Method for producing rice seeds

Abstract

To provide a method for producing rice seeds resistant to bacterial wilt disease of rice in a short period without identifying and introducing a resistance gene and without requiring administration of a microorganism material.SOLUTION: The method for producing rice seeds comprises repeating a process which includes the following steps two or more generations: (1) inoculating a seed or the inside of a plant body of rice with a specific plant-endogenous bacterium; (2) growing the rice seed or plant body after the step (1); and (3) collecting a seed from the rice plant body grown by the step (2).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing rice seeds. [Background technology]

[0002] Bacterial grain rot of rice is a serious disease of rice caused by infection with the rice bacterial grain rot fungus (Burkholderia glumae). The fungus infects rice grains during development and causes them to wither (head blight). However, even when the fungus infects the grains, it may not show symptoms such as withering, and may opportunistically cause seedling rot. As such, because infection with bacterial grain rot does not necessarily cause symptoms, it is difficult to notice the infection and often causes the infection to spread, making it a difficult pathogen to control.

[0003] Measures against such pathogens include, for example, the use of bactericides or antibiotics (e.g., oxolinic acid, etc.) against pathogens, breeding of varieties resistant to pathogens, and use of control materials against pathogens. In breeding of varieties resistant to pathogens, new strains have traditionally been produced by genetic modification through crossing with strains having superior genes (e.g., Non-Patent Document 1). In addition, regarding control materials against pathogens, since there have already been many reports on microorganisms that improve stress resistance (e.g., Non-Patent Document 2), the development of microbial materials such as pesticides using such microorganisms (microbial pesticides) is being promoted.

[0004] In relation to microbial pesticides, for example, Patent Document 1 discloses a microbial pesticide for imparting insect resistance to plants and suppressing quality deterioration, which contains as an active ingredient a Pseudomonas bacterium having an expressible 1-aminocyclopropane-1-carboxylic acid deaminase (ACCd) gene. Patent Document 2 also discloses a pest control agent whose main component is a symbiotic bacterium consisting of bacteria isolated from a naturally occurring plant body, which causes artificially introduced Gramineae plants to express pest resistance. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2015-166344 A [Patent Document 2] JP 2003-300805 A [Non-patent literature]

[0006] [Non-Patent Document 1] Mizobuchi R, et al., “QTLs forResistance to Major Rice Diseases Exacerbated by Global Warming: Brown Spot,Bacterial Seedling Rot, and Bacterial Grain Rot.” Rice (NY). 2016 Dec;9(1):23.doi: 10.1186 / s12284-016-0095-4. Epub 2016 May 13. [Non-Patent Document 2] Liu H, et al., (2017) “Inner Plant Values: Diversity, Colonization and Benefits fromEndophytic Bacteria.” Front. Microbiol. 8:2552. doi:10.3389 / fmicb.2017.02552 Summary of the Invention [Problem to be solved by the invention]

[0007] Conventional breeding methods for resistant varieties require the identification of resistance genes, which then require a long development period through crossbreeding. In addition, when using fungicides or antibiotics against pathogens, the emergence of resistant bacteria reduces their effectiveness. Furthermore, when using control materials such as microbial materials, their effectiveness is unstable, and the only purpose is to confer resistance to contemporary crops.

[0008] Therefore, an object of the present invention is to provide a method for producing rice seeds resistant to bacterial grain rot in a short period of time without identifying and introducing a resistance gene and without the need for administration of a microbial agent. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors discovered that by inoculating rice seeds with a specific endophytic bacterium, growing the seeds, and harvesting the seeds for three generations, the next generation of seeds harvested thereafter will exhibit resistance to rice bacterial grain rot, thus completing the present invention.

[0010] That is, the present invention relates to, for example, the following inventions. [1] A method for producing rice seeds, comprising the steps of: (1) Inoculating rice seeds or plants with endophytic bacteria; (2) After (1) above, growing the rice seeds or plants; and (3) collecting seeds from the rice plant body grown according to (2) above, for two or more generations; A method for producing rice seeds, wherein the plant endophytic bacterium comprises at least one selected from the group consisting of Pseudomonas putida strain RSB15 (accession number: NITE P-02616), Stenotrophomonas maltophilia strain RSB2 (accession number: NITE P-02617), Burkholderia gladioli strain NB6 (accession number: NITE P-03565), and Pantoea dispersa strain BB1 (accession number: NITE P-03365). [2] The method for producing the rice seed or plant body described in [1], wherein the rice seed or plant body is a rice seed. [3] Rice seeds produced by the production method described in [1] or [2]. Effect of the Invention

[0011] According to the present invention, a method for producing rice seeds resistant to bacterial grain rot can be provided, which can be carried out in a short period of time without identifying and introducing a resistance gene and without the need for administration of a microbial material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0013] [Method of producing rice seeds] The rice seed production method of this embodiment (hereinafter also referred to as the "production method") comprises the steps of (1) inoculating a plant endophytic bacterium into a rice seed or plant body, (2) growing the rice seed or plant body after (1), and (3) harvesting seeds from the rice plant body grown by (2) above, for two or more generations.

[0014] The production method according to the present embodiment can produce rice seeds resistant to bacterial grain rot in a short period of time by repeating the above steps (1) to (3) in this order for two or more generations. Furthermore, as shown in the examples described later, the resistance of rice seeds to bacterial grain rot is strengthened as the above steps (1) to (3) are repeated between generations. That is, the production method according to the present embodiment can strengthen the resistance of rice to bacterial grain rot by repeating the above steps (1) to (3) in this order for two or more generations.

[0015] Rice is a plant of the genus Oryza in the family Poaceae, and the variety is not particularly limited, and may be, for example, Asian rice (Oriza sativa) or African rice (Oryza glaberrima), and Asian rice may be selected from the species japonica, indica, and javanica. In this embodiment, the Oryza plant may be paddy rice.

[0016] Rice bacterial grain rot is caused by infection with the rice bacterial grain rot fungus (Burkholderia glumae (hereinafter also referred to as "B. glumae"). Bacterial grain rot of rice occurs around the milky ripe stage (about 6 to 12 days after heading), and the grains wilt and turn pale. At this time, the rachis and stalks of the diseased ears are healthy and green. The diseased grains then turn grayish white or light yellowish brown, and become sterile or pitiful. B. glumae is a gram-negative, seed-borne betaproteobacterium.

[0017] The above (1) refers to inoculating a specific endophytic bacterium into the rice seed or plant body, and containing the specific endophytic bacterium inside the rice seed or plant body. In this specification, unless otherwise specified, "plant body" refers to any organ of a plant, for example, flowers (including buds), buds, leaves, stems, roots, rhizomes, tubers, and underground stems. In the above (1), the plant body is not particularly limited, but is preferably a flower. In the above (1), the rice seed or plant body is preferably a rice seed (rice seed).

[0018] Examples of methods for inoculating a specific endophytic bacterium into rice seeds or plant bodies include immersing rice seeds or plant bodies (e.g., flowers, etc.) in a suspension of endophytic bacteria, wounding the surface of the rice seeds or plant bodies and applying and / or spraying the suspension of endophytic bacteria to the wounded area, and extruding and discharging the suspension into the rice seeds or plant bodies using a syringe or needle, etc. It is preferable to immerse the rice seeds or plant bodies in a suspension of endophytic bacteria, and it is more preferable to immerse the rice seeds or plant bodies in a suspension of endophytic bacteria under reduced pressure conditions. The cell concentration of the endophytic bacteria at the time of inoculation is not particularly limited, and for example, the OD600nm value when suspended in a liquid medium or aqueous medium may be 0.004 or more, preferably 0.01 to 1.0.

[0019] In the above (1), the rice seeds to be inoculated with the specific plant endophytic bacterium are not particularly limited and may be rice seeds that do not exhibit resistance to bacterial grain rot, and further, since the rice seed production method of this embodiment enhances resistance to bacterial grain rot, the rice seeds may be rice seeds that exhibit resistance to bacterial grain rot.

[0020] The plant endophytic bacteria inoculated in (1) above includes at least one selected from the group consisting of Pseudomonas putida RSB15 strain (accession number: NITE P-02616), Stenotrophomonas maltophilia RSB2 strain (accession number: NITE P-02617), Burkholderia gladioli NB6 strain (accession number: NITE P-03565), and Pantoea dispersa BB1 strain (accession number: NITE P-03365).

[0021] Pseudomonas putida (hereinafter referred to as "P. putida") RSB15 strain (Accession Number: NITE P-02616) was isolated from rice infected with B. glume, the causal agent of rice bacterial grain rot, and identified by analysis of 16S ribosomal RNA. It has been deposited at the National Institute of Technology and Evaluation (National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818)) since January 24, 2018. P. putida RSB15 strain is thought to suppress the growth of B. glume, suggesting that it is involved in the suppression of the onset of rice bacterial grain rot.

[0022] Stenotrophomonas maltophilia (hereinafter also referred to as "S. maltophilia") RSB2 strain (Accession Number: NITE P-02617, Identification: SMAL strain (old strain name)) was isolated from rice infected with B. glumae, the causative agent of rice bacterial grain rot, and identified by analysis of 16S ribosomal RNA. It has been deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center since January 24, 2018. It has been confirmed that S. maltophilia RSB2 strain enhances the inhibitory effect of Pseudomonas putida RSB1 strain (accession number: NITE P-02614), Pseudomonas putida RSB10 strain (accession number: NITE P-02615), and P. putida RSB15 strain on the development of rice bacterial grain rot.

[0023] Burkholderia gladioli (hereinafter also referred to as "B. gladioli") NB6 strain (Accession Number: NITE P-03565) is a type of endophytic bacterium that lives symbiotically inside grasses. The B. gladioli NB6 strain was isolated from rice inoculated with B. glume, the pathogen of rice bacterial grain rot, and identified by analysis of 16S ribosomal RNA. It has been deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center since December 2, 2021. The B. gladioli NB6 strain is more likely to grow inside grasses than pathogenic bacteria of grasses, and is therefore thought to competitively inhibit the growth of the pathogenic bacteria and effectively contribute 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.

[0024] Pantoea dispersa (hereinafter referred to as "P. dispersa") BB1 strain (Accession Number: NITE P-03365) was isolated from rice infected with B. glume, the pathogen of rice bacterial grain rot, and identified by analysis of 16S ribosomal RNA. It has been deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center since January 19, 2021. P. dispersa BB1 strain is resistant to a toxin (toxoflavin) produced by B. glume, the pathogen of rice bacterial grain rot, and has an ameliorating effect on rice growth inhibition. The toxicity of toxoflavin is thought to be due to the hydrogen peroxide produced by it. P. dispersa BB1 strain reduces the effects of hydrogen peroxide produced by toxoflavin and can grow in grasses infected with pathogenic bacteria. It is therefore thought to be useful in controlling bacterial diseases such as rice bacterial grain rot, as it competitively inhibits the growth of the pathogenic bacteria.

[0025] The above-mentioned (2) involves growing the rice seeds or plants inoculated with the above-mentioned endophytic bacteria until the seeds can be harvested.

[0026] The growth conditions for the rice seeds or plants in (2) above may be normal conditions for those skilled in the art to grow (cultivate) rice, and may be appropriately set depending on the state of the rice seeds or plants, the type of inoculated plant endophytic fungus, etc. The temperature may be, for example, 20°C to 30°C. The relative humidity may be, for example, 60% to 90%. The photoperiod may be a 14-hour light period and a 10-hour dark period.

[0027] In the above (3), seeds are harvested from the rice grown in the above (2). The seed harvesting method can be a conventional method used by those skilled in the art to harvest seeds from rice.

[0028] The number of generations in which the above steps (1) to (3) are repeated may be two or more generations, counting the first steps (1) to (3) as generation 0 (current generation), and may be three or more generations, four or more generations, or five or more generations from the viewpoint of producing rice seeds having enhanced resistance to bacterial grain rot. The plant endophytic bacteria to be inoculated, the target to be inoculated, and the method may be different for each generation, or may be the same for all generations.

[0029] The rice seeds having resistance to bacterial grain rot can be determined by a method commonly used by those skilled in the art. For example, it can be determined by the method described in the Examples below. More specifically, for example, when rice seeds that are known not to have resistance to bacterial grain rot are inoculated with the rice bacterial grain rot fungus at a cell concentration of OD600nm=0.004 to infect the rice seeds, the rice seeds are determined to have resistance to bacterial grain rot if the plant height of the rice plants that have undergone the same treatment is significantly longer than that of rice plants grown from the rice seeds (for example, rice plants 7 days after sowing the rice seeds). Significantly longer may be a plant height that is 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, or 400% or more compared to that of the control rice.

[0030] The production method according to the present embodiment may further include a step of screening rice seeds having resistance to bacterial grain rot (screening step). The screening step can be carried out by appropriately evaluating resistance to bacterial grain rot. Such a method may be, for example, the above-mentioned method of determining that the rice seeds are resistant to bacterial grain rot.

[0031] [Rice seeds] The rice seeds according to the present embodiment are produced by the rice seed production method according to the present embodiment. Since the rice seeds according to the present embodiment are produced by the production method according to the present embodiment, the rice seeds have resistance to rice bacterial grain rot. EXAMPLES

[0032] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0033] [Test Example 1: Resistance test against rice bacterial grain rot 1] The surface of rice seeds (variety: Nipponbare) was sterilized with a sodium hypochlorite solution for 10 minutes, and 12 of the seeds were inoculated under reduced pressure with 10 mL of culture solution (OD600nm=0.04) of each test strain (RSB1, RSB10, RSB15, RSB2, RSB2+RSB15, NB6, BB1) shown in Table 1. The 0th generation (T0) seeds (12 grains each) inoculated with each test strain were sown in Bonsol No. 1 soil (Sumitomo Chemical Co., Ltd.) and grown under natural light. Next, the first generation (T1) seeds harvested from the grown rice were inoculated with 1 mL of B. glume culture solution (OD600nm=0.0004 or 0.004) under reduced pressure, and the inoculated seeds (10 grains each) were sown on 1 / 2 Murashige-Skoog medium (0.3% Gelrite) and grown under a repeated environment of 14 hours light (28°C) and 10 hours dark (24°C). The height of the rice plants was measured 7 days after sowing, and the average and standard deviation (n=10) were calculated. In addition, the height of rice plants grown from the T1 seeds was also measured as a negative control, except that distilled water (DW) was used instead of the test strain. The results are shown in Table 1. In the table, for example, "T1-RSB1" indicates the T1 seeds harvested from the T0 rice plants inoculated with RSB1 strain.

[0034] [Table 1]

[0035] As shown in Table 1, the rice plants grown from T1-DW, the negative control, had shorter plant heights and their growth was suppressed by inoculation with B. glume. In addition, the plant heights of the rice plants grown from T1 seeds inoculated with the RSB1 or RSB10 strain at T0 (T1-RSB1 or T1-RSB10) were not significantly different from those of the rice plants grown from T1-DW. In other words, even if rice seeds were inoculated with the RSB1 or RSB10 strain, the next generation of rice did not exhibit resistance to bacterial grain rot.

[0036] In addition, when rice seeds were inoculated with B. glumae at a low concentration (OD600nm=0.0004), the plant height of rice plants grown from T1 seeds inoculated with RSB15, RSB2, or RSB2+RSB15 at T0 was longer than that of rice plants grown from T1-DW. The plant height of rice plants grown from T1 seeds inoculated with NB6 or BB1 at T0 was significantly longer than that of rice plants grown from T1-DW. However, when rice seeds were inoculated with B. glumae at a high concentration (OD600nm=0.004), the plant height of rice plants grown from T1-RSB15, T1-RSB2, T1-RSB2+RSB15, or T1-NB6 was not significantly longer than that of rice plants grown from T1-DW. These results indicate that when rice seeds are inoculated with strains RSB15, RSB2, or NB6 at T0, the T1 rice plants acquire partial resistance to bacterial grain rot.

[0037] Even when rice seeds were inoculated with B. glumae at a high concentration, the height of the rice plants grown from T1-BB1 was significantly longer than that of the rice plants grown from T1-DW. This indicates that the seeds (first generation) harvested from individuals inoculated with the BB1 ​​strain and cultivated acquire resistance to bacterial grain rot.

[0038] [Test Example 2: Resistance test against rice bacterial grain rot 2] The same kind of test strain culture solution as T0 was inoculated under reduced pressure to T1-RSB1, T1-RSB10, T1-RSB15, T1-RSB2, or T1-RSB2+RSB15 obtained in Test Example 1. Then, the T1 seeds inoculated with each test strain were sown in soil and grown in the same manner as above. Then, T2 seeds were harvested from the grown T1 rice plants. Next, this operation was repeated for another generation to obtain T3 seeds. The T3 seeds were inoculated with B. glume culture solution in the same manner and then sown on 1 / 2 Murashige-Skoog medium (0.3% Gelrite), and the plant height was measured 7 days after sowing, and the average value and standard deviation (n=10) were calculated. The NB6 strain culture solution was inoculated to T1-NB6 in the same manner as above. Then, the T1 seeds inoculated with each test strain were sown in soil and grown in the same manner as above. Then, T2 seeds were harvested from the grown T1 rice plants. The T2 seeds were inoculated with B. glume culture solution and then sown on 1 / 2 Murashige-Skoog medium (0.3% Gelrite) in the same manner as above. The height of the rice plants was measured on the 7th day, and the average and standard deviation (n=10) were calculated. In addition, the height of the rice plants grown from the T3 seeds was measured as a negative control in the same manner, except that distilled water (DW) was used instead of the test strains. The results are shown in Table 2.

[0039] [Table 2]

[0040] As shown in Table 2, the plant height of rice plants grown from T3-RSB1 or T3-RSB10 was not significantly different from that of rice plants grown from T3-DW. In other words, even after rice seeds were inoculated with the RSB1 or RSB10 strain for three generations, the T3 rice plants did not exhibit resistance to bacterial grain rot.

[0041] On the other hand, when rice seeds were inoculated with B. glumae at low or high concentrations, the plant height of rice plants grown from T3-RSB15, T3-RSB2, T3-RSB2+RSB15, or T2-NB6 was significantly longer than that of rice plants grown from T3-DW. In other words, when rice seeds were inoculated with RSB15, RSB2, or NB6 for three generations, T3 rice plants acquired resistance to bacterial grain rot.

[0042] T1 rice plants were partially resistant to bacterial grain rot, whereas T3 rice plants were completely resistant to bacterial grain rot. These results indicate that resistance to bacterial grain rot induced by inoculation with RSB15, RSB2, and NB6 strains is enhanced by repeated inoculation of the test strains over three generations. In addition, since T1-BB1 also acquired resistance to bacterial grain rot, it is believed that resistance to bacterial grain rot induced by inoculation with the BB1 ​​strain is also enhanced by repeated inoculation of the strain over generations.

[0043] In a known document (JP Patent Publication No. 2019-142847), it was confirmed that when rice seeds were inoculated with B. glume and the RSB2 strain at the same time, the rice grown from the T0 rice seeds did not show resistance to rice bacterial grain rot. On the other hand, as shown in Test Example 2, by repeating the inoculation with the RSB2 strain for three generations, the next generation rice seeds showed resistance to rice bacterial grain rot even without inoculation with the RSB2 strain. This was a surprising result, suggesting that "repeated inoculation with the above specific strain for three generations" contributed to the acquisition of resistance to rice bacterial grain rot in the seeds themselves. Since resistance to rice bacterial grain rot was strengthened by repeated inoculation between generations, it is considered that rice can acquire resistance to rice bacterial grain rot by repeating at least two generations.

Claims

1. A method for producing rice seeds, comprising the steps of: (1) inoculating a rice seed or the inside of a plant body with an endophytic bacterium; (2) after (1), growing the rice seed or plant body; and (3) collecting seeds from the rice plant grown according to (2) above, for two or more generations; The method for producing rice seeds, wherein the plant endophytic bacterium comprises at least one selected from the group consisting of Pseudomonas putida RSB15 strain (Accession Number: NITE P-02616), Stenotrophomonas maltophilia RSB2 strain (Accession Number: NITE P-02617), Burkholderia gladioli NB6 strain (Accession Number: NITE P-03565), and Pantoea dispersa BB1 strain (Accession Number: NITE P-03365).

2. The method of claim 1 , wherein the rice seed or plant is a rice seed.

3. Rice seeds produced by the method according to claim 1 or 2.

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