Novel bacterial strain and use thereof

Abstract

To provide an endophyte involved in the dormancy state of plant seeds.SOLUTION: The present invention is directed to Pseudomonas sp. strains DB4, DB5, and DB9, and these bacterial strains have an effect of inhibiting the germination of plant seeds. These bacterial strains serve not only for inhibition of germination of plant seeds, but also as novel tools in research on dormancy of plant seeds.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to novel bacterial strains and their uses, and particularly to novel bacterial strains related to the dormant state of plant seeds and their uses.

Background Art

[0002] Even when it seems that all the necessary conditions for germination are met, there are cases where plant seeds do not germinate, and this is called "dormancy." Growing in a harsh natural environment and connecting seeds to the next generation, shifting the germination time by "dormancy" has become one of the survival strategies for wild plants. On the other hand, in agricultural crops, it is necessary to germinate the seeds of the target plants all at once in terms of cultivation management, and measures to control "dormancy" have been studied during the process of cultivation. For example, Non-Patent Document 1 describes that the dry heat treatment of rice seeds has a highly stable dormancy-breaking effect, although it requires more treatment days compared to gibberellin treatment and hydrogen peroxide treatment.

[0003] Also, Patent Document 1 describes that a specific strain of Pseudomonas fluorescence suppresses the germination of nobie seeds. Non-Patent Document 2 describes that L-2-amino-4-methoxy-trans-3-butenoic acid secreted from Pseudomonas aeruginosa suppressed the germination of Arabidopsis thaliana seeds. However, these Pseudomonas bacteria are not endophytes, and the relationship between plant endophytes and the dormant state is not described in any literature.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide microorganisms involved in the dormant state of plant seeds.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that specific bacteria of the genus Pseudomonas symbiotic with plants are involved in the dormant state of plant seeds, and have completed the present invention. That is, the present invention provides the following bacterial strains, a composition for suppressing the germination of plant seeds, a method for suppressing the germination of plant seeds, and a method for searching for means for manipulating the dormant state of plant seeds. 〔1〕A bacterial strain which is Pseudomonas sp. strain DB4 (Accession No.: NITE P-03993), strain DB5 (Accession No.: NITE P-03994), or strain DB9 (Accession No.: NITE P-03995). 〔2〕A composition for suppressing the germination of plant seeds, comprising one or more bacterial strains selected from the group consisting of Pseudomonas sp. strain DB4 (Accession No.: NITE P-03993), strain DB5 (Accession No.: NITE P-03994), and strain DB9 (Accession No.: NITE P-03995), or a secretion or extract thereof. 〔3〕The composition according to 〔2〕, wherein the plant includes angiosperms. 〔4〕A method for suppressing the germination of plant seeds, comprising A method comprising the step of applying one or more bacterial strains selected from the group consisting of Pseudomonas sp. strain DB4 (deposit number: NITE P-03993), strain DB5 (deposit number: NITE P-03994), and strain DB9 (deposit number: NITE P-03995), or a secretion or extract thereof, to the plant or its habitat. 〔5〕The method according to 〔4〕 above, wherein the plant includes angiosperms. 〔6〕A method for searching for means to manipulate the dormant state of plant seeds, comprising: preparing one or more bacterial strains selected from the group consisting of Pseudomonas sp. strain DB4 (deposit number: NITE P-03993), strain DB5 (deposit number: NITE P-03994), and strain DB9 (deposit number: NITE P-03995), or a secretion or extract thereof; screening for factors that affect the germination inhibitory effect of the bacterial strain or its secretion or extract; when the factor suppresses the germination inhibitory effect, determining that the application of the factor is effective as a means to break the dormant state of the seeds, and when the factor promotes the germination inhibitory effect, determining that the application of the factor is effective as a means to induce or maintain the dormant state of the seeds; and the method comprising the above steps. 〔7〕The method according to 〔6〕 above, wherein the plant includes angiosperms. 〔8〕The method according to 〔6〕 or 〔7〕 above, wherein the screening step includes the step of applying the bacterial strain or its secretion or extract to the seeds of the plant. 〔9〕The method according to any one of 〔6〕 to 〔8〕 above, wherein the factor includes one or more selected from the group consisting of environmental conditions, compounds, proteins, and microorganisms other than the bacterial strain.

Advantages of the Invention

[0008] According to the present invention, Pseudomonas sp. strains DB4, DB5, and DB9, which are useful as endophytic bacteria for suppressing the germination of plant seeds, are provided. Therefore, by using Pseudomonas sp. strains DB4, DB5, and DB9 or their secretions or extracts as active ingredients, the germination of plant seeds can be suppressed. In addition, Pseudomonas sp. strains DB4, DB5, and DB9 are also useful as new tools in the study of plant seed dormancy. By using the germination inhibitory effect of these bacterial strains or their secretions or extracts, or the growth ability or survival rate of these bacterial strains as indicators, means for manipulating the dormant state of plant seeds can be explored.

Brief Description of the Drawings

[0009]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0010] Hereinafter, the present invention will be described in more detail. As used herein, "dormancy" refers to the state of seeds that have the ability to germinate but do not germinate even when given suitable temperature and moisture for germination. Also, as used herein, "endophyte" (also referred to as "endogenous bacteria") refers to microorganisms that inhabit the interior of plants but do not exhibit pathogenicity to the plants.

[0011] The present invention relates to novel bacterial strains, namely Pseudomonas sp. strain DB4, Pseudomonas sp. strain DB5, or Pseudomonas sp. strain DB9. These bacterial strains were deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, Japan on October 18, 2023, and their accession numbers are as follows.

Table 1

[0012] The bacterial strains of the present invention were found from endophytes of rice. As specifically shown in the examples described below, when the endophytic bacterial flora of rice seeds was compared before and after performing the conventional dry heat treatment as a dormancy-breaking treatment, Pseudomonas sp. strain DB4, strain DB5, and strain DB9 were present only in the endophytic bacterial flora before the dormancy-breaking treatment. And the cells and their secretions of Pseudomonas sp. strain DB4, strain DB5, and strain DB9 showed an inhibitory effect on the germination of seeds of plants not limited to rice. From these facts, it was first demonstrated that endophytes are involved in the dormant state of plant seeds.

[0013] Since the cells of the bacterial strains of the present invention and their secretions have shown a germination inhibitory effect, it is considered that the extract of the cells also shows a germination inhibitory effect. That is, in another aspect, the present invention also relates to a composition for inhibiting the germination of plant seeds, which contains one or more bacterial strains selected from the group consisting of Pseudomonas sp. DB4 strain, DB5 strain, and DB9 strain, or their secretions or extracts. By applying the composition to the target plant (for example, seeds, roots, stems, leaves, and flowers, etc.) or its habitat, the germination of the seeds of the plant can be inhibited. The plant is not particularly limited as long as it forms seeds, and it may be an angiosperm or a gymnosperm. Specifically, examples of the plant include gramineous plants such as rice and barley, and cruciferous plants such as Arabidopsis thaliana.

[0014] The bacterial strains that are the active ingredients of the composition of the present invention are the same as those described above as the bacterial strains of the present invention. The secretions and extracts of the bacterial strains can be appropriately prepared by methods commonly used by those skilled in the art. For example, since the medium after culturing the bacterial strains in a solid medium or a liquid medium by a conventional method contains the secretions of the bacterial strains, this can be used as it is or after removing the cells as the secretions that are the active ingredients of the composition of the present invention. Also, after collecting the cells after culturing the bacterial strains in a solid medium or a liquid medium by a conventional method, the cells can be disrupted in a solvent by a conventional method to remove debris, thereby preparing an extract that is the active ingredient of the composition of the present invention.

[0015] The composition of the present invention may further contain any inert ingredients or additional active ingredients commonly used in the art as long as the object of the present invention is not impaired. The inert ingredients are not particularly limited, but may include, for example, agrichemically acceptable excipients, solvents, buffers, surfactants, and preservatives. Also, the additional active ingredients are not particularly limited, but may include, for example, other ingredients effective for inhibiting the germination of plant seeds.

[0016] In another aspect, the present invention also relates to a method for suppressing the germination of plant seeds, the method comprising applying one or more bacterial strains selected from the group consisting of Pseudomonas sp. strains DB4, DB5, and DB9, or their secretions or extracts, to the plant (e.g., seeds, roots, stems, leaves, and flowers, etc.) or its habitat. The bacterial strain or its secretion or extract is as described above in relation to the bacterial strain of the present invention or the composition of the present invention, and may be used in the form of a composition for suppressing the germination of plant seeds as described above as one aspect of the present invention. Also, the plant to which the suppression method of the present invention is applied is also as described above in relation to the composition of the present invention.

[0017] The suppression method of the present invention may further include any steps commonly used in the art as long as the object of the present invention is not impaired, and may further include the step of applying the additional active ingredient to the seeds of the plant.

[0018] As described above, since it has been clarified that the endophytic Pseudomonas sp. strains DB4, DB5, and DB9 cause the dormant state of plant seeds, a new tool useful for the study of plant seed dormancy has been provided. For example, by using as indicators the germination inhibitory effect of any of the bacterial strains of Pseudomonas sp. strains DB4, DB5, and DB9, or their secretions or extracts, and the growth ability or survival rate of the bacterial strain, it becomes possible to explore means for manipulating the dormant state of plant seeds. That is, in a further aspect, the present invention also relates to a method for exploring means for manipulating the dormant state of plant seeds, the method comprising a step of preparing one or more bacterial strains selected from the group consisting of Pseudomonas sp. strains DB4, DB5, and DB9, or their secretions or extracts, and a step of screening for factors that affect the germination inhibitory effect of the bacterial strain or its secretion or extract, and When the factor suppresses the germination inhibitory effect, it is determined that the application of the factor is effective as a means of breaking the dormant state of the seed, and when the factor promotes the germination inhibitory effect, it includes the step of determining that the application of the factor is effective as a means of inducing or maintaining the dormant state of the seed. The bacterial strain or its secretion or extract is as described above in relation to the bacterial strain of the present invention or the composition of the present invention, and the plant to which the screening method of the present invention is applied is also as described above in relation to the composition of the present invention.

[0019] For example, a factor that inhibits the growth of Pseudomonas sp. strains DB4, DB5, and DB9 or reduces the survival rate can suppress the germination inhibitory effect and thus can be a means of breaking the dormant state of the seed. A factor that promotes the growth of the bacterial strain or increases the survival rate can enhance the germination inhibitory effect and thus can be a means of inducing or maintaining the dormant state of the seed. In addition, a factor that affects the germination inhibitory effect without affecting the growth or survival rate of Pseudomonas sp. strains DB4, DB5, and DB9, for example, a factor that affects the production and / or secretion of the active entity of the germination inhibitory effect or a factor that affects the activity of the active entity itself of the germination inhibitory effect can be screened by confirming the sprout length and / or germination rate when compared with the control group in a test system in which the bacterial strain or its secretion or extract is applied to the seeds of the target plant. That is, in one aspect, the screening step includes the step of applying the bacterial strain or its secretion or extract to the seeds of the plant.

[0020] The factor to be screened in the screening method of the present invention is not particularly limited, and may include, for example, one or more selected from the group consisting of environmental conditions such as temperature or pressure, low-molecular or high-molecular compounds, proteins such as metabolites of plants or microorganisms or plant hormones, and microorganisms other than the bacterial strain.

[0021] The screening method of the present invention may further include any steps usually used in the art as long as the object of the present invention is not impaired.

[0022] Hereinafter, the present invention will be specifically described by way of examples, but the scope of the present invention is not limited to these examples.

Examples

[0023] 1. Identification of endophytes involved in the dormancy of plant seeds Rice seeds dried at 37°C for 5 days immediately after harvest in September were prepared and divided into two groups. One group of rice seeds was subjected to dry heat treatment (dormancy-breaking treatment) at 50°C for 5 days. Then, for each of the untreated group and the dormancy-breaking treatment group of rice seeds, the seed surface was sterilized with antiformin and immersed in distilled water under 30°C in the dark to promote germination. When observing the rice seeds on the 4th day from the start of immersion, most seeds had germinated in the dormancy-breaking treatment group, but there were also many seeds that had not germinated in the untreated group, and even those that had germinated had short sprout lengths. (It was confirmed that when these rice seeds were cultivated as they were, after a certain period of time, the rice seeds in both groups germinated, and they grew similarly after two weeks.)

[0024] On the 4th day from the start of immersion, the rice seeds were collected into tubes, 1 mL of sterilized water was added, and they were crushed with a microsmasher. The crushed liquid was appropriately diluted, spread on a solid medium, and cultured for 3 days. The colonies that appeared on the solid medium were picked up, DNA was extracted, and the sequence of 16S ribosomal RNA was analyzed by a conventional method and subjected to BLAST search to identify the bacterial species of the picked-up colonies. When comparing the bacterial species identified in the untreated group with those identified in the dormancy-breaking treatment group, it was found that the bacteria of the genus Pseudomonas (Pseudomonas sp.) decreased due to the dormancy-breaking treatment. The bacteria of the genus Pseudomonas identified only in the untreated group were isolated, and each strain was named Pseudomonas sp. DB4 strain, Pseudomonas sp. DB5 strain, and Pseudomonas sp. DB9 strain.

[0025] 2. Germination inhibition test by live bacteria In the same manner as in Item 1 above, rice seeds with or without dormancy-breaking treatment were prepared, and their surfaces were sterilized with anti-formalin. Also, Pseudomonas sp. strains DB4, DB5, or DB9 were cultured with shaking overnight (180 rpm, 30 °C) in LB liquid medium, and LB medium was added so that OD = 0.1 to prepare each bacterial solution. Then, 10 rice seeds with dormancy-breaking treatment were immersed in the bacterial solution or distilled water under reduced pressure for 5 minutes. Rice seeds without dormancy-breaking treatment were immersed in distilled water. Each seed was washed with distilled water, added to 3 mL of distilled water, and left standing in an artificial climate chamber at 30 °C. Then, after 6 days, the length of the sprouts was measured. As a result, as shown in Fig. 1, when treated with the bacterial solution, germination was suppressed and the sprout length became shorter in the rice seeds with dormancy-breaking treatment.

[0026] Also, commercially available barley seeds whose dormancy had already been broken were prepared, and their surfaces were sterilized with anti-formalin. To 10 mL of distilled water, 10 μL, 100 μL, and 500 μL of the bacterial solutions of Pseudomonas sp. strains DB4, DB5, or DB9 cultured overnight were added respectively. This diluted bacterial solution or sterilized water was placed in a petri dish together with 20 surface-sterilized barley seeds and left standing in an artificial climate chamber at 22 °C. Then, after 4 days, the germination state was observed and the germination rate was calculated. As a result, as shown in Fig. 2, when treated with the bacterial solution, germination of the barley seeds was suppressed.

[0027] Note that when using the bacterial solution of another endophyte Pseudomonas putida RSB15 strain isolated from rice seeds, no suppression of germination was observed in either rice or barley.

[0028] 3. Germination Inhibition Test by Secretions On the upper part of a solid medium (Murashige and Skoog medium (4.3 g / L), 2-(N-morpholino)ethanesulfonic acid (0.5 g / L), 0.8% (w / v) bacto agar, and 20 mM disodium succinate), Pseudomonas putida RSB15 strain or Pseudomonas sp. DB4 strain, DB5 strain, or DB9 strain was streaked with a width of 5 cm and cultured at 30 °C for 2 days. Arabidopsis thaliana seeds surface-sterilized with antiformin were sown at positions 2 cm, 4 cm, or 6 cm away from the bacteria on the petri dish after culturing (Fig. 3A), and cultivated in a phytotron to observe the germination status over time. As a result, 7 days after sowing, the seeds at all positions germinated in the petri dish of Pseudomonas putida RSB15 strain (Fig. 3B), but germination was suppressed at all positions in the petri dishes of Pseudomonas sp. DB4 strain, DB5 strain, or DB9 strain, and germination was more strongly suppressed the closer to the bacteria (Figs. 3C - 3E).

[0029] In addition, the germination status of seeds on the petri dish was observed in the same manner except that dormant rice seeds (dry heat treatment at 50 °C for 5 days) or commercially available barley seeds whose dormancy had been broken were used instead of Arabidopsis thaliana seeds. The germination rate of rice seeds 7 days after sowing is shown in Fig. 4, and the germination rate of barley seeds 4 days after sowing is shown in Fig. 5. The germination of rice seeds and barley seeds was suppressed at the position 2 cm away from the bacteria.

[0030] From these results, it is considered that the extracellular secretions of Pseudomonas sp. DB4 strain, DB5 strain, or DB9 strain diffused into the medium, and the germination of various seeds was suppressed in a concentration-dependent manner.

[0031] From the above, it was found that Pseudomonas sp. strains DB4, DB5, and DB9 are endophytic bacteria that inhibit the germination of plant seeds. Therefore, by using Pseudomonas sp. strains DB4, DB5, and DB9 or their secretions or extracts as active ingredients, the germination of plant seeds can be inhibited. In addition, Pseudomonas sp. strains DB4, DB5, and DB9 are also useful as new tools in the study of plant seed dormancy. By using the germination inhibitory effect of these bacterial strains or their secretions or extracts, or the growth ability or survival rate of these bacterial strains as indicators, it is possible to explore means to manipulate the dormant state of plant seeds.

Claims

1. A bacterial strain that is Pseudomonas sp. strain DB4 (Deposit Number: NITE P-03993), strain DB5 (Deposit Number: NITE P-03994), or strain DB9 (Deposit Number: NITE P-03995).

2. A composition for suppressing the germination of plant seeds, comprising one or more bacterial strains selected from the group consisting of Pseudomonas sp. strain DB4 (Deposit Number: NITE P-03993), strain DB5 (Deposit Number: NITE P-03994), and strain DB9 (Deposit Number: NITE P-03995), or a secretion or extract thereof.

3. The composition according to claim 2, wherein the plant includes angiosperms.

4. A method for suppressing the germination of plant seeds, comprising applying one or more bacterial strains selected from the group consisting of Pseudomonas sp. strain DB4 (Deposit Number: NITE P-03993), strain DB5 (Deposit Number: NITE P-03994), and strain DB9 (Deposit Number: NITE P-03995), or a secretion or extract thereof, to the plant or its habitat.

5. The method according to claim 4, wherein the plant includes angiosperms.

6. A method for exploring means for manipulating the dormant state of plant seeds, comprising preparing one or more bacterial strains selected from the group consisting of Pseudomonas sp. strain DB4 (Deposit Number: NITE P-03993), strain DB5 (Deposit Number: NITE P-03994), and strain DB9 (Deposit Number: NITE P-03995), or a secretion or extract thereof; screening for factors that affect the germination inhibitory effect of the bacterial strain or its secretion or extract; when the factor suppresses the germination inhibitory effect, determining that the application of the factor is effective as a means for breaking the dormant state of the seeds, and when the factor promotes the germination inhibitory effect, determining that the application of the factor is effective as a means for inducing or maintaining the dormant state of the seeds; and including.

7. The method according to claim 6, wherein the plant includes angiosperms.

8. The method according to claim 6, wherein the screening step includes applying the bacterial strain or its secretion or extract to the seeds of the plant.

9. The method according to any one of claims 6 to 8, wherein the factor includes one or more selected from the group consisting of environmental conditions, compounds, proteins, and microorganisms other than the bacterial strain.

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