WO1992001038A1

WO1992001038A1 - A group of bacteria belonging to the order of actionomycetales, a biocidal compound producible thereof and a process for producing the compound

Info

Publication number: WO1992001038A1
Application number: PCT/DK1991/000186
Authority: WO
Inventors: Hanne Gürtler; Rita Pedersen
Original assignee: Novo Nordisk A/S
Priority date: 1990-07-06
Filing date: 1991-07-02
Publication date: 1992-01-23
Also published as: AU8207191A; DK162990D0

Abstract

A novel group of bacteria belonging to the order Actinomycetales is described. Bacteria of the novel group produces biocidal compounds, which may be used as ingredients in compositions for use within the field of agriculture, and horticulture, especially for controlling insects and/or fungi.

Description

A group of bacteria belonginq to the order of Actinomycetales, a biocidal compound producible thereof and a

process for producing the compound.

TECHNICAL FIELD

This invention relates to a new group of bacteria belonging to the order Actinomycetales, biocidal compounds produced by such bacteria, compositions containing such compounds and use of such compounds within the field of agriculture, and horticulture, especially for controlling insects.

BACKGROUND OF THE INVENTION ACTINOMYCETES

Actinomycetes are Gram-positive bacteria containing DNA that is rich in guanine plus cytosine. They form a distinct group on the basis of nucleic acid sequencing and pairing studies and generally exhibit branched filaments, though some show pleomorphic and even coccoid elements. They are widely distributed in soil, composts, water and other environments. Their basic attributes have been described in "The Biology of the Actinomycetes", eds. M. Goodfellow, M. Mordarski, S.T. Williams, 1984. The current and practical relevance of actinomycetes in biotechnology has been emphasized in "Actinomycetes in Biotechnology" eds. M. Goodfellow, S.T. Williams & M. Mordarski, 1988.

In 1948 there were five actinomycete genera recognized (Breed, R.S. et al. (ed)., Bergey's Manual of Determinative Bacteriology, 6th ed, p. 259-279, 1948). The genera were: Mycobacterium, Nocardia, Actinomyces. Streptomyces. and Micromonospora. Over the span of forty years since that time, an explosive interest in actinomycetes has led to the isolation, characterization, and naming of over eighty genera (A. Dietz, in "Biology of Actinomycetes' 88" Proceedings of Seventh Int. Symp. on Biology of Actinomycetes, eds. Y. Okami, T. Beppu & H. Ogawara, p. 203-209, 1988). Some of these genera have been spun off from those cited above on the basis of development of better methods for studying actinomycetes.

The development and application of new and reliable biochemical, chemical and molecular biological techniques are now challenging the traditional morphological definition of the order Actinomycetales. As a result of these developments established actinomycete genera and species have been defined more precisely, novel taxa have been proposed for new centers of variation and poorly circumscribed species reduced to synonyms of well defined taxospecies. (M. Goodfellow, Sixth. Int. Symp. on Actinomycetes Biology, eds. G. Szabό, S. Birό, M. Goodfellow, p. 487-497, 1985).

Nearly sixty actinomycete genera are now recognized and the fourth volume of Bergey's Manual of Systematic Bacteriology, 1989 is devoted to the actinomycetes.

The genus Streptomyces (Waksman and Henrici, 1943, J. Bacteriol. (46), 337-341) is a member of the family Streptomycetaceae (Waksman and Henrici, 1943) in the bacterial order Actinomycetales. (Buchanan, 1917, J. Bacteriol (2), 155-164).

A detailed discussion of the family Streptomycetaceae can be found in Kutzner (1982) (In "The Prokaryotes" Vol. II, p. 2028-2090, eds. M.P. Starr et al.).

Characterization of the genus Streptomyces and related genera in the family Streptomvcetaceae is in Pridham and Tresner (1974), (In "Bergey's Manual of Determinative Bacteriology" 8th ed. p. 747-845). Streptomyces and related genera can be readily distinguished from a variety of other genera of actinomycetes by chemotaxonomic and micromorphologic properties. These genera have the LL-isomer of diaminopimelic acid (DAP) and glycine in their whole-cell hydrolysates (Becker et al., 1964, Appl. Microbiol. (12), 421-423. Lechevalier et al., 1971, Adv. Appl. Microbiol. (14), 47-72. Lechevalier and Lechevalier, 1980, In "Actinomycetes Taxonomy", eds. A. Dietz & D.W. Thayer, SIM Spec. Publ. No 6, p. 225-291).

They are distinguished morphologically by the production of spores in chains with the exception of two genera which only have spores in sporangia. All the genera are listed in Table I.

Table I

Streptomyces and Related Genera, Genera with L-DAP

and Glycine in Whole-Cell Hydrolysates

Genus Other significant References

characteristics

A: Spores in chains

Streptomyces Chains in various Pridham and

configurations. Tresner(1974) Spores with distinctive Waksman and surface details Henrici (1943) Related Genera

Actinopycnidium Pycnidia Krasilnikow

(1972)

Actinosporangium Slimy sporangia with Krasilnikow

spores. Sporangia and Yuan(1961) lacks walls. Sporangia

formed by merging of

sporophores

Chainia Sclerotic granules Thirumalachar

(1955)

Elytrosporangium Substrate mycelium Falcao de

Morais et al (1966)

Intrasporangium Sporangia intercalary in Kalakoutskii

the mycelia hyphae. et al (1967) Spores not motile. Table 1 (continued)

Streptomyces and Related Genera, Genera with L-DAP and Glycine in Whole-Cell Hydrolysates Genus Other significant References

characteristics

Kitasatoa Club-shaped sporangia. Matsumae et al

Motile zoospores (1968)

Kitasatosporia meso-DAP also found Omura et al Nocardioides Hyphae of primary mycePrauser(1976) lium and aerial mycelium

fragment into rod- to

coccuslike elements

Sporichthya Chains on holdfast. Lechevalier et

Aerial mycelium divides al (1968) into various-shaped

motile elements

Streptoverticilium Chains in verticils Baldacci(1958) B: Spores in sporangialike vesicles

Kineosporia Sporangium with single Pagani and

planospore. Spore motile Parenti(1978) Microellobosporia Sporangium with row of Cross et al

sporangiospores. Spores (1963)

not motile.

From: A. Dietz, 1986, In: "The Bacteria", Vol. IX - Antibiotic producing Streptomyces., Eds: S.W. Queener & L.E. Day

The members of the Streptomycetaceae are aerobic and Gram-positive. Their growth is mycelial with long branched filaments. The mycelium is usually 0.5-2.0μm thick. Hyphae are both aerial and substratal.

The family Streptomycetaceae includes a number of taxa that are mainly morphological in concept. The aerial mycelium of strains of Streptomyces usually bears long chains of spores (>50 spores). The morphology of the spore chains being either rectiflexibiles, rectinaculiaperti or spirales, strains of Chainia typically form sclerotia (Thirumalachar, 1955, Nature, London 174,934-935); Streptoverticillium bears verticils at regular intervals (Baldacci, 1958, Giornale di microbiologia (6), 10-27); Actinosporangium produces sporangia (Krasilinikov & Yuan, 1961 Izvestiya Akademii Nauk SSSR (Seriya Biologicheskaya) 8, 113-116). Actinopycnidium. pycnidia (Krasilnikov, 1962, Mikrobiologiya 31, 204-207), Microellobosporia. sporangia on short sporophores (Cross et al., 1963, J. Gen. Microbiol. 31, 421-429; Rancourt & Lechevalier, 1963, J. Gen. Microbiol. 31, 495-498), Elytrosporangium. podshaped vesicles on the substrate mycelium (Falcao de Morais et al., 1966, Mycopathologia et mycologia applicata, 30, 161-171), and Kitasatoa. clubshaped versicles on the tips of the substrate and aerial hyphae (Matsumae & Hata, 1968, Journal of Antibiotics 21, 616-625).

The status and relationships of these taxa both to one another and to the genus Streptomyces is not clear (Cross & Goodfellow, 1973, In "Actinomycetales: Characteristics and Practical Importance, pp. 11-112. Eds. G. Sykes & F.A. Skinner; Kutzner, 1981, In "The Prokaryotes: A Handbook of Habitats, Isolation and Identification of Bacteria, pp. 2028-2090, Eds. M.P. Starr et al., S.T. Williams et al., 1983, Journal of General Microbiology, 129, 1743-1813; Goodfellow & Cross, 1984, In "The Biology of the Actinomycetes" eds. M. Goodfellow, M. Mordarski, S.T. Williams et al., 1983, Journal of General Microbiology, pp. 7-164).

Intrasporangium (Kalakoutskii et al., 1967, Journal of General Microbiology 48, 79-85), Sporichthya (Lechevalier et al., 1968, Annales de l'Institut Pasteur 114, 277-285) and Nocardioides (Prauser, 1976, International Journal of Systematic Bacteriology 26, 58-65) have been assigned to the family Streptomycetaceae primarily on the basis of wall chemotype. They differ very much in other properties and they may not be members of the Streptomycetaceae at all (Kutzner, H.P., 1981, In "The Prokaryotes, p.p. 2028-2090. Eds. M.P. Starr et al.). In Bergey's Manual of Determinative Bacteriology (Pridham & Tresner, 1974, In Bergey's Manual of Determinative Bacteriology, 8th edn., pp. 747-829, Eds. R.E. Buchanan et al), the genera Microellobosporia. Sporichthya, Streptomyces and Streptoverticillium are recognized; Actinopycnidium, Actinosporanαium and Intrasporangium are listed as genera incertae sedis. while Kitasatoa is classified in the family Actinoplanaceae (Couch & Bland, 1974, In "Bergey's Manual of Determinative Bacteriology", 8th edn. pp. 706-708).

Descriptions of the genus Streptomyces and genera related to it are to be found in Pridham and Tresner (1974) (In "Bergey's Manual of Determinative Bacteriology", 8th ed. pp. 747-845) and in scientific publications prior and subsequent to their publication. Genera that have been considered validly published are listed in the "Approved Lists of Bacterial Names" (Skerman et al., 1980, Int. J. Syst. Bacteriol. 30, 225-420). As new names are validated they are published in the "International Journal of Systematic Bacteriology". Rules and principles to be used in naming cultures are discussed by Lessel (1980) (In "Actinomycete Taxonomy" eds. A. Dietz & D. W. Thayer, SIM Spec. Publ. No. 6, pp. 345-356) Procedures for naming cultures are presented in Lapage et al. (1975) ("International Code of Nomenclature of Bacteria. 1976 Revision". Am. Soc. Microbiol.). Nomenclature of genera in the order Actinomycetales is discussed by Pridham (1971) (Int. J. Syst. Bacteriol. 21, 197-206).

The genus Streptomyces and related genera and reference to their first citation are given in Table I, above. All of the genera listed in Table I are cited in the "Approved Lists of Bacterial Names" (Skerman et al., 1980).

Transfer of the genera Actinopycnidium, Actinosporanqium, Chainia, Elytrosporangium, Kitasatoa and Microellobosporia. to the genus Streptomyces has, however, been suggested by M. Goodfellow et al., 1986, (System.Appl.Microbiol. 8, 48-66). In the current edition of Bergey's Manual ("Bergey's Manual of Systematic Bacteriology, volume 4, 1989) the genera Intrasporangium and Nocardioides are included in section 26 which covers the nocardioform actinomycetes, while the genera Streptomyces, Streptoverticillium, Kinesporia and Sporichthya are included in section 29 which covers the Streptomycetes and related genera. The genera Actinopycnidium. Actinosporangium, Chainia, Elytrosporangium, Kitasatoa, Microellobosporia are included in the genus Streptomyces.

The current and practical relevance of actinomycetes in Biotechnology has been emphasized in "Actinomycetes in Biotechnology" eds. M. Goodfellow, S.T. Williams & M. Mordarski, 1988.

The outstanding property of the actinomycetes, particularly those belonging to the genus Streptomyces, is their capability of producing a variety of antibiotics.

To date, some 6000 antibiotics of microbial origin have been characterized, and about 60% of them are produced by actinomycetes. Among these antibiotics, about seventy compounds, 90% of which originate from Streptomycetes, have found practical application in human and veterinary medicine, in agriculture, and in the fishery industry (S.W. Queener & L.E. Day, 1986, In "The Bacteria" Vol. IX: Antibiotic-Producing Streptomyces; Y. Okami & K. Hotta 1988 (In "Actinomycetes in Biotechnology" eds. M. Goodfellow et al. pp. 33-67)

Antibiotics from actinomycetes include almost all known structural classes of commercially important antibiotics: for example, aminocyclitols, ansamycins, anthracyclins, ß-lactams, macrolides, glutarimides, nucleosides, peptides, peptidolactones, polyenes, polyethers, tetracyclins, and other important antibiotics such as chloramphenicol (Crandall & Hamill (1986), In "The Bacteria, Vol. IX - Antibiotic-Producing Streptomyces. eds. S.W. Queener & L. E. Day. pp. 355-401; Y. Okami & K. Hotta 1988, In "Actinomycetes in Biotechnology" eds. M. Goodfellow et al. pp. 33-67).

Actinomycetes have also proved to be a rich source of vitamins, enzymes and enzyme inhibitors. The use of actinomycetes within agriculture and forestry as agents of biological control, as plant growth enhancers or as producers of agricultural drugs has been reviewed recently by M.P. Lechevalier (In Actinomycetes in Biotechnology, eds. M. Goodfellow, S.T. Williams, M. Mordarski, pp. 328- 358, 1988). From this actinomycete compounds which have been shown to have pesticidal activities include compounds such as avermectin, milbemycin and tetranactin, which have insecticidal activity, bialaphos, which has herbicidal activity, cycloheximide, blasicidin, polyoxins and ecomycin, which have fungicidal activity, and cellocidin, which has antibacterial activity.

SUMMARY OF THE INVENTION

One aspect of the invention relates to the discovery of a new group of actinomycetes having a totally novel and previously undescribed morphology.

The novel and previously undescribed feature of this new group of actinomycetes is its ability to produce ropes on a variety of agar media. These ropes comprise large numbers of spore chains which are fused together forming a rope like structure of 100 μm or more in length and 20 μm wide.

This feature has not previously been described for any actinomycete strain and therefore represents a novel morphological feature within the order Actinomycetales.

This new group of actinomycetes has a mycelium growth form with the production of spore chains. Chemotaxonomic analysis has revealed that the cell wall contains major amounts of LL-DAP and apparently no major amounts of characteristic sugars.

Overall these features indicate close similarity to the family Streptomycetaceae although this has still to be confirmed by more extensive chemotaxonomic analysis.

Two representative isolates of this new group of actinomycetes have been deposited at the Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Mascheroderweg 1b, D-3300 Braunschweig, Federal Republic of Germany, for the pur poses of patent procedure on the dates indicated below. DSM being an International Depositary under the Budapest Treaty affords permanence of the deposits in accordance with rule 9 of said treaty. 1) Deposit date: 16 June 1989

Depositors ref. R-105

DSM designation DSM 5415

2) Deposit date: 15 June 1990

Depositors ref. M9

DSM designation DSM 6012

Another aspect of the invention relates to biocidal compounds produced by this new group of actinomycetes.

This new group of actinomycetes has been found to produce highly active compounds having broad spectrum biocidal activity, good insecticidal activity, and good in vivo activity against plant pathogenic fungi.

The invention consequently also relates to biocidal compounds produced by the novel bacteria, processes for producing such biocidal compounds, and to processes for controlling pests by applying such compounds to the areas where the pests are to be controlled.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described in detail in the following with reference to the accompanying drawings, where

Figure 1 shows the rope formation of the bacteria of the invention in substrate mycelium at the leading edge of a colony of strain DSM 5415 grown on Bennetts agar at 30ºC for four days, as seen with light microscope at × 400.

Figure 2 shows ropes in the substrate mycelium with beginnings of aerial spore chain formation (highly refractive areas) within the body of the mycelium of strain DSM 5415 grown on nutrient agar at 30°C for four days, as seen with light microscopy at × 1600. Figure 3 shows the rope formation in the aerial mycelium of a bacteria of the invention clearly showing ropes comprising intertwined spore chains of up to 100 μm in length and 10 μm in width from strain DSM 5415 grown on oatmeal agar at 30ºC for four days, as seen with light microscope at × 500.

Figure 4 shows the rope formation on aerial mycelium mass of strain DSM 5415 grown on oatmeal agar at 30ºC for two days. Immature sporophores intertwine and initiate ropes prior to spore formation, as seen with scanning electron microscope at × 3.500

Figure 5 shows mature aerial mycelium bearing ropes of varying thickness composed of spore masses derived from intertwined sporophores from strain DSM 5415 grown on oatmeal agar at 30°C for four days, as seen with scanning electron microscope at × 2.000.

Figure 6 shows the detailed structure of rope showing sporechains intertwined to form characteristic rope-like structures on the aerial mycelium of strain DSM 5415 grown on oatmeal agar at 30°C for seven days, as seen with scanning electron microscope at × 10.000. And

Figure 7 shows mature rope formation on aerial mycelium from strain DSM 5415 grown on oatmeal agar at 30°C for seven days, as seen by scanning electron microscope at × 3.500.

DETAILED DESCRIPTION OF THE INVENTION To repeat, this invention relates to a new group of actinomycetes having a totally novel and previously undescribed morphology.

Consequently the present invention in its first aspect relates to a novel group of bacteria that belongs to the order of Actinomycetales, has the ability to produce rope like structures comprising a large number of spore chains which are fused together in said rope like structures, and produces biocidal compounds, or mutants or genetically engineered variants thereof.

A preferred group of these bacteria is one where said biocidal compounds are insecticidal compounds. A further preferred group of these bacteria is one where said biocidal compounds are fungicidal compounds.

A further preferred group is novel microorganism that are morphologically identical to or exhibits the principal morphological features of the bacterial strains deposited under the accession nos. DSM 5415 and DSM 6012 or mutants or genetically engineered variants thereof.

This novel actinomycete strain, DSM 5415, was isolated in our laboratory from a natural substrate using starch casein medium (William, S.T. & E.M.H. Wellington, Methods of Soil

Analysis, Part 2, 969-987 (1982)) and the membrane filter method described by Hirsch et al., Applied and Environmental

Microbiology, 46 (4), 925-929 (1983).

The strain has been characterized by Dr. E.M.H. Wellington, Dept. of Biological Sciences, University of Warwick,

UK.

The strain has a mycelium growth form with the production of spore chains. The mycelium appears to be stable on a variety of media although there are signs of fragmentation in certain rich liquid media during submerged culture.

The strain produces dense, yellow to cream, spore mass on a variety of media including

oatmeal agar (oatmeal (20 g/l), yeast extract (2.5 g/l), agar

(20 g/l), pH 6.5-7.0)

A-37 agar (starch (15 g/l), corn steep liquor (10 g/l), soy flour (10 g/l), NaCl (5 g/l), CaC0₃ (2 g/l), agar (20 g/l), pH

7.0),

modified Bennett's agar (Jones, K.L., J. of Bacteriology, 57,

141-145, 1949),

nutrient agar (Lab-Lemco Powder (1 g/l), yeast extract (2 g/l), peptone (5g/l), NaCl (5 g/1), agar (15 g/l), pH 7.4),

Glycerol-asparagine agar (Wellington, E.M.H. et al., Dev.

Industr. Microbiol. 28: 99-104, 1987)

Basal medium (D-glucose (10 g/l), MgSO₄.7H₂O (0.5 g/l), NaCl (0.5 g/l), FeSO₄.7H₂O (0.01 g/l), K₂HPO₄ (1.0 g/l), agar (12 g/l), pH 7.4), and Minimal agar, supplemented with cellobiose, mannitol and fructose respectively (S.T. Williams et al, J. Gen. Microbiology. 129: 1743-1813, 1983).

Growth is very rapid and sporulation profused on most of these media. The substrate mycelium is stable and ramifying. Spore chains are rectiflexibles and comprise 10 - >100 spores in length.

The spores are 0.5 μm in diameter, and nonmotile, with smooth surfaces.

The novel and previously undescribed morphological feature of the new group of actinomycetes, of which DSM 5415 is a representative strain, is its ability to produce ropes (see figures 1-7) on oatmeal agar, basal medium, and to a lesser extent on the agars described above.

These ropes comprise large numbers of spore chains which are fused together forming a rope-like structure up to 100 μm or more in length and 20 μm wide (figure 6). Using scanning electron microscopy (SEM) it was possible to examine these ropes, and various stages of development could be seen in studies of coverslip cultures.

Coverslips were inserted in agar plates and inoculated with spores of DSM 5415. The coverslips were removed after 2 - 7 days. Coverslips to be studied by light microscopy were used directly. Coverslips to be studied by SEM were dehydrated in formaldehyde vapour and coated with gold-palladium alloy (sputter coated, polaron) and examined with a scanning electron microscope (Jeol Instruments).

Initially the aerial mycelium grow together in ropes, 10 or more hyphae thick (figure 4). These ropes develop further as other hyphae coalesce with the primary rope (figure 5).

These hyphae then begin to show septae and the characteristic spore chain ropes are produced (figures 6 and 7).

The accompanying SEM micrographs demonstrate that this phenomenon is a feature of differentiation and not an artifact of the SEM preparation. Subsequent examination of coverslip cultures by light microscopy also clearly shows this rope development (see accompanying light micrographs , figures 1-3).

This feature has not previously been described for any actinomycete strain and therefore represents a novel morphological feature.

Strain DSM 6012 exhibits the same morphological features as DSM 5415 and belongs to the same group. However, strian DSM 6012 also exhibits certain physiological differences from DSM 5415.

The presence and form of diaminopimelic acid (DAP) in cell walls of strain DSM 5415 were determined by the method of Stanneck & Roberts, 1974 (Chromatography Appl.Microbiol. 28: 226-231) and a carbohydrate analysis of whole cell hydrolysates was performed by the method of Lechevalier, M.P., 1968 (J.Lab.Clin.Med. 71, 934-944).

Thin layer chromatography (TLC) of cell wall material of strain DSM 5415 indicated major amounts of LL-DAP. Carbohydrate analysis of whole cell hydrolysates indicated no major amounts of characteristic sugars.

The DNA of strain DSM 5415 was isolated and preliminary analysis indicated a high GC content of approximately 74%.

Overall these features indicate close similarity to the family Streptomvcetaceae although this has still to be confirmed by more extensive chemotaxonomic analysis (e.g. phospholipids, fatty acids, menaquinones).

A battery of polyvalent actinophages used to delimit the family Streptomycetaceae were applied to lawns of strain DSM 5415 by the method described by E.M.H. Wellington & S.T. Williams (Zbl. Bakt.Abt.I, 1981; Suppl. 11: 93-98).

A number of 6 out of 10 lysed strain DSM 5415 further indicating that this is likely to be a member of this family which includes the genera Streptomyces, Streptoverticillium, Intrasporangium. Kinesporia, Sporichthya, and Nochardioides, (Actinosporangium, Actinopycnidium, Chainia, Elytrosporangium, Microellobosporia, and Kitasatoa). The physiological characteristics of DSM 5415 were determined by the methods described in Williams, S.T. et al., 1983 (Journal of General Microbiology, 129, 1743-1813).

Cellobiose, mannitol and fructose were used as sole carbon source but adonitol, rhamnose, inositol, xylose, raffinose and insulin were not.

Grows on L-histidine, L-asparagine and L-arginine but not on L-hydroxyprolin and DL-amino-n-butyric acid as sole nitrogen source.

Arbutin is degraded but xanthine, pectin and lecithin are not. Hydrogen sulphide is not produced and nitrate is not reduced.

Grows at 30°C and 37°C but not at 45ºC.

Tolerant to phenol (0.1% w/v), sodium chloride (7% NaCl), and rifampicin, variable tolerance to neomycin (depending of growth stage) but sensitive to sodium azide (0.01%).

Shows insecticidal activity towards a number of pests.

Shows fungicidal activity against a number of plant pathogenic fungi.

Accordingly the invention in a further aspect relates to biocidal compounds producible by bacteria belonging to the novel group of bacteria according to the invention.

In a preferred embodiment these compounds are insecticides.

In a further preferred embodiment these compounds are fungicides.

In a still further preferred embodiment the compounds of the invention exhibit activity towards insects of any of the orders Lepidoptera . Coleoptera, and Diptera, and especially towards any of the species Trichoplusia ni, Spodoptera exigua,

Leptinotarsa decemlineata. and/or Aedes aegypti.

In a still further preferred embodiment the compounds of the invention exhibits activity against plant pathogenic phycomycetes, and especially against any of the species Phytophthora infestans, Plasmopara halstedii and

Pseudoperonospora cubensis. In another aspect the invention relates to a process for the production of a compound according to any of the above aspects, by which process bacteria belonging to the novel group of bacteria according to the first aspect of the invention are cultivated in the presence of suitable sources for nitrogen and carbon and essential nutrients, and the biocidal compound is extracted from the culture broth and recovered according to methods known per se.

In a preferred embodiment of this aspect of the invention the bacteria cultivated is morphologically identical to or exhibits the principal morphological features of the bacterial strain deposited under accession no. DSM 5415 mutants or genetically engineered variants thereof.

In this aspect of the invention the compound recovered is preferably an insecticide or a fungicide.

For this aspect it is further preferred that the extraction is performed by use of an organic solvent on the culture broth.

In yet a further aspect of the invention it relates to a process for controlling insects, whereby a compound according to any of the above aspects of the invention is applied to the area, where said insects are to be controlled.

A preferred embodiment of this aspect of the invention is one wherein the insects to be controlled belong to any of the orders Lepidoptera, Coleoptera. and Diptera, and especially to any of the species Trichoplusia ni, Spodoptera exigua,

Leptinotarsa decemlineata, and/or Aedes aegypti.

In yet a further aspect of the invention it relates to a process for controlling plant pathogenic fungi, whereby a compound according to any of the above aspects of the invention is applied to the area, where said pathogens are to be controlled.

A preferred embodiment of this aspect of the invention is one wherein the pathogens to be controlled belong to the Phycomycetes and especially to any of the species Phytophthora infestans, Plasmopara halstedii and/or Pseudoperonospora cubensis. Practice of this invention will be illustrated by the following Examples. Example 1 illustrates the insecticidal activity of DSM 5415. Examples 2-4 illustrate the activity of DSM 5415 against plant pathogenic fungi. EXAMPLES

Example 1

Insecticidal activity

In this example DSM 5415 was tested for insecticidal activity. DSM 5415 was grown for 5 days at 30ºC on agar slants each containing 12 ml of ATCC medium No. 337 (pH adjusted to pH 6.5) 2 ml of a suspension of spores were then transferred to 100 ml of production medium in 500 ml baffle bottom Erlenmeyer flasks. The production medium consisted of the following components in the quantities indicated (expressed as grams per litre).

Soy tone 40

Potato flour 100

Na₂HPO₄, H₂O 12

KH₂PO₄ 6 BAN 120L amylase (Novo-Nordisk a/s) 0.2 ml

Pluronic (antifoaming agent) 0.2 ml

The pH was adjusted to 7.0.

The inoculated flasks were incubated at 30°C for 5 days with shaking (280 rpm).

Prior to testing for insecticidal activity the broth was pretreated in the following ways.

Test sample Pretreatment

1-4 Samples of culture broth were adjusted to pH 3,

7 and 10 respectively, with 1 N HCl or 1 N NaOH. Equal amounts of broth and ethylacetate were mixed and the samples shaken for one hour at room temperature. The samples were then centrifuged at 4000 rpm for 10 minutes at 25°C. The ethylacetate phases were evaporated to dryness and resuspended in 1/10 volume methanol (100%) so that a tenfold higher concentration was obtained. The water phase obtained from the pH 7 ethylacetate extraction was used without any further treatments. The culture broth was adjusted to pH 7. Equal amounts of broth and methanol (100%) were mixed and the mixture was shaken for one hour. Cells were removed by centrifugation at 4000 rpm for

10 minutes at 25°C. The supernatant was then evaporated to dryness and resuspended in 50% methanol to original volume. The cells were removed by centrifugation at 5000 rpm for 20 minutes at 4ºC. The supernatant was used without any further treatments.

Test samples 1-6 were tested for antimicrobial activity against Trichoplusia ni. Spodoptera exigua, Leptinotarsa decemlineata and Aedes aegypti. Lepidoptera:

Trichoplusia ni and Spodoptora exigua.

4 g larvae diet was poured into each of 25 cups made in form pressured transparent plastic in a tray. The diet was left 2 hours for evaporation and cooling. 100 μl of test sample, methanol (control) or water (control) was applied topically on the diet with a micropipette. All applications were duplicated. The tray was tilted and turned to secure that the fluid totally covered the surface in each cup. The tray was covered with paper for 16 hours.

Five second instar larvae were then put into every cup, the trays were covered with a transparent plastic foliage which was ironed on and then pierced to avoid condensation of water in the cups. The trays were placed in temperature and light regulated chambers (30°C, 16/8 light on/off) for four days. The effect of the test samples on the larvae was scored as % mortality after four days.

Coleoptera

Leptinotarsa decemlineata

A potato leaf was cut and positioned on top of two wet blotting papers in a lid of a petri dish (9 cm). A circular hole (3.5 cm) was cut in the bottom of a petri dish (8.5 cm) which was positioned on top of the leaf thus allowing the insect larvae to feed within this area only. 100 μl of test sample, methanol (control) or water (control) was applied topically on the leaf. Each application was duplicated. The petri dish was closed with another lid and the dishes were left for 16 hours to allow evaporation of methanol or water.

Five first instar larvae were then put on top of each leaf and left for four days in a temperature and light regulated chamber (27°C, 16/8 light on/off). The effect of the test samples on the larvae was scored as % mortality and amount of leaf eaten after one and four days. Diptera

Aedes aegyptii

1 mg of powdered dog biscuit was poured into each of 20 cylindrical test-cups (volume 3 ml) in a small tray (Nunc). 100 μl of test sample, methanol (control) or water (control) was applied topically on the powder and allowed to evaporate for 16 hours. 2.0 ml of tap water with five first instar larvae were poured into each cup with a micropipette. The tray was covered with a lid to avoid further evaporation. The effect of the test samples on the larvae was scored as % mortality after 24 hours.

The effect of the test samples on Trichoplusia ni, Spodoptera exigua, Leptinotarsa decemlineata and Aedes aegyptii is shown in table II.

From table II it appears that DSM 5415 gives good control of all four insects. It further appears from table II that the insecticidal activity of DSM 5415 can be extracted from the culture broth by ethyl acetate. Example 2

Control of Potato Late Blight

In this example the test samples described in example 1 were tested for activity against Phythophthora infestans, causal agent of potato late blight. Test samples of DSM 5415 were sprayed (by means of an Aerograph Super 63 sprayer) onto plants of Solanum tuberosum, cultivar SAVA (at the two-four leaf stage). The plants were incubated for 24 hours at 20°C (6 hours in darkness, 18 hours in light) before being inoculated with a sporangium suspension of Phvthophthora infestans. The inoculated potato plantlets were incubated in a humid chamber for 24 hours in darkness, 20°C, and then further incubated with a light period of 18 hours followed by 6 hours darkness. After a total of 4-5 days of incubation the effect of the prophylactic treatments were scored as degree of inhibition of fungal attack as compared to untreated, inoculated controls. A linear scale ranging from 0 (= no inhibition of the disease development) to 9 (= full control of the attack) was used for the scoring. The phytotoxic effect was recorded according to a scale ranging from P₀ to P₃, (0 = no phytotoxicity and 3 = total withering of the plant). Test samples 1, 2 and 4 were all diluted 8 times before they were sprayed onto the plants.

It appears from the results listed in table III that the test samples made from DSM 5415 give good to excellent control of the late blight pathogen.

It further appears from table III that the fungicidal activity of DSM 5415 can be extracted from the culture broth by ethyl acetate. Table III

Control of potato late blight,

Test sample Potato-phytophthora

infestans

No. Pretreatment Control/phytotox

EtAc extraction at

pH 3, EtAc phase 6 (70%)* PO

EtAc extraction at

pH 7, EtAc phase 9 (100%) PO

EtAc extraction at

pH 7, H₂O phase 3 (30%) PO

EtAc extraction at

pH 10, EtAc phase 8 (90%) PO

Methanol extraction at pH 7 8 (90%) PO

Supernatant 4 (40%) PO

Control 50% methanol 0 (0%) PO () % control

Example 3

Control of sunflower downy mildew and cucurbit downy mildew

In this example culture broth of DSM 5415 was produced as described in example 1. The culture broth was adjusted to pH 3.5 and 8, respectively. Equal amounts of culture broth and ethyl acetate were mixed and shaken for twenty hours at 4°C. The ethylacetate phases were evaporated to dryness and resuspended in 50% methanol to original volume.

The test samples were sprayed (by means of a manual atomizer) onto plants of Helianthus annuus (at the four leaf stage) and onto leaf discs of Cucumis sativus. The plants were incubated for 24 hours at room temperature (22 ºC, before inoculated with a zoospore suspension of Plasmopara halstedii, causal agent of sun flower downy mildew, and Pseudoperonospora cubensis causal agent of cucurbit downy mildew, respectively. The inoculated sunflower plantlets were incubated in humid chambers for 16 hours in darkness, 15°C, and then transferred to 20°C with a light period of 16 hours, followed by 8 hours in darkness. The cucumber leaf discs were kept at moist blotters in petri dishes, incubated at 15ºC, 16 hours in darkness followed by a light/darkness period as described above but kept at 15°C. After a total of 10 days of incubation the effect of the prophylactic treatments were scored as degree of inhibition of fungal attack as compared to untreated, inoculated controls. A linear scale ranging from 0 (= no inhibition of the disease development) to 9 (= full control of the attack) was used for the scoring. The phytotoxic effect was recorded according to a scale ranging from P0 to P3, (0 = no phytotoxicity and 3 = total withering of the plant).

It appears from the results listed in table IV that the three preparations made from DSM 5415 give good to excellent control of the two downy mildew pathogens.

Example 4

Control of sun flower downy mildew

In this example DSM 5415 was grown in four different media, all in shake flasks as described in example 1. The production media a to d consisted of the following components in the quantities indicated (expressed as grams per litre tap water), shake flasks were inoculated and incubated as described in example 1.

Medium a: described in example 1 Medium b: starch 15

corn steep liquor 10

soy flour 10

NaCl 5

CaCO₃ 2

Pluronic 0.1 ml

PH 7.0

Medium c: oatmeal 45

yeast extract 2

NaHPO₄.12 H₂O 12

KH₂PO₄ 6

Pluronic 0.2 ml

pH 6.5 - 7.0

Medium d: ATCC medium No. 337 without agar;

pH adjusted to 6.5 - 7.0

Prior to testing for activity against Plasmopara halstedii the culture broths were extract, d with ethanol. Equal amounts of culture broth and ethanol (96%) were mixed and left overnight at 4°C. The cells were removed by centrifugation at 4000 rpm at 5°C for 10 minutes. The resulting supernatants were tested for activity against Plasmopara halstedii as described in example 4. It appears from the results listed in table V that preparations made from DSM 5415 grown on all four media give excellent control of sun flower downy mildew.

Claims

PATENT CLAIMS

1. A novel group of bacteria, characterized in that it belongs to the order of Actinomycetales, has the ability to produce rope like structures comprising a large number of spore chains which are fused together in said rope like structures, and produces biocidal compounds, or mutants or genetically engineered variants thereof.

2. A group of bacteria according to claim 1, further characterized in exhibiting characteristics essentially as described in the specification.

3. A group according to claim 1 or 2, further characterized in that said biocidal compounds are insecticidal compounds.

4. A group according to claim 1 or 2, further characterized in that said biocidal compounds are fungicidal compounds.

5. A novel microorganism belonging to the group of claim 1, 2, 3 or 4, characterized in that it is morphologically identical or exhibits the principal morphological features of the bacterial strain deposited under the accession no. DSM 5415 or mutants or genetically engineered variants thereof.

6. A novel microorganism belonging to the group of claim 1, 2, 3 or 4, characterized in that it is morphologically identical or exhibits the principal morphological features of the bacterial strain deposited under the accession no. DSM 6012 or mutants or genetically engineered variants thereof.

7. An biocidal compound, characterized in that it is producible by bacteria according to any of claims 1 to 6.

8. A compound according to claim 7, further characterized in that it is an insecticide.

9. A compound according to claim 8, further characterized in that it exhibits activity towards insects of any of the orders Lepidoptera, Coleoptera, and Diptera.

10. A compound according to claim 8, further characterized in that it exhibits activity towards Trichoplusia ni,

Spodoptera exigua, Leptinotarsa decemlineata, or Aedes aegypti.

11. A compound according to claim 7, further characterized in that it is a fungicide.

12. A compound according to claim 11, further characterized in that it exhibits activity towards plant pathogenic fungi.

13. A compound according to claim 12, further characterized in that it exhibits activity towards Phytophthora infestans, Plasmopara halstedii, or Pseudoperonospora cubensis.

14. A process for the production of a compound according to any of the claims 7 to 13, characterized in that a bacteria according to any of claims 1 to 6 is cultivated in the presence of suitable sources for nitrogen and carbon and essential nutrients, and the biocidal compound is extracted from the culture broth and recovered according to methods known per se.

15. A process according to claim 14, further characterized in that the bacteria cultivated is morphologically identical to or exhibits the principal morphological features of the bacterial strain deposited under accession no. DSM 5415, mutants or genetically engineered variants thereof.

16. A process according to claim 14, further characterized in that the bacteria cultivated is morphologically identical to or exhibits the principal morphological features of the bacterial strain deposited under accession no. DSM 6012, mutants or genetically engineered variants thereof.

17. A process according to any of the claims 14 to 16, further characterized in that the compound recovered is an insecticide.

18. A process according to any of the claims 14 to 16, further characterized in that the compound recovered is a fungicide.

19. A process according to any of claims 14 to 18, further characterized in that the extraction is performed by use of an organic solvent on the culture broth.

20. An insecticidal preparation, characterized in that it comprises a compound according to any of the claims 8 to 10 in admixture with compatible excipients.

21. A fungicidal preparation, characterized in that it comprises a compound according to any of the claims 11 to 13 in admixture with compatible excipients.

22. A process for controlling insects, characterized in that a compound according to any of the claims 8 to 10, or a preparation according to claim 20 is applied to the area, where said insects are to be controlled.

23. A process according to claim 22, further characterized in that the insects to be controlled belong to any of the orders Lepidoptera, Coleoptera, and Diptera.

24. A process according to claim 22, further characterized in that the insects to be controlled are Trichoplusia ni, Spodoptera exigua, Leptinotarsa decemlineata, or Aedes aegypti.

25. A process for controlling fungi, characterized in that a compound according to any of the claims 11 to 13, or a praparation according to claim 21 is applied to the area, where said fungi are to be controlled.

26. A process according to claim 25, further characterized in that that the fungi to be controlled are plant pathogenic fungi.

27. A process according to claim 25, further characterized in that the fungi to be controlled are Phytophthora infestans, Plasmopara halstedii, or Pseudoperonospora cubensis.