Method for improving the emergence rate of rice by direct sowing under water

Abstract

The application discloses a method for improving the seedling rate of rice in waterlogging direct seeding, and utilizes Camellia sinensis endophytic actinomycete to treat rice seeds, wherein the Camellia sinensis endophytic actinomycete is Streptomyces sp. CoT10, and the strain preservation number is CGMCC No. 22968. In the method, the Camellia sinensis endophytic actinomycete Streptomyces sp. CoT10 can improve the enzyme activity of the antioxidant enzyme system (peroxide dismutase SOD, catalase CAT and antioxidant enzyme POD) of waterlogging rice, reduce the content of malondialdehyde, a membrane lipid peroxidation product, in the waterlogging rice plant, improve the waterlogging stress resistance of the rice, improve the seedling rate of the rice in waterlogging direct seeding, improve the chlorophyll content, plant height, root length and fresh and dry weight of the aboveground part and underground part of the rice under the condition of waterlogging direct seeding, and finally reduce the yield reduction loss of the rice caused by waterlogging.

Description

Technical Field

[0001] This invention belongs to the field of rice direct seeding technology, specifically relating to a method for improving the seedling survival rate of rice direct seeding. Background Technology

[0002] Direct seeding of rice has become a low-cost, high-efficiency planting technique widely used by farmers in recent years. Compared to traditional rice cultivation techniques, direct seeding saves labor, reduces labor costs, and alleviates the seasonal shortage of labor. In direct seeding, not only are birds and rodents prevented from consuming the rice seeds, but the growth of weeds in the field is also suppressed, thus reducing the use of chemical herbicides and improving food safety. However, direct seeding results in the rice seeds being submerged in water. Under these conditions, soil aeration is poor, and the levels of toxic reducing substances increase significantly. The rice seeds are subjected to adverse stresses such as flooding and lack of oxygen, making them highly susceptible to root rot, bud rot, and seedling rot. This leads to low seedling survival rates and reduced yields, severely hindering the application and development of direct seeding technology. Therefore, improving rice seedling survival rates and yields under direct seeding conditions has become an urgent problem to be solved.

[0003] To date, there have been no reports on using endophytic actinomycetes to improve the water stress tolerance of rice. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method to improve the seedling survival rate of rice seedlings directly after flooding.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0006] A method for improving the seedling survival rate of rice seedlings after direct seeding in flooded fields, wherein the method involves treating rice seeds with endophytic actinomycetes from Camellia oleifera; the endophytic actinomycetes from Camellia oleifera is Streptomyces sp. CoT10, and its strain preservation number is CGMCCNo.22968.

[0007] A further improvement to the above method is that the endophytic actinomycetes from Camellia oleifera treat rice seeds in the form of a spore suspension; the concentration of the Camellia oleifera endophytic actinomycete spore suspension is 1×10⁻⁶. 8 CFU / mL ~ 2×10 8 CFU / mL.

[0008] A further improvement to the above method is that the concentration of the Camellia oleifera endophytic actinomycete spore suspension is 1.5 × 10⁻⁶. 8 CFU / mL.

[0009] The above method is further improved by using a suspension of endophytic actinomycete spores from Camellia oleifera to treat rice seeds, including the following steps: mixing the suspension of endophytic actinomycete spores from Camellia oleifera with rice seeds, so that the endophytic actinomycetes from Camellia oleifera are embedded in the rice seeds, thus completing the embedding treatment of the rice seeds.

[0010] In a further improvement to the above method, the ratio of rice seeds to the suspension of Camellia oleifera endophytic actinomycete spores is 1g:51μL.

[0011] In a further improvement to the above method, an adhesive is added to the Camellia oleifera endophytic actinomycete spore suspension; the ratio of rice seeds to adhesive is 1g:463μL.

[0012] In a further improvement to the above method, the adhesive is a xanthan gum solution with a volume percentage concentration of 0.3%.

[0013] The above method is further improved by including the following treatment before the rice seeds are used: disinfecting the rice seeds.

[0014] In a further improvement to the above method, the rice seed is indica rice; the indica rice is one of the following: 9311, Zhongzao 35, Longkezao 1, Longkezao 2, Zhuliangyou 819, Lingliangyou 268, Fengliangyou 4, and Weiliangyou 7148.

[0015] The above method, in a further improvement, includes the following steps after the treatment is completed: drying the treated rice seeds and adding water to promote germination; and culturing the germinated rice seeds under water-flooded conditions.

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] (1) This invention provides a method to improve the seedling survival rate of rice seedlings in flooded direct seeding, by treating rice seeds with endophytic actinomycetes from Camellia oleifera, wherein the endophytic actinomycetes from Camellia oleifera is Streptomyces sp. CoT10. In this invention, the endophytic actinomycete *Streptomyces* sp. CoT10 from *Camellia oleifera* is used. It can increase the enzyme activity of the antioxidant enzyme system (superoxide dismutase SOD, catalase CAT, and antioxidant enzyme POD) in flooded rice, thereby reducing the content of malondialdehyde, a product of lipid peroxidation in the membranes of flooded rice plants. This inhibits lipid peroxidation in the membranes of flooded rice plants, slows down the oxidation rate, and thus helps improve the rice's tolerance to flooding stress. At the same time, under flooded direct seeding conditions, it can improve the rice's tolerance to adverse stresses such as flooding and hypoxia, maintain growth, and significantly increase the seedling rate of rice in flooded direct seeding. Furthermore, treating rice seeds with *Streptomyces oleifera* can also increase the chlorophyll content, plant height, root length, and fresh and dry weight of the above-ground and underground parts of flooded rice, ultimately effectively reducing the yield loss of rice caused by flooding. The method of this invention can effectively improve the seedling rate of direct-seeded rice, thereby ensuring "full seedling emergence" of direct-seeded rice, which is conducive to high and stable yield of direct-seeded rice. Moreover, it can solve problems such as bird damage, rodent damage, weed damage, and seedling burn caused by local high temperature and waterlogging in direct-seeded rice, and provide theoretical basis and technical reference for the large-scale promotion and application of direct-seeded rice.

[0018] (2) The method of the present invention uses Camellia oleifera endophytic actinomycetes to encapsulate rice seeds when the rice is still in the seed stage. The exogenous application of Camellia oleifera endophytic actinomycetes will not cause rice diseases when it re-establishes a symbiotic relationship with the host, ensuring that it can play a role in the early stage of rice growth.

[0019] (3) The method of the present invention uses Streptomyces sp. CoT10, an endophytic actinomycete from Camellia oleifera, which is isolated from the plant and is safer for the environment than chemical substances, without affecting the agricultural ecological environment or human health. In addition, the treatment of endophytic actinomycetes from Camellia oleifera is more convenient, simple and diverse. Compared with other methods, it is both time-saving and labor-saving, and is not limited by region or manpower, making it suitable for widespread promotion. Attached Figure Description

[0020] Figure 1 This is a photograph of the rice growth after the rice seeds were encapsulated with Streptomyces sp. CoT10 and then submerged in water, as shown in Example 1 of this invention.

[0021] Figure 2 This is a comparative chart of data from SPAD treatment of rice seeds after being encapsulated with Streptomyces sp. CoT10 in Example 1 of the present invention.

[0022] Figure 3This is a comparative chart showing the plant height of rice plants after being flooded following the embedding of rice seeds with Streptomyces sp. CoT10 in Example 1 of this invention.

[0023] Figure 4 This is a comparative chart showing the root length of rice plants after being flooded following the embedding of Streptomyces sp. CoT10 in rice seeds in Example 1 of this invention.

[0024] Figure 5 This is a comparative chart showing the data of fresh weight of aboveground parts of rice after being flooded following the embedding of rice seeds with Streptomyces sp. CoT10 in Example 1 of the present invention.

[0025] Figure 6 This is a comparative chart showing the data of fresh weight of the underground parts of rice after being flooded following the embedding of rice seeds with Streptomyces sp. CoT10 in Example 1 of the present invention.

[0026] Figure 7 This is a comparative chart showing the aboveground dry weight of rice seedlings after being encapsulated in Streptomyces sp. CoT10 and then flooded in Example 1 of this invention.

[0027] Figure 8 This is a comparative chart showing the data of dry weight of the underground part of rice after water flooding following rice seed encapsulation with Streptomyces sp. CoT10 in Example 1 of the present invention.

[0028] Figure 9 This is a graph showing the change in malondialdehyde content after rice seeds were wrapped with Streptomyces sp. CoT10 in Example 2 of the present invention.

[0029] Figure 10 This is a graph showing the change in superoxide dismutase (SOD) activity after rice seeds were encapsulated with Streptomyces sp. CoT10 in Example 2 of the present invention.

[0030] Figure 11 This is a graph showing the change in catalase (CAT) activity after rice seeds were encapsulated with Streptomyces sp. CoT10 in Example 2 of the present invention.

[0031] Figure 12 This is a graph showing the change in the activity of the antioxidant enzyme POD after rice seeds were encapsulated with Streptomyces sp. CoT10 in Example 2 of the present invention.

[0032] Figure 13 This is a photograph of the growth of rice seedlings after being encapsulated with Streptomyces sp. CoT10 and then flooded in a field environment, as shown in Example 3 of this invention.

[0033] Figure 14 This is a photograph of the rice plants in Comparative Example 1 of this invention, showing the growth of rice after being submerged following the embedding of Streptomyces hygroscopicus OsiSh-2 in the rice seeds. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0035] In the following examples, all materials and instruments used are commercially available.

[0036] Example 1:

[0037] A method for improving the seedling emergence rate of rice seedlings after direct seeding in flooded areas according to the present invention includes the following steps:

[0038] (1) Preparation of spore suspension using *Streptomyces sp. CoT10*, an endophytic actinomycete from Camellia oleifera:

[0039] (1.1) Inoculate the endophytic actinomycete strain (Streptomyces sp. CoT10) of Camellia oleifera onto ISP-2 solid medium and incubate statically in a constant temperature incubator at 30℃ for 10-14 days.

[0040] (1.2) Collect the mature cells from step (1.1) with an inoculation loop, transfer them to an Erlenmeyer flask containing sterile distilled water and glass beads, shake vigorously and filter to obtain a spore suspension.

[0041] (1.3) Count the spores in the suspension obtained in step (1.2) using a hemocytometer, and adjust its concentration to 1.5 × 10⁻⁶. 8 CFU / mL.

[0042] The endophytic actinomycete from Camellia oleifera used in this invention is Streptomyces sp. CoT10, with the strain preservation number CGMCC No. 22968, the preservation date being July 28, 2021, and it is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0043] (2) Following the ratio of rice seed / xanthan gum solution / spore suspension = 1g / 463μL / 51μL, with a volume percentage concentration of 0.3% for the xanthan gum solution, the spore suspension and xanthan gum solution obtained in step (1.3) were used to embed the sterilized rice seeds. Specifically, the spore suspension and xanthan gum solution were mixed to obtain a mixture, which was then evenly coated onto the surface of the sterilized rice seeds using a sterilized pipette tip. After embedding, the seeds were dried overnight, and an appropriate amount of sterile water was added to promote germination. Once the seeds showed white sprouts, they were transplanted into rice soil cultivation boxes. After 1 day, the seeds were submerged in water. After 21 days, the seedling growth was observed, and their agronomic traits were measured. The treatment group was designated CoT10, and each treatment group was repeated three times, with each group containing 35 rice seedlings. This invention uses xanthan gum as an adhesive, which can improve the ability of the spore suspension to adhere to rice seeds.

[0044] Control group (CK): Sterile water was used to replace the spore suspension, and all other conditions were the same.

[0045] Experimental conditions: Rice was grown in soil in a constant temperature and light incubator at room temperature, with a photoperiod of 16 hours of light / 8 hours of darkness, an incubation temperature of 28-30℃, and an air humidity of 60-70%.

[0046] Experimental crop: Rice (Indica rice - Longkezao No. 1)

[0047] The effects of embedding *Streptomyces* sp. CoT10 in rice seeds in a laboratory environment on changes in SPAD chlorophyll content, plant height, root length, and fresh-dry weight of aboveground and underground parts of rice under flooding treatment were investigated. The measurement results are as follows: Figures 1 to 8 As shown. Combined with Figures 1 to 8 It can be seen that, compared with untreated rice, the present invention, by embedding a suspension of *Streptomyces sp. CoT10* spores on the surface of rice seeds and then culturing them after flooding, increased the SPAD, plant height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of rice by 7.51%, 30.48%, 112.47%, 69.06%, 182.67%, 150.00%, and 210.00%, respectively. These data indicate that the method of the present invention, under laboratory conditions, can effectively improve the SPAD, plant height, root length, and aboveground and underground fresh and dry weights of flooded rice.

[0048] Example 2:

[0049] A method for improving the seedling emergence rate of rice seedlings after direct seeding in flooded areas according to the present invention includes the following steps:

[0050] A spore suspension of *Streptomyces sp. CoT10*, an endophytic actinomycete from *Camellia oleifera*, was prepared with a concentration of 1.5 × 10⁻⁶. 8The concentration of CFU / mL was the same as in Example 1. Rice seeds / xanthan gum solution / spore suspension were prepared at a ratio of 1g / 463μL / 51μL, with a xanthan gum solution volume percentage of 0.3%. Sterilized rice seeds were encapsulated using the spore suspension and xanthan gum solution. After encapsulation, the seeds were dried overnight and then germinated with an appropriate amount of sterile water. Once germination showed signs of sprouting, the seeds were transferred to 50mL centrifuge tubes and immediately submerged. Samples were taken from 6 to 168 hours to measure malondialdehyde (MDA) content and antioxidant enzyme activity in the rice. Each treatment group was designated T10, and each group was repeated five times, with each group containing 10 rice seedlings.

[0051] Control group (CK): Sterile water was used to replace the spore suspension, and all other conditions were the same.

[0052] Experimental conditions: Rice was hydroponically grown in 50mL centrifuge tubes in a room temperature constant temperature and light incubator with a photoperiod of 16 hours of light / 8 hours of darkness, an incubation temperature of 28-30℃, and an air humidity of 60-70%.

[0053] Experimental crop: Rice (Indica rice-9311)

[0054] The effects of encapsulating *Streptomyces* sp. CoT10 in rice seeds under laboratory conditions on the enzyme activities of the antioxidant enzyme system (superoxide dismutase SOD, catalase CAT, and antioxidant enzyme POD) in flooded rice, as well as the content of malondialdehyde (MDA), a lipid peroxidation product in the membranes of flooded rice plants, were investigated. The measurement results are as follows: Figures 9-12 As shown. Combined with Figures 9-12 It is evident that, in this invention, by embedding a suspension of *Streptomyces p. CoT10* spores on the surface of rice seeds and then culturing them after flooding, the content of malondialdehyde (MDA), a lipid peroxidation product of rice plant membranes, decreased by 40.12% after 36 hours; the enzyme activities of superoxide dismutase (SOD), catalase (CAT), and antioxidant enzyme (POD) increased by 39.08%, 104.43%, and 8.27%, respectively. These data indicate that the method of this invention can improve the enzyme activities of the antioxidant enzyme system (SOD, CAT, and POD) in flooded rice and reduce the content of MDA, a lipid peroxidation product of flooded rice plants.

[0055] Example 3:

[0056] A method for improving the seedling emergence rate of rice seedlings after direct seeding in flooded areas according to the present invention includes the following steps:

[0057] A spore suspension of *Streptomyces sp. CoT10*, an endophytic actinomycete from *Camellia oleifera*, was prepared with a concentration of 1.5 × 10⁻⁶. 8The concentration of CFU / mL was the same as in Example 1. Rice seeds were encapsulated in a spore suspension with xanthan gum solution at a ratio of 1g / 463μL / 51μL (xanthan gum solution volume percentage was 0.3%). After encapsulation, the seeds were dried overnight and then germinated with sterile water. Once the seeds showed white sprouts, they were transplanted into rice soil culture boxes. After 1 day, the seeds were flooded. Seedling emergence was observed after 21 days, and the seedling rate was calculated. The treatment group was designated T10. Under laboratory conditions, each treatment group was repeated three times, with each group containing 35 rice seedlings. Under field conditions, each treatment group was repeated three times, with each group containing 200 rice seedlings. Seedlings were sown in the field after germination and flooded after 1 day. Seedling emergence was observed after 21 days, and the seedling rate was calculated.

[0058] Control group (CK): Sterile water was used to replace the spore suspension, and all other conditions were the same.

[0059] Experimental conditions: Rice was grown in soil in a constant temperature and light incubator at room temperature, with a photoperiod of 16 hours of light / 8 hours of darkness, an incubation temperature of 28-30℃, and an air humidity of 60-70%; in the field (sown on June 29, 2023, flooded on June 30, and seedling rate counted on July 21).

[0060] Experimental crops: Rice (Indica rice - Zhongzao 35, Longkezao 1, Longkezao 2, Zhuliangyou 819, Lingliangyou 268, 9311, Fengliangyou 4, Weiliangyou 7148)

[0061] The effects of encapsulating rice seeds with Streptomyces sp. CoT10 followed by flooding on rice emergence rate were investigated in laboratory and field environments. The experimental results are as follows: Figure 13 As shown in Table 1.

[0062] Table 1 Seedling rate of different rice varieties under field and laboratory conditions

[0063]

[0064] Combination Figure 13As shown in Table 1, in this invention, by embedding a suspension of *Streptomyces p. CoT10* spores on the surface of rice seeds, the seedling rate of rice seedlings under flooded direct seeding conditions can be improved under laboratory conditions. Specifically, the seedling rates of Zhongzao 35, Longkezao 1, Longkezao 2, Zhuliangyou 819, Lingliangyou 268, 9311, Fengliangyou 4, and Weiliangyou 7148 were increased by 17.14%, 57.1%, and 10.1%, respectively. 3%, 48.58%, 31.43%, 31.43%, 22.87%, 17.14%, and 34.28%; under field conditions, it can improve the seedling rate of rice when directly sown in flooded areas. Among them, the seedling rates of Longkezao 1, Longkezao 2, Zhuliangyou 819, Lingliangyou 268, 9311, and Fengliangyou 4 were increased by 11.00%, 10.17%, 7.50%, 0.67%, 6.84%, and 6.17%, respectively. Therefore, the method of the present invention, which uses endophytic actinomycetes from Camellia oleifera (Streptomycessp.CoT10) to treat rice seeds, can effectively improve the seedling rate of direct-seeded rice, thus ensuring "full seedling emergence after sowing". This is conducive to high and stable yields of direct-seeded rice, and can also solve problems such as bird damage, rodent damage, weed damage, and seedling burn caused by localized high temperature and waterlogging in direct-seeded rice. It provides a theoretical basis and technical reference for the large-scale promotion and application of direct-seeded rice.

[0065] Comparative Example 1:

[0066] A method for treating rice seeds using rice endophytic actinomycetes (Streptomyces hygroscopicus OsiSh-2) includes the following steps:

[0067] A spore suspension of the rice endophytic actinomycete *Streptomyces hygroscopicus* OsiSh-2 was prepared at a concentration of 1.5 × 10⁻⁶. 8 The concentration of CFU / mL was the same as in Example 1. Rice seeds were encapsulated in a xanthan gum solution at a ratio of 1g / 463μL / 51μL (xanthan gum solution volume percentage was 0.3%). After encapsulation, the seeds were dried overnight and then germinated with sterile water. Once the seeds showed signs of germination, they were transplanted into rice soil culture boxes. After 1 day, the seeds were submerged. Seedling emergence was observed after 21 days, and the seedling rate was calculated. The treatment group was designated OsiSh-2. Each treatment group was repeated three times under laboratory conditions, with each group containing 35 rice seedlings. Seedling emergence was observed after 21 days, and the seedling rate was calculated.

[0068] Control group (CK): Sterile water was used to replace the spore suspension, and all other conditions were the same.

[0069] Experimental conditions: Rice was grown in soil in a constant temperature and light incubator at room temperature, with a photoperiod of 16 hours of light / 8 hours of darkness, an incubation temperature of 28-30℃, and an air humidity of 60-70%.

[0070] Experimental crop: Rice (Indica rice-9311)

[0071] The effect of rice seed burial with Streptomyces hygroscopicus OsiSh-2 followed by flooding on rice emergence rate in a laboratory environment was investigated. The experimental results are as follows: Figure 14 As shown in Table 2.

[0072] Table 2. Seedling rate of rice seeds treated with Streptomyces hygroscopicus OsiSh-2 under laboratory conditions.

[0073] Seedling survival rate CK 42.85％ OsiSh-2 34.29％

[0074] In Comparative Example 1, encapsulating rice seeds with a suspension of endophytic actinomycetes (Streptomyces hygroscopicus OsiSh-2) spores and culturing them after flooding did not improve the seedling rate of rice seedlings directly sown in flooded conditions under laboratory conditions. Compared to the control group (CK), the seedling rate of the OsiSh-2 treatment group decreased by 8.56%. These results indicate that not all endophytic actinomycetes can improve the seedling rate of rice seedlings directly sown in flooded conditions; only treatment with *Streptomyces sp. CoT10* can effectively improve the seedling rate of directly sown rice.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for improving the emergence rate of rice seedlings in waterlogged direct seeding, characterized by, The method is to treat rice seeds by using the endophytic actinomycete of Camellia oleifera Abel. Streptomyces sp . CoT10, the strain preservation number of which is CGMCC No. 22968; the endophytic actinomycete of Camellia oleifera Abel. is in the form of spore suspension to treat rice seeds, including the following steps: mixing the endophytic actinomycete of Camellia oleifera Abel. spore suspension and rice seeds, embedding the endophytic actinomycete of Camellia oleifera Abel. on the rice seeds, and completing the embedding treatment of the rice seeds; the concentration of the endophytic actinomycete of Camellia oleifera Abel. spore suspension is 1.5x10 8 CFU / mL.

2. The method for improving the emergence and seedling rate of waterlogged direct-seeded rice according to claim 1, characterized in that, The ratio of the rice seeds to the spore suspension of the Actinoplanes camponotus is 1g:51μL.

3. The method for improving the emergence and seedling rate of water-seeded rice according to claim 2, wherein An adhesive is further added in the spore suspension of the Actinoplanes camponotus; the ratio of the rice seeds to the adhesive is 1g:463μL.

4. The method for improving the emergence and seedling rate of water-seeded rice according to claim 3, wherein The adhesive is a xanthan gum solution, and the volume percentage concentration of the xanthan gum solution is 0.3%.

5. The method for improving the emergence and seedling rate of water-seeded rice according to any one of claims 1 to 4, wherein The rice seeds further include the following treatment before use: sterilizing the rice seeds.

6. The method for improving the emergence and seedling rate of water-seeded rice according to claim 5, wherein The rice seeds are indica rice; the indica rice is one of 9311, Zhongzao 35, Longkezao No.1, Longkezao No.2, Zhuliangyou 819, Lingliangyou 268, Fengliangyou No.4, and Weiliangyou 7148.

7. The method for improving the emergence and seedling rate of water-logged direct-seeded rice according to any one of claims 1 to 4, wherein The method further includes the following steps after the treatment: drying the treated rice seeds, adding water to germinate the seeds, and culturing the germinated rice seeds under waterlogging conditions.

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