WO2009031874A1

WO2009031874A1 - Strain of bacillus subtilis for agricultural use

Info

Publication number: WO2009031874A1
Application number: PCT/MX2007/000105
Authority: WO
Inventors: Jorge Olmos Soto
Original assignee: Jorge Olmos Soto
Priority date: 2007-09-04
Filing date: 2007-09-04
Publication date: 2009-03-12
Also published as: MX2010002412A; MX337319B

Abstract

The invention relates to a novel strain of Bacillus subtilis NRRL B-50055 which produces secondary metabolites, bioactive substances and degradative enzymes. The aforementioned bacteria and/or the supernatant thereof inhibit the growth of fungi, bacteria, insects and nematodes in plants, fruits, roots and soils contaminated with said pathogens. In addition, said bacteria and/or the supernatant thereof promote seed germination, root growth and plant growth, acting as an organic biofertiliser and inducing the degradation of soil nutrients and the improved assimilation thereof by seeds, roots and plants. The invention also relates to methods for protecting and/or treating plants, fruits and soils in relation to diseases that affect crops, using the bacteria, the supernatant or the pure or semi-pure metabolites. The invention further relates to formulations for treating and protecting plants, fruits and soils in relation to diseases that affect crops, using the bacteria, the supernatant or the pure or semi-pure metabolites.

Description

Bacillus subtilis strain FOR AGRICULTURAL USE

TECHNICAL FIELD OF THE INVENTION

The present invention is in the area of agricultural biopesticides and biofertilizers. Specifically this invention is based on the isolation of a new strain of Bacillus subtilis, NRRL B-50055, which produces secondary metabolites and bioactive substances; which can be used alone or in combination with other organic and / or inorganic compounds, to inhibit the growth of a wide variety of plant, fruit and soil pathogens, including fungi, bacteria, insects and nematodes. Also, this bacterium and / or supernatant has degrading enzymes that confer the properties of organic biofertilizer.

BACKGROUND

In recent years, it has been reported that some microorganisms exhibit biological activity with the ability to control a wide variety of plant pathogens (Asaka, O and Shoda, 1996; Ferrari et al, 1991; Jonson et al, 1993). Although many efforts have been made to find and develop biological pesticides that control plant diseases and do not harm man or the environment, most of the pesticides currently used are highly toxic synthetic compounds. Chemical pesticides are classified as carcinogenic by the EPA, they are toxic to the environment and animals. In addition, plant pathogens generally develop resistance to these compounds (Schwinn eí al, 1991).

Every year hundreds of millions of dollars are used for the purchase of chemical pesticides worldwide. In addition to this, the development of a new chemical pesticide costs an average of 80 to 100 million dollars. In this sense, biological control is a very attractive alternative with respect to chemical pesticides. Biopesticides (living organisms that produce secondary metabolites and bioactive substances), are not toxic to humans, animals or the environment. They are biodegradable and much cheaper for their development (US 5049379; 5061495; 6060051). The development and production of biopesticides using microorganisms is highly recommended for the integrated management of plant pathologies. Bacillus thuringiensis is the best known microorganism for the production of biopesticides, however its range of action is reduced to a certain group of pests, mainly insects (Stabb et al 1990). In this sense, efforts have now been made for the production of biopesticides from Bacillus subtilis, with some commercial examples already found in US 5344647; US 6291426, those that report a wider range of action affecting several groups of pathogens. However, the biopesticide production technology still has various problems that would be very convenient to solve. To date, there is no comprehensive product that can solve most problems in the field.

One of the main problems that arises with current biopesticides is the use of foreign (non-endemic) microbial strains in the agricultural areas where they are applied, which in most cases if the product is not well formulated, gives result in an inefficient biopesticide.

The second problem that exists with the majority of biopesticides of a bacterial nature and that supposes a serious limitation for its practical use, is the low survival of the cells, especially on the surface of the plants and fruits.

In addition, its effectiveness is generally lower than that of chemical biopesticides, which is why high concentrations of the microorganism are required in the formulation. In

In most cases, the effective treatment dose is excessively high for its production on a commercial scale (5X10 ^15'20 ), if the maximum cellular concentration that can be achieved by fermentation is taken into account, which makes the industrial system unfeasible. Reason why, the formulations are the result of dehydration and concentration of the biomass, which complicates and makes the process more expensive. In addition, in general, there is a high loss of bacterial viability during the concentration procedures, due to the elimination of water during dehydration.

In this sense, it is very important to isolate and select Bacillus strains with capacities not only of biopesticide, but also of biofertilizer. In addition, it is of vital importance to have an adequate formulation to ensure the viability and effectiveness of the strain on any surface, with which the over- concentration of the Colony Forming Units (CFU), but rather the stability, viability and efficacy of the strain.

Finally, there are many difficulties for the industrial production of biopesticides, as well as for repeating the quality of the results. However, the main problems are the origin of the strains, the limited spectrum against pathogens, the limited enzyme spectrum; What limits the viability in the field. In addition, there are problems related to the formulation and preservation of the preparation, which also results in a decrease in bacterial viability and efficacy.

In this sense, we have directed our experience of innovation and biotechnological development to the search for a Bacillus strain capable of overcoming the difficulties outlined in the previous paragraphs. These efforts have been culminated with the discovery of a regional strain of Bacillus subtilis, which solves many of the problems in agriculture, by functioning as a broad-spectrum biopesticide against plant pathogens. Likewise, this strain of Bacillus subtilis also functions as an organic biofertilizer, producing a large and diverse amount of degrading enzymes, which generates an efficient assimilation of soil substrates by the plant, stimulating their growth and that of the root. In addition, this new strain of B. subtilis is effective at moderate concentrations (1 x 10 ^6), unconcentrated preparations. Having high viability in plants, fruits and soils. Characteristics that as a whole have not been documented for a Bacillus strain used as a biopesticide or biofertilizer, which is why this strain of Bacillus subtilis is novel.

DESCRIPTION OF THE INVENTION

The characteristic details of the present invention are clearly shown in the following description and in the accompanying figures.

Brief description of the figures:

Figure '1 is a graph showing the growth behavior of bacterial strains in a known culture medium (Olmos eí al, 1997). This figure clearly denotes the great ability to use most substrates by the new strain of B. subtilis NRRL B-50055, unlike other strains used, which make it difficult to use several substrates due to the enzymatic differences between them. In this sense, this graph clearly differentiates the new NRRL B-50055 strain from the other strains used.

Figure 2 is a graph comparing High Performance Liquid Chromatography (HPLC), a fraction of the metabolites produced and secreted into the culture medium by the new β strain. NRRL B-50055 subtilis and the strain of B. subtilis protected by US patent 6060051. It is important to note that the perfijes are very similar, but not the same, which confirms again that these strains are different ecotypes of Bacillus subtilis.

The present invention describes a novel strain of Bacillus subtilis for agricultural use, where said agricultural uses are biopesticides and biofertilizers, both for plants and their organs and their fruits, and in soils.

This strain was deposited with the Agricultural Research Service (ARS) Patent Culture Collection Bank of the United States of America, on August 3, 2007, under the Budapest international treaty for patent purposes, this strain received the NRRL access number B-50055. The novel strain of Bacillus subtilis NRRL B-50055 has the following characteristics: The selected strain was classified as Bacillus subtilis by means of sequencing its 16S rDNA. This strain is Gram positive, has a bacillary shape and has a lateral endospore. Its colonial morphology is rough, has irregular edges and has a white opaque oyster coloration. During its exponential growth it shows chain growth, however, during the stationary phase it presents an individual bacillary form.

EXAMPLES:

The following examples are evidence of an embodiment of the present invention, which should not be considered as limiting to said invention.

1) .- Isolation and phenotypic identification of the strain of B. subtilis For the isolation and selection of bacterial strains that showed potential activity to be used in organic agriculture, samples of Soils and water. From the purified and grown strains in Petri dishes with Bertani (Ib) liuria for 24 h, samples were taken and stained with Gram stain. Gram positive bacteria, in a bacillary manner with endospores, were selected to corroborate their affiliation with the Bacillus genus by sequencing their DNA. These bacteria were preserved at -70 ° C in 1.5 ml Eeppendorf tubes.

2) .- Bacillus subtilis growth curves.

Liquid culture media specifically formulated to determine the growth capacity of the new β strain were used. NRRL subtilis B-50055. The cultures were carried out in a 250 ml flask at 37 ° C, at 250 rpm, for a certain period of time. Samples of the cultures of the different strains were taken at intervals of one hour and their growth was determined, plotting optical density of the culture at 600 nm, against the sampling time (Figure 1) (Olmos and Contreras, 2003). This figure shows that the strain of S. subtilis NRRL B-50055 showed the highest growth of all the strains analyzed, indicating that there was a greater use of the substrates by this strain, which is the subject of patent in this document.

3) .- Molecular identification of the strains

For the isolation of chromosomal DNA, the alkaline and phenol-chloroform method was used (Sambrook et al, 1989). The presence of DNA was verified on a 1.2% agarose gel. From the DNA of the selected strains, the 16S rDNA gene was amplified by means of the PCR technique, with the following reaction conditions: 1 μl of

Strain DNA, 1μl of the sense and antisense universal oligonucleotides for prokaryotes (Arellano and Olmos, 2002). The PCR product of the selected strains was subsequently purified by the phenol-chloroform method

(Sambrook eí al., 1989) and sequence analysis was performed in San Diego State

University, Microchemical Core Facility. Finally, its sequences were compared with known sequences and registered in the Gene Bank of the National Center for

Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The sequenced strain was identified as Bacillus subtilis, containing an identity greater than 95% compared to the Gene Bank sequences (SEQ ID 1).

4) .- HPLC chromatographic profile of the new strain of B. subtilis NRRL B-50055

In order to compare the secondary metabolites secreted to the culture medium between the new strain of Bacillus subtilis NRRL B-50055 and other strains of Bacillus subtilis; the supernatant obtained from the growth of the strains was filtered and subjected to reverse phase liquid chromatography (HPLC). The typical chromatogram of the supernatants is shown in Figure # 2. Other strains of Bacillus grown in the same culture medium, presented a similar, but not the same chromatogram (data not shown). In this sense, this chromatogram together with the other characteristics described in the previous paragraphs, represents an excellent fingerprint for the recognition and typing of the strain, since it produces a type chromatographic profile, depending on the culture conditions and the medium used , making its recognition and differentiation from other strains 100% safe.

5) .- In-vitro and in-vivo biopesticide activity;

In solid media containing a wide variety of plant pathogens, aliquots of the precipitate and the supernatant were tested, as well as the complete culture broth of the fermentations of the selected Bacillus strains. The strains that presented the best bioactivity against the pathogens, were selected to develop the field experiments with different crop varieties (grape, basil, tomato, onion, cucumber, pumpkin, chili peppers of different varieties, strawberry, etc). The cultures were inoculated by dripping and by spraying with the selected Bacillus subtilis strains, several presented the ability to inhibit the growth of fungi, bacteria, insects and nematodes, producing a disease-free culture. However, all the cultures treated with the new strain of Bacillus subtilis NNRL B-50055, were the only ones that did not present any disease in the open field or in the greenhouse (see Table 1).

6) .- Biofertilizer activity of the strain of B. subtilis NRRL B-50055 In addition to presenting a broad spectrum as a biopesticide, the new strain of B. subtilis B-50055, presented an excellent ability to aid in nutrient degradation of the soil, in the germination of the seeds, in the growth of the root and of the plants; compared to cultures treated with other Bacillus and with untreated controls. In addition, the cultures treated with the new strain of B. subtilis B-50055 prolonged their production time and crop yield.

For all of the above, the Bacillus subtilis B-50055 strain reason for this patent is novel, since it has biopesticide and biofertilizer activity at the same time and is effective at moderate concentrations of 5X10 ⁶ . In addition, it has the ability to survive for a long time on the surface of the plants and fruits, increases the germination of the seed, increases the growth of the root and the plants. Prevents the appearance of a large number of diseases, protecting 100% of all the crops evaluated.

Table 1.- Crops and the diseases that affect them. The novel strain of S. subtilis NRRL B-50055, showed both in-vitro and in-vivo activity for all pathogens evaluated and shown in this table. Unassessed cultures are shown in bold.

REFERENCES

Arellano, C.F., Olmos, SJ. , (2002). Thermostable α-1, 4- and α-1-6-glucosidase enzymes from Bacillus sp. Solated from a marine environment. Word Journal of Microbiol and Biotechnol. 18: 791-795.

Asaka, O. And Shoda, M. (1996). Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl Environ Microbiol. 62: 4081-4085. Ferrari et al., (1991). Biological control of Eutypa can on grapevine by an antagolistic strain of Bacillus subtllis. Phytopathology 81: 283-287. Johnson eta al., (1993). Insecticidal activity of EG4961, a novel strain of Bacillus thuringiensis toxic to larvae and adults of Southern corn rootworm (Coleóptera: Chrysomelldae) and Colorado potato beetle (Coleóptera: Chrysomelidae). J.

Econ. Entomol 86: 330-333. Olmos SJ'and Contreras FR (2003). Genetic system constructed to overproduce and secrete prolnsulln in Bacillus subtilis. Appl Microbiol Biotechnol 62: 369-373 Olmos, SJ. , De Anda, R., Ferrari, E., Bolívar, F., Valle, F., 1997. Effects of the sinR and degU32_ {Hy) mutations on the regulation of the aprE in Bacillus subtilis. Mol.

Gen. Genet. 253: 562-567. Sambrook KT, Frisch EF, Maniatis T (1989) Molecular cloning. A laboratory Manual,

2nd edn. CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY. Schwinn et al., (1991) Control with chemicals. Advances in Plant Pathology: VoI. 7, Phytophtohora ¡nfestans, The cause of late blight of potato. Academic Press, San

Diego Chapter 8, pp. 225-265.

Claims

1. An isolated strain of Bacillus subtilis for agricultural use, deposited under accession number NRRL B-50055, as well as its mutants.

2. A culture of the strain of claim 1, which exhibits biopesticide and biofertilizer activity for plants and soil.

3.-The supernatant obtained from the fermentation of the strain of claim 1, which exhibits fungicidal, bactericidal and insecticidal activity, against diseases and pests in plants and soil.

4. The supernatant obtained from the fermentation of the strain of claim 1, which exhibits biofertilizer properties.

5.-The secondary metabolites and pure or partially pure bioactive substances, produced by the strain of claim 1, which exhibit biopesticidal and biofertilizing properties in plants and soil.

6. -A formulation for agricultural use, characterized in that it contains: at least one of the components from the culture of the strain of claim 1; and at least one organic and / or inorganic compound.

7. A formulation, as claimed in claim 6, wherein the organic and / or inorganic compound is a chemical and / or biological pesticide.

8. A formulation, as claimed in claim 6, wherein the organic and / or inorganic compound is a microorganism and / or its parts.

9.- A formulation, as claimed in claim 6, wherein the organic and / or inorganic compound is a plant and / or animal extract.

10. A formulation, as claimed in claim 6, wherein the organic and / or inorganic compound is a combination with the compounds mentioned in claims 7, 8 and 9.

11.-A method to treat or protect plants, fruits, roots and / or soils, of pathologies caused by bacteria, fungi, insects and / or nematodes, characterized in that an effective amount of the culture of the strain NRRL B-50055 is applied, of claim 2; or at least one component of said culture, of claims 1, 3 and 5.

12.-The method of claim 11, wherein the culture component is only strain NRRL B-50055.

The method of claim 11, wherein the culture component is only the supernatant of claim 3.

14. The method of claim 11, wherein the crop component is only the metabolites of clause 5.

15.- A method to treat or protect plants, fruits, roots and / or soils, of pathologies caused by bacteria, fungi, insects and / or nematodes, characterized in that an effective amount of the formulations of clauses 6, 7, is applied, 8, 9 and 10.

16. The methods of claims 11-15, wherein the products used are applied in the form of liquid, powder, granules, microencapsulated and / or any other presentation.

17. A method for fertilizing plants and soils, characterized in that an effective amount of the culture of the strain of Bacillus subtilis of claim 2, or at least one of the components of said crop, mentioned in claims 1, 3 is applied and 5, be

18. A method for fertilizing plants and soils, characterized in that an effective amount of the formulations of claims 6, 7, 8, 9 and 10 is applied.

19. The methods of claims 17 and 18, wherein the products used are applied in the form of liquid, powder, granules, microencapsulated and / or any other presentation. SEQUENCE LIST

SEQ ID NO: 1

No. OF SEQUENCES: 1

INFORMATION FOR THE SEQ. ID NO: 1

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1500 base pairs

(B) TYPE: nucleic acid

(C) CHAIN TYPE: double

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA (16S rDNA gene) (vi) ORIGINAL SOURCE

(A) ORGANISM: Bacillus subtilis

(B) CEPA: B-50055

(viii) POSITION IN THE GENOME (ix) CHARACTERISTICS

(A) NAME / KEY: rDNA

(B) LOCATION: 1-1500 base pairs

(C) IDENTIFICATION METHOD: Sequencing

(D) OTHER INFORMATION: Isolated bacteria in the region of Baja California, Mexico.

GTACGCTGGC GTGCGTGACT RRWACAGTGC AAGTCGAGCG GACAGATGGG AGCTTGCTCC 60

CTGATGTTAG CGGCGGACGG GTGAGTAACA CGTGGGTAAC CTGCCTGTAA GACTGGGATA 120

ACTCCGGGAA ACCGGGGCTA ATACCGGATG GTTGTCTGAA CCGCATGGTT ₄ CAGACATAAA 180

AGGTGGCTTC GGCTACCACT TACAGATGGA CCCGCGGCGC ATTAGCTAGT TGGTGAGGTA 240

ACGGCTCACC AAGGCGACGA TGCGTAGCCG ACCTGAGAGG GTGATCGGCC ACACTGGGAC 300

TGAGACACGG CCCAGACTCC TACGGGAGGC AGCAGTAGGG AATCTTCCGC AATGGACGAA 360

AGTCTGACGG AGCAACGCCG CGTGAGTGAT GAAGGTTTTC GGATCGTAAA GCTCTGTTGT 420

TAGGGAAGAA CAAGTGCCGT TCAAATAGGG CGGCACCTTG ACGGTACCTA ACCAGAAAGC 480

CACGGCTAAC TACGTGCCAG CAGCCGCGGT AATACGTAGG TGGCAAGCGT TGTCCGGAAT 540

TATTGGGCGT AAAGGGCTCG CAGGCGGTTT CTTAAGTCTG ATGTGAAAGC CCCCGGCTCA 600

ACCGGGGAGG GTCATTGGAA ACTGGGGAAC TTGAGTGCAG AAGAGGAGAG TGGAATTCCA 660

CGTGTAGCGG TGAAATGCGT AGAGATGTGG AGGAACACCA GTGGCGAAGG CGACTCTCTG 720

GTCTGTAACT GACGCTGAGG AGCGAAAGCG TGGGGAGCGA ACAGGATTAG ATACCCTGGT 780

AGTCCACGCC GTAAACGATG AGTGCTAAGT GTTAGGGGGT TTCCGCCCCT TAGTGCTGCA 840

GCTAACGCAT. TAAGCACTCC GCCTGGGGAG TACGGTCGCA AGACTGAAAC TCAAAGGAAT 900

TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT TCGAAGCAAC GCGAAGAACC 960

TTACCAGGTC TTGACATCCT CTGACAATCC TAGAGATAGG ACGTCCCCTT CGGGGGCAGA 1020

GTGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT TAAGTCCCGC 1080

AACGAGCGCA CCCTTGATCT TAGTGCCAGC ATTCAGTTGG GCACTCTAAG .GTGACTGCCG 1140

GTGACAAACC GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGACCTGGGC 1200

TACACACGTG CTACAATGGA CAGAACAAAG GGCAGCGAAA CCGCGAGGTT AAGCCAATCC 1260

CACAAATCTG TTCTCAGTTC GGATCGCAGT CTGCAACTCG ACTGCGTGAA GCTGGAATCG 1320

CTAGTAATCG CGGATCAGCA TGCCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC 1380

CGTCACACCA CGAGAGTTTG TAACACCCGA AGTCGGTGAG GTAACCTTTT AGGAGCCAGC 1440

CGCCGAAAGG TGGGACAGAT GATTGGGGTG AAGTCGTACA SRRGGGTGGA ATCCCGAAGA 1500