Insecticidal Bacillus Thuringiensis Strains With Activity Against Lepidoptera

The present invention relates to novel bacterial strains, and in particular to novel strains of the bacterium Bacillus thuringiensis and uses therefor.

The organism Bacillus thuringiensis produces a protein crystal endotoxin which kills insects. It is not however toxic to mammals. It is thus very useful as an agricultural insecticide, in particular against Lepidoptera, and strains of Bacillus thuringiensis have been used as agricultural insecticides for a number of years.

The strain of Bacillus thuringiensis which is most commonly used commercially is HD-1 (available from the collection of Bacillus thuringiensis strains maintained by the U.S. Department of Agriculture). We have now discovered novel strains of Bacillus thuringiensis having generally similar properties to HD-1, but distinguished therefrom by improved insecticidal activity against a range of lepidopteran pests.

According to the present invention we provide the novel strains A20 and A29 of Bacillus thuringiensis, deposited on Oct. 20, 1987 at the National Collection of Industrial and Marine Bacteria (Torry Research Station, P.O. Box 31, 135 Abbey Rd., Aberdeen, AB9 8DG, United Kingdom) under the accession numbers NCIB 12570 and NCIB 12571, respectively. We further provide novel insecticidal compositions characterised in that they contain a .delta.-endotoxin produced by said strains A20 or A29, and a method of protecting plants from insect attack which comprises exposing the insects to a .delta.-endotoxin produced by said strains A20 or A29.

The strain A20 was isolated from soil found under a cedar tree in Coburg, Ontario. The strain A29 was isolated from a colorado potato beetle larva. In morphology and general biochemical properties they are both generally similar to HD-1. The morphology of the strains is compared in Table 1.

Biochemical properties of the strains are compared in Tables 2-4.

In view of these similarities, it is surprising that A20 and A29 show significantly improved insecticidal activity over a range of lepidopteran pests as compared with HD-1.

The strains according to the invention may be prepared in any quantity required by fermenting a sample of NCIB 12570 or NCIB 12571 obtained from the National Collection of Industrial and Marine Bacteria under suitable conditions in an appropriate medium. Such conditions and media are well known to the art. The media will, for example, generally contain a nitrogen source (e.g. fish protein) and a carbohydrate source such as starch. Suitable conditions include a temperature in the range 15.degree.-45.degree. C., and an approximately neutral pH. Fermentation most conveniently carried out in batches, typically for periods of 1-3 days.

Insecticidal compositions according to the invention may be obtained from the fermentation liquor by concentration, for example by centrifugation or filtration followed by addition of any desired and appropriate formulating agents. Formulating agents which may be useful include for example surface active agents, e.g., wetting agents: solid diluents, dispersing agents and UV stabilisers. If desired, solid formulations may be prepared by known methods.

The process of the invention is generally carried out by spraying on to plants infested with or liable to infestation by insects insecticidal compositions as described above diluted with a diluent such as water. The insecticidal agent is the toxic endotoxin: if desired this may be applied to the plants or insects infesting them independently of the bacteria that produce it, but separation of the crystalliferous protein from the bacteria is generally not necessary.

One method of carrying out the process of the invention is to arrange for the plant susceptible to insect attack to produce the .delta.-endotoxin in situ. This is done by cloning a .delta.-endotoxin gene from either of the strains NCIMB 12570 or 12571, by known methods, providing it with a suitable promoter (for example the CaMV35S promoter) which will cause expression of the gene in plants, and transforming the plant by known methods (e.g., the use of Ti plasmids). Such processes are described in more detail in EPA 142924 (Agrigenetics), the disclosure of which incorporated herein by reference.

Insects which are most effectively combatted by the process of the invention are lepidoptera, for example, those in Table 5 below.

The process of the invention may be used to protect a wide variety of plants prone to infestation by lepidoptera. Specific examples of commercially important plants to be protected by the invention are maize (corn) and conifers: as well as rice, small grain cereals such as wheat and barley, and vegetables including lettuce, tomatoes and sugar beet.

The following Examples illustrate the invention.

The soil sample found under a cedar tree in Coburg, Ontario was diluted by placing 0.5 g of the sample into a dilution bottle containing 45 ml of 0.05% peptone to give a 10.sup.-1 dilution. The sample was then heat treated by transferring it to a 60.degree. C. water bath for 10 minutes. Sequential dilutions were made by taking 0.5 ml of the sample with the highest dilution and placing it into 4.5 ml of 0.05% peptone (e.g. 0.5 ml of the 10.sup.-1 dilution into 4.5 ml peptone diluent gives a 10.sup.-2 dilution); this was repeated until a 10.sup.-5 dilution was obtained. B.cereus selective medium (Bacillus cereus Agar base, Code CM617 from Oxoid, Canada) and esculin agar (in g/L of water: esculin 1.0; ferric citrate 0.5; peptone 10; NaCl 5; Agar 20) were used to plate 10.sup.-3 to 10.sup. -6 dilutions. The plated samples were incubated at 30.degree. C. for 5 days and then examined for potential B.t. colonies. Slides were made of the chosen colonies using the Smirnoff staining procedure and were examined under the microscope (at a magnification of 1000x using the oil immersion lens) for the presence of darkly staining parasporal crystals which were usually but not necessarily bipyramidal in shape. Crystal-positive colonies were streaked onto nutrient agar in order to ensure a pure culture, and incubated for another 5 days at 30.degree. C. The Smirnoff staining procedure was repeated to confirm crystal presence, and to check for purity. Purified colonies were transferred to nutrient agar slants and stored in a refrigerator at 40.degree. C. for further use.

The source of the A29 strain was a dead Colorado Potato Beetle Larva. The insect was ground in a sterilised mortar and a pestle with a small amount of the peptone diluent. The liquid was recovered using a pasteur pipette, and placed into a test tube containing 4.5 ml of the peptone diluent (0.05% peptone in water), this was considered a 10.sup.-1 dilution. The sample was then heat treated by transferring it to a 60.degree. C. water bath for 10 minutes. Sequential dilutions were made by taking 0.5 ml of the sample with the highest dilution and placing it into 4.5 ml of 0.05% peptone (e.g., 0.5 ml of the 10.sup.-1 dilution into 4.5 ml peptone diluent gives a 10.sup.-2 dilution); this was repeated until a 10.sup.-5 dilution was obtained. B. cereus selective medium (Bacillus cereus agar base, Code CM617 from Oxoid, Canada) and esculin agar (in g/1 of water: esculin 1.0; ferric citrate 0.5; peptone 10; NaCl 5; Agar 20) were used to plate 10.sup.-3 to 10.sup.-6 dilutions. The plated samples were incubated at 30.degree. C. for 5 days and then examined for potential B.t. colonies. Slides were made of the chosen colonies using the Smirnoff staining procedure and were examined under the microscope (at a magnification of 1000x using the oil immersion lens) for the presence of darkly staining parasporal crystal which was usually but not necessarily bipyramidal in shape. Crystal-positive colonies were streaked into nutrient agar in order to ensure a pure culture, and incubated for another 5 days at 30.degree. C. The Smirnoff staining procedure was repeated to confirm crystal presence, and to check for purity. Purified colonies were transferred to nutrient agar slants and stored in a refrigerator at 4.degree. C. for further use.

Inoculum of A20 was transferred from a slant to a 250 ml Erlenmeyer flask containing 100 ml of CRL No. 1 medium (in g or ml/liter of water: nutrient broth 8; glucose 6; yeast extract 5; xylose 0.5; cotton seed flour extract 30 ml; corn steep liquor 3.2 ml; Mary Mendel's salt mixture 1 ml) and incubated with agitation at 30.degree. C. and 300 rpm. After 24 hours, the entire 100 ml was used to inoculate 1 liter of the same medium in a 2L flask; this was incubated with agitation for 5 days at 300 rpm at 30.degree. C.

A29 was propagated in an identical manner.

Upon completion of the fermentation cycle, A20 bacteria was harvested by first separating the B.t. spores and crystals from the fermentation broth by centrifugation or microfiltration. The recovered spores and crystals were resuspended in 100 ml of water and formulated into a liquid concentrate by adding 4.9 g of Morwet D-425 (dispersing agent), 4.9 g of Veegum HV (suspending agent), 4.9 ml of Tween 80 (wetting agent) and 24.4 ml of Sorbo (anti-freezing agent). Each ingredient as added separately in order stated above. The product was kept at 40.degree. C. prior to use. A similar formulation was made up using A29, and also HD-1; the latter was used as a control.

Using the formulations described in Example 4, A20 and A29 B.t. strain spores and crystals mixtures, obtained as described in Example 3, were tested against Spruce budworm in a foliage assay. Branches of fir-tree naturally infested with the insect (4-5th instar larvae) were sprayed using a backpack sprayer model MRY-2 (from Hartvig Jensen & Co., Denmark). For A20 and HD-1, the size of the drops (in diameter) were ranging from 20 to 120 um for an average of 32 .mu.m. For A29 the drop diameter ranged from 20 to 60 .mu.m for an average of 30 um. In all cases there were approximately 11 drops per needle. All strains were sprayed at a concentration of 30.times.10.sup.9 IU/hectare. The treated branches were incubated in a growth chamber until pupation. The results were expressed by the percentage of mortality ([number of dead insects/number of dead insects+number of living insects].times.100).

In this example, A20 was 5.1.times.more active, and A29 3.53.times.more active than HD-1, based on their potency ratios.

After the 5 day incubation carried out in Example 3, the cells, spores and crystals were harvested by centrifugation and acetone-precipitated. The resulting powder was incorporated into an artificial diet suitable for the tested insect. The diet was fed to the insects and mortality recorded using standard methodology. Comparative tests were carried out using HD-1.