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Bacillus thuringiensis insecticides in crop protection — reality and prospects
Crop Protection 19 (2000) 669}676
Bacillus thuringiensis insecticides in crop protection * reality and prospects Amos Navon* Department of Entomology, ARO, The Volcani Center, Bet Dagan 50250, Israel
Abstract Bacillus thuringiensis (Bt) has been the leading biopesticide against lepidopterous pests since 1959. In the 1990 s the following developments contributed to increased rational uses of Bt: (1) natural and recombinant Bt products were developed to broaden the insect host range in pest management programs; (2) new formulations based on conventional or genetically engineered encapsulation of the toxins and/or feeding stimulants to increase ingestion and, in turn, the e$cacy of the microbe; (3) screening of the interactions of Bt with insect herbivores and plant allelochemicals or natural enemies of the pests, to aid the formulation of biological control strategies; and (4) knowledge and management of insect resistance to Bt. The prospects for Bt insecticides will be described and discussed. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Bacillus thuringiensis products; Bioinsecticides; Crop protection; Lepidoptera; Coleoptera; Agricultural insect pests
1. Introduction
2. Constraints in the use of Bt products
The market for biopesticides based mostly on Bt products still forms less that 1% of the crop protection market. Since the 1990s the intoduction of new Bt strains and formulations, and the development of recombinant Bt products has furthered the growth of the Bt market, mainly by extending the insect host range of the microbial products and enhancing the insecticidal potencies of the toxins. In forestry, Bt has e!ectively replaced chemical insecticides and has become established in control programs against defoliator larval moths in Europe and North America (van Frankenhuyzen, 2000). In Israel, since 1987, Bt has e!ectively replaced chemical insecticides against Thaumatopoea pityocampa in pine forests, mainly because of the public use of forests as recreation areas. In agriculture, the considerations and possibilities involved in using Bt in pest management strategies are complicated. Some of the current issues concerning the use of Bt, and its future prospects are discussed below.
Two major constraints a!ect "eld e!ectiveness: (1) the toxic protein is an oral insecticide and therefore has to be consumed by the larva; and (2) environmental conditions reduce the e$cacy of the product. More speci"cally, the following problems reduce the insecticidal power of Bt:
* Corresponding author. Tel.: #972-3-968-3832; fax: #972-39683835.
(1) the narrow host range of Bt strains; (2) Bt is an oral insecticide; (3) bollworm and borer larvae avoid a lethal dose of Bt by penetrating into the plant; (4) the lethal dose of Bt is instar dependant, and the susceptibility of mature larvae is very low; (5) solar irradiation (300}380 nm) inactivates the crystal protein, mostly through the destruction of tryptophan; (6) wash-o! of the Bt product by rain and dew dilute the microbe dose on the plant; (7) phylloplanes and allelochemicals can adversely a!ect the Bt protein activity; (8) application of Bt products in row crops is partially e!ective. Some of these constraints have been minimized by a rational pest management strategies which are detailed below.
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3. Development of Bt products Screening of Bt products based on the spore crystal complex has been conducted since the 1950 s, but it was only in 1970 that the HD-1 strain of Bt subspecies kurstaki, which is active against a large number of moth species, was discovered (Dulmage, 1970) and produced commercially by many agrochemical and fermentation companies (Table 1). Screening programs by means of potency bioassays (Navon, 2000) resulted in highly potent new strains. Between 1970 and 1990, strains of Bt subspecies aizawai, active against armyworms such as Spodoptera spp., and subspecies subspecies tenebrionis (Krieg et al., 1983) and san-diego (Hernstadt et al., 1986, active against beetles were introduced into the market, and these new products extended the means of control to more insects. It has been shown in lepidopterous insects that the spore potentiated (Moar et al., 1989) and synergized (Dubois and Dean, 1995) the insecticidal activity of the crystal protein. Improved knowledge of the molecular biology of Bt (Cannon, 1996) led to the use of Bt genes for manipulation of the Bt toxin (Baum et al., 1999) (Table 1). During the 1990 s genetic manipulations became a new tool for combining the toxic proteins (Cry proteins) of two or more Bt strains in one product. Transconjugation and recombination were used to develop the genetically manipulated Bt products. In addition, encapsulation of the Cry proteins in the cell walls of Pseudomonas yuorescens was used to protect the toxin against environmental inactivation. These genetic manipulations simpli"ed and improved pest control mainly in row crops, where they enabled a single genetically modi"ed Bt product to control all the lepidopterous pests infesting the plant. Another contribution to the widening of Bt uses in agriculture was the manufacture of Bt products in developing countries, where domestic strains were incorporated into commercial products (Shah and Goettel, 1999) (Table 1).
In a di!erent approach to photoprotection of Bt, a melanin-producing mutant of the microbe provided increased UV resistance and insecticidal activity (Patel et al., 1996). Rainproo"ng was increased by Bt encapsulation in biopolymers, to reduce washing of the product from the plant (Ramos et al., 1998). Selection of environmentally safe and cost-e!ective formulations to increase the residual activity of commercial Bt products is one of the major needs for widening the use of the microbe.
5. Application In agricultural use Bt is applied mostly with the same ground sprayers used for the application of chemical insecticides. Since the crystalline toxin a!ects the larva through feeding, high volumes of aqueous spray per unit area are required for adequate coverage of the plant canopy, and this is very costly because substantial amounts of the product are lost by dripping o! the plants. In addition, the use of high volumes of aqueous spray can be impracticable in areas where water is not available. In recent years, e!orts have been made to reduce the spray volume and to achieve better control of the droplets: avocado orchards (Wysoki, 1989) were effectively air sprayed from a helicopter, to control Boarmia (Ascotis) selenaria (M. Wysoki, ARO, Israel, unpublished), and aerial spraying of Bt in ultra-low volume (ULV) application in the control of Anarsia lineata in almond orchards also seemed promising (Roltsch et al., 1994). The use of an air-assisted sleeve boom (Degania Sprayers, Degania Bet, Israel) on row crops increased spray penetration and plant coverage, and reduced drift. Also, locating the sprayer nozzles at the height of the plant canopy markedly improved delivery to the lower surfaces of cotton leaves and to the #ower buds and bolls (A. Navon, unpublished).
6. Residual activity 4. Formulations A substantial amount of information on development of Bt formulations has been published (Burges and Jones, 1999). The formulation additives include wetting agents, stickers, sunscreens, synergists and phagostimulants. It is widely accepted that UV inactivation of the crystalline toxin is the major cause of the rapid loss of Bt activity; nevertheless, in practice, the commercial spray formulations lack photoprotection of the spores and crystals. Several chromophores have been selected to shield Bt preparations against inactivation by sunlight (Dunkle and Shasha, 1989; Cohen et al., 1991), but environmental safety and palatability to the insects are prerequisites for using these dyes and pigments in commercial products.
A promising approach to improving residual activity is exempli"ed in the use of starch-encapsulated Bt in the control of Ostrinia nubilalis in corn (Dunkle and Shasha, 1989), or in the embedding of Bt in a dusting formulation based on wheat #our for the control of lepidopteran pests of vegetable and "eld crops (Navon et al., 1997). These protective materials included phagostimulants which encouraged the larvae to feed on the granule and ingest lethal amounts of the microbe. Low persistence of the spore}crystal product on the plant is one of the main problems in Bt application: low persistence (48 h) of Bt products on cotton was observed with products of Bt subsp. kurstaki (Fuxa, 1989). Leaf growth indirectly causes a reduction in Bt persistence as it causes natural dilution of the product on the plant,
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Table 1 Natural and genetically modi"ed Bt products registered for agricultural use Bt strain
Company
Product
Target insects
Crop
Abbott Laboratories Chicago IL, US
Biobit, Dipel, Foray,
Lepidoptera
Field and vegetable crops, greenhouse, orchard fruits and nuts, ornamentals, forestry, stored products
Tenebrionis
Abbott
Javelin, Steward, Thuricide, Vault Bactospeine, Futura, Able, Costar, Florbac, Xentari Novodor
Lepidoptera
Kurstaki Kurstaki Aizawai
Thermo Trilogy Corp. Columbia MD, US Abbott Thermo Trilogy Abbott
(a) Natural Kurstaki HD-1
Kurstaki HD-1
Tenebrionis Kurstaki Kurstaki Galleriae YB-1520 * CT-43
Thermal Trilogy BioDalia, Dalia, Israel Rimi, Tel Aviv, Israel Tuticorin Alkali Chemicals & Fertilizers Ltd. India Huazhong Agric. University, China Scient. & Technol. Develop. China Huazhong Agric. University, China
Trident Bio-Ti
Lepidoptera Lepidoptera Lepidoptera armyworms Colorado Potato Beetle, Elm Bark Beetle Coleoptera Lepidoptera
Bitayon (granular feeding baits) Spicturin
Btrachedra amydraula Lepidoptera
Mainfeng pesticide
Lepidoptera
Row crops, fruit trees
Bt 8010 Rijin
Lepidoptera
Shuangdu
Lepidoptera, Coleoptera, Diptera
Row crops, rice, maize, fruit trees, forests, ornamentals Row crops, garden plants, forests
Lepidoptera (Resistant P.xylostella) Lepidoptera
Row crops
Lepidoptera
Vegetables, horticultural, ornamental Turf, hay, row crops, sweet corn
Row crops Potato, tomato, eggplant Ornamentals, shade trees Potato, tomato, eggplant Avocado, tomato, vineyards, pine forests Date palms Cruciferous crop plants
(b) Genetically modi"ed Aizawai recipient kurstaki donor
Thermo Trilogy
Agree, Design (transconjugant)
Kurstaki recipient aizawai donor Kurstaki
Ecogen, Inc. Langhorne PA, US Ecogen
Condor, Cutlass (transconjugant) CRYMAX, Leptinox,
Kurstaki
Ecogen
Leptinox (recombinant)
Kurstaki recipient
Ecogen
d-endotoxin encapsulated in Pseudomonas yuorescens
Mycogen, Corp. San Diego, CA, USA
Raven (recombinant) MVP MATTCH MTRACK (CellCap)
Lepidoptera armyworms Lepidoptera Coleoptera Lepidoptera Lepidoptera Coleoptera
Row crops
Row crops Potato, tomato, eggplant Row crops * armyworms Potato, tomato, eggplant
Based on Baum et al. (1999), Shah and Goettel (1999).
especially in broad leaf crops such as cotton and leaf vegetables. Low residual activity of Bt subsp. tenebrionis products used against the Colorado potato beetle on potatoes in the "eld persisted for only 48 h (Ferro et al., 1993). The e!ectiveness of Bt application depends strongly on its timing; the following guidelines can be useful: (1) Application early in the season, before high "eld populations of parasitoids and predators on the pests
have been reduced by chemical insecticides. It has been shown in the US that Bt use against heliothine infestation early in the cotton season, followed by traditional insecticides (Hand and Luttrell, 1997), e!ectively reduced the Heliothis virescens populations. (2) Timing the Bt application according to monitoring of egg hatching: the most e!ective control of the Colorado potato beetle in potato was achieved up to 4 days after 30% of the eggs had hatched, whereas premature or delayed application
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allowed signi"cantly more defoliation and larval recovery from the Bt intoxication (Ghidiu and Zehnder, 1993). This timing can be determined by monitoring the pest population with the aid of traps based on sexpheromones or other insect attractants: a signi"cant increase in trap catches may indicate the onset of egg laying. (3) Bt application after sunset instead of in the morning can increase the persistence of the Bt product in warm countries, where activity of the microbe persists for only 2}3 days (Fuxa, 1989).
7. Larval age One of the most important economic aspects of pest management with Bt is the use of the microbe against young larvae, preferably neonates, because it has been shown in laboratory and "eld bioassays that third instar larvae of Lepidopteran (Navon et al., 1990) and Coleopteran (Ferro and Lyon, 1991) are less susceptible to the Bt products than younger larvae. Thus, the costs of controlling 1st instar larvae can be a fraction of that of controlling older larvae. These di!erences in larval susceptibility to Bt are probably aspects of a more general phenomenon among lepidopteran and coleopteran pests. Furthermore, the initial nibbling of Bt by neonate larvae is followed by cessation of feeding and gut paralysis within minutes (Gould et al., 1991), so that there is negligible damage to plants, whereas mature larvae feeding on Bt-treated plants may still cause su$cient damage to the crop to reduce the quality of the agricultural product. Also, mature larvae may recover from the Bt intoxication (Bryant, 1994), and even complete the developmental cycle; this situation can be aggravated by incomplete spray coverage, by rapid degradation of the crystalline toxin by UV radiation, or by washing o! of the Bt spray by rain or overhead irrigation.
8. Insect feeding behaviour The larval feeding habits markedly a!ect the e$cacy of pest management with the microbe, as they determine the availability of the crystalline toxin to the insect (Navon, 1993). Defoliators are e!ectively controlled by Bt, but the control of bollworms, borers and feeders on hypogeaic plant parts has been marginal, because the amounts of Bt available to them are commonly below lethal doses. Knowledge of feeding behaviour is a fundamental requirement in the development of new formulations and the optimization of the utilization of biopesticides. Bt dose-transfer modelling, based on monitoring the e!ects on larval feeding behaviour and locomotion (Hall et al., 1995), has identi"ed factors that need to be improved to ensure better control of lepidopteran larvae. The use of a phagostimulant mixture, such as COAX (Trader Oil
Mill Co., USA) in a sprayable starch encapsulation (McGuire and Shasha, 1995), or a yeast extract in a dustable granular formulation, as a feeding stimulant (Navon et al., 1997) are promising new tactics for manipulating the feeding behaviour of bollworm and borer larvae. The principles behind the use of such formulations, in addition to increasing residual toxic activity, are: (1) the larvae are attracted to feed selectively on the Bt product and therefore feed less on the plant; (2) for the same Bt dose per acre, the amounts of spore}crystal materials available to the larvae are several times greater than in a leaf meal with the Bt spray. With these approaches, the insect-control e$cacies of the new Bt formulations can exceed those obtained with earlier ones.
9. Insect resistance to Bt In recent years, an increasing number of reports on the development of resistance to Bt in agricultural pests have been published. Most of the reports concern laboratory inducement of resistance (Gelernter, 1997) (Table 2). Field resistance to Bt in Plutella xylostella (Tabashnik et al., 1990), can reduce the e!ectiveness of the insect control. In practice, however, one or two Bt sprays per season are not considered to impose e!ective selection pressure to develop resistance to the microbe. Furthermore, in Bt crops (Bt transgenic plants) the Cry proteins are expressed in all the plant cells, and larvae are continuously exposed to the toxic protein. In contrast to this, when sprayed on the plants, the Bt has a short residual activity in row crops, and the coverage of the plant with the Bt product is very often incomplete. As a result of these pest control constraints, natural &refuge' sites are created on the plant, to the bene"t of the defoliator larvae, and even more so of the bollworm and borer larvae. Thus, the larvae are exposed to sublethal doses of the Bt product. Nevertheless, excessive use of Bt in pest control may induce resistance in the pests and it is necessary to apply bioassays for recording "eld resistance in the insect populations, to avoid control failures because of resistant pests.
10. Safety Extensive toxicity studies have shown that Bt isolates devoid of b-exotoxin are not toxic or pathogenic to mammals (McClintock et al., 1995). Nevertheless, people working with spore}crystal products should take precautions against spore infection by avoiding contact with open wounds and protecting the eyes against spray or dust (Sample and Buettner, 1983). In the western world, Bt strains which produce b-exotoxin cannot be registered for use in plant protection, because of their toxicity to mammals (Sebesta et al., 1981). In veterinary, b-exotoxin
A. Navon / Crop Protection 19 (2000) 669}676
formulations are used against #ies in di!erent breeding sites such as poultry manure.
11. Combination of Bt with other means of pest management 11.1. Introduction The use of Bt in IPM programmes o!ers the advantage of replacing undesired chemical insecticides with safe bioinsecticides, which could be combined with other biological means, provided that Bt is synergistic or compatible with them. The rationale of such a strategy is that biological means such as entomopathogenic microbes and nematodes, natural enemies of the pests, and/or natural insecticides, in combination with Bt can improve pest control, especially when the e$cacy of Bt is suboptimal. In this review, laboratory experiments and "eld uses of Bt, in combination with other pest control agents are described, and considerations of compatibility and antagonistic interactions involved in this strategy are discussed. 11.2. Entomopathogenic microbes The combination of Bt with baculoviruses has shown mainly an additive e!ect (Navon, 1993); although this combination may not be justi"ed in terms of cost-e!ectiveness, the termination of larval feeding caused by Bt may reduce the amounts of virus needed to kill the larva. The combination of Bt with fungi has not been evaluated in any detail but, in view of the high e$cacy of commercial insecticidal fungi against sucking insects, combinations of such fungi with Bt in suitable formulations might provide a useful strategy in complex pest situations, where moths and white#ies or aphids infest the same crop. The combination of Bt and insecticidal nematodes was tried and seemed not suitable for the control of resistant P. xylostella (Bauer et al., 1998): the two biopesticides applied together provided little advantage over the use of either one of them alone. 11.3. Natural enemies Egg parasitoides have been found highly compatible with Bt, as the egg is not a target stage for the microbe: successful examples include Trichogramma platneri against B. senenaria in avocado (Wysoki, 1989) and T. cacoeciae for the control of O. nubilalis (Hassan, 1983). The release of Trichogramma in a Bt-based IPM program in tomato contributed to the improved pro"tability of this program, compared with the use of chemical insecticides (Trumble and Alvarado-Rodriguez, 1993). The combination of Bt with natural enemies of larval pests
673
necessitates careful screening for compatibility; the level of host intoxication has a direct impact on the parasitoid performance. For example, longer exposures of the pest to lethal levels of Bt resulted in lower survival of Myiopharus doryphorae, the tachinid parasitoid of the Colorado potato beetle (Lopez and Ferro, 1995) and of the braconid, Microplitis croceipes in Helicoverpa armigera (Blumberg et al., 1997). Such incompatibility would result in undesired loss of a parasitoid progeny in the host larvae and, therefore, in reduced parasitoid populations. Sequential use of Bt and parasitoids, instead of simultaneous application, is the recommended strategy: it is more useful when the parasitoids infest 3rd instar and older larvae, which have survived Bt application against neonate larvae. The parasitoid Cotesia marginiventris emerged more successfully from Heliothis virescens when exposure to Bt was delayed for 48 h following parasitization (Atwood et al., 1997a). In Bt-sensitive P. xylostella the microbe had a negative e!ect on the host parasitoid Cotesia plutella, but this competition between Bt and the parasitoid improved the performance of C. plutellae if the P. xylostella was highly resistant to Bt (Atwood et al., 1997a). In contrast to the observed adverse e!ects of Bt on immature stages of larval parasitoids, Bt products in aqueous mixtures consumed by adult parasitoids of a target (Blumberg et al., 1997) or a non-target pest (Wysoki, 1989) led to increased parasitoid longevity, probably because of the nutritious fermentation residues in the commercial product. The control of the Colorado potato beetle in the "eld showed synergy between Bt and Prillus bioculatus (Pentatomidae), a predator of the eggs and larvae of the pest (Cloutier and Jean, 1998). Bt had no detrimental e!ects on natural predators of the heliothine populations in cotton (Young et al., 1997).
11.4. Plant allelochemicals and natural insecticides Limonoids isolated from citrus, which tend to suppress feeding, seemed to be antagonistic to Bt in its activity against the Colorado potato beetle (Murray et al., 1993). Also, cotton condensed tannins were found antagonistic to Bt, probably because they deterred the larvae and/or inactivated the toxin (Navon et al., 1993). Since neem has antifeedant e!ects (Walter, 1999), which stop larval feeding, it may impair the e!ect of Bt on some insects. On the other hand, plant phenols (Ludlum et al., 1991) and ca!eine (Morris et al., 1994) increased the toxicity of Bt. The screening of phytochemical e!ects at tritrophic levels is useful for the following purposes: (1) to evaluate the compatibility of Bt with allelochemicals used in crop breeding programs for developing natural resistance to insects; (2) to select plant allelochemicals that synergize with Bt products and that could be used in the microbe formulations for pest control; and (3) to develop
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Table 2 Laboratory and "eld-derived insect resistance to d-endotoxin and Cry proteins (references listed in Gelernter, 1977). (LC50520 times greater in selected than in unselected populations) No.
Resistant insect species
Common name
Resistance to Bt strain/toxin
1 2 3 4 5 6 7 8 9
Plodia interpunctella Heliothis virescens Plutella xylostella Leptinotarsa decemlineata Trichoplusia ni Spodoptera exigua Chrysomela scripta Spodoptera littoralis Plutella xylostella
Indian meal moth Tobacco budworm Diamondback moth Colorado potato beetle Cabbage looper Beet armyworm Cottonwood leaf beetle Cotton leafworm Diamondback moth
kurstaki, HD-1 CryIAb, CryIAc Kurstaki HD-1 tenebrionis CryIAb (activated CryIC Cry3A Cry1C Cry1C
Cross resistance. Resistance was derived in the "eld.
rotational uses of Bt and insecticides of botanical origin, to overcome insect resistance to the microbe. 11.5. Chemical insecticides Mixtures of chemical insecticides and Bt have been evaluated mostly to enhance the e!ectiveness of the microbe. Several constraints on the use of such mixtures were found: (1) most of the chemical insecticides were not compatible with biological control based on egg and larval parasitoids and predators, whereas Bt did not a!ect several bene"cial insects (Atwood et al., 1997b; Hassan and Graham-Smith, 1995; Kring and Smith, 1995; Muckenfuss and Shepard, 1994; Young et al., 1997); (2) mixing of Bt with the chemical instead of using them sequentially does not prevent the imposition of continuous selection pressure for resistance to the microbe; (3) some chemical insecticides have antifeeding e!ects on the pest, probably rendering them antagonistic in combination with Bt, as was probably the case when the pyrethroid esfenvalerate was tested with Bt against P. xylostella (Hoy and Hall, 1993). In such cases, the chemical may reduce the ingestion of the microbe and so reduce its e$cacy; (4) Bt products reduce larval feeding and thereby minimize the e$cacy of chemical insecticides which a!ect the insects per-os.
12. Future prospects The future prospects of Bt seems to be positive, for the following reasons. 1. The number of new pesticides registered by the EPA in 1987}1991 was half that in 1972}1976, because of high costs of registration under the new regulations. 2. The forecast annual growth rate for biopesticides over the next 10 years is 10}15%, compared with 2% for chemical insecticides.
3. The validity of biopesticide registrations lasts for 1 yr compared with 3}4 yr for conventional chemical insecticides. 4. The cost of development of a Bt insecticide is $3}5 million, compared with $50}80 million for a chemical insecticide. 5. The cost of manufacturing a Bt product by local industry is half that of an imported product ($7/kg compared with $15/kg (16,000 IU/mg in liquid form)). In contrast to this prospect for Bt, the use of chemical insecticides seems likely to decline in the future, and increasing restrictions on their registration may result in a smaller market for these products. Also, insect resistance to the chemicals will continue. This will further the increased use of Bt, but competition from natural and synthetic selective chemicals will not be avoided, and costs of Bt products may limit the crop range recommended for pest control with the microbe. The e!ects of Bt transgenic crops on the market for Bt products cannot be accurately predicted at this stage. Increasing acreage under Bt row crops may reduce the use of Bt sprays, but niche markets for the microbe will increase for use on cash crops which are free of Bt genes. Annual variations in agricultural crop areas and in levels of pest infestation can introduce wide uncertainties in market calculations, with consequently wide variations in sales. Two groups of factors will a!ect market growth: 1. Social and public interactions within the framework detailed in Fig. 1. The continuing development of new Bt products, #ow of information, progress with legislation and registration, and availability of funding among the various public and social disciplines are essential to the support of the annual increase in the market for Bt products and their use in insect control strategies. 2. Developments and knowledge that will be essential for the improvement of microbial pest management with Bt include: (1) novel formulations that will increase the
A. Navon / Crop Protection 19 (2000) 669}676
Fig. 1. Schematic structure of social and public activities linked to increasing the use of Bt.
residual activity of the microbe in the "eld; (2) improved application technologies, developed with consideration of the environmental constraints and mode of action of Bt; (3) a wider choice of IPM strategies which combine Bt with selective chemicals, natural enemies and other microbial agents; (4) biotechnological e!orts to increase Bt activities and insect host range. The determination of cost-e!ective levels of Bt coin for use in agriculture has been slow, because of the constraints discussed in this review. A helpful route for rapid integration of the microbe into pest control programs, compatible with those using chemical control agents might be through transferring to conventional agriculture the knowledge gained from successful IPM programs in bioorganic farming. References Atwood, D.W., Young III, S.Y., Kring, T.J., 1997a. Development of Cotesia (Hymenoptera: Braconidae) in tobacco budworm (Lepidoptera: Nocuidae) larvae treated with Bacillus thuringien and thiodicarb. J. Econ. Entomol. 90, 751}756. Atwood, D.W., Young Jr, S.Y., Kring, T.J., 1997b. Impact of Bt and thiodicarb alone and in combination on tobacco budworm, mortality and emergence of the parasitoid Microplitis croceipes, Vol 2. In: Proceedings of the Beltwide Cotton Conference. National Cotton Council, New Orleans, USA, pp. 1305}1310. Bauer, M.E., Kaya, H.K., Tabashnik, B.E., Chilcutt, C.F., 1998. Suppression of Diamondback moth (Lepidoptera: Plutellidae) with an entomopathogenic nematode (Rhabditida: Steinernematidae) and Bacillus thuringiensis. J. Econ. Entomol. 91, 1089}1095.
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Baum, J.A., Timothy, B.J., Carlton, B.C., 1999. Bacillus thuringiensis. In: Hall, F.R., Menn, J.J. (Eds.), Biopesticide Use and Delivery. Humana Press, NJ. USA, pp. 189}209. Blumberg, D., Navon, A., Keren, S., Goldenberg, S., Ferkovich, S.M., 1997. Interactions among Helicoverpa armigera (Lepidoptera: Noctuidae), its larval endoparasitoid Microplitis croceipes (Hymenoptera: Braconidae), and Bacillus thuringiensis. J. Econ. Entomol. 90, 1181}1186. Bryant, J.E., 1994. Application strategies for Bacillus thuringiensis. Agric. Ecosys. Environ. 49, 65}75. Burges, H.D., Jones, K.A., 1999. Formulation of bacteria, viruses and protozoa to control insects. In: Burges, H.D. (Ed.), Formulation of microbial biopesticides. Kluwer Academic Publisher, Dordrecht, The Netherlands, pp. 34}127. Cannon, R.J.C., 1996. Bacillus thuringiensis use in agriculture: a molecular perspective. Biol. Rev. 71, 561}636. Cloutier, C., Jean, C., 1998. Synergism between natural enemies and biopesticides: a test case using stinkbug Perillus bioculatus (Hemiptera: Pentatomidae) and Bacillus thuringiensis tenebrionis against Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 91, 1096}1108. Cohen, E., Rozen, H., Joseph, T., Margulis, L., 1991. Photoprotection of Bacillus thuringiensis var. kurstaki from ultra-violet irradiation. J. Invertebr. Pathol. 57, 343}351. Dubois, N.R., Dean, D.H., 1995. Synergism between Cry1A insecticidal crystal proteins and spores of Bacillus thrinigiensis, other bacterial spores, and vegetative cells against Lymantria dispar (Lepidoptera: Lymantriidae) larvae. Environ. Entomol. 24, 1741}1747. Dulmage, H.D., 1970. Insecticidal activity of HD-1, a new isolate of Bacillus thuringiensis var. alesti. J. Invertebr. Pathol. 15, 232}239. Dunkle, R.L., Shasha, B.S., 1989. Response of starch encapsulated Bacillus thuringiensis containing UV screens to sunlight. Environ. Entomol. 18, 1035}1041. Ferro, D.H., Lyon, S.M., 1991. Colorado potato beetle (Coleoptera: Chrysomelidae) larval mortality: operative e!ects of Bacillus thuringiensis subsp. san diego. J. Econ. Entomol. 84, 806}809. Ferro, D.H., Yuan, Q.C., Slocombe, A., Tutle, A., 1993. Residual activity of insecticides under "eld conditions for controlling the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 86, 511}516. Fuxa, J., 1989. Fate of released entomopathogens with reference to risk assessment of genetically engineered microorganisms. Bull. Entomol. Soc. Am. 35, 12}24. Gelernter, W.D., 1997. Resistance to microbial insecticides: The scale of the problem and how to manage it. In: Microbial Insecticides: Novelty or Necessity. The British Crop Protection Council Symposium Proceedings No. 86, Surrey, UK, pp. 201}212. Ghidiu, G.M., Zehnder, G.W., 1993. Timing of the initial spray application of Bacillus thuringiensis for control of the Colorado potato beetle (Coleoptera: Chrysomelidae) in potatoes. Biol. Control 3, 348}352. Gould, F., Anderson, A., Landism, D., van Mellert, H., 1991. Feeding behavior and growth of Heliothis virescens larvae on diets containing Bacillus thuringiensis formulations or endotoxins. Entomol. Exp. Appl. 58, 199}210. Hall, F.R., Chapple, A.C., Taylor, R.A.J., Downer, R.A., 1995. Modeling the dose acquisition process of Bacillus thuringiensis. In: Hall, F.R., Barry, J.W. (Eds.), Biorational Pest Control Agents Formulation and Delivery. ACS Symposium Series, London, UK, pp. 68}78. Hand, S.S., Luttrell, R.G., 1997. Strategies for foliar application of Bacillus thuringiensis in cotton. Proceedings of the Beltwide Cotton Conference National Cotton Council, New Orleans, USA, Vol. 2, pp. 1151}1157. Hassan, S.A., 1983. Results of laboratory testing of a series of pesticides on egg parasites of the genus Trichogramma (Hymenoptera:
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Trichogrammatidae) Nachrichtenbl. Dtsch P#kanzenschutzdienst (Braunschw.) 35, 21}25. Hassan, E., Graham-Smith, S., 1995. Toxicity of endosulfan, esfenvalerate and Bacillus thuringiensis on adult of Microplitis demolitor Wilkinson and Trichogrammatoidea bactrae Nagaraja. Z. P#anzenk. P#anzensch. 102, 428}442. Hernstadt, C., Soares, G.G., Wilcox, E.R., Edwards, D.L., 1986. A new strain of Bacillus thuringiensis with activity against coleopteran insects. Bio/Technology 4, 305}308. Hoy, C.W., Hall, F.R., 1993. Feeding behaviour of Plutella xylostella and Leptinotarsa decemlineata on leaves treated with Bacillus thuringiensis and esfenvalerate. Plant Sci. 38, 335}340. Krieg, A., Huger, A.M., Langenbruch, G.A., Schnetter, W., 1983. Bacillus thuringiensis var. tenbrionis: Ein neuer, gegenuK ber Glarven von Coleoptaran Wirksamer Pathotype. Z. angew. Entomol. 96, 500}508. Kring, T.J., Smith, T.B., 1995. Trichogramma pretiosum e$cacy in cotton under Bt-insecticide combinations. Proceedings of the Beltwide Cotton Conference, National Cotton Council, Vol. 2. San Antonio, TX, USA, pp. 856}857. Lopez, R., Ferro, D.N., 1995. Larviposition response of Myiopharus doryphorae (Diptera: Tachinidae) to Colorado potato beetle (Coleoptera: Chrysomelidae) larvae treated with lethal and sublethal doses of Bacillus thuringiensis Berliner subsp. tenebrionis. J. Econ. Entomol. 88, 870}874. Ludlum, C.T., Felton, G.W., Du!ey, S.S., 1991. Plant defenses: chlorogenic acid and polyphenol oxidase enhance toxicity of Bacillus thuringiensis subsp. kurstaki to Heliothis zea. J. Chem. Ecol. 17, 217}237. McClintock, J.T., Scha!er, C.R., Sjoblad, R.D., 1995. A comparative review of the mammalian toxicity of Bacillus thuringiensis-based pesticides. Pest. Sci. 45, 95}105. McGuire, M.R., Shasha, B.S., 1995. Starch encapsulation of microbial pesticide. In: Hall, F.R., Barry, J.W. (Eds.), Biorational Pest Control Agents Formulation and Delivery. ACS Symposium Series 595, London, UK, pp. 229}237. Moar, W.J., Trumble, J.T., Federici, B.A., 1989. Comparative toxicity of spores and crystals from the NRD-12 and HD-1 strains of Bacillus thuringiensis subsp. kurstaki to neonate beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 82, 1593}1603. Morris, O.N., Trorrier, M., McLaughlin, N.B., Converse, V., 1994. Interaction of ca!eine and related compounds with Bacillus thuringiensis ssp. kurstaki in Bertha armyworm (Lepidoptera: Nuctuidae). J. Econ. Entomol. 87, 610}617. Muckenfuss, A.E., Shepard, B.M., 1994. Seasonal abundance and response of Diamondback moth, Plutella xylostella (L,) (Lepidoptera: Plutellidae), and natural enemies to esfenvalerate and Bacillus thuringiensis subsp. kurstaki Berliner in coastal South Carolina. J. Agric. Entomol. 11, 361}373. Murray, K.D., Alford, A.R., Groden, E. et al., 1993. Interactive e!ects of an antifeedant used with Bacillus thuringiensis var. san diego deltaendotoxin on Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 86, 1793}1801. Navon, A., 1993. Control of lepidopteran pests with Bacillus thuringiensis. In: Entwistle, P.F., Cory, J.S., Bailey, M.J., Higgs, S. (Eds.),
Bacillus thuringiensis, an Environmental Biopesticide: Theory and Practice. Wiley, New York, USA, pp. 125}146. Navon, A., 2000. Bioassays of Bacillus thuringiensis. In: Bioassays of Entomopathogenic Microbes and Nematodes. CABI Publishing, UK. Navon, A., Klein, M., Braun, S., 1990. Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets. J. Invertebr. Pathol. 55, 387}393. Navon, A., Hare, J.D., Federici, B.A., 1993. Interactions among Heliothis virescens larvae, cotton condensed tannin and the CryIA(c) endotoxin of Bacillus thuringiensis. J. Chem. Ecol. 19, 2485}2499. Navon, A., Keren, S., Levski, S., Grinstein, A., Riven, J., 1997. Granular feeding baits based on Bacillus thuringiensis products for the control of lepidopterous pests. Phytoparasitica 25 (suppl), 101S}110S. Patel, K.R., Wyman, J.A., Patel, K.A., Burden, B.J., 1996. A mutant of Bacillus thuringiensis producing a dark-brown pigment with increased UV resistance and insecticidal activity. J. Invertebr. Pathol. 67, 120}124. Ramos, L.M., McGuire, M.R., Galan Wong, L.J., 1998. Utilization of several biopolymers for granular formulations of Bacillus thuringiensis. J. Econ. Entomol. 91, 1109}1113. Roltsch, W.J., Zalom, F.G., Barry, J.W., Kirfman, G.W., Edstrom, J.P., 1994. Ultra-low volume aerial application of Bacillus thuringiensis variety kurstaki for the control of peach twig borer in almond trees. Appl. Eng. Agric. 11, 25}30. Sample, J.R., Buettner, H., 1983. Ocular infection caused by a biological insecticide. J. Infec. Dis. 148, 614. Sebesta, K., Farkas, J., Horska, K., Vankova, J., 1981. Thuringiensin, the beta-exotoxin of Bacillus thuringiensis. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Diseases 1970}1980. Academic Press, London, UK, pp. 249}282. Shah, P.A., Goettel, M.S. (Eds.), 1999. Directory of microbial control products and services. Society of Invertebrate Pathology, Gainesville, FL 32614-7050, USA, pp. 31. Tabashnik, B.E., Cushing, N.L., Finson, N., Johnson, M.W., 1990. Field development of resistance to Bacillus thuringiensis in diamonback moth. J. Econ. Entomol. 83, 1671}1676. Trumble, J., Alvarado-Rodriguez, B., 1993. Development of economic evaluation of an IPM program for fresh market tomato production in Mexico. Agric. Ecosys. Environ. 43, 267}284. van Frankenhuyzen, K., 2000. Application of Bt in forestry. In: Entomopathogenic Bacteria: from Laboratory to Field Application. Francois, J., Delecluse, A., Nielsen-LeRoux, C. (Eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands (in press). Walter, J.F., 1999. Commercial experience with neem products. In: Hall, F.R., Menn, J.J. (Eds.), Biopesticide Use and Delivery. Humana Press, NJ. USA, pp. 155}163. Wysoki, M., 1989. Bacillus thuringiensis preparations as a means for the control of lepidopterous pests in Israel. Isr. J. Entomol. 23, 119}129. Young, S.Y., Kring, T.J., Johnson, D.R., Klein, C.D., 1997. Bacillus thuringiensis alone and in mixtures with chemical insecticides against heliothines and e!ects on predator densities in cotton. J. Entomol. Sci. 32, 183}191.
Bacillus thuringiensis insecticides in crop protection * reality and prospects Amos Navon* Department of Entomology, ARO, The Volcani Center, Bet Dagan 50250, Israel
Abstract Bacillus thuringiensis (Bt) has been the leading biopesticide against lepidopterous pests since 1959. In the 1990 s the following developments contributed to increased rational uses of Bt: (1) natural and recombinant Bt products were developed to broaden the insect host range in pest management programs; (2) new formulations based on conventional or genetically engineered encapsulation of the toxins and/or feeding stimulants to increase ingestion and, in turn, the e$cacy of the microbe; (3) screening of the interactions of Bt with insect herbivores and plant allelochemicals or natural enemies of the pests, to aid the formulation of biological control strategies; and (4) knowledge and management of insect resistance to Bt. The prospects for Bt insecticides will be described and discussed. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Bacillus thuringiensis products; Bioinsecticides; Crop protection; Lepidoptera; Coleoptera; Agricultural insect pests
1. Introduction
2. Constraints in the use of Bt products
The market for biopesticides based mostly on Bt products still forms less that 1% of the crop protection market. Since the 1990s the intoduction of new Bt strains and formulations, and the development of recombinant Bt products has furthered the growth of the Bt market, mainly by extending the insect host range of the microbial products and enhancing the insecticidal potencies of the toxins. In forestry, Bt has e!ectively replaced chemical insecticides and has become established in control programs against defoliator larval moths in Europe and North America (van Frankenhuyzen, 2000). In Israel, since 1987, Bt has e!ectively replaced chemical insecticides against Thaumatopoea pityocampa in pine forests, mainly because of the public use of forests as recreation areas. In agriculture, the considerations and possibilities involved in using Bt in pest management strategies are complicated. Some of the current issues concerning the use of Bt, and its future prospects are discussed below.
Two major constraints a!ect "eld e!ectiveness: (1) the toxic protein is an oral insecticide and therefore has to be consumed by the larva; and (2) environmental conditions reduce the e$cacy of the product. More speci"cally, the following problems reduce the insecticidal power of Bt:
* Corresponding author. Tel.: #972-3-968-3832; fax: #972-39683835.
(1) the narrow host range of Bt strains; (2) Bt is an oral insecticide; (3) bollworm and borer larvae avoid a lethal dose of Bt by penetrating into the plant; (4) the lethal dose of Bt is instar dependant, and the susceptibility of mature larvae is very low; (5) solar irradiation (300}380 nm) inactivates the crystal protein, mostly through the destruction of tryptophan; (6) wash-o! of the Bt product by rain and dew dilute the microbe dose on the plant; (7) phylloplanes and allelochemicals can adversely a!ect the Bt protein activity; (8) application of Bt products in row crops is partially e!ective. Some of these constraints have been minimized by a rational pest management strategies which are detailed below.
0261-2194/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 0 ) 0 0 0 8 9 - 2
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3. Development of Bt products Screening of Bt products based on the spore crystal complex has been conducted since the 1950 s, but it was only in 1970 that the HD-1 strain of Bt subspecies kurstaki, which is active against a large number of moth species, was discovered (Dulmage, 1970) and produced commercially by many agrochemical and fermentation companies (Table 1). Screening programs by means of potency bioassays (Navon, 2000) resulted in highly potent new strains. Between 1970 and 1990, strains of Bt subspecies aizawai, active against armyworms such as Spodoptera spp., and subspecies subspecies tenebrionis (Krieg et al., 1983) and san-diego (Hernstadt et al., 1986, active against beetles were introduced into the market, and these new products extended the means of control to more insects. It has been shown in lepidopterous insects that the spore potentiated (Moar et al., 1989) and synergized (Dubois and Dean, 1995) the insecticidal activity of the crystal protein. Improved knowledge of the molecular biology of Bt (Cannon, 1996) led to the use of Bt genes for manipulation of the Bt toxin (Baum et al., 1999) (Table 1). During the 1990 s genetic manipulations became a new tool for combining the toxic proteins (Cry proteins) of two or more Bt strains in one product. Transconjugation and recombination were used to develop the genetically manipulated Bt products. In addition, encapsulation of the Cry proteins in the cell walls of Pseudomonas yuorescens was used to protect the toxin against environmental inactivation. These genetic manipulations simpli"ed and improved pest control mainly in row crops, where they enabled a single genetically modi"ed Bt product to control all the lepidopterous pests infesting the plant. Another contribution to the widening of Bt uses in agriculture was the manufacture of Bt products in developing countries, where domestic strains were incorporated into commercial products (Shah and Goettel, 1999) (Table 1).
In a di!erent approach to photoprotection of Bt, a melanin-producing mutant of the microbe provided increased UV resistance and insecticidal activity (Patel et al., 1996). Rainproo"ng was increased by Bt encapsulation in biopolymers, to reduce washing of the product from the plant (Ramos et al., 1998). Selection of environmentally safe and cost-e!ective formulations to increase the residual activity of commercial Bt products is one of the major needs for widening the use of the microbe.
5. Application In agricultural use Bt is applied mostly with the same ground sprayers used for the application of chemical insecticides. Since the crystalline toxin a!ects the larva through feeding, high volumes of aqueous spray per unit area are required for adequate coverage of the plant canopy, and this is very costly because substantial amounts of the product are lost by dripping o! the plants. In addition, the use of high volumes of aqueous spray can be impracticable in areas where water is not available. In recent years, e!orts have been made to reduce the spray volume and to achieve better control of the droplets: avocado orchards (Wysoki, 1989) were effectively air sprayed from a helicopter, to control Boarmia (Ascotis) selenaria (M. Wysoki, ARO, Israel, unpublished), and aerial spraying of Bt in ultra-low volume (ULV) application in the control of Anarsia lineata in almond orchards also seemed promising (Roltsch et al., 1994). The use of an air-assisted sleeve boom (Degania Sprayers, Degania Bet, Israel) on row crops increased spray penetration and plant coverage, and reduced drift. Also, locating the sprayer nozzles at the height of the plant canopy markedly improved delivery to the lower surfaces of cotton leaves and to the #ower buds and bolls (A. Navon, unpublished).
6. Residual activity 4. Formulations A substantial amount of information on development of Bt formulations has been published (Burges and Jones, 1999). The formulation additives include wetting agents, stickers, sunscreens, synergists and phagostimulants. It is widely accepted that UV inactivation of the crystalline toxin is the major cause of the rapid loss of Bt activity; nevertheless, in practice, the commercial spray formulations lack photoprotection of the spores and crystals. Several chromophores have been selected to shield Bt preparations against inactivation by sunlight (Dunkle and Shasha, 1989; Cohen et al., 1991), but environmental safety and palatability to the insects are prerequisites for using these dyes and pigments in commercial products.
A promising approach to improving residual activity is exempli"ed in the use of starch-encapsulated Bt in the control of Ostrinia nubilalis in corn (Dunkle and Shasha, 1989), or in the embedding of Bt in a dusting formulation based on wheat #our for the control of lepidopteran pests of vegetable and "eld crops (Navon et al., 1997). These protective materials included phagostimulants which encouraged the larvae to feed on the granule and ingest lethal amounts of the microbe. Low persistence of the spore}crystal product on the plant is one of the main problems in Bt application: low persistence (48 h) of Bt products on cotton was observed with products of Bt subsp. kurstaki (Fuxa, 1989). Leaf growth indirectly causes a reduction in Bt persistence as it causes natural dilution of the product on the plant,
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671
Table 1 Natural and genetically modi"ed Bt products registered for agricultural use Bt strain
Company
Product
Target insects
Crop
Abbott Laboratories Chicago IL, US
Biobit, Dipel, Foray,
Lepidoptera
Field and vegetable crops, greenhouse, orchard fruits and nuts, ornamentals, forestry, stored products
Tenebrionis
Abbott
Javelin, Steward, Thuricide, Vault Bactospeine, Futura, Able, Costar, Florbac, Xentari Novodor
Lepidoptera
Kurstaki Kurstaki Aizawai
Thermo Trilogy Corp. Columbia MD, US Abbott Thermo Trilogy Abbott
(a) Natural Kurstaki HD-1
Kurstaki HD-1
Tenebrionis Kurstaki Kurstaki Galleriae YB-1520 * CT-43
Thermal Trilogy BioDalia, Dalia, Israel Rimi, Tel Aviv, Israel Tuticorin Alkali Chemicals & Fertilizers Ltd. India Huazhong Agric. University, China Scient. & Technol. Develop. China Huazhong Agric. University, China
Trident Bio-Ti
Lepidoptera Lepidoptera Lepidoptera armyworms Colorado Potato Beetle, Elm Bark Beetle Coleoptera Lepidoptera
Bitayon (granular feeding baits) Spicturin
Btrachedra amydraula Lepidoptera
Mainfeng pesticide
Lepidoptera
Row crops, fruit trees
Bt 8010 Rijin
Lepidoptera
Shuangdu
Lepidoptera, Coleoptera, Diptera
Row crops, rice, maize, fruit trees, forests, ornamentals Row crops, garden plants, forests
Lepidoptera (Resistant P.xylostella) Lepidoptera
Row crops
Lepidoptera
Vegetables, horticultural, ornamental Turf, hay, row crops, sweet corn
Row crops Potato, tomato, eggplant Ornamentals, shade trees Potato, tomato, eggplant Avocado, tomato, vineyards, pine forests Date palms Cruciferous crop plants
(b) Genetically modi"ed Aizawai recipient kurstaki donor
Thermo Trilogy
Agree, Design (transconjugant)
Kurstaki recipient aizawai donor Kurstaki
Ecogen, Inc. Langhorne PA, US Ecogen
Condor, Cutlass (transconjugant) CRYMAX, Leptinox,
Kurstaki
Ecogen
Leptinox (recombinant)
Kurstaki recipient
Ecogen
d-endotoxin encapsulated in Pseudomonas yuorescens
Mycogen, Corp. San Diego, CA, USA
Raven (recombinant) MVP MATTCH MTRACK (CellCap)
Lepidoptera armyworms Lepidoptera Coleoptera Lepidoptera Lepidoptera Coleoptera
Row crops
Row crops Potato, tomato, eggplant Row crops * armyworms Potato, tomato, eggplant
Based on Baum et al. (1999), Shah and Goettel (1999).
especially in broad leaf crops such as cotton and leaf vegetables. Low residual activity of Bt subsp. tenebrionis products used against the Colorado potato beetle on potatoes in the "eld persisted for only 48 h (Ferro et al., 1993). The e!ectiveness of Bt application depends strongly on its timing; the following guidelines can be useful: (1) Application early in the season, before high "eld populations of parasitoids and predators on the pests
have been reduced by chemical insecticides. It has been shown in the US that Bt use against heliothine infestation early in the cotton season, followed by traditional insecticides (Hand and Luttrell, 1997), e!ectively reduced the Heliothis virescens populations. (2) Timing the Bt application according to monitoring of egg hatching: the most e!ective control of the Colorado potato beetle in potato was achieved up to 4 days after 30% of the eggs had hatched, whereas premature or delayed application
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allowed signi"cantly more defoliation and larval recovery from the Bt intoxication (Ghidiu and Zehnder, 1993). This timing can be determined by monitoring the pest population with the aid of traps based on sexpheromones or other insect attractants: a signi"cant increase in trap catches may indicate the onset of egg laying. (3) Bt application after sunset instead of in the morning can increase the persistence of the Bt product in warm countries, where activity of the microbe persists for only 2}3 days (Fuxa, 1989).
7. Larval age One of the most important economic aspects of pest management with Bt is the use of the microbe against young larvae, preferably neonates, because it has been shown in laboratory and "eld bioassays that third instar larvae of Lepidopteran (Navon et al., 1990) and Coleopteran (Ferro and Lyon, 1991) are less susceptible to the Bt products than younger larvae. Thus, the costs of controlling 1st instar larvae can be a fraction of that of controlling older larvae. These di!erences in larval susceptibility to Bt are probably aspects of a more general phenomenon among lepidopteran and coleopteran pests. Furthermore, the initial nibbling of Bt by neonate larvae is followed by cessation of feeding and gut paralysis within minutes (Gould et al., 1991), so that there is negligible damage to plants, whereas mature larvae feeding on Bt-treated plants may still cause su$cient damage to the crop to reduce the quality of the agricultural product. Also, mature larvae may recover from the Bt intoxication (Bryant, 1994), and even complete the developmental cycle; this situation can be aggravated by incomplete spray coverage, by rapid degradation of the crystalline toxin by UV radiation, or by washing o! of the Bt spray by rain or overhead irrigation.
8. Insect feeding behaviour The larval feeding habits markedly a!ect the e$cacy of pest management with the microbe, as they determine the availability of the crystalline toxin to the insect (Navon, 1993). Defoliators are e!ectively controlled by Bt, but the control of bollworms, borers and feeders on hypogeaic plant parts has been marginal, because the amounts of Bt available to them are commonly below lethal doses. Knowledge of feeding behaviour is a fundamental requirement in the development of new formulations and the optimization of the utilization of biopesticides. Bt dose-transfer modelling, based on monitoring the e!ects on larval feeding behaviour and locomotion (Hall et al., 1995), has identi"ed factors that need to be improved to ensure better control of lepidopteran larvae. The use of a phagostimulant mixture, such as COAX (Trader Oil
Mill Co., USA) in a sprayable starch encapsulation (McGuire and Shasha, 1995), or a yeast extract in a dustable granular formulation, as a feeding stimulant (Navon et al., 1997) are promising new tactics for manipulating the feeding behaviour of bollworm and borer larvae. The principles behind the use of such formulations, in addition to increasing residual toxic activity, are: (1) the larvae are attracted to feed selectively on the Bt product and therefore feed less on the plant; (2) for the same Bt dose per acre, the amounts of spore}crystal materials available to the larvae are several times greater than in a leaf meal with the Bt spray. With these approaches, the insect-control e$cacies of the new Bt formulations can exceed those obtained with earlier ones.
9. Insect resistance to Bt In recent years, an increasing number of reports on the development of resistance to Bt in agricultural pests have been published. Most of the reports concern laboratory inducement of resistance (Gelernter, 1997) (Table 2). Field resistance to Bt in Plutella xylostella (Tabashnik et al., 1990), can reduce the e!ectiveness of the insect control. In practice, however, one or two Bt sprays per season are not considered to impose e!ective selection pressure to develop resistance to the microbe. Furthermore, in Bt crops (Bt transgenic plants) the Cry proteins are expressed in all the plant cells, and larvae are continuously exposed to the toxic protein. In contrast to this, when sprayed on the plants, the Bt has a short residual activity in row crops, and the coverage of the plant with the Bt product is very often incomplete. As a result of these pest control constraints, natural &refuge' sites are created on the plant, to the bene"t of the defoliator larvae, and even more so of the bollworm and borer larvae. Thus, the larvae are exposed to sublethal doses of the Bt product. Nevertheless, excessive use of Bt in pest control may induce resistance in the pests and it is necessary to apply bioassays for recording "eld resistance in the insect populations, to avoid control failures because of resistant pests.
10. Safety Extensive toxicity studies have shown that Bt isolates devoid of b-exotoxin are not toxic or pathogenic to mammals (McClintock et al., 1995). Nevertheless, people working with spore}crystal products should take precautions against spore infection by avoiding contact with open wounds and protecting the eyes against spray or dust (Sample and Buettner, 1983). In the western world, Bt strains which produce b-exotoxin cannot be registered for use in plant protection, because of their toxicity to mammals (Sebesta et al., 1981). In veterinary, b-exotoxin
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formulations are used against #ies in di!erent breeding sites such as poultry manure.
11. Combination of Bt with other means of pest management 11.1. Introduction The use of Bt in IPM programmes o!ers the advantage of replacing undesired chemical insecticides with safe bioinsecticides, which could be combined with other biological means, provided that Bt is synergistic or compatible with them. The rationale of such a strategy is that biological means such as entomopathogenic microbes and nematodes, natural enemies of the pests, and/or natural insecticides, in combination with Bt can improve pest control, especially when the e$cacy of Bt is suboptimal. In this review, laboratory experiments and "eld uses of Bt, in combination with other pest control agents are described, and considerations of compatibility and antagonistic interactions involved in this strategy are discussed. 11.2. Entomopathogenic microbes The combination of Bt with baculoviruses has shown mainly an additive e!ect (Navon, 1993); although this combination may not be justi"ed in terms of cost-e!ectiveness, the termination of larval feeding caused by Bt may reduce the amounts of virus needed to kill the larva. The combination of Bt with fungi has not been evaluated in any detail but, in view of the high e$cacy of commercial insecticidal fungi against sucking insects, combinations of such fungi with Bt in suitable formulations might provide a useful strategy in complex pest situations, where moths and white#ies or aphids infest the same crop. The combination of Bt and insecticidal nematodes was tried and seemed not suitable for the control of resistant P. xylostella (Bauer et al., 1998): the two biopesticides applied together provided little advantage over the use of either one of them alone. 11.3. Natural enemies Egg parasitoides have been found highly compatible with Bt, as the egg is not a target stage for the microbe: successful examples include Trichogramma platneri against B. senenaria in avocado (Wysoki, 1989) and T. cacoeciae for the control of O. nubilalis (Hassan, 1983). The release of Trichogramma in a Bt-based IPM program in tomato contributed to the improved pro"tability of this program, compared with the use of chemical insecticides (Trumble and Alvarado-Rodriguez, 1993). The combination of Bt with natural enemies of larval pests
673
necessitates careful screening for compatibility; the level of host intoxication has a direct impact on the parasitoid performance. For example, longer exposures of the pest to lethal levels of Bt resulted in lower survival of Myiopharus doryphorae, the tachinid parasitoid of the Colorado potato beetle (Lopez and Ferro, 1995) and of the braconid, Microplitis croceipes in Helicoverpa armigera (Blumberg et al., 1997). Such incompatibility would result in undesired loss of a parasitoid progeny in the host larvae and, therefore, in reduced parasitoid populations. Sequential use of Bt and parasitoids, instead of simultaneous application, is the recommended strategy: it is more useful when the parasitoids infest 3rd instar and older larvae, which have survived Bt application against neonate larvae. The parasitoid Cotesia marginiventris emerged more successfully from Heliothis virescens when exposure to Bt was delayed for 48 h following parasitization (Atwood et al., 1997a). In Bt-sensitive P. xylostella the microbe had a negative e!ect on the host parasitoid Cotesia plutella, but this competition between Bt and the parasitoid improved the performance of C. plutellae if the P. xylostella was highly resistant to Bt (Atwood et al., 1997a). In contrast to the observed adverse e!ects of Bt on immature stages of larval parasitoids, Bt products in aqueous mixtures consumed by adult parasitoids of a target (Blumberg et al., 1997) or a non-target pest (Wysoki, 1989) led to increased parasitoid longevity, probably because of the nutritious fermentation residues in the commercial product. The control of the Colorado potato beetle in the "eld showed synergy between Bt and Prillus bioculatus (Pentatomidae), a predator of the eggs and larvae of the pest (Cloutier and Jean, 1998). Bt had no detrimental e!ects on natural predators of the heliothine populations in cotton (Young et al., 1997).
11.4. Plant allelochemicals and natural insecticides Limonoids isolated from citrus, which tend to suppress feeding, seemed to be antagonistic to Bt in its activity against the Colorado potato beetle (Murray et al., 1993). Also, cotton condensed tannins were found antagonistic to Bt, probably because they deterred the larvae and/or inactivated the toxin (Navon et al., 1993). Since neem has antifeedant e!ects (Walter, 1999), which stop larval feeding, it may impair the e!ect of Bt on some insects. On the other hand, plant phenols (Ludlum et al., 1991) and ca!eine (Morris et al., 1994) increased the toxicity of Bt. The screening of phytochemical e!ects at tritrophic levels is useful for the following purposes: (1) to evaluate the compatibility of Bt with allelochemicals used in crop breeding programs for developing natural resistance to insects; (2) to select plant allelochemicals that synergize with Bt products and that could be used in the microbe formulations for pest control; and (3) to develop
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Table 2 Laboratory and "eld-derived insect resistance to d-endotoxin and Cry proteins (references listed in Gelernter, 1977). (LC50520 times greater in selected than in unselected populations) No.
Resistant insect species
Common name
Resistance to Bt strain/toxin
1 2 3 4 5 6 7 8 9
Plodia interpunctella Heliothis virescens Plutella xylostella Leptinotarsa decemlineata Trichoplusia ni Spodoptera exigua Chrysomela scripta Spodoptera littoralis Plutella xylostella
Indian meal moth Tobacco budworm Diamondback moth Colorado potato beetle Cabbage looper Beet armyworm Cottonwood leaf beetle Cotton leafworm Diamondback moth
kurstaki, HD-1 CryIAb, CryIAc Kurstaki HD-1 tenebrionis CryIAb (activated CryIC Cry3A Cry1C Cry1C
Cross resistance. Resistance was derived in the "eld.
rotational uses of Bt and insecticides of botanical origin, to overcome insect resistance to the microbe. 11.5. Chemical insecticides Mixtures of chemical insecticides and Bt have been evaluated mostly to enhance the e!ectiveness of the microbe. Several constraints on the use of such mixtures were found: (1) most of the chemical insecticides were not compatible with biological control based on egg and larval parasitoids and predators, whereas Bt did not a!ect several bene"cial insects (Atwood et al., 1997b; Hassan and Graham-Smith, 1995; Kring and Smith, 1995; Muckenfuss and Shepard, 1994; Young et al., 1997); (2) mixing of Bt with the chemical instead of using them sequentially does not prevent the imposition of continuous selection pressure for resistance to the microbe; (3) some chemical insecticides have antifeeding e!ects on the pest, probably rendering them antagonistic in combination with Bt, as was probably the case when the pyrethroid esfenvalerate was tested with Bt against P. xylostella (Hoy and Hall, 1993). In such cases, the chemical may reduce the ingestion of the microbe and so reduce its e$cacy; (4) Bt products reduce larval feeding and thereby minimize the e$cacy of chemical insecticides which a!ect the insects per-os.
12. Future prospects The future prospects of Bt seems to be positive, for the following reasons. 1. The number of new pesticides registered by the EPA in 1987}1991 was half that in 1972}1976, because of high costs of registration under the new regulations. 2. The forecast annual growth rate for biopesticides over the next 10 years is 10}15%, compared with 2% for chemical insecticides.
3. The validity of biopesticide registrations lasts for 1 yr compared with 3}4 yr for conventional chemical insecticides. 4. The cost of development of a Bt insecticide is $3}5 million, compared with $50}80 million for a chemical insecticide. 5. The cost of manufacturing a Bt product by local industry is half that of an imported product ($7/kg compared with $15/kg (16,000 IU/mg in liquid form)). In contrast to this prospect for Bt, the use of chemical insecticides seems likely to decline in the future, and increasing restrictions on their registration may result in a smaller market for these products. Also, insect resistance to the chemicals will continue. This will further the increased use of Bt, but competition from natural and synthetic selective chemicals will not be avoided, and costs of Bt products may limit the crop range recommended for pest control with the microbe. The e!ects of Bt transgenic crops on the market for Bt products cannot be accurately predicted at this stage. Increasing acreage under Bt row crops may reduce the use of Bt sprays, but niche markets for the microbe will increase for use on cash crops which are free of Bt genes. Annual variations in agricultural crop areas and in levels of pest infestation can introduce wide uncertainties in market calculations, with consequently wide variations in sales. Two groups of factors will a!ect market growth: 1. Social and public interactions within the framework detailed in Fig. 1. The continuing development of new Bt products, #ow of information, progress with legislation and registration, and availability of funding among the various public and social disciplines are essential to the support of the annual increase in the market for Bt products and their use in insect control strategies. 2. Developments and knowledge that will be essential for the improvement of microbial pest management with Bt include: (1) novel formulations that will increase the
A. Navon / Crop Protection 19 (2000) 669}676
Fig. 1. Schematic structure of social and public activities linked to increasing the use of Bt.
residual activity of the microbe in the "eld; (2) improved application technologies, developed with consideration of the environmental constraints and mode of action of Bt; (3) a wider choice of IPM strategies which combine Bt with selective chemicals, natural enemies and other microbial agents; (4) biotechnological e!orts to increase Bt activities and insect host range. The determination of cost-e!ective levels of Bt coin for use in agriculture has been slow, because of the constraints discussed in this review. A helpful route for rapid integration of the microbe into pest control programs, compatible with those using chemical control agents might be through transferring to conventional agriculture the knowledge gained from successful IPM programs in bioorganic farming. References Atwood, D.W., Young III, S.Y., Kring, T.J., 1997a. Development of Cotesia (Hymenoptera: Braconidae) in tobacco budworm (Lepidoptera: Nocuidae) larvae treated with Bacillus thuringien and thiodicarb. J. Econ. Entomol. 90, 751}756. Atwood, D.W., Young Jr, S.Y., Kring, T.J., 1997b. Impact of Bt and thiodicarb alone and in combination on tobacco budworm, mortality and emergence of the parasitoid Microplitis croceipes, Vol 2. In: Proceedings of the Beltwide Cotton Conference. National Cotton Council, New Orleans, USA, pp. 1305}1310. Bauer, M.E., Kaya, H.K., Tabashnik, B.E., Chilcutt, C.F., 1998. Suppression of Diamondback moth (Lepidoptera: Plutellidae) with an entomopathogenic nematode (Rhabditida: Steinernematidae) and Bacillus thuringiensis. J. Econ. Entomol. 91, 1089}1095.
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