Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2442—Chitinase (3.2.1.14)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

• the field of invention is drawn to pesticidal strains producing novel enzymes with applications in plant protection, more specifically controlling insects and phytopathogenic fungi of economic significance to agriculture and forestry sectors.
• This invention relates to antifungal activity of chitinases to control phytopathogenic fungi and synergistic interaction of chitinases with the insecticidal proteins of Brevibacillus laterosporus to exhibit greater efficacy and enhanced toxicity to achieve a higher degree of insect control.
• the present invention can be applied to the rational design and development of new generation biopesticides based on Brevibacillus laterosporus for agricultural and forest pests.
• biopesticides may effectively address most of the challenges related to the use of pesticides and as a result they received ample attention in current research programs on sustainable crop protection.
• Bacterial, agents are environmentally safe due to their host specificity, required in very low dosage, easy to prepare commercially in large-scale and are less costly. Since the 1950s, strains of Bacillus thuringiensis have been used as potent insecticides to control economically important agricultural insect pests. Formulations of B. thuringiensis var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. But the toxins of B. sphaericus and B. thuringiensis var israelensis in particular, do not persist long in nature and require frequent application which is a limiting factor for these organisms to be most successful and potent biolarvicide.
• Chitin is an unbranched polysaccharide polymer consisting of N-acetyl-D-glucosamine units ("GluNAc”) joined by beta-1,4 glycosidic linkages. Chitin is insoluble in water, dilute mineral acids and bases but can be broken down enzymatically by chitinase, the degradation products being soluble monomers or multimers of GluNAc. Chitinases are a class of hydrolytic enzymes which degrade chitin by endolytic or exolytic mechanisms.
• Chitinase is produced by certain naturally occurring bacteria, fungi, actinomycetes, nematodes, insects, crustaceans, plants and some vertebrates and there have been reports of the role of chitinase in the suppression of pathogens.
• fungi actinomycetes
• nematodes insects, crustaceans, plants and some vertebrates
• US patent 6329504 discloses an antifungal polypeptide and methods for controlling plant pathogenic fungi.
• Brevibacillus laterosporus is reported to have a very wide spectrum of biological activity compared to the most popular insecticidal bacteria Bacillus thuringiensis and Bacillus sphaericus, its biological control potential has not been fully explored since the attempts to isolate this organism from different ecological niches was not successful since the distribution of strains of Brevibacillus laterosporus is limited compared to the strains of Bacillus thuringiensis and Bacillus sphaericus (Oliveira E J D, L Rabinovitch L, Monnerat RG, Passos LKJ and Zahner V (2004) Molecular characterization of Brevibacillus laterosporus and its potential use in biological control. Appl. Environ.Microbiol. 70 (1 1): 6657-64).
• U.S pat 80761 19 discloses an invention which relates to a method for biological control of insects belonging to the order Diptera (flies, mosquitos, horseflies and midges) and especially against the species Musca domestica.
• Brevibacillus laterosporus strain compositions containing the same and method for the biological control of dipterans.
• a small number of research articles have been published about the enzymatic profile and effects of toxicity from Brevibacillus laterosporus strains.
• the literature on chitinase from Brevibacillus laterosporus and its biotechnological applications, in particular agricultural and environmental applications is scarce. It is an object of the present invention to provide a new, wild type (i.e naturally occurring) chitinolytic, insecticidal strain of Brevibacillus laterosporus LAK1210 which produces a novel chitinase.
• the chitinolytic enzymes from this new biocontrol strain synergises the insecticidal activity and enhances the entomotoxicity of the said strain making it effective against a wide spectrum of insects and plant pathogenic fungi.
• the invention broadly pertains to a novel isolate of Brevibacillus laterosporus, Brevibacllus laterosporus LAK 1210 that exhibits strong chitinolytic and insecticidal activity.
• the new strain has been deposited at Microbial Type Culture Collection MTCC), an International repository at IMTECI I, INDIA and can be accessioned as 5487.
• MTCC Microbial Type Culture Collection
• IMTECI I International repository
• INDIA International repository at IMTECI I
• the subject of invention also concerns a natural strain that could be quickly developed into an effective, safe, natural biopesticide/ biocontrol formulation.
• the present invention further relates to the discovery of a new chitinase and antifungal metabolites (polypeptides) that exhibit broad spectrum antifungal activity against phytopathogenic fungi.
• the novel polypeptides represent a new group of protein antifungals obtainable from the new strain, Brevibacillus laterosporus LAK1210.
• the present invention provides method of effecting or modulating phytopathogenic infection, in particular, fungi and insects, using cell suspensions of Brevibacillus laterosporus LAK 1210 or supernatant containing metabolites ( antifungal chitinases) or purified metabolites to all or part of a plant or a plant seed, under conditions effective to control insect pests and plant diseases of agronomic and horticultural importance.
• synergistic effect of the chitinases potentiate the insecticidal action resulting in additive enhancement of the toxicity, efficacy and substantially broadens the spectrum of activity against the insects to be controlled and also allows lower application rates.
• the chitinases according to the invention also exhibit broad spectrum antifungal activity which may contribute significantly to the enhanced, persistent fungicidal action against a wide range of phytopathogenic fungi.
• the invention has particular utility for a new chitinase from a new insecticidal strain as an agriculturally beneficial biocontrol agent in controlling/ inhibiting/modulating phytopathogenic infection caused by insects, phytopathogenic fungi, bacteria and nematodes.
• This invention is directed at antifungal combinations of chitinolytic enzymes and use thereof for topical or internal application to inhibit germination or replication of phytopathogenic fungi and synergistic action of chitinases to enhance the insecticidal effectiveness. More specifically, the invention provides methods and compositions for preventing and controlling fungal diseases and combating insects of economic significance to agriculture and forestry sectors.
• Brevibacillus laterosporus LAK 1210 has lepidopteran activity as tested against diamond backmoth (Plutella xylostella), a devastating pest of crucifers.
• An aspect of the present invention relates to a biologically pure culture of a new strain of Brevibacillus laterosporus designated as Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487, a variant or a mutant thereof.
• Another aspect of the present invention relates to an isolated 16S rRNA gene sequence of the strain Brevibacillus laterosporus LAK 1210 having accession number MTCC 548, wherein the nucleotide sequence of gene is as set forth in SEQ ID NO: 1 having GenBank accession number HQ412764.
• Yet another aspect of the present invention relates to "a chitinolytic enzyme having molecular weight selected from a group consisting of 25 kDa, 55 kDa, 70 kDa, 75 kDa, 90 kDa, wherein the enzyme is active at a pH ranging from 3.0 to 1 1.0 and at a temperature ranging from 30°C to 90°C.
• FIG. 1210 having accession number MTCC 5487 in a culture medium comprising 0.1-1 % (w/v) colloidal chitin or 0.1-1% (w/v) marine chitinous waste to obtain cell culture, and obtaining the supernatant comprising chitinases and insecticidal proteins from the cell culture.
• Fig. 1 shows phylogenetic relationship of Brevibacillus laterosporus Lakl210 to selected species from the genera Bacillus, Paenibacillus and Brevibacillus, based on 16S RNA genes.
• the tree was constructed with the Treebuilder tool provided by the Ribosomal Database Project (see method section for details).
• Brevibacillus laterosporus Lak 1210 is indicated with an arrow.
• Fig. 2 shows a) chitinolytic activity of Brevibacillus laterosporus Lak.1210.
• the plate contained 0.2 % colloidal chitin as the only carbon source, b) Chitin plate stained with 1% Congo Red after 7 days of incubation (30°C). Chitin degradation is shown as a clear halo around the colony.
• the figure also shows the morphology of a typical older Lakl210 colony, filamentous with fringed margins, c) Photomicrograph of the 72 h sporulating culture of Brevibacillus laterosporus LAK 1210.
• Fig.3 shows a) SDS PAGE. Proteins precipitated from the supernatant of chitin-induced culture (lane 1) and chitin affinity purification (lane 2). Protein bands that were subjected to characterization by trypsination and mass spectroscopy are numbered. M, protein size markers b) Activity staining. Proteins precipitated from the supernatant of chitin-induced culture (lane 1) and chitin affinity purification (lane 2).
• Fig. 4 is characterization of the chitinase mixture using 4MU-NAG2 as substrate. Each point represents the average of three measurements. Standard deviations are not shown because they were below 2 % of the value and thus hidden by the data points, (a) pH activity profile.
• the buffers used were: Citrate phospate (pH 3.0 - 5.0), sodium acetate (pH 5.0 - 6.0), sodium phosphate (pH 6.0 - 8.0), Tris-HCl (pH 8.0 - 9.0), Carbonate- bicarbonate (pH 9.0 -1 1.0).
• Fig. 5 shows a) Antagonism against Fusarium equiseti b) Antagonism against Fusarium oxysporium f.sp lycopersici c) Antagonism against Fusarium moniliforme d) Antifungal assay showing antagonism against Rhizoctonia solani. Paper discs were treated with 100, 200 and 500 ⁇ of the mixture of purified chitinases, in 10 mM sodium phosphate, pH 6.0; the samples are marked 1 to 3 respectively. . C indicates a control disc impregnated with sodium phosphate buffer. The dotted line indicates a clear border of fungal growth for sample 3.
• Fig. 6 shows Agar cup assays for scoring of the larvae of Plutella xylostella a) Control (Third and fourth instar larvae), b) Dead larvae (showing discolouration from green to brownish- black), c) Feeding assay experiment in a tissue culture tray.
• Fig. 7 is the effect of chitinases on insecticidal activity.
• the graph shows survival of second and third instars larvae on cabbage leaves treated with sterile water or with a suspension of non-induced Brevibacillus laterosporus Lakl210 supplemented with 0- 500 ⁇ of supernatant from a chitin induced culture of Brevibacillus laterosporus Lakl210. Each point represents the average survival from three independent measurements (10 larvae per measurement).
• SEQ ID NO: 1 shows 16 s rDNA sequence (1-1500 bp) having NCBI Genbank Accession No HQ_412764.
• SEQ ID NO: 2 to 18 shows peptide sequence present in the chitinase(s) produced by a new strain of Brevibacillus laterosporus designated as Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487.
• SEQ ID NO: 19 shows universal forward primer sequence
• SEQ ID NO: 20 shows universal reverse primer sequence
• Table 1 depicts peptide sequence present in the chitinase(s).
• the sequence information was derived for a limited number of the six proteins (usually only one), as listed in the second column marked "protein".
• Peptides are scored positive (1) on the basis of corresponding peptide masses.
• the sequence ASVPTNYK was determined twice, while analyzing proteins 3 and 6, and belongs to a peptide with a molecular mass that was detected in the peptide maps of proteins 2, 3 & 6. These analyses are not absolute. Certain peptides may remain undetected in certain samples, whereas other peptides may be detected unexpectedly in certain samples due to cross-contamination (e.g. between proteins 2 & 3, which are very close to each other on the gel).
• X unknown amino acid.
• the new strain of Brevibacillus lalersporus LAK 1210 has been deposited at Microbial Type Culture Collection, an International repository at IMTECH, INDIA with the accession number MTCC 5487 under the Budapest Treaty.
• This new strain encode novel polypeptides of agricultural interest, with biological activity against insects and phytopathogenic fungi and in particular embodiments, these polypeptides synergises and enhance the insecticidal activity relative to the activity of insecticidal proteins alone.
• the embodiments involve the discovery of a rarely distributed, natural insecticidal strain producing a novel chitinase and the genes that encode the novel polypeptides.
• the embodiments further relate to the identification of fragments and variants of the naturally-occurring coding sequence that encode biologically active polypeptides with chitinolytic activity.
• the nucleotide sequences of the embodiments find direct use in methods for impacting pests, particularly insect pests such as pests of the order Lepidoptera. Accordingly, the embodiments provide new approaches for impacting insect pests that do not depend on the use of conventional insecticides.
• the embodiments encompass a wild type (i.e naturally occurring) bacterial strain which finds use in compostions and methods for impacting insect pests of the plant, such as, for example, lepidopteran pests and also protecting the plants from phytopathogenic fungi.
• Insect pests may be tested for insecticidal activity of compositions of the embodiments in early developmental stages, e.g., as second, third and fourth larval instars.
• the synergism of chitinolytic enzymes potentiates the insecticidal activity effecting changes in insect feeding, growth, and/or behaviour at any stage of development, including but not limited to killing the insect retarding growth, preventing reproductive capability, anti-feedant activity and the like.
• a biologically pure culture of a new strain of Brevibacillus laterosporus designated as Brevibacillus later osporus LAK 1210 having Accession number MTCC 5487, a variant or a mutant thereof.
• Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487, a variant or a mutant thereof, wherein the strain produces chitinases and insecticidal proteins.
• An embodiment of the present invention provides a biologically pure culture of a new strain of Brevibacillus laterosporus designated as Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487, a variant or a mutant thereof, wherein the strain exhibits antifungal activity against phytopathogenic fungi.
• Another embodiment of the present invention provides a biologically pure culture of a new strain of Brevibacillus laterosporus designated as Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487, a variant or a mutant thereof, wherein the strain exhibits an inhibitory effect on insects.
• Yet another embodiment of the present invention provides an isolated 16S rRNA gene sequence of the strain Brevibacillus laterosporus LAK 1210 having accession number MTCC 548, wherein the nucleotide sequence of gene is as set forth in SEQ ID NO: 1 having GenBank accession number HQ412764.
• compositions and methods of the embodiments comprise chitinolytic enzymes encoded by a naturally-occurring nucleic acid of the embodiments. More specifically, the embodiments provide novel polypeptides having antifungal activity comprising the amino acid sequences set forth in (Table 1).
• the present invention provides a process for producing a supernatant comprising chitinases and insecticidal proteins, wherein the process comprises culturing liquid culture of novel strain of Brevibacillus laterosporus designated as LAK 1210 having accession number MTCC 5487 in a culture medium comprising 0.1-1 % (w/v) colloidal chitin or 0.1-1% (w/v) marine chitinous waste to obtain cell culture, and obtaining the supernatant comprising chitinases and insecticidal proteins from the culture broth.
• marine chitinous wastes wherein the example of the marine chitinous wastes includes but is not limited to shrimp shell powder, crab shells, squid pen powder.
• the present invention provides a process for producing a supernatant comprising chitinases and insecticidal proteins, wherein the process comprises culturing liquid culture of novel strain of Brevibacillus laterosporus designated as LAK 1210 having accession number MTCC 5487 in a culture medium comprising 0.1-1 % (w/v) colloidal chitin or 0.1-1% (w/v) marine chitinous waste to obtain cell culture, obtaining the supernatant comprising chitinases and insecticidal proteins from the cell culture, and purifying chitinases from the supernatant using purification method comprising ammonium sulphate precipitation followed by affinity or ion exchange chromatography.
• Yet another embodiment of the present invention provides a chitino lytic enzyme having molecular weight selected from a group consisting of 25 kDa, 55 kDa, 70 kDa, 75 kDa, 90 kDa.
• Another embodiment of the present invention relates to chitinolytic enzymes which are active at a pH range of 3.0-11 .0, with two pH optima (pH 6.0 and 8.0).
• the enzymes are alkaline active under alkaline conditions (at a pH above 7.0 and in a pPI range of pH 9.0-1 1.0) but may also be active under neutral and/or acid conditions.
• the chitinolytic enzymes of the present invention are particularly effective in controlling insects, because they are active under alkaline conditions. As a result, these enzymes can. be ingested by insects and then attack the insects by degrading their chitin-containing, alkaline digestive tracts.
• a chitinolytic enzyme having molecular weight selected from a group consisting of 25 kDa, 55 kDa, 70 kDa, 75 kDa] '9 TkDa, wherein the enzyme is active at a temperature rangin from 30°C to 90°C) herein the enzyme has a temperature optimum of " 70 o C
• the present invention further provides relates to chitinolytic enzymes with high thermostability, with an optimum temperature of 70°C for the mixture of chitinases.
• the temperature activity curve coincidences with the thermostability curve, indicating that enzyme activity at high temperatures is impaired by the stability of one or several of the proteins in the mixture.
• the high thermostability of the chitinases is remarkable considering that the thermostable chitinases from a mesophilic bacterium adapted to a mangrove forest biome with moderate temperatures is a rare find and there are very few reports on thermostable enzymes from mesophiles.
• thermostability is an important parameter for industrial use of enzymes and the high thermostability of the chitinases from Brevibacillus laterosporus Lakl210 could thus be an advantage in further utilization of the strain in biomass utilization, biofuel research and in other industrial processes where the harsh environment demands higher pH and temperature.
• the present invnetiuon provides a composition comprising cells of the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 or a metabolite obtained from the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 and a carrier.
• Yet another embodiment of the present invention provides a composition the supernatant obtained from the culture of .
• the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 or a metabolite obtained from the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 and a carrier.
• Still another embodiment of the present invention provides a composition comprising the purified chitinases obtained from the supernatant obtained from the cell culture of the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 or a metabolite obtained from the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487.
• a synergistic composition comprising the chitinases obtained from the supernatant obtained from the cell culture of the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 or a metabolite obtained from the Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487 and insecticidal proteins selected from the group consisting of i) culture supernatant of Brevibacillus laterosporus LAK 1210 having accession number MTCC 5487 ii) cells of strain Brevibacillus laterosporus LA 1210 having accession number MTCC 5487 and iii) one or more derivatives of Brevibacillus laterosporus LAK 1210 having accession number MTCC 5487, wherein the derivatives are selected from a group consisting of spores, inclusions, enzymatic fractions and cellular extracts: and a carrier.
• compositions as disclosed in the present invention can be formulated in the form of wettable powders, dust, pellets, granules, seed treatment products, emulsions, sprayable solutions, aqueous solutions, oil or water based dispersions, ULV formulations or microencapsulations.
• compositions as disclosed in the present invention can be a biopesticide, biofungicide, insecticide or nematici.de.
• Further embodiment of the present invention provides a method for protecting or treating or modulating phytopathogenic infection in plant or a part thereof, wherein the method comprises applying synergistically effective amounts of the composition as disclosed in the present invnetion to the plant or the part thereof, natural or artificial soil or planting media.
• the combinations are compositions, particularly compositions for use in treating or modulating phytopathogenic infection, more specifically phytopathogenic fungi and insects.
• the above mentioned combinations may also be formulated into compositions with an agriculturally acceptable shelf life.
• phytopathogenic infection means infection of plants by pathogenic fungi, bacteria, insects and nematodes.
• Preferred combinations can be formulated with an acceptable carrier into a biopesticide/biocontrol agent. Furthermore, these combinations of this invention have an advantage of being formulated as an adjuvant, a colloid, a wettable powder, an emulsifiable concentrate, an aerosol or spray, a dusting powder, a dispersible granule or pellet, an impregnated granule, a suspension, a solution, an emulsion and also microencapsulations.
• the compositions can be formulated by conventional methods such as those described in, for example, Winnacker-Kuchler (1986), "Chemische TECH" [Chemical Technology], Vol. 7, C. Mauser Verlag Kunststoff, 4th Ed.
• Necessary formulation aids include such as carriers, inert materials, surfactants, solvents, and other additives.
• Such formulated compositions may be prepared by concentration of a culture of cells comprising the polypeptides, desiccation, extraction, filtration, centrifugation, sedimentation, homogenization and lyophilization.
• the invention is additionally directed to a method of treating or modulating the phytopathogenic infection, in particular phytopathogenic fungi and insects.
• These formulations can be applied to the environment hosting a target insect pest/pathogenic fungus, e.g., soil, water or a plant or a part thereof wherein seeds, plantules, plants, foliage of plants of the plant are treated with an effective amount of the formulation.
• the phytopathogenic infection as described in the present invention is a plant disease caused by at least one fungus selected from the group consisting of Fusarium. Rhizoclonia, Pythium, Phylophthora, Cercospora. Puccinia. Ventiiria, Alternaria, Uncinula, Ustilago, Colletotrichum, Erysiphe, Bolrytis. Sclerotium and Monilinia which comprises contacting such fungus with purified antifungal chitinases. effective to obtain said inhibiting.
• the method of the present invention involving application of a chitinolytic enzyme can be carried out through a variety of procedures when all or part of the plant is treated, including seeds, roots, stems and leaves etc.
• Fusarium species include Fusarium oxysporum f. sp. lycopersici, Fusarium moniliforme or Fusarium equiseti.
• insects include economically important agronomic, forest, greenhouse, nursery, ornamentals, food and fiber, public and animal health, domestic and commercial structure, household, and stored product pests.
• Insect pests include insects selected from the orders Diptera, Lepidoptera, Coleoptera, Hymenoptera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Isoptera, more specifically insects from Diptera , Coleoptera and Lepidoptera.
• the phytopathogenic infection as described in the present invention may be caused by at least one insect belonging to Lepidoptera, Diptera, Coleoptera, Homoptera or Hymenoptera.
• the novel strain of Brevibacillns laterosporus LAK 1210 exhibits an "improved pesticidal activity" which refers to synergistic action of chitinolytic enzymes of the embodiments that has enhanced insecticidal activity where a wider range of insects is impacted relative to the range of insects that is affected by activity of its corresponding non-chitinolytic strains of Brevibacillus laterosporus strains and may be other wild- type non-chitinolytic insecticidal strains.
• the formulations of the embodiments can be used in combination with other Bt toxins or other insecticidal proteins to increase insect target range for insect resistance management and control. Furthermore, the formulations can also be used in combination with other biocontrol agents or chemical fungicides for integrated pest management.
• an improved method for protecting or treating or modulating phytopathogenic infection in plant or a part thereof comprising applying the composition as disclosed in the present invention concurrently with the Bacillus-based insecticides, wherein the method enhances the insecticidal effectiveness of the Bacillus-based insecticides for insect control.
• Bacillus species includes Bacillus thuringiensis or Bacillus sphaericus.
• Example 1 Screening of insecticidal bacteria for novel chitinolytic enzymes
• Brevibacillus laterosporus Lakl210 was isolated from mangrove, marshy soil in Palasa located in Srikakulam district of Andhra Pradesh, India, as part of an effort to isolate novel pesticidal strains with chitinolytic potential.
• the active strain was identified as a member of Brevibacillus laterosporus, based on morphological, and physiological characteristics, and 16 s r RNA sequence (SEQ ID NO: 1 ).
• the strain was accessioned into the Microbial Type Culture Collection, MTCC 5487.
• the 16S rRNA was amplified from genomic DNA using the universal primers p27f (SEQ ID NO: 19 - 5-AGA GTT TGA TCM TGG CTC AG-3and pl 525r (SEQ ID NO: 20 - 5-AAG GAG GTG WTC CAR CC-3).
• Genomic DNA was extracted using DNAzol (Invitrogen, California USA).
• the genomic DNA was subjected to PCR in a 50 ⁇ reaction containing 1 ⁇ DNA (0.2 ng), 1 ⁇ each of forward and reverse primers (25 ⁇ ), 2 ⁇ of each of the dNTPs, 2 ⁇ MgC12, 2.5 ⁇ 10X PCR-Buffer II (without MgC12), and 0.2 ⁇ Taq-DNA Polymerase (2.5 U).
• PCR conditions were as follows: initial activation of the Taq-DNA Polymerase for 30s at 94°C, followed by 30 cycles comprising of a denaturation step, for at 94°G, an annealing step for 1 min at 58°C, and an extension step for 50 sec at 72°C, followed by a final elongation step for 5 min at 72°C.
• PCR products were excised from a 1% agarose gel after electrophoresis, purified with the QIA quick Gel Extraction kit (QIAGEN, Inc.) and sequenced directly using a Biotech Diagnostic Big Dye sequencing kit (Biotech Diagnostics, Website Niguel, CA,USA) and an ABI 377 sequencer (Applied Biosystems, Foster City, CA,USA).
• the 16S rRNA sequence of Brevibacillus laterosporus Lakl ' 21 ' 0 (1500 bp) did not exhibit 100% identity with any 16S rRNA sequences in Genbank or the RDP database (Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed- Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucl Acid Res 37 (Database issue):D141-145).
• the closest neighbours are Brevibacillus laterosporus BPM3 and Brevibacillus laterosporus S62-9, both with more than 99 % identity.
• the strain exhibited 96.5-97.0 % identity with the type strains of Brevibacillus ginsengisoli, Brevibacillus invocatus, Brevibacillus reuszeri, Brevibacilus formosus and Brevibacillus agri ( Figure 1).
• Phenotypic studies showed that the isolated bacterium was aerobic and Gram-positive. Cells appear as small rods, 0.7-1.0 ⁇ wide by 3.0-5.0 ⁇ long, occurring singly or in short chains. We observed formation of ellipsoidal spores in swollen sporangia, laterally positioned with a canoe-shaped parasporal body ( Figure 2 c). Colonies of Brevibacillus laterosporus Lakl210 grown on nutrient agar were opaque, granular, jagged and 1.0- 2.0 mm in diameter after incubation for 2 days at 30°C. The pH and temperature range for growth were 7.0-1 1.0 and 25-37°C, and the optimum growth pH and temperature were 7.0-8.0 and 30-32°C, respectively.
• Example 3 Culturing of Brevibacillus laterosporus LAK1210 (MTCC5487) and production of chitinolytic enzymes
• the bacterial cultures were maintained on nutrient agar slants and plates [Lab-Lamco powder, 10.0 g, Bacto peptone (Difco), 10.0 g, sodium chloride, 5.0 g and Bacto agar (Saveen Werner AB, Limhamn, Sweden), 2.0 g per liter of water], at 30°C.
• the medium (pH 7.0) was composed as follows (g per liter): K 2 HP0 4 , 3.0 g, (NH 4 )2S0 4 , 3.0 g, MgS0 4 7H 2 0, 0.03g, peptone 2.0 g, yeast extract, 1.0 g, CaCl 2 2H 2 0, 18 mg, FeS0 4 7H 2 O,0.75 mg, MnS0 4 7H 2 0, 50 mg, CuS0 4 5H 2 0, 7.5 mg, ZnS0 4 7H 2 0, 7.5 g and optionally, 0.5 % (w/v) colloidal chitin.
• sundried shrimp shell powder or other marine chitinous wastes were also used as a source of carbon and nitrogen.
• the cultures were grown in flasks with constant shaking at 30°C at 180 rpm for 7 days.
• chitinolytic activity cells were grown on colloidal chitin agar plates (Sampson MN, Gooday GW (1998) Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects. Microbiology 144:2189-2194) containing a semi-minimal medium.
• the medium consisted of a l : lmixture of minimal medium containing 0.1% ammonium sulphate, 0.03% MgS0 4 7H 2 0, 0.6% K 2 HP0 4 , 1% K 2 HP0 4 , pH 7.0) and nutrient broth (Oxoid, UK), supplemented with colloidal chitin (0.2%) and solidified with 1.5% agar.
• Example 6 Enzyme assays for chitinolytic activity
• Chitinolytic activity was measured by using the fluorogenic chitin derivatives, 4 methylumbelliferyl-N-acetyl- ⁇ -D-glucosaminide (4MU-NAG), 4- methylumbelliferyl- -D-N,N"-diacetylchitobioside (4MU-NAG 2 ) and 4- methylumbelliferyl- -d-N,N',N"-triacetylchitotriose (4MU-NAG 3 ) (Sigma Chemicals, St.Louis, USA).
• 4 methylumbelliferyl-N-acetyl- ⁇ -D-glucosaminide 4 methylumbelliferyl- -D-N,N"-diacetylchitobioside
• 4MU-NAG 3 4- methylumbelliferyl- -d-N,N',N"-triacetylchitotriose
• SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
• SDS-PAGE analysis of the chitinolytic fractions obtained from chitin-affinity chromatography of ammonium sulfate precipitates from 96 h chitin- induced cultures revealed three major and several less prominent bands.
• Cultures were grown at 30°C for 7 days with a constant shaking (180 rpm). The biomass was removed from the broth by centrifugation at 6000 X g for 30 min. 4°C. The supernatant was filtered through mira cloth, then adjusted to 80% saturation with ammonium sulfate to isolate total protein. The precipitate was collected by centrifugation (4°C, 15,000 x g, 20 min), dissolved in 20 mM sodium phosphate buffer pH 6.0 and dialyzed against the same buffer.
• Protein bands were excised from silver or coomassie stained gels, transferred to individual 0.2 ml PCR tubes and dehydrated twice by washing with 100 % acetonitrile.
• the gel pieces were submersed in 50 ⁇ of a freshly made DTT solution (10 mM dithiothreitol in 100 mM ammonium bicarbonate) and incubated at 56°C for 30 min. After removal of the DTT solution, 50 ⁇ IAA solution (55 mM 2- iodoacetamide, 100 mM ammonium bicarbonate) was added, followed by 30 minutes incubation in the dark. Then the gel pieces were washed once with 100 mM ammonium bicarbonate and once with acetonitrile.
• the gel pieces were covered with 30-50 ⁇ (depending on the size of the gel piece) Trypsin solution (10 ng/ ⁇ sequence grade trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate containing 10 % acetonitrile). After incubation on ice for 90 minutes, 25 mM ammonium bicarbonate containing 10 % acetonitrile (no trypsin) was added as needed to completely hydrate the gels (typically 30 - 50 ⁇ ), followed by incubation overnight at 37°C.
• Trypsin solution 10 ng/ ⁇ sequence grade trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate containing 10 % acetonitrile. After incubation on ice for 90 minutes, 25 mM ammonium bicarbonate containing 10 % acetonitrile (no trypsin) was added as needed to completely hydrate the gels (typically 30 - 50 ⁇ ), followed by
• the gel pieces were extracted by sonication for 10 minutes (in a Vibracell 505, Sonics, Newtown, USA) in 50 ⁇ 1 % formic acid in dH 2 0, followed by three extractions with 25 ⁇ of 5% formic acid in 50% acetonitrile (each extraction step included 10 minutes of sonication).
• the four extracts were pooled, and after vacuum-drying of the solution, the peptides were dissolved in 25 ⁇ of 0.1% trifluoroacetic acid.
• Lys-tag Lys-tag
• the size of the catalytic domains of family 18 chitinases varies from about 30 kDa in typical endochitinases such as ChiC from Serratia marcescens (Synstad B, Vaaje-Kolstad G, Cederkvist FH, Saua SF, Horn SJ, Eijsink VG, Sorlie M (2008) Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method. Biosci Biotechnol Biochem 72(3):715-723) to about 45 kDa in processive exochitinases such as ChiA and ChiB from 5.
• typical endochitinases such as ChiC from Serratia marcescens (Synstad B, Vaaje-Kolstad G, Cederkvist FH, Saua SF, Horn SJ, Eijsink VG, Sor
• marcescens van Aalten DM, Synstad B, Brurberg MB, Hough E, Riise BW, Eijsink VG, Wierenga RK (2000) Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution. Proc Natl Acad Sci USA 97(11):5842-5847).
• Example 10 Novel characteristics of chitinolytic enzymes from Brevibacillus laterosporus LAK1210
• the pH optimum for the chitinase activity was measured using 4MU-NAG 2 under standard assay conditions, using the following buffers (all at 50 mM): citrate phosphate (pH 3.0-5.0), sodium acetate (pH 5.0-6.0) sodium phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0), and carbonate - bicarbonate buffer (pH 9.0-11.0).
• the temperature optimum for chitinase activity was determined by carrying out standard activity assays in 50 mM Tris-HCl buffer, pH 8.0 at temperatures ranging from 25°C to 100°C- Thermal stability of the enzyme was determined by preincubating the enzyme samples without the substrate in 50 mM Tris-HCl, pH 8.0, at temperatures ranging from 25°C to 100°C for 30 min, after which the residual enzyme activity of 10- fold diluted samples was measured under standard assay conditions.
• the pH-activity profile revealed two pH optima, one at pH 6.0 and the other at pH 8.0 (Fig 4a).
• the optimum temperature for chitinolytic activity for the mixture of chitinases was found to be 70°C (Fig 4b).
• thermostability curve coincidences with the thermostability curve (Fig 4c), indicating that enzyme activity at high temperatures is impaired by the stability of one or several of the proteins in the mixture.
• the high thermostability of the chitinases is remarkable considering that they originate from bacterium adapted to a mangrove forest biome with moderate temperatures. Thermostability is an important parameter for industrial use of enzymes and the high thermo'stability of the chitinases from Lakl210 could thus be an advantage in further utilization of the strain.
• Chitinolytic enzymes which are alkaline active are of interest because the alkaline chitinases function well in the alkaline environment of insect gut to enable the insecticidal proteins to permeate through easily.
• U.S Pat 391 1 1 10 discloses an invention on chitinases that facilitate the action of insecticidal proteins, when used in conjunction with the entomopathogenic organisms.
• Example 11 Antifungal Assays and antagonistic activity of Brevibacillus laterosporus LAK 1210 against Fusarium species and other phytopathogenic fungi
• PDA Potato Dextrose Agar
• Sterile filter paper discs (5 mm in diameter) impregnated with varying amounts of chitinase solutions in 20 mM sodium phosphate buffer (pH 6.0) were then placed on the agar surface at a distance of 0.5 cm from the front edge of The growing fungal colony. Control discs were saturated with sodium phosphate buffer (pH 6.0). The plates were then further incubated at 28°C and the fungal growth inhibition was visually assessed by the presence of a zone of inhibition near the chitinase-treated discs.
• Figure 5a shows that the enzymes indeed inhibited the fungus in a dose-dependent manner.
• the control disc is overgrown, disc 1 (with 100 ⁇ chitinase solution) and disc 2 (with 200 ⁇ chitinase solution) seem less overgrown, whereas disc 3 (500 ⁇ ) shows a clear zone of inhibition demarcated as an arc (Fig 5d).
• Brevibacillus laterosporus LAK 1210 exhibits antagonistic activity against other Fusarium species also, Fusarium oxysporium f.sp. lycopersici (Fig 5b), Fusarium moniliforme (Fig 5c) and the other phytopathogenic fungi, such as Rhizoctonia solani (Fig 5d).
• Example 12 Bioefficacy of larval bioassays and synergistic action of chitinases on insecticidal activity
• Bioassays were conducted to evaluate the effects of the chitinolytic enzymes set forth in (Table 1) on insecticidal activity, on a lepidopteran insect pest.
• larval Plutella xylostella (diamond backmoth) were reared on a high wheat germ-based diet as per the method of Webb et al (1988) Laboratory rearing of the imported Cabbageworm, NY Food Life Sci. Bull., 122.
• Each bioassay included 3 treatments replicated three times. All larvae were weighed when controls reached the ultimate in star and then monitored daily for developmental changes.
• Feeding assays were conducted by incorporating the culture broth containing the chitinolytic enzymes (0, 0.25%, 0.5%, and 1% of chitinolytic enzyme mixture (Table 1 ) and the insecticidal proteins into lepidopteran-specific artificial diet placed in a 10 well- tissue culture tray ( Figure 6c).
• the protein is suspended in Tris-HCl buffer at a pH of 8.0.
• One third instar larva was placed in each well to feed on the artificial diet for 5 days.
• agar cup assays were performed to study the effect of the chitinases on the insecticidal activity of Brevibacillus laterosporus Lakl210 against larvae of diamond backmoth.
• Larvae of Plutella xylostella (diamond backmoth) were obtained from cultures raised on Brassica oleracea var. capitata.
• Agar cup assays were performed excised cabbage leaf discs were treated with 5 ml of uninduced cell suspension (1 mg/ml) mixed with varied amounts (0, 100, 250 and 500 ⁇ ) of the supernatant containing from a culture of Lak 1210 grown in standard growth medium supplemented with 1 % (w/v) colloidal chitin.
• Second and third instar larvae of Plutella xylostella were placed in agar cups containing excised cabbage leaf discs (3.5 cm) which had been spread with the four different test suspensions. In each of the four treatments, 10 larvae were used in three replicates (in total 30 larvae), and mortality was recorded every 24 h for 7 days. Cabbage leaf discs sprayed with sterile distilled water were used as a negative control. The agar cups were kept at 21°C, > 40% R. H. and continuous light. After the treatment with different test suspensions, the larvae were scored for developmental changes.
• Fig.7 shows that supematants from chitin-induced cultures increased the lethal effect of Brevibacillus laterosporus Lakl210 on the larvae in a dose-dependent manner. Upon addition of 500 ⁇ supernatant, 100% mortality was observed on day 5, compared to day 7 without this addition. All treatments with Brevibacillus laterosporus Lakl210 caused death of 100% of the larvae within 7 days. Fig 7 shows that the LT50 value decreased with increasing amounts of chitinolytic enzymes.
• Brevibacillus laterosporus LAK 1210 (MTCC 5487) is a novel pesticidal strain that meets all the requirements of enzyme prospecting in terms of the organism and the enzyme characteristics of an industrial enzyme.
• the chitinolytic enzymes of the present invention are particularly effective in controlling insects insecticidal proteins are active under alkaline conditions.
• Chitinase from Brevibacillus laterosporus LAK 1210 exhibits higher activity at alkaline pH (pH 8.0), alkaline active in the range of pH 9.0-1 1.0.
• Synergistic action of the novel chitinase potentiates the insecticidal activity making the strain Brevibacillus laterosporus LAK 1210 (MTCC 5487) highly efficacious for biocontrol and a possible, viable alternative to Bt and Bt control.
• Brevibacillus laterosporus LAK 1210 (MTCC 5487) is a new, natural strain that could be quickly developed into a safe biopesticide/biocontrol formulation.
• the present invention that uses a new strain of Brevibacillus laterosporus LAK 1210 (MTCC 5487) provides significant economic benefits to agriculture and forestry sectors and environment.
• Brevibacillus laterosporus LAK 1210 demonstrates lepidopteran activity of economically and agriculturally important lepidopteran insect pests, which so far have not been successfully reported for strains of Brevibacillus laterosporus.
• the chitinase is alkaline active, thermoactive and thermostable, exhibiting activity over a wide range of pH (3.0 -1 1.0) with two pH optima (pH 6.0 and 8.0) and an optimum temperature of 70°C and these unique characteristics of the novel chitinase from the new strain oi Brevibacillus laterosporus LAK 1210 (MTCC 5487) can be exploited in industrial processes with harsh environment, in particular biomass utilization in a biofuel industry.
• Table 1 peptide sequence present in the chitinase(s) produced by a new strain of Brevibacillus laterosporus designated as Brevibacillus laterosporus LAK 1210 having Accession number MTCC 5487

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