International # Contributions to Tobacco Research Volume 20 # No. 1 # February 2002

Bacillus thuringiensis (Bt) for the Control of Insect Pests in Stored Tobacco: a Review* by

Michel Blanc, Pascale Kaelin1 and Ferruccio Gadani2

Philip Morris International, Research and Development, c/o Philip Morris Products SA, CH-2000 Neuchâtel, Switzerland 1Present address: 2 Crestbury Court, Durham, NC 27713, USA 2Present address: Philip Morris USA, RD&E Department, PO Box 26583, Richmond, VA 23261, USA

The present paper is dedicated to the memory of the late Line Zaugg whose excellent professional skills made the work reported here possible.

SUMMARY smoke evaluations did not show any significant difference between the test and control cigarettes. Among the insect species causing infestations and serious Although the tested Bt strains and products did not yield damages to stored commodities, the cigarette beetle, Lasio- satisfactory levels of mortality at the required times and derma serricorne (F.) and the tobacco moth, Ephestia elu- doses, the experimental results summarized in the present tella (Hübner) are the major pests of both raw and manu- review indicate that B. thuringiensis has potential for the factured tobacco. Post-harvest tobacco control is achieved control of the cigarette beetle. through sanitation, insect monitoring, and fumigation with The integration of conventional control approaches with phosphine. However, insect resistance to phosphine and novel systems based on biological agents with different control failures have been reported, and increasing regulatory modes of action should offer new avenues for the effective pressure is being exerted on fumigants. management of stored tobacco pests in line with integrated Biological control agents such as Bacillus thuringiensis (Bt) pest management (IPM) concepts. [Beitr. Tabakforsch. Int. appear to be environmentally sound and potentially viable 20 (2002) 15–22] alternatives to chemical control. Bt is a bacterium that produces insecticidal crystal proteins during the sporulation phase and has been, for more than 40 years, the micro- ZUSAMMENFASSUNG organism of choice for the biocontrol of phytophagous insect pests. It produces insecticidal crystal proteins that display Der bei Rohtabak und verarbeitetem Tabak am häufigsten specific activity against certain orders of insects and become auftretende Schädlingsbefall durch Insekten, der Infestation active upon ingestion by the insect. und ernsthafte Lagerschäden verursacht, wird durch den Our laboratory has conducted extensive research and world- Tabakkäfer Lasioderma serricorne (F.) und die Tabakmotte wide surveys to evaluate the presence of Bt in stored tobacco Ephestia elutella (Hübner) hervorgerufen. Schädlings- and has confirmed previous findings indicating that Bt may bekämpfung beim Tabak nach der Ernte wird durch Hygie- be considered part of the naturally occurring phylloplane nemaßnahmen, Insektenmonitoring und Begasung mit microflora. Several Bt strains were isolated from tobacco and Phosphin gewährleistet. Es wurden jedoch Resistenzaus- characterized by DNA and protein profiling. The insecticidal bildung der Insekten gegenüber der Phosphinbehandlung activity of selected strains and of two commercial products und Versagen von Schädlingsbekämpfungsmaßnahmen against the larvae of L. serricorne was determined by diet beobachtet, des weiteren unterliegen die Begasungsmittel incorporation assays. Moreover, the stability of Bt spores and zunehmendem Druck durch Reglementierungen. crystal proteins on cured tobacco leaves was assessed over Biologische Schädlingsbekämpfungsmittel wie z.B. Bacillus a storage period of time of 30 months. Cigarette prototypes thuringiensis (Bt) scheinen eine umweltfreundliche und were made with Bt-treated tobacco. Standard cigarette and potentiell entwicklungsfähige Alternative zum Einsatz

*Received: 13th August 2001 – accepted: 28th November 2001 Presented, in part, at the 27th Congress of the Colombian Society of Entomology (SOCOLEN), Medellin, Colombia, July 26–28, 2000, and at the CORESTA Congress, Lisbon, Portugal, October 15–19, 2000. 15 chemischer Pflanzenschutzmittel zu sein. Bt ist ein Bakteri- Notre laboratoire a conduit des recherches importantes et des um, das insektizide kristalline Proteine während der Sporula- études sur des échantillons du monde entier pour évaluer la tion produziert und ist im biologischen Pflanzenschutz seit présence naturelle de Bt dans la microflore du phylloplan. mehr als 40 Jahren der Mikroorganismus der Wahl bei Plusieurs souches de Bt ont été isolées du tabac et caracté- Schädlingsbefall durch phytophage Insekten. Bt produziert risées par leur profil d’ADN et protéique. L’activité insecti- insektizide kristalline Proteine, welche eine spezifische cide de souches sélectionnées et de deux formulations Aktivität gegenüber bestimmten Insektenarten entwickeln commercialisées a été déterminée contre L. serricorne par und bei Aufnahme durch das Insekt aktiv werden. titrage biologique (survie des larves sur tabac imprégné In unserem Labor wurden extensive Forschungstätigkeiten d’une préparation de Bt). De plus, la stabilité des spores de und weltweite Untersuchungen durchgeführt, um das Bt et des cristaux de protéines sur les feuilles de tabac Vorhandensein von Bt bei gelagertem Tabak zu untersuchen. séchées a été suivie sur une durée d'entreposage de 30 mois. Dabei wurden frühere Ergebnisse bestätigt, wonach Bt als Des cigarettes prototypes ont été confectionnées avec des Teil der natürlich vorkommenden Mikroflora auf der tabacs traités par Bt. Les caractéristiques de ces cigarettes et Blattfläche von Pflanzen zu betrachten ist. Mehrere Bt les analyses de la fumée ne révèlent pas de différences Stämme wurden aus Tabak isoliert und mittels DNA-Ana- significatives par rapport aux cigarettes témoins. lyse und anhand des Proteinprofils charakterisiert. Die insek- Bien que les souches et les produits testés ne donnent pas des tizide Aktivität ausgewählter Bt Stämme und zweier handels- taux de mortalité satisfaisants aux doses et dans les délais üblicher Produkte für den Einsatz gegen die Larven von L. requis, les résultats expérimentaux montrent que Bacillus serricorne wurden durch biologische Titration bestimmt. thuringiensis est un agent potentiel pour la lutte contre L. Darüber hinaus wurde die Stabilität von Bt Sporen und serricorne. L’intégration d’approches de lutte convention- kristallinen Proteinen auf getrockneten Tabakblättern wäh- nelle avec de nouveaux systèmes basés sur des agents biolo- rend einer Lagerungszeit von 30 Monaten ermittelt. Cigaret- giques présentant différents modes d’action devraient ouvrir ten-Prototypen mit Bt-behandeltem Tabak wurden her- de nouvelles voies pour combattre efficacement les rava- gestellt. Der Vergleich mit Standard-Cigaretten und Rauch- geurs du tabac entreposé, conformément aux principes de la untersuchungen zeigten keinen signifikanten Unterschied lutte intégrée. [Beitr. Tabakforsch. Int. 20 (2002) 15–22] zwischen Test- und Kontrollcigaretten. Obwohl die untersuchten Bt Stämme und Produkte zu INTRODUCTION keinen befriedigenden Mortalitätsraten nach der vorgegebe- nen Zeit und Dosierung führten, weisen die in dieser Über- Insects are the most significant pests of stored products since sicht zusammengefaßten Ergebnisse darauf hin, dass B. ancient times (32). They are not easily excluded from ware- thuringiensis ein mögliches Agens zur Bekämpfung des houses by physical barriers and are well adapted to life in the Tabakkäfers ist. Die Kombination einer konventioneller storage environment. Economic damage caused by insect Schädlingsbekämpfung mit neuen, auf biologischen Ansät- pests include weight loss due to direct feeding, and quality zen beruhenden Methoden mit unterschiedlicher Wirkungs- reduction by contamination with excreta, dead insects and weise, basierend auf dem Konzept einer integrierten Schäd- waste products. Moreover, insects may facilitate product lingsbekämpfung, bietet neue Wege zu einer effektiveren deterioration due to contamination with molds, which can Kontrolle der Lagerschädlinge bei Tabak. [Beitr. Tabak- grow well in the moist and warm microhabitat of the infested forsch. Int. 20 (2002) 15–22] product. It is estimated that insects account for 10 to 30% of the losses recorded in stored grains and pulses. RESUME Tobacco is subject to the attack of stored product insects during aging or storage of leaf and throughout manufactur- Parmi les insectes responsables d’infestations et de dommages ing and marketing of tobacco products. Two insect species, importants causés aux denrées entreposées, le coléoptère des the cigarette beetle (CB) (Lasioderma serricorne (F.); cigarettes, Lasioderma serricorne (F.), et la pyrale du tabac, Coleoptera: Anobiidae) and the tobacco moth (Ephestia Ephestia elutella (Hübner), sont les principaux ravageurs du elutella (Hbn.); Lepidoptera: Pyralidae), account for heavy tabac brut et du tabac manufacturé. La lutte contre ces in- post-harvest losses and quality deterioration of tobacco sectes après la récolte est assurée par l’assainissement, la dé- (36,45). Beetles feed on stored products as both adults and tection de la présence d’insectes et la fumigation à la phos- larvae, whereas moths feed on them only as larvae (45). phine. Cependant, la résistance des insectes à la phosphine et Although native of the Mediterranean region, L. serricorne les échecs des traitements ont été rapportés, et la législation is particularly suited to tropical regions and is known to exerce une pression croissante sur l’usage des fumigants. infest and consume tobacco at all stages of the manufactur- Les agents de lutte biologique comme Bacillus thuringiensis ing process, resulting in spoilage of at least 1% of tobacco (Bt) offrent une alternative aux pesticides chimiques respec- stocks per annum. Commodities such as grains, beans, tueuse de l'environnement. Depuis 40 ans, Bt est considéré spices, dried fruit and vegetable, dried fish and leather can comme le micro-organisme de choix pour la lutte contre les also be infested by the cigarette beetle (4). insectes phytophages. Il s'agit d'une bactérie qui produit des The tobacco moth, E. elutella, also known as the cocoa or cristaux de protéines insecticides en phase de sporulation. Ces warehouse moth, is indigenous to the Northern Hemi- cristaux de protéines deviennent actifs dès leur ingestion et sphere, being adapted to temperate climates and intolerant présentent une toxicité spécifique contre certains ordres to long exposures to high temperatures. Similarly to L. d’insectes. serricorne, larvae of E. elutella can breed on a wide variety

16 of stored products, such as flue-cured and Oriental tobacco, ans; cry3 genes encoding proteins toxic to coleopterans; grains, cocoa beans, dried fruit, coffee and pepper (3). and cry4 genes encoding proteins toxic to dipterans alone. The control of infestations can be achieved by sanitation and Additional classes have since been added. To date, the phosphine fumigation treatments (36,45). However, despite number of sequenced crystal protein genes has grown from constant monitoring and comprehensive pest management 14 to well over 100 and the Bt nomenclature has been programs, it is conservatively estimated that the tobacco in- revised recently (reviewed in 10). dustry loses approximately US$300 million worth of tobacco Powerful biochemical and molecular techniques have been lamina annually due to the infestations of both pests. employed to enlarge the narrow spectrum of activity of Bt In recent years, some stored product fumigants and protec- in order to address multiple insect infestation problems tants have been phased out, either because of regulatory faced by farmers in the field. The potential of B. thurin- actions or due to the development of insecticidal resistance giensis strains exhibiting novel biological activities has in important pest species. Resistance to phosphine and lead to the development of transgenic plants as efficient control failures have been reported for L. serricorne in delivery systems for ICPs (15,43) and other insecticidal tobacco warehouses (44,53). This may affect the long-term proteins, such as protease and alpha-amylase inhibitors efficacy of the current insect control practices in tobacco (21). Tobacco was the first experimental Bt transgenic storage, also considering that viable alternatives to phos- plant developed in 1987 (reviewed in 16) and the approach phine treatment are in short supply (54). The increasing has been adopted subsequently by farmers of certain costs of research and development efforts and registration countries for crops such as cotton and potato. Insect- for new active molecules have indeed limited the interest of resistant crops expressing Bt genes are today grown the agrochemical industry towards specific developments commercially on 8.2 million hectares worldwide (23). for the relatively small stored-product treatment market. In the past it was hoped that insects would not develop Microbial biopesticides, consisting of, or derived from resistance to Bt toxins, since Bt and insects had coevolved. naturally occurring organisms (bacteria, fungi, and viruses) From the mid-1980s, however, different levels of insect appear to be an alternative to chemical insecticides for the resistance to Bt were reported in laboratory selection control of storage pests (18,14,49). The insecticidal experiments (reviewed in 48). Since then, other cases of bacterium Bacillus thuringiensis (Bt) (reviewed in 9,40,48) field-selected resistance to Bt have been recorded in is the most widely used biocontrol agent, marketed world- numerous regions of the world. Various resistance manage- wide for the control of important field crop and forestry ment strategies, e.g. the use of multiple toxins and spatial pests, including field tobacco pests such as Heliothis or temporal refuges, have been proposed and are under virescens (F.) and Manduca sexta (F.). Bt has also been field evaluation at present. used against storage pests, e.g. Plodia interpunctella The objective of our research program was the isolation of (Indian meal moth) and Cadra cautella (almond moth) Bt strains from the stored tobacco environment and the (1,2,7,29,31,34,37,39,46,47,52). evaluation of their efficacy against L. serricorne, with the Bt is a ubiquitous Gram-positive, spore-forming bacterium perspective of developing alternative or integrated tools for characterized by the abundant production of insecticidal the biological control of stored tobacco pests (18). This crystal protein (ICP) inclusions during sporulation, which review summarizes the results of investigations and can account for 20 to 30 percent of the dry weight of the insecticide bioassays conducted in our laboratory from sporulated cell. A variety of Bt strains have been isolated 1993 to 2000. worldwide from many habitats, including soil (13,35,51), insects and stored-product environments (28,30,38). Isola- tion from the phylloplane of many plants including cured INVESTIGATIONS ON THE NATURAL tobacco (11,12,24,28,33,41,42,50) suggests that Bt is part MICROFLORA OF THE TOBACCO LEAF of their natural microflora. After ingestion by a susceptible insect, the ICPs, commonly The isolation of bacteria from cured tobacco leaves proved designated as “Cry” proteins, require solubilization and pro- to yield a low diversity of genera: the bacterial populations teolysis in order to become biologically active. The toxin were shown to consist almost exclusively of Gram-positive, then acts in a highly specific manner by binding to gly- spore-forming bacteria belonging to the genus Bacillus coprotein receptors of the midgut, inserting into the apical (>95%) (19,20,25,26). Our studies indicated total counts of membrane and creating ion channels or pores, which lead to bacteria of burley, flue-cured and Oriental tobacco types to cell lysis and death of the host (reviewed in 9 and 48). Bt be in the range of 103–107 bacteria (colony-forming units toxins are considered nontoxic to mammals, as demon- [cfu]) per gram, with B. pumilus being the predominant strated by numerous toxicology studies (for a review, see 8). species in all studied tobaccos. Three other species were Many Bt crystal proteins (and the genes coding for them) found to occur at high frequency, with proportions greatly have been described with an activity spectrum including varying according to the tobacco types: B. subtilis, B. primarily insect species of the order Lepidoptera, some megaterium, and B. licheniformis. Diptera and, more recently, species of Coleoptera. The first The vast majority of the bacterial community consisted of systematic effort to organize the genetic nomenclature of dormant spores of Bacillus spp. Their predominance on Bt was carried out in the late 1980s and was based upon the dried tobacco leaves can be explained by the resistance insecticidal activities of crystal proteins (22): cry1 genes which allow them to survive through the successive heat encoding proteins toxic to lepidopterans; cry2 genes treatments and desiccation of post-harvest tobacco process- encoding proteins toxic to both lepidopterans and dipter- ing (25,26).

17 Table 1. Insecticidal activity of Bt spore/crystal prepara- Table 2. Insecticidal activity of Bt crystal proteins against L.

tions (10 mg/g tobacco) against L. serricorne (compilation serricorne larvae expressed as LC50 (concentration of crystal of selected data from [27,28] and unpublished results). Data are proteins to achieve 50% mortality over 7 d). Data were based on based on a minimum of 4 replicate bioassays of 16 larvae each. 4 to 13 replicates of 16-larvae bioassays (see ref. 27 for calcula- Bt strains are differentiated into 3 main subgroups on the basis tion details). Bt strains PME1401 and PME3904 were shown to be of Western blot analyses and PCR-based DNA analysis. immunologically homologous to Bt subsp. tenebrionis, the Cry3- anticoleopteran reference (28).

Immuno/PCR match Insecticid. activity Bt strain with Btta profile after 7 db LC 95% fiducial Strain Sample size (n) 50 (mg/g) limits Btt (Cry3 – anticoleopt. ref.) + 62 76 Bt tenebrionis 1088 2.84 2.225–3.580 Btkc (Cry1 – antilepidopt. ref.) 20 PME1401 382 4.43 3.374–5.836 PME3904 1112 3.41 2.704–4.296 Cry1-related isolates 618 Cry2-related isolates 212 Cry3-related isolates + 3483 Raven® (Ecogen) N.D.d 5387 In contrast to the previous study, which investigated the Foil® (Ecogen) N.D. 7394 presence of Bt in the stored-tobacco habitat, the leaf survey results showed: aBt subsp. tenebrionis b% Mortality; control mortality 6%. a higher proportion of isolates harboring Cry1-type c d Bt subsp. kurstaki N.D. = not determined. proteins (the predominance of isolates producing anti- lepidopteran type proteins has also been reported for Bt strains recovered from other plants [50]). ISOLATION AND IDENTIFICATION OF Bt ASSOCI- a much lower incidence of Bt strains on the cured ATED WITH THE STORED TOBACCO ENVIRONMENT tobacco phylloplane when compared to Bt levels in dust and scraps. Following the investigation of microbial populations asso- Some strains showed characteristics of Bt subsp. kurstaki ciated with cured leaves, we conducted a survey to evaluate (Btk) HD-1. This could point at residues of insecticidal the worldwide occurrence, distribution and diversity of Bt in sprays, Bt being used in certain countries as a foliar the stored tobacco environment (28). A total of 126 samples insecticide in the field control of tobacco budworms and of tobacco dusts and scraps, processed tobacco or dead to- hornworms. Nevertheless, we observed a genetic diversity bacco beetles were collected in tobacco warehouses and that confirmed our previous findings of diverse populations manufacturing facilities of 15 countries (28). Twenty-seven of Bt in the phyllosphere of tobacco (28). This habitat percent of the samples yielded a total of 107 Bt strains that clearly represents a possible source of Bt isolates for the were subsequently differentiated into 3 main subgroups on control of both stored-tobacco insect pests, L. serricorne the basis of crystal morphology and crystal protein character- and E. elutella. ization by sodium dodecyl sulfate – polyacrylamide gel elec- trophoresis (SDS–PAGE) and Western blot analyses (28): ASSESSMENT OF THE INSECTICIDAL ACTIVITY OF – Cry1, lepidopteran-specific related strains (18%) Bt ON THE CIGARETTE BEETLE (Lasioderma serricorne) – Cry2, lepidopteran/dipteran-specific related strains (23%) Based on biochemical and molecular characterizations, we – Cry3, coleopteran-specific related strains (59%). selected tobacco Bt isolates and evaluated their insecticidal The above grouping was confirmed by a molecular charac- activity on L. serricorne first-instar larvae (average size terization that was carried out by polymerase chain reaction and weight: less than 1 mm long, about 0.1 mg). Bioassays (PCR)-based analysis and nucleotide sequencing of were carried out with both spore/crystal and purified selected Cry3-type strains: they harbored a 65-kDa crystal crystal protein preparations using in vivo single-organism toxin encoded by genes identical to the gene of Bt subsp. assays. tenebrionis (27). Table 1 shows a summary of the results obtained with our Bt isolates as well as data obtained with two commercial Bt ISOLATION AND IDENTIFICATION OF Bt products registered for agricultural use. ASSOCIATED WITH CURED TOBACCO LEAVES At the screening dose of 10 µg/mg diet (total protein concentration of spore/crystal suspensions), larval mortality A second worldwide survey focused on the frequency and reached a maximum of 83% for certain tobacco Bt strains distribution of Bt populations present on the surface of and 94% for the commercial product Foil®. In general, cured tobacco leaves during post-harvest storage (24). A spore/crystal mixtures exhibited a slightly higher insecti- total of 133 tobacco samples of different types (flue-cured, cidal activity than purified protein preparations after 7 d burley, Oriental, and Maryland) and various geographical (27). This could be due to synergistic effects between the origins (18 countries) were analyzed. Bt could be isolated proteins and the spores. The effectiveness of commercial from 9% of the samples and 24 strains were characterized. products Raven® and Foil® was comparable to that PCR analysis revealed seven distinct cry1 profiles and one recorded for tobacco Bt strains (27) and thus indicates cry3 profile (24). potential use of commercially available Bt products if

18 Figure 1. Viability of spores of Bacillus thuringiensis subsp. tenebrionis applied to dry tobacco leaves and stored at 23 °C and 60% R.H. in the dark (cfu, colony-forming units) toxicity potentiation can be achieved against the cigarette quality and subjective characteristics of tobacco products. beetle. Five hundred ppm of Raven® were sprayed onto strips The Cry3-related isolates showed morphological, biochemi- prior to processing into cut-filler. Experimental cigarettes cal, and molecular characteristics similar to those of the were manufactured therewith and submitted to chemical, reference strain Bt subsp. tenebrionis (Btt). Accordingly, physical, and sensory evaluations. based on quantification of larval mortality (Table 2), ciga- The results of the analytical investigations showed that the rette beetle larvae appeared to have similar degrees of presence of Bt products did not significantly modify the sensitivity to Btt and to selected tobacco Bt Cry3-related chemical or physical characteristics of the product (8a). isolates. It should also be noted that the calculated LC50 Moreover, B. thuringiensis cells and crystal proteins values may be overestimated because a dramatic reduction appeared to be remarkably stable throughout all steps of the of insect feeding rate was observed when the larvae were experimental cigarette manufacturing process. In the exposed to crystal proteins (27). sensory evaluation, no significant difference was found between control cigarettes and experimental cigarettes STABILITY OF Bt CRYSTAL PROTEINS AND treated with Raven®. SPORES ON CURED TOBACCO LEAVES CONCLUSIONS Prior to possible application for long-term storage protection, the stability of spores and crystal proteins of Btt had Microbial investigations and insect bioassays conducted in to be evaluated over time on stored tobacco leaves. Flue- our laboratory have shown that some Bt isolates associated cured tobacco leaves were sprayed with water suspensions with dried tobacco leaves have an insecticidal activity of spores (108 per leaf) and/or crystals (50 µg per leaf) against the larvae of L. serricorne, indicating a potential from Btt and stored at 23 °C and 60% R.H. in the dark. use of Bt in the biological control of the cigarette beetle SDS-PAGE analyses and bacterial counts were performed on (17,18,27). These results along with those recently reported flue-cured tobacco leaves to monitor the degradation of the by other investigators (33,39,41) for coleopteran pests and crystal proteins and the evolution of the population of Bt Ephestia elutella appear to confirm the potential of Bt as spores. The crystal proteins showed a slight degradation after alternative or integrated pest control tool in stored products 16 months of storage, and after 30 months all the proteins (18). appeared almost completely degraded. However, Bt spore We showed that diverse Bt isolates were present in tobacco viability showed a very good stability over time (Fig. 1). samples collected in warehouses and processing plants of Taking into consideration that the average storage time for 15 different countries, as well as in cured leaf samples of tobacco ranges from 12 to 30 months (5), the observed per- different types and origins. Our findings are consistent with sistence of the spore-crystal product on the leaf surface the results of several studies reporting that Bt is a naturally suggests that Bt would maintain its larvicidal efficacy occurring organism on the phylloplane of many plants throughout the storage period. On the other hand, the stabi- (11,12,42,50). In particular, LIU and coworkers of the lity of Bt in the stored product environment may favor the Yunnan Academy of Tobacco Science (China) have buildup of insect resistance due to continuous exposure of recently isolated Bt strains from stored tobacco leaf, which the larvae to the toxic proteins. produce rhomboidal crystals belonging to the Cry3-type (33). Also, ORDUZ and coworkers (Biological Research PHYSICOCHEMICAL AND SENSORY EVALUATION Corporation, Medellin, Colombia) have isolated various Bt OF Bt-TREATED TOBACCO PRODUCTS strains from stored tobacco and dead cigarette beetles (41). These results confirm that Bt is indigenous to the stored- Experimental trials were carried out to assess the impact of tobacco environment. Importantly, our experimental data a Bacillus thuringiensis-based insecticide (Raven®) on the with insecticidal doses of Bt applied to cured leaf suggest

19 that Bt may retain its efficacy over a typical leaf storage advantages of Bacillus thuringiensis-protected plants to period of time. control insect pests; Reg. Toxicol. Pharmacol. 32 Nevertheless, neither the B. thuringiensis strains isolated (2000) 156–173. from stored tobacco nor the tested commercial products 8a. Blanc, M. and F. Gadani: Bacillus thuringiensis as a have yet shown satisfactory levels of mortality and potency candidate for the biocontrol of stored tobacco insect on L. serricorne at the required times and doses. Conse- pests; 2000 Congress of the Cooperation Center for quently, research efforts should be undertaken to screen Scientific Research Relative to Tobacco (CORESTA), further the tobacco and other environments and to assess Lisbon, Portugal, Oct. 15–19, 2000, Conference academic and industrial collections for new isolates of Proceedings, Abstract no. AP10, p. 60. higher potency to be tested on the cigarette beetle. Modern 9. 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