Novel strain, composition containing same, and method for controlling bacterial diseases of grasses

Abstract

To provide a novel bacterial strain useful for controlling a bacterial disease of gramineous plants in order to increase the range of microbial material variations.SOLUTION: The present invention relates to Burkholderia gladioli NB6 strain (accession number: NITE P-03565), a composition including the bacterial strain or culture supernatant thereof, a method for controlling a bacterial disease of gramineous plants which includes a process of applying the bacterial strain or culture supernatant thereof to a place where Burkholderia glumae grows or a place where the growth of Burkholderia glumae is suspected, and a seed of gramineous plant including the bacterial strain.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to novel strains and compositions containing same and methods for controlling bacterial diseases of grasses, and also to seeds of grasses. [Background technology]

[0002] Bacterial diseases such as rice bacterial grain rot and rice bacterial seedling blight are known as diseases that occur during the seedling raising period 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. Non-Patent Document 1 describes that a specific strain such as Burkholderia gladioli 1064A strain has a control effect against rice bacterial grain rot. On the other hand, Non-Patent Document 1 does not describe the control effect of the culture supernatant of this strain against rice bacterial grain rot. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Journal of the Japanese Society of Plant Pathology (2000), 66(3), 232-238 Summary of the Invention [Problem to be solved by the invention]

[0004] Toward the realization of 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, but microbial materials have problems in that they are applicable only to limited diseases and crops, and their production costs are high. Therefore, the present invention aims to provide a novel strain that is useful for controlling bacterial diseases in grasses in order to expand the variety of microbial materials. [Means for solving the problem]

[0005] As a result of intensive research aimed at solving the above problems, the present inventors have found that a novel strain of B. gladioli is particularly useful for controlling rice bacterial grain rot, and have completed the present invention. That is, the present invention provides the following strains and compositions containing the same, methods for controlling bacterial diseases in grasses, and seeds of grasses. [1] Burkholderia gladioli strain NB6 (accession number: NITE P-03565). [2] A composition comprising Burkholderia gladioli strain NB6 (accession number: NITE P-03565) or its culture supernatant. [3] The composition described in [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] The composition described in [4] above, wherein the bacterial disease is rice bacterial grain rot. [6] Seeds of a grass family plant containing Burkholderia gladioli strain NB6 (accession number: NITE P-03565). [7] A method for controlling bacterial diseases of grasses, comprising a step of applying Burkholderia gladioli NB6 strain (accession number: NITE P-03565) or a culture supernatant thereof to a place where Burkholderia glumae is growing or where the growth of Burkholderia glumae is suspected. Effect of the Invention

[0006] According to the present invention, the B. gladioli NB6 strain or a culture supernatant thereof exerts an ameliorative effect against the growth inhibition of grass plants caused by pathogenic bacteria, and therefore, the B. gladioli NB6 strain or a culture supernatant thereof is useful for controlling bacterial diseases of grass plants. [Brief description of the drawings]

[0007] [Figure 1]1 shows the control effect of live B. gladioli strain NB6 (the present invention), B. gladioli strain MAFF212204 (conventional 1), and B. gladioli strain MAFF301064 (conventional 2) against rice bacterial grain rot. Different letters in the figure indicate significant differences by Tukey's analysis of variance (p<0.05). [Diagram 2] 1 shows the growth curve of B. glumae depending on the amount of culture supernatant of B. gladioli strain NB6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The present invention will now be described in further detail. The present invention relates to a novel strain, B. gladioli NB6, and a composition containing the strain or its culture supernatant. B. gladioli is a type of endophytic fungus that lives symbiotically inside grasses. The B. gladioli NB6 strain is a novel strain isolated from rice infected with B. glume, a pathogen of rice bacterial 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 December 2, 2021 (Accession Number: NITE P-03565). Without being bound by any particular theory, it is believed that the B. gladioli NB6 strain is more likely to grow inside grasses than pathogenic bacteria of grasses, and therefore antagonistically inhibits the growth of the pathogenic bacteria, thereby effectively contributing to the control of bacterial diseases such as rice bacterial grain rot. In addition, it is believed that the culture supernatant of the B. gladioli NB6 strain contains antibacterial components, and that the inhibition of the growth of the above-mentioned pathogenic bacteria by these antibacterial components also contributes effectively to the control of bacterial diseases in grasses.

[0009] That is, the B. gladioli NB6 strain of the present invention and a composition containing the strain or its culture supernatant may be used to suppress the growth of pathogenic bacteria such as B. gladioli or to control bacterial diseases of grasses. Therefore, the B. gladioli NB6 strain of the present invention and a composition containing the strain or its culture supernatant may be applied to grasses and the like. Since B. gladioli is originally an endophytic bacterium of grasses, the B. gladioli NB6 strain of the present invention and a composition containing the strain or its culture supernatant are expected to have low occurrence of drug-resistant bacteria and low environmental load. 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, and may be, for example, rice bacterial grain rot, rice bacterial seedling blight, and rice brown stripe disease. "Rice bacterial grain rot" and "rice bacterial seedling blight" are bacterial diseases of grasses caused by pathogenic bacteria of the Burkholderia genus. The pathogens of the former are B. glume and some B. gladioli, and the pathogens of the latter are B. plantarii. "Rice brown stripe" is a bacterial disease of grasses caused by Pseudomonas avenae.

[0010] The composition of the present invention may further contain any component that may be commonly used in the art, as long as it does not impair the object of the present invention, and may further contain other components that are effective in controlling bacterial diseases of grasses.

[0011] In another aspect, the present invention also relates to a seed of a grass family plant containing the B. gladioli NB6 strain. 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 B. gladioli NB6 strain before germination. The means for incorporating the B. gladioli NB6 strain into the seed of a grass family plant is not particularly limited, and for example, the B. gladioli NB6 strain may be applied to an untreated seed of the grass family plant in a state suspended in a liquid medium or various aqueous solvents, so that the B. gladioli NB6 strain is 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.

[0012] In another aspect, the present invention also relates to a method for controlling bacterial diseases of grasses, the method comprising a step of applying the B. gladioli NB6 strain or a culture supernatant thereof to a place where B. glume grows or where B. glume is suspected to grow. The method for applying the B. gladioli NB6 strain or a culture supernatant thereof is not particularly limited, and for example, the B. gladioli NB6 strain or a culture supernatant thereof may be applied to the seeds (rice seeds) or ears of the grasses in a state of being suspended or diluted in a liquid medium or various aqueous solvents. When the B. gladioli NB6 strain or a culture supernatant thereof is applied to the rice seeds, for example, the rice seeds may be immersed in a liquid medium or various aqueous solvents containing the B. gladioli NB6 strain or a culture supernatant thereof at a temperature of about 15°C to about 35°C for about 1 minute to about 10 minutes under a reduced pressure environment. The bacterial cell concentration at the time of application is not particularly limited. For example, the OD600nm value when suspended in a liquid medium or various aqueous solvents may be about 0.004 or more, and is preferably about 0.01 to about 1.0.

[0013] The method of the present invention may further include any step that may be commonly used in the art, as long as it does not impair the object of the present invention, and may further include a step of applying other components that are effective in controlling bacterial diseases of grasses.

[0014] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples. EXAMPLES

[0015] [Test Example 1: Isolation of strains] The surface of NonaBokra or Bleiyo seeds, known to be resistant to bacterial grain rot, was sterilized and soaked in water, and then grown at 30°C for 3 days to germinate. B. glume, the causative agent of rice bacterial grain rot, was inoculated under reduced pressure. As a control, distilled water was used instead of B. glume. The germinated seeds were then planted in sterilized soil (Bonsol No. 2, Sumitomo Chemical Co., Ltd.) and cultivated in an artificial climate chamber for 4 to 7 days. NonaBokra rice plants grew well even when inoculated with B. glume, whereas B. glume leaves hardly grew when inoculated with B. glume using the method described here.

[0016] In addition, rice plants cultivated under the above conditions for 4 days were collected in tubes, 1 mL of sterilized water was added, and the plants were 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 selected bacterial species. Among the identified bacteria, B. gladioli was not detected in Breyo rice, but was specifically detected in Nona Bocra rice. Moreover, B. gladioli accounted for about half of the endophytic bacteria contained in Nona Bocra rice, and the proportion of endophytic bacteria did not change much depending on whether or not it was infected with B. glume. Therefore, we focused on B. gladioli (especially B. gladioli NB6 strain) and used it in the subsequent experiments.

[0017] [Test Example 2: Control effect against rice bacterial grain rot 1] The surface of Nipponbare rice seeds was sterilized with sodium hypochlorite solution for 10 minutes, and 10 seeds were inoculated with 1 mL of B. glume culture (OD600nm=0.004) and 1 mL of culture solution of the test strain (OD600nm=0.04) or its culture supernatant (viable bacteria were removed by centrifugation and filtration) under reduced pressure. The seeds inoculated with each bacterium (10 seeds each) were sown on 1 / 2 Murashige and Skoog medium (0.3% Gelrite) and grown under a repeated environment of 14 hours light (28℃) and 10 hours dark (24℃). The height of the rice plants was measured 7 days after sowing, and the average and standard deviation (n=8) were calculated. The test strains used were distilled water as a control, B. gladioli NB6 strain, or the known B. gladioli MAFF301064 strain (a strain that is pathogenic to onions and the like but not to rice) or its pathogenicity-deprived strain 1064A strain (MAFF212204 strain) (a strain that is not pathogenic to either onions or rice). The results are shown in Table 1.

[0018] [Table 1]

[0019] When inoculated with B. gladioli, the growth of rice was suppressed, and the plant height did not grow much even 8 days after sowing the seeds. However, when live bacteria of B. gladioli were inoculated together, the plant height increased, and in particular, when live bacteria of B. gladioli NB6 strain were inoculated, the plant height tended to grow well. In addition, when only the culture supernatant of B. gladioli NB6 strain was applied, the plant height could be increased well. On the other hand, the plant height hardly grew when the culture supernatant of B. gladioli MAFF212204 strain or B. gladioli MAFF301064 strain was used. Therefore, B. gladioli NB6 strain, which has a control effect not only in the live bacteria but also in the culture supernatant, exerts a remarkable effect in controlling rice bacterial grain rot that could not be predicted from the conventional technology.

[0020] [Test Example 3: Control effect against rice bacterial grain rot 2] In order to compare in more detail the control effects of live bacteria, B. gladioli strain NB6 (present invention), B. gladioli strain MAFF212204 (conventional 1), and B. gladioli strain MAFF301064 (conventional 2), against bacterial grain rot of rice, a test similar to Test Example 2 was carried out using an increased number of samples. The height of rice plants was measured 7 days after sowing, and the average and standard deviation (n=15) were calculated, followed by Tukey's analysis of variance.

[0021] As shown in Figure 1, when live bacteria of B. gladioli strain NB6 (the present invention) were used, the plant height grew at a level equivalent to that of the group not inoculated with the pathogen. The plant height at this time was significantly longer than when live bacteria of B. gladioli strain MAFF212204 (conventional 1) or live bacteria of B. gladioli strain MAFF301064 (conventional 2) were used. Therefore, the control effect exerted by live bacteria of B. gladioli strain NB6 is significantly superior to that of the conventional technology.

[0022] [Test Example 4: Growth inhibition effect of B. glumae by culture supernatant] B. gladioli NB6 strain was cultured overnight at 30°C in 3mL of LB medium. The culture solution was centrifuged, and the supernatant was filtered to remove live bacteria, obtaining the culture supernatant of B. gladioli NB6 strain. Then, 100μL of LB medium or appropriately diluted culture supernatant of B. gladioli NB6 strain was added to 100μL of B. glumae bacterial solution adjusted to OD600nm = 0.1 with LB medium, and OD600nm was measured over time.

[0023] [Table 2]

[0024] As shown in Figure 2, the culture supernatant of B. gladioli NB6 strain inhibited the growth of B. glumae in a concentration-dependent manner. Therefore, it is considered that the culture supernatant of B. gladioli NB6 strain contains antibacterial components against B. glumae.

[0025] From the above, it was found that the B. gladioli NB6 strain or its culture supernatant has an ameliorating effect on the growth inhibition of grass plants caused by pathogenic bacteria. Therefore, the B. gladioli NB6 strain or its culture supernatant is useful for controlling bacterial diseases of grass plants.

Claims

1. Burkholderia gladioli strain NB6 (accession number: NITE P-03565).

2. A composition comprising Burkholderia gladioli NB6 strain (accession number: NITE P-03565) or a culture supernatant thereof.

3. The composition according to claim 2 for inhibiting the growth of Burkholderia glumae.

4. The composition according to claim 2 or 3 for controlling bacterial diseases of grasses.

5. The composition according to claim 4, wherein the bacterial disease is rice bacterial grain rot.

6. Seeds of a grass family plant containing Burkholderia gladioli strain NB6 (accession number: NITE P-03565).

7. A method for controlling bacterial diseases in grasses, comprising the step of applying Burkholderia gladioli NB6 strain (Accession Number: NITE P-03565) or a culture supernatant thereof to a place where Burkholderia glumae grows or is suspected of growing.

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