A rhizobium bacterium and its application in promoting plant growth and increasing fruit sugar content

Abstract

The present invention relates to a Rhizobium bacterium and its application in promoting plant growth and increasing the sugar content of fruit. The Rhizobium bacterium is specifically tropical rhizobium (Rhizobium sp.) Rhiz_8E1, which was deposited in the China Center for Type Culture Collection on December 3, 2024, with a deposit number of CCTCC M 20242702. Experiments have shown that applying Rhiz_8E1 to the roots of citrus and tomatoes can promote plant growth and increase the sugar content of fruit, mainly manifested in significantly promoting the increase in plant height and increasing the content of soluble solids, sucrose, glucose and fructose in plant fruits, thereby improving fruit quality. The present invention has important application value.

Description

Technical Field

[0001] The present invention relates to the field of methods for promoting plant growth and increasing the sugar content of plant fruits, and particularly to a Rhizobium bacterium Rhiz_8E1 and its application in promoting plant growth and increasing the sugar content of fruit. Background Art

[0002] Fruit sugar content is a key factor in determining fruit flavor and quality. Its concentration and ratio have a direct impact on fruit sweetness, making it a crucial indicator for consumers to evaluate fruit quality. During fruit ripening, the accumulation and conversion of sugars underpin the formation of a fruit's distinctive flavor, with sweetness being the most intuitive sensory attribute experienced by consumers. Therefore, increasing fruit sugar content not only enhances fruit sweetness and improves its taste, but also significantly improves the consumer experience, thereby enhancing the market competitiveness of horticultural products. In the genetic improvement of modern horticultural crops, cultivating high-quality horticultural crops through genetic engineering and traditional breeding methods has been a hot topic. While these methods can improve fruit quality to a certain extent, they are often time-consuming, labor-intensive, and inefficient. Therefore, the exploration and discovery of microbial resources that can increase fruit sugar content is attracting increasing attention as a potential solution. Microbial products can promote sugar accumulation in fruit through interactions between microbes and plants, thereby improving fruit quality without altering plant genes. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide Rhizobium bacteria Rhiz_8E1 and its application in promoting plant growth and increasing fruit sugar content in response to the above shortcomings.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] A Rhizobium bacterium, wherein the Rhizobium bacterium is tropical rhizobium (Rhizobium sp.) Rhiz_8E1, which was deposited in the China Center for Type Culture Collection on December 3, 2024, with a deposit number of CCTCC M 20242702.

[0006] The application of the Rhizobium bacteria Rhiz_8E1 in promoting plant growth and increasing the sugar content of fruits.

[0007] Furthermore, the application method specifically comprises treating plant roots with a bacterial agent containing the aforementioned Rhizobium bacteria.

[0008] Furthermore, the OD600nm value of the inoculum containing Rhizobium bacteria was 0.2.

[0009] The beneficial effects of the present invention are as follows: Experiments have shown that the Rhiz_8E1 strain provided by the present invention can promote the growth of citrus and tomatoes and increase the sugar content of their fruits, thereby improving the quality of the fruits. The present invention has important application value.

[0010] The present invention is described in detail below with reference to the accompanying drawings and implementation examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a growth comparison chart of citrus (red mandarin) seedlings treated with a fungus containing the strain Rhiz_8E1 and untreated control plants;

[0012] Figure 2 The statistical results of the plant height of citrus seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0013] Figure 3 The statistical results of the net photosynthetic rate of citrus seedling leaves after being treated with the inoculant containing the strain Rhiz_8E1;

[0014] Figure 4 The statistical results of root length of citrus seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0015] Figure 5 The statistical results of the root surface area of ​​citrus seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0016] Figure 6 This is a growth comparison chart of Micro-Tom tomato seedlings treated with the fungus containing the strain Rhiz_8E1 and untreated control plants;

[0017] Figure 7 The statistical results of the plant height of Micro-Tom tomato seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0018] Figure 8 This is the statistical result of fresh weight of Micro-Tom tomato seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0019] Figure 9 The dry weight statistics of Micro-Tom tomato seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0020] Figure 10 This is a comparison of 5-year-old fruiting potted Newhall citrus fruits treated with the inoculant containing the strain Rhiz_8E1 and the untreated control;

[0021] Figure 11The statistical results of sucrose (Suc), fructose (Fru), glucose (Glu), total sugar (SS) and soluble solids (TSS) in Newhall fruit after being treated with the inoculum containing the strain Rhiz_8E1;

[0022] Figure 12 This is a comparison of the fruiting period of Majiayou plants in the field treated with the fungus containing the strain Rhiz_8E1 and the untreated control plants;

[0023] Figure 13 The statistical results of the solidification of pomelo fruit in the field after being treated with the inoculant containing the strain Rhiz_8E1;

[0024] Figure 14 This is a comparison of the fruiting period of Micro-Tom tomatoes treated with the fungus containing the strain Rhiz_8E1 and the untreated control plants;

[0025] Figure 15 This is a comparison of the fruiting period of Micro-Tom tomatoes treated with the fungus containing the strain Rhiz_8E1 and the untreated control plants. DETAILED DESCRIPTION

[0026] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0027] LB liquid medium: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride in 1 L of distilled water, then sterilize at 121°C for 15 min and cool before use.

[0028] LB solid medium: Add agar to LB liquid medium to a concentration of 15 g / L; sterilize at 121°C for 15 minutes. Cool the liquid medium to approximately 55°C into a Petri dish and allow to cool naturally.

[0029] Citrus rootstock red mandarin (Citrus reticulata Blanco, cv. Hongju) is recorded in the following literature: Sun Yi, Hu Yibo, Ye Junli, et al. Analysis of genetic diversity of hybrid offspring of red mandarin × trifoliate orange [J]. Journal of Huazhong Agricultural University, 2023, 42(4): 150-157. Citrus rootstock red mandarin (Citrus reticulata Blanco, cv. Hongju) is referred to as red mandarin in the following text.

[0030] Five-year-old potted seedlings with Newhall navel orange (Citrus sinensis (L.) Osbeck 'Newhall') as scion and trifoliate orange (Poncirus trifoliata (L.) Raf.) as rootstock are described in the following literature: Chen C., Lyon MT, Malley D., Claire T., et al. Origin and frequency of 2n gametes in Citrus sinensis×Poncirus trifoliata and their reciprocal crosses. Plant Science. 2008, 1: 1-8. Five-year-old potted seedlings with Newhall navel orange (Citrus sinensis (L.) Osbeck 'Newhall') as scion and trifoliate orange (Poncirus trifoliata (L.) Raf.) as rootstock are hereinafter referred to as Newhall.

[0031] Citrus maxima (L.) Osbeck'Majiayou' is recorded in the following literature: Xu Chenyu, Tang Qizheng, Liu Huiyu, et al. Comprehensive evaluation of Majiayou fruit quality of 6 hybrid pollination combinations based on principal component analysis [J]. Journal of Fruit Science, 2024, 41(02): 282-293. Citrus maxima (L.) Osbeck'Majiayou' is referred to as Majiayou in the following text.

[0032] The wild-type tomato (Solanum lycopersicum) Micro-Tom is described in the following literature: Aoki, K., Yano, K., Suzuki, A. et al. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics. BMC Genomics 11, 210 (2010). The wild-type tomato (Solanum lycopersicum) Micro-Tom is hereinafter referred to as tomato.

[0033] (1) Isolation, identification and preservation of Rhizobium tropici Rhiz_8E1.

[0034] I. Isolation of Rhiz_8E1

[0035] 1. Add 5 g of rhizosphere soil sample (collected from the rhizosphere soil of healthy citrus trees with Poncirus trifoliata as rootstock and Newhall as scion in the orchard of Ganzhou Citrus Research Institute) to 45 mL of phosphate buffer, stir for 15 minutes, let it stand for 10 minutes, then take 1 mL of the supernatant and add it to a sterile test tube containing 9 mL of phosphate buffer and mix thoroughly (the dilution at this time is recorded as 10). -1 ), then draw 1 mL from the test tube and add it to another sterile test tube containing 9 mL of phosphate buffer and mix thoroughly. Repeat this process to make 10 -2 , 10 -3 , 10 -4 , 10 -5 Take 0.1 mL of bacterial suspension from each dilution gradient and evenly spread it on LB solid medium. Incubate at 28°C for 2-3 days.

[0036] 2. After completing step 1, pick a single colony from the LB solid medium and repeat purification three or more times. Name the selected bacterial strain Rhiz_8E1.

[0037] 2. Identification of Bacterial Rhiz_8E1

[0038] 16sRNA sequence homology analysis

[0039] The 16sRNA of bacterial Rhiz_8E1 was compared with the sequences in NCBI. The results showed that the bacterial Rhiz_8E1 had the highest homology with Rhizobium tropici, reaching 99.7%.

[0040] 3. Preservation

[0041] Based on the above 16sRNA sequence homology analysis results, the Rhiz_8E1 isolated and purified in step 1 was identified as Rhizobium.

[0042] Preservation of the strain: Inoculate a single colony of Rhiz_8E1 into LB liquid medium and culture at 28°C, 200 rpm, and shake for 8 hours to obtain a bacterial culture solution. Mix 1 volume of the bacterial culture solution with 1 volume of 60% (v / v) glycerol and store at -80°C.

[0043] (2) Preparation of microbial agents

[0044] 1. Activate the Rhiz_8E1 bacterial solution stored at -80°C in Example 1 on LB solid culture medium.

[0045] 2. Pick a single colony of LB solid medium coated with Rhiz_8E1 strain and inoculate it into a sterilized conical flask (specification: 250 mL) containing 100 mL LB liquid medium. Incubate at 28°C and 200 r / min for 8 h. The obtained bacterial liquid is centrifuged and the cells are dissolved in sterile water and the OD value is calculated. 600nm The value is adjusted to 0.2, and the bacterial solution is the prepared Rhiz_8E1 bacterial agent.

[0046] (3) Application of Rhiz_8E1 in promoting plant growth and increasing fruit sugar content

[0047] 1. Culture medium preparation:

[0048] Specifications of culture dish: 10cm×10cm

[0049] MS solid medium: Dissolve 4.46 g of MS minimal medium powder (Coolaber), 20 g of sucrose, and 3 g of plant gel in distilled water, then dilute to 1 L with distilled water. Adjust the pH to 5.8, sterilize at 121°C for 15 minutes, and use after cooling.

[0050] 2. Effects of Rhiz_8E1 on the growth and fruit sugar accumulation of citrus and Mic-Tom tomatoes The incubator culture conditions were: 22°C; 14h light / 10h dark; light intensity of 12000Lx.

[0051] Both the potted Newhall and the field pomelo were inoculated in open air locations.

[0052] 1. Take red tangerine seeds, treat them with 1 mol / L NaOH solution for 15 minutes, then wash them three times with sterile water, then treat them with 2.6% (v / v) sodium hypochlorite aqueous solution for 15 minutes, and then wash them three times with sterile water.

[0053] 2. After completing step 1, bury the tangerine seeds in moist sterilized vermiculite, cover the surface with a double layer of moistened sterilized gauze, and culture them in a 28°C constant temperature biochemical incubator in the dark. Transplant the seeds after they have grown two true leaves.

[0054] 3. Transplant the tangerine seedlings that have completed step 2 into pots filled with substrate soil, 3 plants per pot, and obtain 20 pots of tangerine seedlings, totaling 60 plants.

[0055] 4. Take Mic-Tom tomato seeds, treat them with 75% alcohol solution for 30 seconds, then wash them three times with sterile water, then treat them with 2.6% (v / v) sodium hypochlorite aqueous solution for 6 minutes, and finally wash them three times with sterile water.

[0056] 5. After completing step 4, sow the Mic-Tom tomatoes on MS solid culture medium. After vernalization at 4°C for two days, place them in a constant temperature incubator at 22°C, 14 hours of light / 10 hours of darkness, and a light intensity of 5000Lx until the tomato seedlings grow two cotyledons and then transplant them.

[0057] 6. Transplant the Mic-Tom tomato seedlings that have completed step 5 into pots filled with substrate soil, 3 plants per pot, and obtain 20 pots of Mic-Tom tomato seedlings, totaling 60 plants.

[0058] 7. Use Rhiz_8E1 strain OD 600nm Five pots of Mic-Tom tomatoes (three plants per pot), five pots of red mandarins (three plants per pot), five pots of fruiting Newhall navel oranges (one plant per pot), and ten Majia pomelo plants were inoculated with a 0.2 inoculum via root irrigation. Each red mandarin and Mic-Tom tomato seedling was inoculated with 5 mL of inoculum, each Newhall plant was inoculated with 3 L of inoculum, and each Majia pomelo plant was inoculated with 15 L of inoculum. The mandarins and tomatoes were inoculated once every two weeks for a total of three times, while the potted Newhall navel oranges and field-grown Majia pomelo plants were inoculated once a month for a total of three times. Five additional pots of Mic-Tom tomatoes, five red mandarins, five fruiting Newhall navel oranges, and ten field-grown Majia pomelo plants were used as controls. The red mandarins and Mic-Tom tomatoes were treated with 5 mL of water every two weeks, the Newhall navel oranges were treated with 3 L of water every month, and the Majia pomelo plants were treated with 15 L of water every month for a total of three inoculations.

[0059] 8. Observe the growth and development phenotypes of red orange and Mic-Tom tomatoes in each treatment group. Figure 1 、 Figure 6 , statistics of red orange plant height ( Figure 2 ), leaf net photosynthetic rate ( Figure 3 ), root length ( Figure 4 ) and root surface area ( Figure 5 ), calculate the height of Mic-Tom tomato plants ( Figure 7 )、Fresh weight( Figure 8 ) and dry weight ( Figure 9 ).

[0060] 9. Observe the growth of Newhall fruit in various treatments. Figure 10 , and test the fruit sucrose, fructose, glucose, and total sugar content. Figure 11 ; The growth of Majiayou fruit in the field is as follows Figure 12 , the fruit solid content is as follows Figure 13 ; Mic-Tom tomato fruit growth Figure 14 , and test the contents of sucrose, fructose, glucose, total sugar and soluble solids in its fruit. Figure 15 .

[0061] 10. The results showed that, compared with the control, Rhiz_8E1 treatment significantly promoted the growth of red mandarin orange plants, enhanced photosynthetic intensity, and promoted root growth. It also significantly promoted the growth of Mic-Tom tomato plants and increased their biomass. This shows that Rhiz_8E1 can promote plant growth.

[0062] 11. By Figure 11 、 Figure 13 and Figure 15 It can be seen that compared with the control, Rhiz_8E1 can promote sugar accumulation in Newhall navel orange, Majia pomelo and Mic-Tom tomato fruits.

[0063] The foregoing is an example of the best mode of carrying out the present invention. Any portion not described in detail herein is common knowledge within the skill of one of ordinary skill in the art. The scope of protection of the present invention shall be determined by the claims. Any equivalent transformation based on the technical teachings of the present invention shall also be within the scope of protection of the present invention.

Claims

1. A Rhizobium bacterium, characterized in that The Rhizobium bacteria are tropical Rhizobium ( Rhizobium tropicali ) Rhiz_8E1, deposited in the China Center for Type Culture Collection on December 3, 2024, with the deposit number CCTCCNo: M 20242702.

2. Use of the Rhizobium bacteria according to claim 1 in promoting the growth of citrus and tomatoes and increasing the sugar content of fruits.

3. The use according to claim 2, characterized in that The application method specifically comprises using the bacterial agent containing the Rhizobium bacteria according to claim 1 to treat the root systems of citrus and tomatoes by root irrigation.

4. The use according to claim 3, characterized in that OD of the inoculum containing Rhizobium bacteria 600 The value is 0.2.

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