WO2013042900A2

WO2013042900A2 - Novel strain bacillus vallismortis bso7m capable of promoting growth of plant and confering cold resistance on plant and microbial formulation comprising the same

Info

Publication number: WO2013042900A2
Application number: PCT/KR2012/007348
Authority: WO
Inventors: Kyung Seok Park
Original assignee: Republic Of Korea(Management : Rural Development Administration)
Priority date: 2011-09-22
Filing date: 2012-09-13
Publication date: 2013-03-28
Also published as: WO2013042900A3; JP5567634B2; KR20130032100A; JP2013066467A; KR101624628B1

Abstract

Disclosed are a novel strain, identified as Bacillus vallismortis BS07M, and a microbial formulation comprising the same. The novel strain anchors a gene having the nucleotide sequence of SEQ ID NO: 1 and very effectively defends plants from pathogenesis and promotes plant growth. Thanks to its functions of potentiating the immunity of plants as well as effectively inhibiting plant pathogens, the novel microorganism or the microbial formulation can reduce the occurrence of plant diseases and promote crop growth, thus making a great contribution to environmentally friendly agriculture.

Description

NOVEL STRAIN BACILLUS VALLISMORTIS BSO7M CAPABLE OF PROMOTING GROWTH OF PLANT AND CONFERING COLD RESISTANCE ON PLANT AND MICROBIAL FORMULATION COMPRISING THE SAME

The present invention relates to Bacillus vallismortis BS07M, a novel strain of Bacillus vallistmortis capable of promoting the growth of plants and augmenting cold resistance in plants and a microbial formulation comprising the same. More particularly, the present invention relates to Bacillus vallismortis BS07M that has the function of inhibiting the growth of plant pathogens, activating a plant defense system to promote the growth of plants, and augmenting cold resistance in plants, and a microbial formulation comprising the same.

Extravagant use of agricultural chemicals has resulted in significant pollution in the agricultural ecosystem. With the increasing amount of social attention being paid to agricultural pollution and safe agricultural products, trials have been made of environment-friendly pest controlling methods, such as biological control, for various diseases and pests which are difficult to control with synthetic chemicals.

In the past, microorganisms inhibitory of pathogens were employed as biological controls. In recent years, induced systemic resistance (ISR) has newly arisen as an effective weapon. ISR is a state of enhanced defensive capacity against pathogens as a result of the activation of latent innate immune responses that are expressed upon subsequent, challenge inoculation with a microorganism.

Various different bacterial microorganisms are known to induce resistance to pathogenesis in plants. Representative among these microorganisms are Bacillus spp. There are not many strains that are applicable in practice in spite of their ability to induce systemic resistance in plants. In addition, strains, although belonging to the same genus, significantly differ from one species to another in terms of their immune activity related to induced systemic resistance. Thus, the selection of strains that are pertinent is required.

In this context, the strains must not only confer stable induced systemic resistance against diseases on plants but must guarantee the plants excellent immunity until a late growth stage after their application to plants at the seedling stage.

It is therefore an object of the present invention to provide a novel Bacillus vallismortis BS07M strain that has functions of being inhibitory against plant pathogens, inducing plants to be resistant to diseases, promoting the growth of plants by immunopotentiation, and conferring cold-resistance on plants.

It is another object of the present invention to provide a microbial formulation comprising the novel Bacillus vallismortis BS07M strain.

The technical objectives to be achieved by the present invention are not limited to those stated above, and other objectives not yet stated may be understood to those skilled in the art from the following description.

In accordance with an aspect thereof, the present invention provides a novel strain of Bacillus vallismortis, identified as Bacillus vallismortis BS07M KCTC11991BP, which anchors a gene having the nucleotide sequence of SEQ ID NO: 1 and is effective at promoting the growth of a plant, particularly by conferring defensive activity against a plant pathogen, immunopotentiation against a plant disease, and high cold-resistance on the plant.

In one preferred embodiment of the present invention, the plant may be selected from among pepper, cucumber and tobacco.

According to another preferred embodiment of the present invention, the plant pathogen may be selected from among Colletotrichum acutatum, Alternaria alternata, Botrytis Cinerea, Penicillium italicum, Penicillium expansum, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum.

According to a further preferred embodiment of the present invention, the plant disease may be selected from among diseases caused by Phytophthora capsici, Pectobacterium carotovora, Colletotrichum acutatum, and cucumber mosaic virus.

The novel Bacillus vallismortis BS07M strain KCTC11991BP of the present invention is characterized by a gene having the nucleotide sequence of SEQ ID NO: 1 which functions to confer cold-resistance on plants.

The novel microorganism can be used as an active ingredient in a microbial formulation selected from among biopesticides, microbial fertilizers, seed coatings, soil improvers, compost decomposers, foliage sprays, drench sprays and a combination thereof.

Accordingly, a microbial formulation comprising the novel Bacillus vallismortis BS07M strain KCTC11991BP or a culture thereof as an active ingredient forms another aspect of the present invention.

In accordance with a further aspect thereof, the present invention provides a method of promoting growth of a plant by spraying the plant or drenching the soil around the plant with the microbial formulation of the present invention for the plant.

In accordance with still a further aspect thereof, the present invention provides a method of controlling a plant pathogen by spraying the plant or drenching the soil with the microbial formulation of the present invention for the plant.

In accordance with still another aspect thereof, the present invention provides a method of increasing cold-resistance in a plant by spraying the plant or drenching the soil with the microbial formulation of the present invention for the plant.

When applied to a plant, the novel microorganism of the present invention, identified as Bacillus vallismortis BS07M, increases cold-resistance of the plant so that it can tolerate low temperature. In addition, the novel microorganism Bacillus vallismortis BS07M very effectively defends plants from pathogenesis and promotes plant growth.

Thanks to its functions of potentiating the immunity of plants as well as effectively inhibiting plant pathogens, therefore, the novel microorganism or the microbial formulation comprising the same in accordance with the present invention can reduce the occurrence of plant diseases and promote crop growth, thus making a great contribution to environmentally friendly agriculture.

FIG. 1 is a phylogenetic tree showing the classification of the novel strain Bacillus vallismortis BS07M on the basis of the 16S rRNA gene sequence;

FIG. 2 photographically shows the promoting effect of Bacillus vallismortis BS07M on the growth of pepper seedlings after soil drenching.

FIG. 3 photographically shows the promotional effect of Bacillus vallismortis BS07M on the growth of pepper fruits after soil drenching.

FIG. 4 photographically shows the inhibitory activity of Bacillus vallismortis BS07M on plant pathogens.

FIG. 5 is HPLC chromatograms showing the purification of a BS07 portion (YC329B8k).

FIG. 6 shows preparative HPLC results of a BS07 portion (YC329B8k).

FIG. 7 is a proton NMR spectrum of iturin A2.

FIG. 8 is a proton NMR spectrum of iturin A3.

FIG. 9 is a proton NMR spectrum of iturin A4.

FIG. 10 is a schematic view showing chemical structures of iturin A2, A3 and A4.

FIG. 11 shows the expression of defense genes induced by Bacillus vallismortis BS07M,

1. Col O: wild-type Arabidopsis thaliana

2. etr3-5: mutant A. thaliana

3. nah G: mutant A. thaliana unable to express PR

4. npr1: mutant A. thaliana

FIG. 12 photographically shows the lignification of the cucumber seedling root treated with Bacillus vallismortis BS07M.

FIG. 13 photographically shows the ISR of pepper seedlings induced by Bacillus vallismortis BS07M against Pectobacterium carotovora SCC1.

FIG. 14 is a graph showing the suppression of anthracnose pathogenesis by Bacillus vallismortis BS07M in pepper plants grown in an open field condition.

FIG. 15 photographically shows the suppressive effect of Bacillus vallismortis BS07M on cucumber mosaic virus disease.

FIG. 16 photographically shows the induction of cold resistance in cucumber plants by Bacillus vallismortis BS07M.

FIG. 17 photographically shows the induction of cold resistance in tobacco seedlings by Bacillus vallismortis BS07M.

Unless otherwise defined, the terms and techniques described in the specification are intended to encompass the meanings generally accepted in the art to which the present invention pertains.

Below, a detailed description will be given of the present invention.

The present invention addresses a novel microorganism which is capable of promoting the growth of plants, endowing plants with weather tolerance, and controlling plant pathogens.

Homology analysis of 16S rRNA identified the novel microorganism as a strain of Bacillus vallismortis. It was named Bacillus vallismortis BS07M and deposited with the KCTC (the Korean Collection for Type Cultures) on July 28, 2011, under accession No. KCTC11991BP.

The novel microorganism Bacillus vallismortis BS07M of the present invention is found to anchor a gene having all or a part of the nucleotide sequence of SEQ ID NO: 1 with an open reading frame (ORF) consisting of 1478 bp.

Field experiments demonstrated that the novel microorganism or a microbial formulation comprising a culture of the novel microorganism induces growth promotion, immunopotentiation and cold-resistance in plants irrespective of the kind of plants with the greatest effect being exhibited in peppers, cucumbers and tobaccos. By way of example, peppers were harvested at 30% higher yield when treated with the novel microorganism or the microbial formulation of the present invention.

In addition, the novel microorganism was found to produce a potent antibacterial agent which acts against plant pathogens causing plant diseases including anthracnose. Also, the novel microorganism was observed to increase the defense of plants against such diseases caused by Phytophthora capsici, Pectobacterium carotovora, Colletotrichum acutatum and cucumber mosaic virus as Phytophthora blight, soft rot, anthracnose, green mottle mosaic, etc. The agent responsible for the antibacterial and antiviral activity, produced by the novel microorganism of the present invention, was identified as the three iturin derivatives of A2, A3 and A4.

Further, it was discovered that the novel microorganism of the present invention, Bacillus vallismortis BS07M KCTC11991BP, expresses a disease-resistant gene which potentiates the immunity of plants through the salicylic acid-mediated signaling pathway.

Furthermore, the novel strain of the present invention increases the resistance of plants to low temperature as all of the plants treated with the strain survived the temperature of 4 C or less for 24 hours.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1: Isolation and Selection of Strain

To isolate a strain capable of potentiating the defense system of plants against diseases, rhizosphere soil was collected from various agricultural farms across Korea.

The samples thus obtained were dissolved in sterile water and shaken. Serial dilutions of the solution were made and spread over TSA plates.

After 2 days of incubation at 30℃, colonies appeared on the media. Microbial isolates were obtained from single colonies and maintained in broths.

They were assayed for their ability to evoke induced systemic resistance to Colletotrichum orbiculare in plants.

In this regard, cucumber seeds were treated with 1% sodium chloride to sterilize their surfaces and were then sowed in plastic pots two weeks after which the seeds germinated and were drenched with suspensions of the isolated strains.

One week after the drenching, the germinated leaves were sprayed with a spore suspension of Colletotrichum orbiculare, followed by examination of lesions so as to select the strain which was the most resistant to the disease.

EXAMPLE 2: Identification of Novel Strain

To identify the strain selected in Example 1, 16S rRNA sequencing analysis was conducted.

(1) 16S rRNA sequencing analysis

The microbial isolates were identified by analyzing the 16S rRNA sequence.

For this, DNA was purified from the microbial isolates using a DNA extraction kit and served as a template to amplify a 16S rRNA gene via PCR using the universal primers fD1(5'-AGAGTTTGATCCTGGCTCAG-3') and rP2(5'-ACGGCTACCTTGTTACGACTT-3').

The PCR product thus obtained was treated with a DNA sequencing kit before running the sequencing products on the 3100 Genetic Analyzer. A BLAST search of the NCBI database revealed the genus of the bacterial isolates.

Phylogenetic relationships were analyzed on CLUSTA W alignments of the 16S rDNA sequence with base sequences of standard strains. Phylogenetic trees were constructed from the dataset using the MEGA version program. Bootstrap analysis was based on 1000 resamplings of the sequence alignment.

As seen in the phylogenetic tree of FIG. 1, the microbial isolate is closely related to Bacillus subtilis, Bacillus mojavensis and Bacillus vallismortis, with the highest phylogenetical relevance being to Bacillus vallismortis. Therefore, the microbial isolate of interest was identified as a novel strain of Bacillus vallismortis and named Bacillus vallismortis BS07M. It was deposited with the Korean Collection for Type Cultures on July 28, 2011, under accession No. KCTC11991BP.

EXAMPLE 3: Culturing of the Novel Strain Bacillus vallismortis BS07M

A stock of Bacillus Vallismortis BS07M in a deep freezer (-80℃) was thawed, spread over sterile TSA (Tryptic Soy Agar) plates and incubated at 30℃ for 48 hours (optionally, the colonies might be boosted in TSB broth at 30℃ for 48 hours).

The cells were harvested and suspended at a density of 10⁹~10¹⁰ cfu/mL in 1 L of sterile water or 0.2 M phosphate buffered saline before use in the subsequently conducted soil drenching or application to the plants.

EXPERIMENTAL EXAMPLE 1: Assay of Bacillus vallismortis BS07M for Ability to Promote Growth of Pepper

1) In greenhouse

A suspension of Bacillus vallismortis BS07M was prepared as in Example 3.

Under greenhouse conditions, the rhisosphere soil of peppers in a seedling stage was drenched with the cell suspension.

In detail, a 100-fold dilution of the cell suspension (10⁹ cfu/mL) was used to drench pepper plants in the 2~3-leaf stage and then the rhizosphere soil was drenched with it twice at regular intervals of 10 days.

The results are shown in FIG. 2 and summarized in Tables 1 and 2, below.

As can be seen in FIG. 2, the pepper plants treated with Bacillus vallismortis BS07M had overgrown the control during the seedling and subsequent stages.

Table 1

*LSD: least significant difference

In addition, as is apparent from the data of Table 1, pepper plants did not only grow very much taller and thicker but also bore much more fruit, when treated with Bacillus vallismortis BS07M, as compared to the control and the plants treated with 0.1 mM BTH, a disease resistance inducer.

Table 2

There were large differences in plant growth promotion between the strains of Bacillus, as shown in Table 2. Fruits of the pepper plants treated with Bacillus vallismortis BS07M weighed more than those of the other pepper plants, indicating that the novel strain of the present invention can effectively promote plant growth.

2) On the Bare Ground

The rhizosphere soil was drenched with the cell suspension during the seedling stage under greenhouse conditions, after which the plants were transplanted into the bare ground.

The results are summarized in Table 3, below.

Table 3

*LSD: least significant difference

As seen in Table 3, the pepper plants treated with Bacillus vallismortis BS07M were superior to the control in terms of plant height, weight of the fruit, and thickness of fruit, and bore much more fruit, compared to the control.

This is confirmed in FIG. 3. As seen in FIG. 3, treatment with Bacillus vallismortis BS07M made pepper plants thicker and longer.

EXPERIMENTAL EXAMPLE 2: Assay of Bacillus vallismortis BS07M for Inhibitory Activity against Plant Pathogens

Inhibitory activity of the novel strain Bacillus vallismortis BS07M against major plant pathogens is shown in FIG. 4.

Bacillus vallismortis BS07M was found to inhibit the growth of a broad spectrum of plant pathogens including Colletotrichum acutatum, Alternaria alternata, Botrytis cinerea, Penicillium italicum, Penicillium expansum KC08001, Penicillium expansum JVV81, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotirum.

Higher inhibitory effects were detected on Colletotrichum acutatum, Alternaria alternata, Botrytis cinerea and Sclerotinia sclerotirum than the other strains.

EXPERIMENTAL EXAMPLE 3: Isolation, Identification and Activity of Antibacterial Agent

To isolate an antibacterial agent therefrom, the BS07M strain was spread over 10 L of TSA (tryptic soy agar) solidified in petri dishes (each 15.0 x 1.5 cm) and incubated for 7 days. Extraction by two rounds of deposition in 80% ethanol was followed by filtration through a celite filter to remove cell mass. After concentration, C-18, silica G60, and LH20 column chromatography were conducted to purify antibacterial agents. Their activities were monitored with Rhizoctonia solani.

In this regard, a gradient of methanol (MeOH/H₂O) from 0 to 100% and a gradient of butanol from 0 to 100% were used to elute fractions active to R. solani. The active fractions were purified by HPLC (FIGS. 5 and 6).

NMR analysis identified the active agents in the three fractions thus obtained as iturin A2, A3 and A4, as shown in FIGS. 7 to 10.

Inhibitory effects that the iturin A2 identified from the novel strain had on plant pathogens are summarized in Table 4, below.

Table 4

As seen in Table 4, the Iturin A2 at 500 ppm had inhibitory activity against plant pathogens such as R. solani, C. acutatum, B. cinerea, and S.sclerotinia. Inhibitory activity was also detected when using iturin A3 and A4 (data not shown).

EXPERIMENTAL EXAMPLE 4: Induced Systemic Resistance by Bacillus vallismortis BS07M

1) Method

(1) Culturing of Arabidopsis thaliana

To examine whether treatment with Bacillus vallismortis BS07M has an influence on the expression of antifungal genes responsible for ISR in plants, wild-type Arabidopsis thaliana (Col 0) and mutant plants (nah G: SA: unable to express a PR gene; etr3-5; npr1) were employed.

The wild-type was obtained from the center and the mutant plant nah G (A. thaliana) is known to express salicylate dehydrogenase which functions to degrade SA.

Seeds of the plants were surface sterilized (immersed in 70% ethanol for 2 min and then in 1% sodium hypochlorite for 20 min), washed three times with sterile distilled water and inoculated on the concentration of half strength MS (Murashige-Skoog) media containing 1.5% sucrose and gelled with 0.8% agar having pH 5.7. In a dark condition, the seeds were allowed to germinate at 4℃ for 2 days. The seedlings were incubated at a relative humidity of 50~60% and a temperature of 22±1℃ under a 40W fluorescent lamp in growth chambers, with a cycle of 12 h light/12 h darkness.

After two weeks of incubation, the seedlings were transplanted to 60 mL pots containing potting soil which had been autoclaved for 1 hour twice at regular intervals of 24 hours.

Before the transplantation, the potting soil was treated with either a suspension of Bacillus vallismortis BS09 or an equal volume of sterile distilled water. Thereafter, the plants were cultivated at a relative humidity of 70% in growth chambers under a 9-hour light (200 μE/m²s, 24℃)/15-hour darkness (20℃) photocycle.

The plants were irrigated with water every other day and treated with a half-strength Hoagland nutrient solution every week. Two weeks later, the Arabidopsis thaliana plants were soil drenched with the suspension of Bacillus vallismortis BS07M.

(2) Expression of defense gene by treatment with Bacillus vallismortis BS07M

The Arabidopsis thaliana plants were soil drenched with a suspension of Bacillus vallismortis BS07M at a density of 10⁸ cfu/mL. One week later, leaves were taken from the plants to analyze the RNA.

For total RNA extraction, at least 2 g of each of the frozen plant samples was homogenized in an equal volume of the extraction buffer (0.35 M Glycine, 0.048 M NaOH, 0.34 M NaCl, 0.04 M EDTA, 4% (w/v) SDS), followed by the treatment of the homogenate with phenol and chloroform. RNA was precipitated with LiCl.

For RT-PCR, Ex Taq polymerase was used.

The PCR mixture was prepared to contain cDNA 0.1 μg, a forward and a reverse primer each 10 pMol, dNTP 250 nM, and 0.5U Ex Taq polymerase in 20 μL of buffer.

PCR was initiated at 94℃ for 5 min and performed with 25 cycles of 94℃ for 1 min, 57℃ for 1 min and 72℃ for 1 min followed by final extension at 72℃ for 10 min.

Primers for amplifying defense genes were forward 5'-TGCGGTAACACCGAACCATAC-3' (SEQ ID NO: 1) and reverse 5'-CGACAGTTGCATTGGTCCTCT-3' (SEQ ID NO: 2) for PDF1.2 and forward 5'-AACCGCCAAAAGCAAACGCA-3' (SEQ ID NO: 3) and reverse 5'-TCACGGAGGCACAACCAAGTC-3' (SEQ ID NO: 4) for PR-1.

The PCR products thus obtained were run on 1.2% agarose gel.

Pathogen attack or other stress factors cause plants to express ISR and exhibit a hypersensitive reaction. Plant reactions to these factors involve the activation of a set of genes encoding pathogenesis-related (PR) proteins such as glucanase, chitinase, the lignification of plant cells, and the production of phytoalexin and phenolic compounds which subsequently prevent invasion from various pathogens.

Likewise, rhizosphere treatment with the non-pathogen Bacillus vallismortis BS07M evoked the immunity of plants.

2. Results

When treated with Bacillus vallismortis BS07M, as can be seen in FIG. 11, wild-type Arabidopsis thaliana expressed the defense gene PR1 whereas the Nah G mutant did not, indicating that the novel strain of the present invention activates the immune system of plants through the SA-mediated signaling pathway.

To confirm that Bacillus vallismortis BS07M intensifies the defense system of plants, reactions in the roots were examined. As seen in FIG. 12, lignification of the roots was induced upon treatment with the novel strain of the present invention. Lignification, shown in red in FIG. 12, is one of the intensified defensive mechanisms, reflecting the physical strengthening of the cell wall. Therefore, Bacillus vallismortis BS07M strengthens the cell wall of the roots to subsequently prevent attack by pathogens.

EXPERIMENTAL EXAMPLE 5: Suppression of Pathogenesis in Plants by Bacillus vallismortis BS07M-Induced ISA

To examine the suppressive effect of Bacillus vallismortis BS07M-induced ISA on pathogenesis in plants, laboratory and field experiments were conducted.

The expression of a defensive system was examined in pepper plants after their leaves and fruits were taken in the seedling stage and from under field conditions in which they had been treated with Bacillus vallismortis BS07M.

For laboratory experiments of soft rot, leaves were taken from pepper plants in the seedling stage and from pepper plants grown in an open field after they had or had not received Bacillus vallismortis BS07M treatment.

Then, they were analyzed for ability to resist Pectobacterium carotovora SCC1.

The results are summarized in Table 5 and depicted in FIG. 13.

Table 5

*LSD: least significant difference

As is apparent from Table 5 and FIG. 13, no lesions were found in the plants treated with Bacillus vallismortis BS07M whereas the control had a lesion area as great as 42%, indicating that the novel strain of the present invention effectively suppresses pathgenesis.

Leaves from plants grown under the open field condition were analyzed and the results are given in Table 6, below.

Table 6

*LSD: least significant difference

As seen in FIG. 6, soft rot was generated at a rate of 71.7% and 53.7% in primary and secondary tests, respectively, in the control while the infection rates were remarkably reduced to 20.0% and 0.0% in the plants treated with the novel strain of the present invention.

For laboratory experiments of anthracnose, fruits which had been taken from pepper plants grown in the open field condition were used.

Spores of Colletotrichum acutatum were suspended at a density of 10⁵ spores/mL in sterile water and mixed with a spreader before being sufficiently sprayed.

The plants were incubated at relative humidity of 100% for 24 hours and left in a greenhouse for 7 days. Then, pathogenesis was examined in the pepper fruits that had or had not been treated with Bacillus vallismortis BS07M.

As seen in FIG. 6, the infection rate of anthracnose was 15% in the control, but decreased to as low as 1% in the Bacillus vallismortis BS07M-treated plants, indicating the novel strain of the present invention most effectively suppresses the generation of anthracnose.

As for the field experiment, it was conducted under open field conditions.

Spores of Colletotrichum acutatum were suspended at a density of 10⁵ spores/mL in sterile water and mixed with a spreader before being sufficiently sprayed over the leaves and fruits of pepper plants. Pathogenesis of anthracnose was examined four times at regular intervals of 15 days.

Treatment with Bacillus vallismortis BS07M was found to suppress the pathogenesis of anthracnose maximally under the open field condition as well, as shown in FIG. 14.

Inhibitory activity against cucumber mosaic virus (CMV) was examined.

In this regard, each leaf taken from the 2-week-old cucumber seedlings which were drenched with a spore suspension of the BS07M strain was inoculated with CMV by rubbing it with a CMV-diseased cucumber leaf juice together with carborundum powder. The plants were left for one week in a greenhouse after which CMV infection was examined. Non-treated plants were drenched with sterile water and inoculated with CMV in the same manner.

As seen in FIG. 15, No CMV infections were detected in the BS07M-treaed cucumber seedlings, demonstrating that the novel strain of the present invention has excellent ability to control viral infections.

EXPERIMENTAL EXAMPLE 6: Induction of Cold Resistance by Bacillus vallismortis BS07M

Cucumber and tobacco seedlings were examined for cold resistance after treatment with Bacillus vallismortis BS07M.

The plants were subjected to cold stress at 4℃ for 24 hours and then left in a greenhouse for 6 hours, followed by examining discoloration in leaves with the naked eye.

As can be seen in FIGS. 16 and 17, leaves of the control curled up and became off-white as chlorophylls were destroyed by the cold stress. In contrast, the leaves of the plants treated with Bacillus vallismortis BS07M remained green as a result of the cold resistance increased by the novel strain of the present invention.

Accordingly, the novel strain of the present invention provides plants with enhanced cold resistance so that plants can survive a low temperature condition such as 4℃. That is, Bacillus vallismortis BS07M can be effective as an agent for preventing cold damage in plants.

In addition, thanks to its ability to inhibit the growth of a broad spectrum of pathogens and to augment the defense mechanism of plants, Bacillus vallismortis BS07M according to the present invention can protect plants from diseases and promote the growth of plants, making great contributions to eco-friendly agriculture.

While the present invention has been particularly shown and described with reference to the foregoing preferred and alternative embodiments, it should be understood by those skilled in the art that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention without departing from the spirit and scope of the invention as defined in the following claims. It is intended that the following claims define the scope of the invention and that the method and apparatus within the scope of these claims and their equivalents should be covered thereby. This description of the invention should be understood to include all novel and non-obvious combinations of elements described herein, and claims may be presented in this or a later application to any novel and non-obvious combination of these elements. The foregoing embodiments are illustrative, and no single feature or element must be essentially included in all possible combinations that may be claimed in this or a later application.

Depositary Authority: Korean Collection for Type Cultures

Accession No.: KCTC11991BP

Date of Deposition: July 28, 2011

Claims

Bacillus vallismortis BS07M strain KCTC11991B, anchoring a gene having a nucleotide sequence of SEQ ID NO: 1, which is able to promote growth of a plant.
The Bacillus vallismortis BS07M strain KCTC11991B of claim 1, wherein the plant is selected from among pepper, cucumber and tobacco.
Bacillus vallismortis BS07M strain KCTC11991B of claim 1, anchoring a gene having a nucleotide sequence of SEQ ID NO: 1, which is inhibitory against a plant pathogen.
The Bacillus vallismortis BS07M strain KCTC11991B of claim 3, wherein the plant pathogen is selected from the group consisting of Colletotrichum acutatum, Alternaria alternata, Botrytis cinerea, Penicillium italicum, Penicillium expansum, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and a combination thereof.
Bacillus vallismortis BS07M strain KCTC11991B, anchoring a gene having a nucleotide sequence of SEQ ID NO: 1, which is able to evoke a defense system against a plant disease.
The Bacillus vallismortis BS07M strain KCTC11991B of claim 5, wherein the plant disease is selected from the group consisting of diseases caused by Phytophthora capsici, Pectobacterium carotovora, Colletotrichum acutatum, and cucumber mosaic virus.
The Bacillus vallismortis BS07M strain KCTC11991B, anchoring a gene having a nucleotide sequence of SEQ ID NO: 1, which is able to confer cold resistance on a plant.
The Bacillus vallismortis BS07M strain KCTC11991B of any one of claims 1 to 7, being used as an active ingredient in a microbial formulation selected from among biopesticides, microbial fertilizers, seed coatings, soil improvers, compost decomposers, foliage sprays, drench sprays and a combination thereof.
A microbial formulation, comprising Bacillus vallismortis BS07M strain KCTC11991B or a culture thereof, said strain anchoring a gene having a nucleotide sequence of SEQ ID NO: 1.
A microbial formulation, comprising an iturin compound produced from Bacillus vallismortis BS07M strain KCTC11991B or a culture thereof, said strain anchoring a gene having a nucleotide sequence of SEQ ID NO: 1.
A method of promoting growth of a plant, comprising spraying the plant or drenching soil around the plant with the microbial formulation of claim 9 or 10.
A method of controlling a plant pathogen, comprising spraying the plant or drenching soil around the plant the microbial formulation of claim 9 or 10.
A method of augmenting cold resistance of a plant, comprising spraying the plant or drenching soil around the plant with the microbial formulation of claim 9 or 10.