阅读说明：本技术 生物杀虫剂 (Biological insecticide ) 是由 本·雷蒙德 尼尔·克里克莫尔 于 2018-08-09 设计创作，主要内容包括：本发明涉及苏云金芽孢杆菌(Bacillus thuringiensis)菌株,所述菌株在表型上是稳定的并且与野生型菌株相比具有增加的毒力,其中,所述增加的毒力通过在一次或多次传代期间将菌株暴露于诱变剂中而获得。这样的菌株特别适合用作生物杀虫剂。还描述了增加微生物杀虫剂中毒力的方法。(The present invention relates to a Bacillus thuringiensis (Bacillus thuringiensis) strain which is phenotypically stable and has increased virulence compared to a wild-type strain, wherein the increased virulence is obtained by exposing the strain to a mutagen during one or more passages. Such strains are particularly suitable for use as biopesticides. Methods of increasing the toxicity of microbial pesticides are also described.)

1. A bacillus thuringiensis strain that is phenotypically stable and has increased virulence compared to a wild-type strain, wherein the increased virulence is obtained by exposing the strain to a mutagen during one or more passages.

2. A bacillus thuringiensis strain according to claim 1, wherein the mutagen comprises Ethyl Methane Sulfonate (EMS).

3. A bacillus thuringiensis strain according to claim 1 or 2, for use as a pesticide.

4. A bacillus thuringiensis strain according to claim 3, wherein the insecticide is used to control one or more of: aphids, other hemipteran pests, thrips, lepidopteran larvae, dipteran larvae, coleopteran larvae, leaf mites, locusts, crickets, ants, cockroaches, flies, wasps, termite wood worms, wood ants, book worms, silverfish, bark beetles, clothiantus gibsoniana, chiggers, mange, ticks, mites, lice, fleas, bugs, mosquitoes, tsetse flies, bug bugs, root knot nematodes, soybean cyst nematodes, potato cyst nematodes, slugs, and snails.

5. A bacillus thuringiensis strain according to claim 3, wherein the insecticide is used for controlling Plutella xylostella and larvae thereof.

6. A Bacillus thuringiensis strain according to any one of claims 3 to 5, wherein the insecticide is applied to the soil of crucifers, in the vicinity of the soil or in the soil.

7. A Bacillus thuringiensis strain according to any one of the preceding claims, wherein the Bacillus thuringiensis strain comprises a Bacillus thuringiensis Enterobacter insecticidal subspecies, a Bacillus thuringiensis Clarias subspecies or a Bacillus thuringiensis Kustak subspecies.

8. A composition comprising a bacillus thuringiensis strain according to any one of the preceding claims.

9. The composition of claim 8, wherein the bacillus thuringiensis strain is in spore or crystal form.

10. The composition according to claim 8 or 9, characterized in that it is in solid or liquid form.

11. The composition according to claims 8 to 10, characterized in that it further comprises one or more of the following ingredients: wetting agents, thickeners, viscosity regulators, stabilizers and surfactants and dispersion media, for example water or edible oils.

12. Composition according to claims 8 to 11, characterized in that it is used as a pesticide.

13. Composition according to claims 8 to 12, characterized in that it is intended to be applied by spraying, spreading or sprinkling to plants, to the soil surface and/or to the soil itself.

14. A method of improving the virulence of a microbial pesticide, the method comprising:

(a) dividing pest populations into subpopulations;

(b) contacting a subpopulation of pests with one or more microbial pesticides;

(c) selecting a pest carcass from the subpopulation having the highest mortality rate;

(d) inoculating the sporulation medium with the pest carcass to provide sufficient inoculum for easy quantification of subsequent infestation;

(e) purifying and quantifying microbial spores from selected cadavers to identify one or more microbial pesticides with improved virulence; and is

Wherein the microbial insecticide is exposed to a mutagen.

15. The method of claim 14, further comprising infecting one or more second pest populations with the microbial spores from step (e).

16. The method of claim 14 or 15, wherein steps (a) to (e) are repeated at least once and optionally the microbial pesticide is repeatedly exposed to the mutagen.

17. The method of claims 14 to 16, wherein during step (c), the carcass is selected based on the total mortality rate of the pest population reaching 25% to 30%.

18. The method of claims 14 to 17, wherein a single clone is selected from the purified microbial spores of step (e) to infect other pest populations.

19. The method of claim 18, wherein the monoclonal is selected such that it is further infected at least twice every 5 generations.

20. The method of claims 15-19, wherein the one or more pest population populations comprise a genetically variant population.

21. The method of claims 15 to 20, wherein the pest population comprises different levels of resistance to the microbial pesticide or one or more toxic compounds produced by the microbial pesticide.

22. The method of claims 15 to 20, wherein the pest population comprises a moderate level of resistance to the microbial pesticide or one or more toxic compounds produced by the microbial pesticide.

23. The method of claims 14 to 22, wherein the method is used to provide a microbial pesticide library.

24. The method of claim 23, wherein the library is formed from a plurality of microbial pesticides having increased virulence compared to the virulence of a wild-type microbial pesticide.

25. The method of claims 14 to 24, wherein the mutagen comprises Ethyl Methanesulfonate (EMS).

26. The method of claim 25, wherein the microbial insecticide is incubated with 0.1% to 0.5% EMS.

27. The method of claim 25, wherein the microbial insecticide is incubated with about 0.2% EMS.

28. The method of claims 14 to 24, wherein the mutagen is incubated with the selected pest carcass prior to inoculation with the sporulation medium.

29. The method of claim 28, wherein the mutagenic agent is removed prior to inoculating the sporulation medium.

30. The method of claims 14 to 29, wherein the microbial pesticide comprises a bacterium.

31. The method according to claim 30, wherein the bacterium is mutated by inactivating or disrupting its mutS gene.

32. The method of claim 30 or 31, wherein the bacteria comprise bacillus thuringiensis.

33. The method of claim 32, wherein the bacillus thuringiensis comprises bacillus thuringiensis enterobacter subspecies insecticidally, bacillus thuringiensis kustak subspecies, or bacillus thuringiensis catzu subspecies.

34. The method of claims 14 to 33, wherein the pests include one or more of: aphids, other hemipteran pests, thrips, lepidopteran larvae, dipteran larvae, coleopteran larvae, leaf mites, locusts, crickets, ants, cockroaches, flies, wasps, termite wood worms, wood ants, book worms, silverfish, bark beetles, clothiantus gibsoniana, chiggers, mange, ticks, mites, lice, fleas, bugs, mosquitoes, tsetse flies, bug bugs, root knot nematodes, soybean cyst nematodes, potato cyst nematodes, slugs, and snails.

35. The method as claimed in claims 14 to 35, wherein the pests include plutella xylostella and larvae thereof.

36. A microbial insecticide library comprising a plurality of microbial insecticides according to claims 1 to 13 and/or microbial insecticides produced by the method of claims 14 to 35.

Technical Field

The present invention relates to a strain of Bacillus thuringiensis (Bacillus thuringiensis) and its use, in particular its use as an insecticide. The invention also relates to compositions comprising the bacillus thuringiensis strains. Furthermore, the present invention relates to methods of using selection among host pest populations to passage microbial pesticides to increase toxicity of the microbial pesticides.

Background

Existing pest control technologies have limitations. The efficacy of microbial pesticides and Genetically Modified (GM) crops is constantly threatened by the evolution of resistance as are their chemical or synthetic counterparts. Prior to selection, some important pests have relatively low susceptibility to the existing bacillus thuringiensis (Bt) toxins, while several species of the order lepidoptera, especially Plutella xylostella (Plutella xylostella), have developed resistance in response to the use of microbial Bt insecticides. Increased resistance to GM crops, associated with field control failure, also began to emerge in some cases (1, 2).

One strategy to combat resistance is to rely on the discovery of novel toxins. A large number of Bt strains with a very diverse Cry toxins have been isolated and characterized. However, although many toxins are known, there are few good toxins that can be matched to the potency and broad host range of one particular class (Cry1 group). Most field resistance develops in response to these toxins; the Cry1 toxin is the most common type of toxin engineered for GM plants (2). If such efficacy defeats resistance, there are few similarly effective alternatives. Thus, an alternative approach is to try and modify existing well-characterized proteins to increase toxicity to resistant or tolerant pests (3, 4). A second commercially important benefit of modifying currently utilized proteins is that licensing is often easier and cheaper than completely new products.

In response to this preference for modified toxins, several companies have used random toxin mutagenesis ("directed evolution") as a means to discover toxin variants with desirable properties. Older methods of screening mutant libraries have relied on a combination of cumbersome bioassays and sequencing. Recently, a successful approach has improved the "phage display" approach to developing synthetic in vitro screens for mutagenized Cry proteins (5). However, this in vitro method does have the limitation of producing Cry proteins (5) sensitive to insect larvae gut proteases. This is also a process bound to single protein-protein interactions, however, in general, protein engineering-based methods can only be applied to biocontrol methods based on the expression of transgenes that modify proteins. The original and still very successful application of Bt is as a microbial product based on spores and toxins. Methods of artificial selection for improving biocontrol products at the organism level may still be valuable for strain improvement in the traditional biocontrol field.

Microbial experimental evolution, while still a small area, has had a significant impact on the biology of evolution and solved fundamental co-evolution, ecological and population genetics problems (6-9). Many microorganisms, including Bt, can rapidly acquire a large number of beneficial mutations under laboratory conditions (8, 10, 11). Experimental evolution has provided a solution for overcoming insect resistance without control and may be applicable to many insect pests and many pathogens.

Experimental passaging of insect viruses has been successfully used to increase the virulence of biocontrol agents and to identify genotypes with improved effects against resistant hosts (12). The high mutation rate of the virus makes it a suitable target for improvement by selection. One way to increase the rate at which a bacterial pathogen (e.g., Bt) adapts to a resistant or tolerant host is to increase its mutation rate. In an evolving bacterial population, highly mutated strains or "mutators" (13) (deficient proofreading genes) are associated with a large proportion of new beneficial mutations. The theory that increased mutation rates confer increased adaptation rates to resistant hosts is widely supported. For example, incremental variants are often favored through strong selection (14, 15) and co-evolution military competition (16). The mutator variants evolve higher virulence in model insect hosts compared to the wild type strain (17). Under difficult selection, i.e. when the phenotype of an organism must exceed a certain threshold in order for it to reproduce, an augmented variant may be more capable of conferring enhanced fitness. This type of selection may be common among pathogens, which may have to express some threshold level of virulence factors in order to enter the host (18, 19). In small populations with limited mutation supply, high mutation rates are expected to bring more benefits (18, 19). Bt experiences a very narrow population bottleneck (20, 21) in each round of infestation, so the effective population size can be small. Mutator variants have a higher recombination rate (22), which may facilitate large-scale genetic changes. Although mutator variants can acquire deleterious mutations and are expected to be eliminated by long-term selection, experimental evolution suggests that they can persist for thousands of generations in populations without being eliminated by reversion or recombination (19).

An important recent development in understanding microbial virulence is the application of the theory of "social evolution", i.e. the notion associated with maintaining cooperation or his traits (7, 24, 25). Microorganisms participate in a form of cooperative behavior known as public goods production. From an economic perspective, public goods are goods or services that benefit the entire community but are costly to individuals. In bacteria, this public availability occurs through long-range secretion of metabolites into the extracellular space. Bacterial public goods include biofilms, non-spore components of fruiting bodies, virulence factors and enzymes important for nutrient acquisition (25-28). Importantly, this includes the Cry toxins produced by Bt and to some extent a series of quorum-sensing regulated nutrient-producing factors (21, 28) that are critical to enabling these bacteria to invade the insect host. Two key factors are important for maintaining synergistic virulence: high correlation (or low diversity within a group) and population structure that promotes competition between groups (7). Relevance is critical, as competition between genotypes will favor "cheater" mutants, which have nullified expensive virulence production in favor of rapid growth, by diversified invasion. Population structure is the division of a population into uniform groups, which facilitates group-level selection of traits that favor groups over individuals.

It is an object of the present invention to overcome one or more of the problems associated with current pest control. It is another object to provide an improved pest control. Preferably, pest control will utilize microorganisms such as bacillus thuringiensis, and the strains will have higher virulence characteristics. It would be desirable to develop a method that would enable bacillus thuringiensis to evolve into diverse strains and to select the most effective/most virulent strain from the diverse pool and improve virulence.

Disclosure of Invention

According to one aspect of the invention there is provided a bacillus thuringiensis strain which is phenotypically stable and has increased virulence compared to a wild-type strain, wherein the increased virulence is achieved by exposing the strain to a mutagen in one or more passages.

According to another aspect of the present invention there is provided a strain of Bacillus thuringiensis comprising Bacillus thuringiensis subspecies NCTC17080301 or Bacillus thuringiensis subspecies NCTC17080302 or a mutant or derivative thereof.

Preferably, the bacillus thuringiensis strain has increased virulence relative to its ancestor. The increase in virulence of the bacillus thuringiensis strain may be at least 10-fold greater than its ancestral virulence. More preferably, the increase in virulence of the bacillus thuringiensis strain may be at least 100-fold greater than the virulence of its ancestor.

The mutagen may include Ethyl Methanesulfonate (EMS).

The bacillus thuringiensis strain can be used as an insecticide. Preferably, the bacillus thuringiensis strain has increased virulence against the pest relative to its ancestor.

It has been found that the strains of the invention advantageously have enhanced killing ability against target pests, including those pests having at least some degree of pesticide resistance.

The bacillus thuringiensis strains are useful for controlling pests, including insects, mites, nematodes and gastropods. The bacillus thuringiensis strain may be used to control one or more of: lepidoptera, diptera, coleoptera, and nematodes.

Bacillus thuringiensis is preferably used to control one or more of: aphids, other hemipteran pests, thrips, lepidopteran larvae, dipteran larvae, coleopteran larvae, leaf mites, locusts, crickets, ants, cockroaches, flies, wasps, termite wood worms, wood ants, book worms, silverfish, bark beetles, clothianidin gypsy, chiggers, mange mites (Sarcoptes scabies), ticks, mites, lice, fleas, bed bugs, mosquitoes, tsetse flies, bugs, root knot nematodes, soybean cyst nematodes, potato cyst nematodes, slugs, and snails.

The bacillus thuringiensis strain can be used for controlling insects. Preferably, the bacillus thuringiensis strain is used for controlling lepidopterans. The Bacillus thuringiensis strain can be used for preventing and treating diamondback moth (Plutella xylostella) and larva thereof.

The bacillus thuringiensis strains are useful for controlling pests of plants, including agricultural crops. Preferably, the bacillus thuringiensis strain is useful for controlling pests of crucifer (Brassicaceae) plants. In particular, the bacillus thuringiensis strain can be used for controlling pests of crucifers. The bacillus thuringiensis strain can be used for controlling pests of the following plants: horseradish, cress (land crop), brassica juncea, kale, green cabbage, kale, cabbage, brussels sprouts, kohlrabi, broccoli, cauliflower, wild broccoli, cabbage, komatsuna, japanese turnip, watercress, cabbage, chinese cabbage, turnip root, turnip cabbage (swedish), siberian kale, rape/rapeseed, cabbage mustard, mustard seed, white mustard seed, black mustard seed (black mustard seed), white cabbage, wild sesame, arugula, field bell pepper, maca, cress, watercress (watercress), radish (small radish), radish root (daikon), and horseradish.

The bacillus thuringiensis strain may be applied to the soil of the plant, near the soil or in the soil. The bacillus thuringiensis strain may be applied to the leaves, stems, flowers and/or roots of the plant.

Bacillus thuringiensis strains have similar levels of potency to Cry-susceptible insects as their ancestors. Thus, an effective application rate for Cry-susceptible insects can be per cm²The surface area of the crop is 0.01mg to 0.1mg of Bacillus thuringiensis strain or per cm²The surface area of the crop is 10,000 to 100,000 Bacillus thuringiensis spores. Although the effective application rate to Cry1A resistant insects will depend on the exact level and efficacy of resistance of the bacillus thuringiensis strain to that genotype, it can be in the range of each cm²0.01mg to 0.5mg of Bacillus thuringiensis strain per cm of surface area of the crop²The surface area of the crop is 10,000 to 500,000 Bacillus thuringiensis spores.

Preferably, the Bacillus thuringiensis strain may comprise Bacillus thuringiensis entomococci, Bacillus thuringiensis catuazawai, or Bacillus thuringiensis kurstaki.

According to a further aspect of the invention there is provided a strain of bacillus thuringiensis comprising SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or related sequences having 95% or more homology thereto.

The bacillus thuringiensis strain may be a bacillus thuringiensis subspecies custarkii strain.

If the Bacillus thuringiensis strain comprises a related sequence, it is preferred that the related sequence has up to 96% or more homology with SEQ ID No.2 and/or SEQ ID No.3 and/or SEQ ID No.4, more preferably that the related sequence has 97% or more homology with SEQ ID No.2 and/or SEQ ID No.3 and/or SEQ ID No. 4. Even more preferably, the related sequences have 98% or more homology with SEQ ID No.2 and/or SEQ ID No.3 and/or SEQ ID No. 4. Most preferably, the related sequences have 99% or more homology with SEQ ID No.2 and/or SEQ ID No.3 and/or SEQ ID No. 4.

It will be apparent to those skilled in the art that any feature, integer, property, or compound described in connection with certain aspects of the invention may be equally applied to other aspects of the invention unless incompatible therewith.

According to another aspect of the invention there is provided a composition comprising a strain of bacillus thuringiensis comprising bacillus thuringiensis subspecies NCTC17080301 or bacillus thuringiensis subspecies NCTC17080302, or a mutant or derivative thereof.

According to another related aspect of the invention, there is provided a composition comprising a strain of bacillus thuringiensis comprising SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or related sequences having 95% or more homology thereto.

The bacillus thuringiensis strain may be added to the composition in the form of spores or crystals.

The composition may be in the form of a solid or a liquid.

The composition may further comprise one or more of the following ingredients: wetting agents, thickeners, viscosity modifiers, stabilizers and surfactants. The composition may comprise a dispersion medium, such as water or an edible oil.

The composition may be used as or as an insecticide.

The composition may be applied to (or near) the surface of the plant and/or soil. Alternatively, the composition may be incorporated into the soil itself. The composition may be applied to the plant and/or soil by spraying, spreading or sprinkling. The composition may be applied to the foliage, stems, flowers and/or roots of the plant.

In another aspect of the present invention, there is provided a method of improving the toxicity of a microbial pesticide, the method comprising:

(a) dividing pest populations into subpopulations;

(b) contacting a subpopulation of pests with one or more microbial pesticides;

(c) selecting a pest carcass from the subpopulation having the highest mortality rate;

(d) inoculating the sporulation medium with the pest carcass to provide sufficient inoculum for easy quantification of subsequent infestation;

(e) purifying microbial spores from the selected cadavers and quantifying the microbial spores to identify microbial pesticides with improved virulence; and is

Wherein the microbial insecticide is exposed to a mutagen.

The method can further comprise infecting one or more second pest populations with the microbial spores from step (e).

Steps (a) to (e) may be repeated by infecting one or more pest populations with the purified microbial spores of step (e). Steps (a) to (e) may be defined as passaging, and the resulting microorganism may be defined as a passaged microorganism. Optionally or preferably, the microbial pesticide is repeatedly exposed to the mutagen at each passage. Alternatively, the microbial pesticide may be exposed to the mutagen during alternate passages or only during primary passage. The purified spores from step (e) can be used to infest at least 2 pest populations. Preferably, steps (a) to (e) are repeated at least once. Steps (a) to (e) may be repeated at least 5 times.

Cadavers may be selected during step (c) based on total mortality of the pest population reaching 20% to 40%. Preferably, the carcass is selected during step (c) based on the total mortality rate of the pest population reaching 25% to 30%.

The above method provides group level competition for mortality in insect herds, which advantageously means that the pathogen is selected only from groups with high mortality levels for further passage.

The one or more pest populations can include a genetically variant population. This enables the pathogen to adapt to hosts that are difficult to kill. Pest populations may include varying levels of resistance to microbial pesticides or one or more toxic compounds produced by microbial pesticides. The pest population may include a moderate level of resistance to the microbial pesticide or one or more toxic compounds produced by the microbial pesticide.

The method may be used to provide a microbial pesticide library. In certain embodiments of the invention, there is provided a microbial pesticide library produced by the above method. The library may be formed from a plurality of microbial pesticides having increased virulence compared to the virulence of the wild-type microbial pesticide.

The mutagen may comprise Ethyl Methanesulfonate (EMS). In this method, the microbial pesticide may be incubated with 0.05% to 1.0% EMS. The microbial pesticide may be incubated with 0.1% to 0.5% EMS, 0.1% to 0.4% EMS, 0.1% to 0.3% EMS, 0.1% to 0.2% EMS. Most preferably, the microbial insecticide is incubated with about 0.2% EMS.

The mutagen is preferably incubated with the selected pest cadavers prior to inoculation with the sporulation medium. Also preferably, the mutagen is substantially removed prior to inoculation with the sporulation medium. Removal can be by a variety of methods, such as centrifugation.

The purified microbial spores from step (e) can be used as a pesticide.

The microorganism may be a pesticide. The microorganism may be a pesticide with a defined pathogenicity. Microbial pesticides may include bacteria. Preferably, the bacteria comprise bacillus thuringiensis. The Bacillus thuringiensis strain may be Bacillus thuringiensis Enterobacter insecticidal subspecies, Bacillus thuringiensis Kustak subspecies or Bacillus thuringiensis Clazis subspecies. Preferably, the bacillus thuringiensis strain comprises bacillus thuringiensis subsp. kustax, bacillus thuringiensis catus, bacillus thuringiensis tenebrionis, bacillus thuringiensis israelensis or any other insect or nematode pathogenic bacillus thuringiensis strain.

The pest may be an insect, mite, nematode or gastropod. Preferably, the pests are aphids, other hemipteran pests, thrips, lepidopteran larvae, dipteran larvae, coleopteran larvae, leaf mites, locusts, crickets, ants, cockroaches, flies, wasps, termite wood worms, wood ants, book moths, silverfish, bark beetles, spiderworm gypsophila, chiggers, scabies (Sarcoptes scabies), ticks, mites, lice, fleas, bed bugs, mosquitoes, tsetse flies, lygus bugs, root knot nematodes, soybean cyst nematodes, potato cyst nematodes, slugs, and snails.

Preferably, the pests are of the lepidoptera order. The pest may be diamondback moth (Plutella xylostella) and its larvae.

The microorganism may be mutated. Microorganisms can be mutated by chemical mutagenesis. Preferably, the microorganism is a bacterium mutated by inactivating the mutS gene. Advantageously, high mutants or "mutagens" are associated with a high proportion of new beneficial mutations, and an increased mutation rate may confer an increased adaptation rate to a resistant host. Mutators also have higher recombination rates, which can facilitate large-scale genetic changes.

In another aspect of the present invention, there is provided a method for screening for improved virulence in microorganisms comprising:

growing the purified microbial pesticide spores;

infesting pest populations; and

the number of pest deaths was recorded.

The pest population can be infested with at least 1 dose of the microbial pesticide spores. Preferably, the pest population is infested with at least 3 doses of microbial pesticide spores. May be present at 10 per microliter¹To 1.2X 10⁵And (4) spores.

In another aspect of the invention, there is provided a microbial and/or biopesticide produced or identified by using the above method.

Detailed Description

Embodiments of the invention will now be described, by way of example only, in which:

FIG. 1 is a schematic diagram showing the steps of infecting an insect population with bacterial spores according to the group selection and pool selection methods.

FIG. 2 is a bar graph showing early larval mortality of Cry1Ac resistant plutella xylostella larvae exposed to the ancestors Bacillus thuringiensis subspecies kustoke (wt anc), undeveloped Bacillus thuringiensis subspecies kustoke, and evolutionary processing. Data are mean and SE pooled across 2 replicate bioassays (4 replicate lineages treated with 3 doses each);

fig. 3 is a panel of 6 charts showing the total mortality of Cry1Ac resistant plutella xylostella larvae exposed to the ancestors bacillus thuringiensis subspecies (wt anc), undeveloped bacillus thuringiensis subspecies, and evolutionary treatment. Data are the proportion of deaths per dose (n-50 to 60 larvae) with different symbols representing repeat lineages in the treatment. The line is a fitted statistical model (inverse transformed into probabilities) fitted for each lineage and each biometric. The line type represents (dashed or solid) an independent biometric. The dose is viable spores per microliter;

fig. 4 is 2 graphs showing the efficacy of the best performing wild type clone (p3c2) against plutella xylostella resistant and susceptible to bacillus thuringiensis subspecies in comparison to its ancestor and an alternative commercial Bt isolate (XenTari) for resistant larvae. Data are 5 days of initial mortality from bioassay (60 larvae per dose);

FIG. 5 is 2 graphs showing the relative fitness of selected evolved clones p3c1 (Bacillus thuringiensis subspecies Custak NCTC 17080301) and p3c2 (Bacillus thuringiensis subspecies Custak NCTC17080302) with increased virulence relative to the ancestor over the range of initial frequencies. The data is based on the ratio of the estimated markas parameters; the dashed line represents the fitted linear model, where SE is grey. Each treatment N ═ 40 to 50 larvae;

fig. 6 is 2 graphs showing data against Cry1Ac resistant and susceptible larvae.

Fig. 7 is a graph showing alternate background clonal bioassays for Cry1Ac susceptible larvae.

FIG. 8 is 3 graphs showing bioassays of resistance and co-evolving passaging treatments for co-evolving larvae, Cry1Ac resistant larvae and Cry1Ac susceptible larvae; and

fig. 9 is a graph showing the results of bioassays to evaluate the toxin classes of the initial mixture, HCO and HCO + EMS in a toxin library selection experiment.