INSECTICIDAL BACTERIA

FIELD OF THE INVENTION

The present invention is generally in the field of insecticides and concerns preparations and methods for combatting crop insect infestations. More specifically, the present invention provides such preparations and methods where the active ingredient is an insecticidal bacteria, dead bacterial cells or components thereof. The invention further concerns novel bacteria possessing an insecticidal activity.

BACKGROUND OF THE INVENTION Infestation of crops by insects is a major cause of reduction of crop yield. While chemical insecticides have been used for decades to combat such infestation, it is today a growing tendency, given the environ¬ mental hazards associated with the use of such chemicals, to shift towards biological control means. Bacteria of the species Photorhabdus have the capability to infect insects with the infection having the effect of killing their insect host. Photorhabdus bacteria are carried by nematodes and their infectious life cycle involves the use of the nematodes as an infection vector. Indeed, it has been believed in the art that the Photorhabdus infection depends on the nematode host. Accordingly, it has been suggested to use Photorhabdus carrying nematodes as insecticidal agents (Bedding, R.A. and Miller, L.A. Env. Entomυl, 10:449-453, 1981).

SUMMARY OF THE INVENTION

In accordance with the invention it has surprisingly been found that contrary to previous belief, certain Photorhabdus luminescens (P.luminescens; also known as Xenorhabdus luminescens) bacteria have the capacity to infect insects without dependency on a nematode carrier as an infection vector. In accordance with the invention some novel P.luminescens strains have been isolated and obtained in a pure form and found to be highly effective as insecticidal agents. Such bacteria, to be referred to herein at times as "insecticidal bacteria " ', may be used to combat insect infestation of crops in agriculture or horticulture.

In addition it was found in accordance with the invention that the insecticidal activity is also, at least partially, retained by inactivated bacterial cells, namely the insecticidal activity does not necessarily depend on the viability of the insecticidal bacteria. Still further, it was found that at least some insecticidal activity is also manifested by broken inactivated insecti¬ cidal bacteria, as well as by the supernatant obtained from a culture of the insecticidal bacteria. The active ingredient in the supernatant may be a protein or a proteinaceous substance, as evidenced by the loss or partial loss of activity after exposure to a protease. The invention thus provides, by one of its aspects, an insecticidal composition, comprising insecticidally effective amounts of an active ingredient selected from the group consisting of: i. bacteria having insecticidal activity belonging to the species Photorhabdus luminescens, the insecticidal activity being manifested without the need for a nematode as an infection vector; ii. inactivated P.luminescens cells; iii. fragments of inactivated P.luminescens cells; and iv. supernatant of cultures of P.luminescens cells, a fraction thereof possessing the insecticidal activity, or insecticidally active substance derived from said cells.

A bacteria having insecticidal activity, in the context of the invention, is used to denote a bacteria which can infect an insect and cause its death, inhibition of its growth, effect its motility, etc. The end result of such insecticidal activity is a reduction for inhibition of insect infestation of the plants or crops. The insecticidal bacteria of the invention are unique in that the insecticidal activities are also manifested if applied by themselves without a nematode vector.

Also provided by the invention is a method for treatment of insect infestation of plants comprising administering to plants or to their surround- ings an effective amount of said active ingredient.

The term "treatment" in the context of the invention should be understood as encompassing both treatment of an acute infestation as well as treatment intended for prevention of insect infestation prior to its occurrence. The bacteria may be administered on either or both of the plants' aerial parts as well as to the ground. The administration may be by spraying, by delivering the composition through the irrigation water, as well as by administration of a composition in a dry particulate or powder form.

The composition may either contain said active ingredient alone or in combination with one or more additional insecticidal agents such as another insecticidal microorganism or chemical insecticide, in both cases together with a suitable carrier. Such a carrier may be any one of those known in the art, e.g. natural or regenerated mineral substances, diluents, dispersants, wetting agents, tackifires, binders or fertilizers. The present invention also provides by a further of its aspects, a pure culture of novel insecticidal bacteria of the species P.luminescens. Particularly preferred are such bacteria belonging to a new strain purified in accordance with the invention which is termed herein as "XP01 ". The XP01 strain has been deposited in the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25, rue du Dr. Roux, Paris 15eme having the CNCM Accession No. 1-1760 with the deposit date being July 23, 1996. It will be appreciated that this strain is but an example and

other strains of P.luminescens which possess an insecticidal activity may also be used in accordance with the invention.

For treatment, an effective amount of said active ingredient will be applied onto plants, plant parts, or introduced into the plant environment, particularly into the soil. An effective amount is to be understood as an amount of said active ingredient sufficient to cause mortality of a substantial portion of the insects, an amount effective in reducing the level of activity of the insects, an amount effective in reducing the average weight of the insect, an amount effective in reducing the insect biomass, etc. Where said active ingredient is used for prevention of insect infestation, an effective amount would be an amount which is effective in inhibiting development of the insects to yield a population which will be damaging to the plants or the plants' crops.

Said active ingredient may be applied in either acute or preventive treatment, in a number of administration modes. For example, said active ingredient may be mixed with the irrigation water and thus be applied to either aerial parts of plants or to the soil (depending on the type of irrigation) via the irrigation water. By another example, liquid formulation comprising said active ingredient may be sprayed, particularly onto aerial plant parts, e.g. using conventional sprayers. By another example, said active ingredient may be mixed with soil, e.g. pot soil, for the purpose of prevention of insecticidal infestation of the plants after planting. By a further example, plant parts, and particularly seeds, are impregnated with a solution comprising said active ingredient. The active ingredient may also be used for post-harvest protection of crops, which may be achieved by spraying or impregnating the crops with the active ingredient, either pre or after harvest.

As will be appreciated by the artisan, the treatment regime of said active ingredient will vary depending on the kind of insects to be controlled, the nature of the plants to be treated, the method of application of the composition comprising the bacteria, etc. In some cases, a single treatment may suffice, while in other cases, the infected plants will have to be treated

- -> -

over a period of time by recurrent application of the composition. The dose of the administered active ingredient within a treatment will also vary depending on the above and other factors.

The bacteria of the invention may be obtained by isolation from cadavers of larvae of insects which were incubated with nematodes carrying insecticidal bacteria. Haemolymph obtained from the larvae cadavers may be incubated in a suitable medium wherein bacterial colonies develop and single bacterial colonies may then be selected and purified.

The insecticidal bacteria may be cultivated to obtain large amounts by fermentation in a suitable growth medium for various periods of time. The fermented broth which is harvested after fermentation and contains large amounts of the selected bacteria will hereinafter be referred to as "ferment".

Said active agent was found to be effective in particular against Lepidoptera such as Mamestra brassicae, Spodoptera littoralis, Helicoverpa armigera, Agrotis ipsilon, Scotia segetum and Lobesia botrana.

The invention will be illustrated further by some specific embodiments described in the following examples and in the annexed drawings:

DESCRIPTION OF THE DRAWING

The Figure shown in the annexed drawing is a graphic representation of results of an experiment in which the insecticidal activity of different preparations derived from various dilutions of P.luminescens XPOl bacteria was tested. The effect which was tested was a change in weight of 5-days old Mamestra brassicae larvae five days after adminis¬ tration of the preparation (the initial weight of the larvae averaged 6.33 mg). The P.luminescens ferment from which the preparation were derived was a 19 hour culture. Different samples were tested as follows: Sample A19: Cells from an XPOl ferment washed three times in a basic

Ringer solution (pH 9);

Sample B19: Cells from an XPOl ferment washed three times in an acid

Ringer solution (pH 5); Sample C19: Cells from an XPOl ferment washed three times in a neutral

Ringer solution (pH 7); Sample D19: Supernatant of an XPOl ferment filtered on a 22 micron filter (to remove bacterial cells from the suspension); Sample E19: Non treated whole XPOl culture. Sample F19: R5 medium not containing XPOl cells incubated for 19 hours at 25°C.

EXAMPLES

In some examples below, the insecticidal activity against the various insects was determined by measuring the mortality score (%) of the neonate larvae incubated with the tested preparation. The scored mortality of the treated larvae was corrected to take into consideration the mortality of untreated control larvae usin *ge Abbott's formula as follows:

% Corrected mortality = % test mortaUty - % control mortality _{χ m}

100 - % control mortality

Example 1

The XPOl strain of Photorhabdus luminescens XPOl was isolated from cadavers of Galleria mellonella as follows: i. 10 last instar larvae of G. mellonella were placed into a petri dish with moist filter paper and approximately 1500 Dauer Juveniles of Heterorhabditis bacteriophora strain were added. The petri dish containing the larvae and the nematodes was then incubated at 25°C in the dark. ii. At the moment of insect death (after approx. 2 days of incuba¬ tion), the cadavers were washed in a petri dish with 70% methanol for 5 mins. and then rinsed in sterile demineralized water.

iii. Each inseci cadaver was opened with the help of a scissor and a needle. A drop of haemolymph from each cadaver was streaked with a sterile loop on a nutrient agar plate, iv. The nutrient agar plates were incubated at 25°C in the dark for 48 hours. After morphological observation of the developed colonies and microscopical observation of the bacteria the single P.luminescens colonies were selected and streaked on NBTA, McConkey and nutrient agar plates. v. The sub-culture of colonies as described in Step No. iv above was repeated several times in order to avoid contaminants and to ensure selection of a single colony of Photorhabdus. A pure culture of a novel strain of P.luminescens was obtained. One of such culture, termed "XPOl ", was deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on July 23, 1996, under Accession No. 1-1760.

Example 2

Serial dilutions of XPOl ferments were prepared using demineral- ized water as the diluent and the whole culture and each of the dilutions were incubated with Mamestra brassicae neonates. Mortality of the larvae was scored after 4 days of incubation at 27°C. The results are shown in the following Table 1:

Table 1

Insecticidal efficacy of the raw bacterial suspension against neonate of Mamestra brassicae at several dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control — 64 2 3.1 (untreated)

XPOl ferment non-diluted 31 31 100

XPOl ferment 1:10 32 30 93.6

XPOl ferment 1:100 32 8 22.6

XPOl ferment 1:1000 32 1 0

As seen in the above Table 1, the XPOl ferment showed insecticidal activity against the neonate larvae of M. brassicae as measured by the percent mortality of the neonate larvae.

Example 3

Samples of ferments of XPOl fermented in R5 medium at 25°C, were taken at 19, 24 and 40 hours from the beginning of the fermentation.

Part of the sample taken at 19 hours was centrifuged on a bench centrifuge at 15000 rpm for 5 mins. and then the supernatant was separated from the bacterial pellet.

The pellets from the 19 hours ferment were divided into six samples:

Sample A19: Was washed three times in a basic Ringer solution (pH 9);

Sample B19: Was washed three times in an acid Ringer solution (pH 5); Sample C19: Was washed three times in a neutral Ringer solution (pH 7);

Sample D19: The supernatant was filtered through an 0.22 micron filter to remove bacterial cells from the suspension; Sample E19: Original ferment (whole bacterial culture);

Sample F19: R5 medium which did not contain XPOl culture incubated for 19 hours at 25°C.

The samples were bioassayed against 5 day old Mamestra brassicae larvae similarly as in Example 2, and incubated for 6 days after which mortality was scored and each dose group of the surviving larvae was weighed and the average weight of the larvae calculated. As seen in the

Figure, a reduction in the weight of the larvae treated with the Samples A19,

B19, C19 and E19 was detected which was inversely proportional with the dilution of the samples. No reduction was observed using Samples D19 and F19.

The 19 hours ferment (Sample E19), the 24 hours ferment (Sample E24) and the 40 hours ferment (Sample E40) were bioassayed against neonate Mamestra brassicae similarly as above, the mortality was scored after 6 days and Lethal Dilution Rate 50% (LDR50) was calculated by probit analysis or estimated by graphical interpolation. As seen in Tables 2-4 below, the insecticidal activity of the XPOl whole culture was not affected by the fermentation length of the culture and there were no significant differences between the cultures fermented for 19, 24 or 40 hours.

Table 2

(Neonates - 19 hours)

Insecticidal efficacy of the raw bacterial suspension (harvested after

19 hours of incubation) against neonate of

Mamestra brassicae at several dilution rates

Treatment Dilution # insects % dead % mortality (corrected by Abbott's formula except control)

Control - 64 7 10.9 (untreated)

E19 non-diluted 32 32 100

E19 1:2 32 31 96.5

E19 1:4 32 32 100

E19 1:8 32 32 100

E19 1:16 31 15 42.1

E19 1 :32 32 9 19.3

E19 1:64 32 9 19.3

E19 1:128 31 5 5.9

Table 3

(Neonates - 24 hours)

Insecticidal efficacy of the raw bacterial suspension

(harvested after 24 hours of incubation) against neonate of Mamestra brassicae at several dilution rates

Treatment Dilution # in¬ # dead % mortality (corrected sects by Abbott's formula except control)

Control - 64 12 18.8 (untreated)

E24 non-diluted 32 32 100

E24 1:2 32 30 92.3

E24 1 :4 32 30 92.3

E24 1:8 32 28 84.6

E24 1:16 32 25 73.1

E24 1 :32 32 10 15.3

E24 1:64 32 6 0

E24 1 :128 32 4 0

Table 4

C - (Neonates - 40 hours)

Insecticidal efficacy of the raw bacterial suspension

(harvested after 40 hours of incubation) against neonate of Mamestra brassicae at several dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control - 64 2 3.1 (untreated)

E40 non-diluted 32 32 100

E40 1 :2 32 32 100

E40 1 :4 32 29 903

E40 1:8 32 30 93.6

E40 1:16 32 19 58.1

E40 1:32 32 8 22.6

E40 1:64 32 2 3.2

E40 1: 128 32 1 0

The approximate LDR50 for the different preparations was as follows:

E19 - about 1:17.2 dilutions E24 - about 1 : 18.5 dilutions E40 - about 1 : 18.2 dilutions

Example 4

The insecticidal activity of XPOl ferments was tested against neonate larvae of four different Lepidopterian species: (S. littoralis, H. armigera, A. ipsilon and S. segetum).

Several samples, each containing 6 ml of 24 hour XPOl ferments, (the fermentation being for 24 hours at 25°C in an R5 medium) were prepared. Diluted and non diluted samples were incubated with neonates of the four Lepidopterian species.

As seen in Tables 5-8 below, XPOl ferments showed an insecticidal activity against all four Lepidopterian species.

Table 5

(Spodoptera littoralis neonates)

Insecticidal efficacy of the raw bacterial suspension against neonate of Spodoptera littoralis at four dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control - 64 0 3.1 (untreated)

E24 non-diluted 32 22 67.8

E24 1:4 32 15 45.2

E24 1: 16 32 2 3.2

E24 1 :64 32 1 0

Table 6

(Agrotis ipsilon neonates)

Insecticidal efficacy of the raw bacterial suspension against neonate oi Agrotis ipsilon at four dilution rates

treatment dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control — 64 1 1.6 (untreated)

E24 non-diluted 32 30 93.6

E24 1 :4 32 18 55.5

E24 1 :16 32 8 23.8

E24 1 :64 32 4 11.1

Table 7

(Helicoverpa armigera neonates)

Insecticidal efficacy of the raw bacterial suspension against neonate of Helicoverpa armigera at four dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control - 64 1 1.6 (untreated)

E24 non-diluted 32 31 96.8

E24 1 :4 32 26 11.1

E24 1:16 32 4 0

E24 1:64 32 0 0

Table 8

(Scotia segetum neonates)

Insecticidal efficacy of the raw bacterial suspension against neonate of Scotia segetum at four dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula)

Control - 64 0 0 (untreated)

E24 non-diluted 32 32 100

E24 1:4 32 31 96.9

E24 1:16 32 8 25

E24 1:64 32 3 9.4

Example 5

Ferments of XPOl, fermented for 24 hours at 25°C in R5 medium were obtained and the following samples were prepared: Sample A: Non treated ferments; Sample B: R5 medium containing no bacteria but incubated for 24 hours at 25°C; Sample C: Supernatant of the ferments filtrated through a 22 micron filter; Sample D: Bacterial pellets of the ferments washed three times in isotonic solution and then resuspended in isotonic solution; Sample E: Bacterial pellets as in D resuspended in a medium having a pH 4.5; Sample F: Demineralized water; Sample G: Bacterial pellets of the ferments crushed in liquid nitrogen in a mortar with pestle;

Sa ple H: Supernatant of a resuspended pellet of the whole bacterial culture which was crushed in liquid nitrogen in a mortar with pestle (Sample G) filtered on a 22 micron filter.

All the above samples were incubated with neonate larvae of

M. brassicae at 27°C for a period of 5 days. The results are shown in the following Table 9 and the Probit analysis in Table 10 below:

As seen in Table 9 below, the highest insecticidal activity detected was that of the whole XPOl culture. The supernatant of the whole bacterial culture which was filtrated through a 22 micron filter (Sample C) showed no insecticidal activity against these neonate larvae. Against this, the supernatant produced from the bacterial cell pellet of the cells which were lightly damaged by liquid nitrogen treatment (Sample H) showed an insecticidal activity. This supports the possibility that the bacterial cells comprise an insecticidal component which upon cell damage leaks into the supernatant.

Table 9

Insecticidal efficacy of the raw bacterial suspension and some derivatives derivatives against neonate of Mamestra brassicae at several dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control - 64 5 7.8 (untreated)

A non-diluted 32 32 100

A 1:2 32 31 96.6

A 1:4 32 30 93.2

A 1:8 32 29 89.8

A 1:16 32 15 42.4

A 1:32 32 6 11.9

A 1:64 32 0 0

A 1:128 32 0 0

B non-diluted 32 0 0

B 1:2 32 0 0

B 1:4 32 0

B 1:8 32 1 0

B 1:16 32 0 0

B 1:32 32 0 0

B 1:64 32 0 0

B 1:128 32 1 0

C non-diluted 32 2 0

C 1:2 32 1 0

C 1:4 32 0 0

C 1:8 32 1 0

C 1:16 32 0 0

C 1:32 32 0 0

C 1:64 32 1 0

C 1:128 32 0 0

Treatment Dilution # insects #dead % mortality (corrected by Abbott's formula except control)

D non-diluted 32 31 96.6

D 1:2 32 29 89.8

D 1:4 32 66.1

D 1:8 32 24 72.9

D 1:16 32 11 28.8

D 1:32 32 4 5.1

D 1:64 32 0 0

D 1:128 32 0 0

E non-diluted 32 32 100

E 1:2 31 26 79.7

E 1:4 31 22 66.1

E 1:8 32 19 55.9

E 1:16 32 8 18.7

E 1:32 32 5 8.5

E 1:64 32 1 0

E 1:128 32 o 0

F non-diluted 32 5 8.5

F 1:2 32 1 0

F 1:4 32 1 0

F 1:8 32 1 0

F 1:16 32 1 0

F 1:32 32 1 0

F 1:64 32 0 0

F 1:128 32 2 0

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

G non-diluted 32 28 86.4

G 1:2 32 26 79.7

G 1:4 32 25 76.3

G 1:8 32 12 32.2

G 1 :16 32 7 15.3

G 1 :32 32 2 0

G 1:64 32 1 0

G 1 :128 32 2 0

H non-diluted 32 18 52.5

H 1:2 32 11 28.8

H 1 :4 32 2 0

H 1:8 32 1 0

H 1:16 32 3 0

Table 10

Probit analysis of results of Table 9

subjects 256 slope = 3.0441.353

A

LDRδO-sO.068 limits: .043 to .104 subjects 256 slope = 1.1691.233

D

LDR50=.113 limits: 0.68 to .183 subjects 254 slope = 2.2001.287

E

LDR50=.136 limits: .101 to .177 subjects 256 slope = 2.0331257

G

LDR50=.185 limits: .122 to .272

Estimated LDR50 of treatment H= .89 limits: .7 to 1.5

Example 6

The insecticidal activity of a whole bacterial culture of XPOl bacteria was tested against neonates of H. armigera. XPOl culture was fermented for 24 hours at 25°C in R5 medium and from the ferments the following samples were prepared:

Sample 1: Bacterial pellet was prepared and exposed to osmotic shock by resuspension in 60 ml of demineralized water for 2 hours. Sample 2: A pellet of the XPOl ferment was dried at 50°C for 9 hours, resuspended in an isotonic solution and sonicated to disrupt the cell walls;

Sample 3: A pellet of the ferment was treated as described for Sam¬ ple 3 with a difference that the resuspension was in an acidic solution at pH 4.5.

Sample 4: The original ferment was tested against neonate larvae of H. armigera in an eight-dose bioassay.

The results from Samples 1-3 are shown in the following Table 11 and those of Sample 4 in Table 13 below. The Probit analysis is shown in Tables 12 and 14, respectively.

Table 11

Insecticidal efficacy of processes samples from the raw bacterial suspension against neonate of Mamestra brassicae at eight dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott s formula)

Control - 64 0 0 (untreated)

Sample 1 non-diluted 32 29 90.6

Sample 1 1:2 32 25 78.1

Sample 1 1:4 32 10 31.2

Sample 1 1:8 32 5 15.6

Sample 1 1: 16 32 0 0

Sample 1 1:32 31 3 9.7

Sample 1 1:64 32 o 6.2

Sample 1 1:128 32 0 0

Sample 2 non-diluted 32 20 62.5

Sample 2 1:2 32 24 75

Sample 2 1:4 32 15 46.9

Sample 2 1:8 32 9 28.1

Sample 2 1 : 16 32 3 9.4

Sample 2 1 :32 31 0 0

Sample 2 1 :64 32 0 0

Sample 2 1 :128 32 0 0

Sample 3 non-diluted 32 24 75

Sample 3 1:2 32 21 65.6

Sample 3 1:4 32 16 50

Sample 3 1:8 32 9 28.1

Sample 3 1 :16 32 o 6.2

Sample 3 1 :32 31 0 0

Sample 3 1:64 32 0 0

Sample 3 1:128 32 1 3.1

_ 0 _

Table 12

Probit analysis of results of Table 11

subjects 256 slope = 1.8751.211

SI

LDR50=.291 limits: .149 to .799 subjects 256 slope = 1.7181.212

LDR50=.343 limits: .219 to .622 subjects 256 slope = 1.578+183

S3

LDR50=.260 limits: .131 to .692

Table 13

Insecticidal efficacy of the raw bacterial suspension against neonate of Helicoverpa armigera at eight dilution rates

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula except control)

Control - 64 0 0 (untreated)

Sample 4 non-diluted 32 28 87.5

Sample 4 1:2 32 26 81.2

Sample 4 1:4 32 26 81.2

Sample 4 1:8 32 7 21.9

Sample 4 1 : 16 32 14 43.8

Sample 4 1 :32 31 7 21.9

Sample 4 1 :64 32 0 0.0

Sample 4 1 :128 32 2 6.2

Table 14

Probit analysis of results of Table 13

subjects 256 slope = 1.4831.160

Sample 4

LDR50=0.135 limits: 0.068 to 0.293

As seen in Table 11, the XPOl cell culture shows an insecticidal activity against larvae of M. brassicae in correlation with the dilution of the tested composition (Samples 1, 2, 3).

As seen in Table 13, an XPOl whole culture used in an 8-dose bioassay showed insecticidal activity against neonates of Helicoverpa armigera (Sample 4) although to a different extent than the insecticidal activity of the same bacteria against neonates of M. brassicae.

Example 7

The insecticidal activity of XPOl cells inactivated by formaline against neonates of M. brassicae was tested.

XPOl was fermented in an R5 medium at 25°C, samples of the ferment were taken and treated as follows:

Sample A Untreated ferment. Sample B Ferment treated with 3μl/ml of 30% (v/v) formaline. Sample C Ferment treated with 6μl/ml of 30% (v/v) formaline.

The activity of these samples was tested versus the following control preparations: Control A: Deionized water. Control B: Deionized water with 3μl/ml of 30% (v/v) formaline. Control C: Deionized water with 6μl/ml of 30% (v/v) formaline.

In order to control the efficacy of formaline to kill the bacteria, cells from Sample A, B and C, were streaked on 3 nutrient agar plates that

were incubated at 25°C in the dark for 2 days. This test confirmed that all bacterial cells were killed by the formaline.

The above controls and samples were tested in a bioassay against neonates of M. brassicae.

The results summarized in Table 15 show that XPOl cells inactivated by formaline maintain their insecticidal activity against neonates of M. brassicae.

Table 15

Treatment Dilution # insects # dead % mortality (corrected by Abbott's formula)

Control A — 32 0 0

Control B — 32 0 0

Control C - 32 0 0

Sample A 1:1 32 24 75.0

Sample A 1:2 32 27 84.4

Sample A 1:4 31 17 54.8

Sample A 1:8 32 15 46.9

Sample A 1:16 32 1 3.1

Sample A 1:32 31 o 6.2

Sample A 1:64 32 3 9.4

Sample A 1:128 32 1 3.1

Sample B 1:1 32 28 87.5

Sample B 1:2 32 27 84.4

Sample B 1:4 32 28 87.5

Sample B 1:8 32 20 62.5

Sample B 1:16 32 3 9.4

Sample B 1:32 32 1 3.1

Sample B 1:64 32 0 0

Sample B 1:128 32 0 0

Sample C 1:1 32 30 93.8

Sample C 1:2 32 29 90.6

Sample C 1:4 32 21 65.6

Sample C 1:8 32 14 43.7

Sample C 1:16 30 3 10.0

Sample C 1:32 31 1 3.2

Sample C 1:64 32 0 0

Sample C 1:128 32 0 0

Example 8

The insecticidal activity of XPOl fermentate against neonates of L. botrana was tested as follows:

XPOl culture was fermented in R5 medium at 25°C for 24 hours, after which a sample of the ferment was taken and used in one-dose bioassays against L. botrana neonates.

As seen in Table 16 below, the whole culture of XPOl showed insecticidal activity against neonates of L. botrana.

Table 16

Treatment Dilution # insects # insects % mortality (corrected dead by Abbott's formula except control)

Control 31 2 6.5 XPOl 1 :1 32 24 733

Example 9

The insecticidal activity of whole culture of XP05 and XP98 against neonate larvae of M. brassicae, H. armigera, A. ipsilon, S. segetum, S. littoralis L. botrana was tested and compared to the insecticidal activity of whole culture of XPOl against larvae of the same kind.

A bacteria strain termed "XP05", was isolated, as described in Example 1 for strain XPOl, from haemolymph oi Galleria mellonella larvae infected by Dauer Juveniles of Steinernema feltiae strain, UK. The bacteria XP98 was isolated in a similar manner also from haemolymph of Galleria mellonella larvae infected by Dauer Juveniles oi Steinernema sp. strain 98.

XPOl, XP05, XP98 were fermented in R5 media at 25°C in a 24 hour fermentation run.

The ferments were harvested and used without any dilution in a one-dose bioassays against neonates of M. brassicae, H. armigera, A. ipsilon, S. segetum, S. littoralis L. botrana. The results are shown in the following Tables 17-22.

Table 17

One-dose bioassays against neonates of M. brassicae

Treatment Dilution # insects # insects % mortality (corrected dead by Abbott's formula)

Control — 32 0 0

XP05 1:1 32 1 3.1

XP98 1:1 32 3 9.4

XPOl 1:1 32 24 75.0

Table 18

One-dose bioassays against neonates of H. armigera

Treatment Dilution # insects # insects % mortality (corrected dead by Abbott's formula)

Control — 32 0 0

XP05 1:1 32 0 0

XP98 1:1 32 0 0

XPOl 1:1 32 24 75.0

Table 19

One-dose bioassays against neonates of L. botrana

Treatment Dilution # insects # insects % mortality dead (corrected by

Abbott's formula except control)

Control — 31 2 6.5

XP05 1:1 32 3 3.1

XP98 1 :1 32 1 0

XPOl 1: 1 32 24 73.3

Table 20

One-dose bioassays against neonates of A. ipsilon

Treatment Dilution # insects # insects % mortality dead (corrected by

Abbott's formula except control)

Control — 32 1 3.1

XP05 1:1 32 4 9.7

XP98 1 :1 32 1 0

XPOl 1:1 32 30 93.6

Table 21

One-dose bioassays against neonates of 5. segetum

Treatment Dilution # insects # insects % mortality dead (corrected by

Abbott's formula)

Control — 32 0 0

XP05 1:1 31 5 16.1

XP98 1: 1 32 3 9.4

XPOl 1: 1 32 31 96.9

Table 22

One-dose bioassays against neonates of S. littoralis

Treatment Dilution # insects # insects % mortality dead (corrected by

Abbott's formula except control)

Control — 31 1 3.2

XP05 1 :1 32 2 3.1

XP98 1 : 1 32 4 9.4

XPOl 1: 1 32 24 74.2

As seen in Tables 17-22, while the insecticidal activity of XPOl against the 6 Lepidoterian species was apparent, the XP05 and XP98 strains have a small activity against the 6 Lepidoterian species.

Example 10

Whole culture of XPOl fermented for 24 hours at 25°C in R5 medium was concentrated by centrifugation at 9000 rpm for 20 minutes. The concentrate obtained was freeze dried to produce the Technical Powder of XPOl fermented broth.

The following samples were prepared: Sample A: Whole culture of XPOl fermented for 24 hours at 25 °C in

R5 medium. Sample B: Suspension of Technical Powder resuspended at a concen¬ tration of 0.0028g/ml in demineralized water. All the above samples were incubated with neonate larvae of M.brassicae at 27°C for a period of 5 days. The results are shown in the following Table 23:

Table 23

Insecticide efficacy of raw bacterial suspension and its

Technical Powder against neonate of M.brassicae at several dilution rate

Treatment Dilution # insects #dead

Control (untreated) - 64 1

Sample A non-diluted 32 29

Sample A 1:4 32 27

Sample A 1:16 32 3

Sample A 1:64 32 0

Sample B non-diluted 32 22

Sample B 1:4 32 6

Sample B 1:16 32 2

Sample B 1:64 32 0

As seen in Table 23 above, the Technical Powder of XPOl has insecticidal activity. This supports the notion that the bacteria strain XPOl contains an insecticidal component which can be used in insect control.

Example 11

The insecticidal activity of a supernatant from a suspension of Technical Powder of XPOl was tested against neonate larvae oi M.brassicae (Lepidopterian). A suspension of Technical Powder of XPOl (rate 0.0028g/ml dem. water) was stirred for 30 mins. The suspension was then centrifuged at 3500 φm for 5 mins. The supernatant was withdrawn and filtered on a 22 micron filter.

The following samples were prepared: Sample A: Whole culture of XPOl fermented for 24 hours at 25°C in

R5 medium. Sample B: Supernatant of resuspended Technical Powder filtered on a 22 micron filter. All the above samples were incubated with neonate larvae of M.brassicae at 27°C for a period of 5 days. The results are shown in the following Table 24:

Table 24

Insecticide efficacy of raw bacterial suspension and supernatant from XPOl Technical Powder against neonate of M.brassicae at several dilution rates

Treatment Dilution # insects #dead

Control (untreated) - 64 10

Sample A non-diluted 32 30

Sample A 1:2 32 27

Sample A 1 :4 32 26

Sample A 1:8 32 19

Sample A 1:16 32 13

Sample A 1:32 32 14

Sample A 1:64 32 8

Sample B non-diluted 32 17

Sample B 1:2 32 11

Sample B 1:4 32 8

Sample B 1:8 32 6

Sample B 1:16 32 2

Sample B 1:32 32 5

Sample B 1:64 32 5

As seen in Table 24 above, the supernatant of Technical Powder of XPOl has insecticidal activity, and that the insecticidal component is smaller than 22 micron.

Example 12

The insecticidal activity of a Whole Culture of XPOl was tested against neonate larvae of M.brassicae (Lepidopterian) in a dip-leaf assay.

Several dilutions of whole culture of XPOl fermented for 24 hours at 25°in R5 medium were placed in plastic cups. Cabbage leaf discs

were dipped in each dilution ior 5 seconds, then they were air dried. 0.02% of the surfactant Triton CS-7 was included in each solution.

The treated leaf discs were incubated with neonate larvae of M.brassicae at 20°C for a period of 5 days. The results are shown in Table 25 below.

Table 25

Insecticide efficacy of raw bacterial suspension on leaf-dip assay against neonate of M.brassicae at several dilution rates

Treatment Dilution # insects # dead

Control (untreated) - 32 1

Sample non-diluted 32 32

Sample 1:2 32 32

Sample 1:4 32 31

Sample 1:8 32 26

Sample 1:16 31 15

Sample 1 :32 31 17

Sample 1 :64 32 12

As seen in Table 25 above, whole culture of XPOl have an insecticidal activity also when applied onto leaf surfaces.

Example 13

The effect of two protease digestions on the insecticidal activity of suspension of Technical Powder of XPOl was tested.

From a suspension of Technical Powder of XPOl (at a concentra¬ tion of 0.0028 g/ml in dem. water) in TRIS solution 100 mM pH 7.5, the following samples were prepared:

Sample A: 9 ml of XPOl suspension administered with 1 ml solution of protease from Streptomyces caespitosus in TRIS 100 M at pH 7.5 (final protease concentration 0.0028 g/ml).

Sample B: 9 ml of XPOl suspension administered with 1 ml solution of protease from Bovine pancreas in TRIS 100 mM at pH 7.5 (final protease concentration of 0.0028 g/ml). Sample C: 9 ml of XPOl suspension administered with 1 ml solution of TRIS 100 mM at pH 7.5.

All the above samples were incubated at 37°C for a period of 4 hours. To assess the proteolytic digestion, gel SDS PAGE was made with the incubated samples and with non treated XPOl suspension.

All the above samples were incubated with neonate larvae of M.brassicae at 27°C for a period of 5 days. Additional controls correspond¬ ing to the above samples were tested as follows: Control A: Solution of Protease from Streptomyces caespitosus in TRIS

100 mM at pH 7.5 (protease concentration 0.0028 g/ml). Control B: Solution of Protease from Bovine pancreas in TRIS 100 mM at pH 7.5 (protease concentration 0.0028 g/ml.

Control C: Solution of 100 mM TRIS at pH 7.5. The results are shown in Table 26 below:

Table 26

Insecticide efficacy of suspension of XPOl Technical Powder protease treated against neonate of M.brassicae at several dilution rate

Treatment Dilution # insects #dead

Control (untreated) - 64 10

Sample A non-diluted 32 30

Sample A 1:2 31 18

Sample A 1:4 32 13

Sample A 1:8 32 1

Sample A 1:16 32 7

Control A 1:1 32 5

Sample B non-diluted 32 5

Sample B 1:2 32 1

Sample B 1 :4 32 1

Sample B 1 :8 32 1

Sample B 1:16 32 0

Control B 1:1 32 0

Sample C non-diluted 32 32

Sample C 1 :2 32 31

Sample C 1:4 32 23

Sample C 1:8 32 4

Sample C 1:16 32 0

Control C 1:1 32 0

As seen in Table 26 above, the protease from Bovine pancreas can reduce dramatically the insecticidal activity of XPOl. This points to the possibility that the active ingredient in XPOl preparation can be a protein that is severely damaged by Bovine pancreas protease and less damaged by S. caespitosus protease.