JP7623708B2 - Novel strain, composition containing same, and agent for controlling bacterial diseases of grass family plants - Google Patents

Description

NPMD NITE P-03365

The present invention relates to a novel strain, a composition containing the same, and an agent for controlling bacterial diseases of grasses, and also relates to seeds of grasses.

Bacterial diseases such as rice bacterial grain rot and rice bacterial seedling blight are known to occur during the seedling stage of paddy rice. In recent years, such bacterial diseases have been increasing worldwide with the spread of heated seedling raising devices and the progress of global warming. Patent Document 1 describes that non-pathogenic Pantoea ananatis bacteria are useful for controlling rice bacterial grain rot and other diseases.

Furthermore, Non-Patent Documents 1 and 2 state that certain strains of Pantoea dispersa have antifungal activity against certain types of filamentous fungi. On the other hand, none of the prior art documents state that P. dispersa is useful for controlling bacterial diseases of grasses.

JP 2015-059090 A

Jiang et al. Sci Rep (2019), 9(1), 16354. doi: 10.1038/s41598-019-52804-3 Verma et al. J App Microbiol (2017), 122(6), 1680-1691. doi: 10.1111/jam.13463

To realize sustainable agriculture, there is a demand for the development of microbial materials that reduce the occurrence of drug-resistant bacteria and have a low environmental impact. However, microbial materials are limited in the diseases and crops they can be used for, and production costs are high. Therefore, the present invention aims to provide a new bacterial strain that is useful for controlling bacterial diseases in grasses in order to expand the variety of microbial materials.

As a result of intensive research aimed at solving the above problems, the present inventors have found that bacteria such as P. dispersa exhibit resistance to a toxin (toxoflavin) produced by Burkholderia glumae, a pathogen of rice bacterial grain rot, and have an ameliorative effect on rice growth inhibition, thereby completing the present invention. That is, the present invention provides the following bacterial strains and compositions containing the same, a control agent for bacterial diseases of grasses, and seeds of grasses.
[1] Pantoea dispersa BB1 strain (accession number: NITE P-03365).
[2] A composition comprising Pantoea dispersa BB1 strain (accession number: NITE P-03365) or a culture supernatant thereof.
[3] The composition according to [2] above, for inhibiting the growth of Burkholderia glumae.
[4] The composition according to [2] or [3] above, for controlling bacterial diseases of grasses.
[5] An agent for controlling bacterial diseases of grasses, comprising a toxoflavin-resistant bacterium or a culture supernatant thereof.
[6] The control agent according to [5], wherein the toxoflavin-resistant bacteria include bacteria of the genus Pantoea and/or bacteria of the genus Paenibacillus.
[7] The control agent according to [5] or [6], wherein the toxoflavin-resistant bacteria includes Pantoea dispersa BB1 strain (accession number: NITE P-03365).
[8] The control agent according to any one of [5] to [7], wherein the bacterial disease is rice bacterial grain rot.
[9] Seeds of a grass family plant containing a toxoflavin-resistant bacterium.
[10] The seed according to [9], wherein the toxoflavin-resistant bacterium comprises Pantoea dispersa strain BB1 (accession number: NITE P-03365).

According to the present invention, toxoflavin-resistant bacteria such as P. dispersa strain BB1 have an ameliorating effect on the growth inhibition of grass plants caused by pathogenic bacteria. Therefore, toxoflavin-resistant bacteria such as P. dispersa strain BB1 are useful for controlling bacterial diseases of grass plants.

1 shows the growth curve of P. dispersa strain BB1 in the absence or presence of toxin (toxoflavin). 1 shows the growth curve of P. dispersa strain BB44 in the absence or presence of toxin (toxoflavin). NCCP-568 in the absence or presence of toxin (toxoflavin). 1 shows the growth curve of Paenibacillus hunanensis in the absence or presence of toxin (toxoflavin). 1 shows the growth curve of B. glumae in the presence or absence of culture supernatant of P. dispersa strain BB1.

The present invention will now be described in further detail.
The present invention relates to a novel strain, P. dispersa BB1, and a composition containing the P. dispersa BB1 strain or its culture supernatant. P. dispersa is a type of endophytic fungus that lives symbiotically inside a grass plant. P. dispersa BB1 is a novel strain isolated from rice infected with B. gourmet, a pathogen of rice grain rot, and identified by analysis of 16S ribosomal RNA, and has been deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center since January 19, 2021 (Accession Number: NITE P-03365). P. dispersa BB1 exhibits resistance to a toxin (toxoflavin) produced by B. gourmet, a pathogen of rice grain rot, and has an improving effect on rice growth inhibition. Without being bound by any particular theory, it is believed that the toxicity of toxoflavin is due to the hydrogen peroxide produced by it, and since P. dispersa BB1 strain reduces the effects of hydrogen peroxide produced by toxoflavin and can grow in grasses infected with pathogenic bacteria, it is believed that it is useful for controlling bacterial diseases such as rice bacterial grain rot by competitively inhibiting the growth of the pathogenic bacteria.

That is, the composition containing the P. dispersa BB1 strain of the present invention or the culture supernatant thereof may be used to suppress the growth of pathogenic bacteria such as B. gourmet, or to control bacterial diseases of grasses. Therefore, the composition containing the P. dispersa BB1 strain of the present invention or the culture supernatant thereof may be applied to grasses. Since P. dispersa is originally an endophytic bacterium of grasses, it is expected that the composition containing the P. dispersa BB1 strain of the present invention or the culture supernatant thereof will cause less drug-resistant bacteria and have a low environmental impact. The grasses are not particularly limited, and may be, for example, rice, wheat, barley, barnyard millet, and corn. The bacterial disease is not particularly limited, and may be, for example, a bacterial disease caused by a pathogenic bacterium of the genus Burkholderia, such as rice bacterial grain rot, rice bacterial seedling blight, and rice brown stripe. It is known that "rice bacterial grain rot" and "rice bacterial seedling blight" are bacterial diseases of grasses caused by pathogenic bacteria of the genus Burkholderia, and that the pathogen of the former is B. glume and some B. gladioli, and that the pathogen of the latter is B. plantarii. It is known that "rice brown stripe" is a bacterial disease of grasses caused by Pseudomonas avenae.

In another aspect, the present invention also relates to a control agent for bacterial diseases of grasses, comprising a toxoflavin-resistant bacterium. Toxoflavin is a toxin produced by pathogenic bacteria of the genus Burkholderia. In the presence of this toxin, the growth of other bacteria is significantly restricted, but the toxoflavin-resistant bacterium reduces the effect of hydrogen peroxide produced by the toxin and can still grow in its presence. The toxoflavin resistance of the target bacterium can be appropriately confirmed by a method commonly used in the art. For example, if the target bacterium is cultured in a liquid medium, the concentration is adjusted to OD600nm = 0.1, and toxoflavin is added to the liquid medium at a final concentration of about 100 μM. If an increase in the OD600nm value is observed, the target bacterium can be confirmed to be a toxoflavin-resistant bacterium. Preferably, the toxoflavin-resistant bacteria are capable of starting to grow within 48 hours, and more preferably within 24 hours, when cultured in the presence of about 100 μM toxoflavin. Without being bound by any particular theory, it is believed that when the toxoflavin-resistant bacteria grow in a grass plant infected with a pathogenic bacterium, the growth of the pathogenic bacterium is competitively inhibited, making it possible to control bacterial diseases caused by the pathogenic bacterium.

In one embodiment, the toxoflavin-resistant bacteria include endophytic bacteria of grasses, more specifically, bacteria of the genus Pantoea such as P. dispersa and/or bacteria of the genus Paenibacillus such as Paenibacillus hunanensis, preferably P. dispersa BB1 strain. The bacteria of the genus Pantoea and the bacteria of the genus Paenibacillus can be appropriately identified by a method commonly used in the art. For example, the type of the target bacteria can be identified by analyzing the base sequence of the 16S ribosomal RNA of the target bacteria and performing a homology search and phylogenetic analysis against a database.

The method of applying the toxoflavin-resistant bacteria to the grass family plant is not particularly limited, and for example, the toxoflavin-resistant bacteria may be applied to the seeds (rice seeds) or ears of the grass family plant in a state suspended in a liquid medium or various aqueous solvents. When applying the toxoflavin-resistant bacteria to rice seeds, for example, the rice seeds may be immersed in a liquid medium or various aqueous solvents containing the toxoflavin-resistant bacteria in a reduced pressure environment at a temperature of about 15°C to about 35°C for about 1 minute to about 10 minutes. The concentration of the bacteria at the time of application is not particularly limited, and for example, the OD600nm value when suspended in a liquid medium or various aqueous solvents may be about 0.004 or more, and preferably about 0.01 to about 1.0.

The composition or control agent of the present invention may further contain any component that is commonly used in the art, and may further contain other components that are effective in controlling bacterial diseases of grasses, so long as the purpose of the present invention is not impaired.

In another aspect, the present invention also relates to a seed of a grass family plant containing a toxoflavin-resistant bacterium. It is expected that the occurrence of bacterial diseases in grass family plants can be prevented by protecting the seed of a grass family plant with the toxoflavin-resistant bacterium before germination. The means for including the toxoflavin-resistant bacterium in the seed of a grass family plant is not particularly limited, and for example, the toxoflavin-resistant bacterium may be applied to an untreated seed of the grass family plant in a state of being suspended in a liquid medium or various aqueous solvents, and the toxoflavin-resistant bacterium may be attached to the surface of the seed or penetrated into the inside of the seed. The seed of the present invention can be germinated and cultivated by a method commonly used in the art.

The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

Test Example 1: Isolation of strains
The surface of the rice seeds was sterilized and soaked in water, and the seeds were grown at 30° C. for 3 days to germinate. B. glume, the causative agent of rice bacterial grain rot, was inoculated under reduced pressure. The germinated rice seeds were then planted in sterilized soil (Bonsol No. 2, manufactured by Sumitomo Chemical Co., Ltd.) and cultivated in an artificial climate chamber for 4 days.

Cultivated rice was collected in a tube, 1 mL of sterilized water was added, and the rice was crushed using a microsmash. The crushed liquid was appropriately diluted, applied to a solid medium, and cultured for 3 days. Colonies that appeared on the solid medium were picked up, DNA was extracted, and the 16S ribosomal RNA sequence was analyzed to identify the picked bacterial species. Two species of Pantoea dispersa (strain BB1 and BB44), Pantoea sp. NCCP-568, and Paenibacillus funanensis, which are endophytic bacteria of rice and increased due to pathogen infection, were used in subsequent experiments.

[Test Example 2: Toxoflavin Resistance Test]
Each isolated strain was inoculated into LB medium to give an OD600nm of 0.1, toxoflavin (final concentration 100 μM) was added, and the mixture was cultured at 25°C for 46 hours. The absorbance at 600 nm was measured periodically to check the degree of bacterial growth. The results are shown in Figures 1A to 1D.

P. dispersa strain BB1 (Fig. 1A), P. dispersa strain BB44 (Fig. 1B), Pantoea sp. NCCP-568 (Fig. 1C), and P. funanensis (Fig. 1D) were all able to grow even in the presence of toxoflavin. In particular, P. funanensis was highly resistant to toxoflavin. Since B. glumae produces toxoflavin as a toxin, it was expected that each of the isolated strains would grow antagonistically with B. glumae in rice plants infected with B. glumae and weaken the pathogenicity of B. glumae.

[Test Example 3: Test against rice bacterial grain rot]
The surface of the Nipponbare rice seeds was sterilized with a sodium hypochlorite solution for 10 minutes, and 25 seeds were inoculated with 10 mL of B. gourmet culture solution (OD600nm = 0.0004 or 0.004) and 10 mL of test strain culture solution (OD600nm = 0.04) under reduced pressure. The seeds inoculated with each bacterium (25 grains each) were sown in Bonsol No. 1 soil (manufactured by Sumitomo Chemical Co., Ltd.) and grown under a repeated environment of 14 hours of light (28°C) and 10 hours of darkness (24°C). 10 days after sowing, the length of the longest part of the leaves (above ground) and roots was measured, and the average value (n = 25) was calculated. The results are shown in Tables 1 and 2.

When B. gourmete was inoculated, rice growth was suppressed, and even 10 days after sowing the seeds, the roots and leaves did not grow much, but when the endophytic fungus isolated in Test Example 1 above was inoculated together, root and leaf growth was observed. In particular, when P. dispersa BB1 strain was inoculated, a significant improvement was confirmed compared to the other test strains.

[Test Example 4: Growth Inhibitory Effect of Culture Supernatant on B. glumae]
P. dispersa BB1 strain was cultured overnight at 30 ° C. in 3 mL of LB medium. The culture solution was centrifuged, and the supernatant was subjected to filter filtration to remove live bacteria, and the culture supernatant of P. dispersa BB1 strain was obtained. Then, 100 μL of the culture supernatant of P. dispersa BB1 strain (BB1Sup50%) diluted to 50% with LB medium (LB), distilled water (DW), or distilled water was added to 100 μL of B. gourmet bacterial solution adjusted to OD600 nm = 0.1 in LB medium, and OD600 nm was measured over time.

As shown in Figure 2, the culture supernatant of P. dispersa strain BB1 inhibited the growth of B. glumae. Therefore, it is believed that the culture supernatant of P. dispersa strain BB1 contains antibacterial components against B. glumae.

From the above, it is expected that toxoflavin-resistant bacteria such as P. dispersa strain BB1 or their culture supernatants will have an ameliorating effect on the growth inhibition of grass plants caused by pathogenic bacteria. Therefore, toxoflavin-resistant bacteria such as P. dispersa strain BB1 or their culture supernatants are useful for controlling bacterial diseases of grass plants.

Claims (9)

Pantoea dispersa strain BB1 (accession number: NITE P-03365). A composition containing Pantoea dispersa strain BB1 (accession number: NITE P-03365) or its culture supernatant. The composition according to claim 2 for inhibiting the growth of Burkholderia glumae. The composition according to claim 2 or 3 for controlling bacterial diseases of grasses. 1. A control agent for bacterial diseases of grasses , comprising a toxoflavin-resistant bacterium or a culture supernatant thereof, wherein the toxoflavin-resistant bacterium comprises a bacterium of the genus Pantoea dispersa and/or Paenibacillus . The control agent according to claim 5, wherein the toxoflavin-resistant bacteria include Pantoea dispersa BB1 strain (accession number: NITE P-03365) and/or Paenibacillus funanensis . The control agent according to claim 5 or 6, wherein the toxoflavin-resistant bacteria includes Pantoea dispersa strain BB1 (accession number: NITE P-03365). The control agent according to any one of claims 5 to 7, wherein the bacterial disease is rice bacterial grain rot. 1. A seed of a grass family plant containing a toxoflavin-resistant bacterium, wherein the toxoflavin-resistant bacterium contains Pantoea dispersa strain BB1 (accession number: NITEP-03365) .

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