Rhizobium bacterium and application thereof in promoting plant growth and increasing fruit sugar content

Abstract

The invention relates to a rhizobium bacterium and application of the rhizobium bacterium in promoting plant growth and increasing fruit sugar content, the rhizobium bacterium is specifically rhizobium tropicum (Rhiz8E1), the rhizobium bacterium is preserved in China Center for Type Culture Collection on December 3, 2024, and the preservation number is CCTCC M 20242702. Experiments prove that the plant growth can be promoted and the sugar content of fruits can be increased by applying the Rhiz8E1 to the root systems of the citrus and the tomatoes, which is mainly shown in that the plant height increase is obviously promoted, the contents of soluble solids, cane sugar, glucose, fructose and the like of the plant fruits are increased, and the fruit quality is improved. The method has an 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 in particular to a Rhizobium bacterium Rhiz_8E1 and an application thereof in promoting plant growth and increasing the sugar content of fruit. Background Art

[0002] As one of the key factors that determine the flavor and quality of fruits, the concentration and ratio of fruit sugar content have a direct impact on the sweetness of fruits, and thus become an important indicator for consumers to evaluate the quality of fruits. During the ripening process of fruits, the accumulation and transformation of sugars are the basis for the formation of the unique flavor of fruits, among which sweetness is the most intuitive attribute in the sensory experience of consumers. Therefore, increasing the sugar content of fruits can not only enhance the sweetness of fruits and improve their taste, but also significantly improve consumers' eating experience, thereby enhancing the market competitiveness of horticultural products. In the genetic improvement of modern horticultural crops, the cultivation of high-quality horticultural crops through genetic engineering and traditional breeding methods has always been a hot topic of research. Although these methods can improve the quality of fruits to a certain extent, they are often accompanied by problems such as time-consuming, laborious and inefficient. Therefore, exploring and discovering microbial resources that can increase the sugar content of fruits, as a potential solution, has attracted more and more attention from scientific researchers. Using microbial products, the accumulation of sugar in fruits can be promoted through the interaction between microorganisms and plants without changing plant genes, thereby improving the quality of fruits. 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 view of 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 above-mentioned 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 the bacterial agent 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 bacterial agent 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 treated with the bacterial agent containing the strain Rhiz_8E1;

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

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

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

[0019] Fig. 9 The statistical results of dry weight of Micro-Tom tomato seedlings after being treated with the inoculant containing the strain Rhiz_8E1;

[0020] Fig.10 This is a comparison of 5-year-old fruit-bearing potted Newhall citrus fruits treated with the bacterial agent containing the strain Rhiz_8E1 and the untreated control;

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

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

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

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

[0025] Fig.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 are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

[0027] LB liquid culture 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 use after cooling.

[0028] LB solid medium: Add agar to the LB liquid medium to a concentration of 15 g / L; then sterilize at 121°C for 15 min. Pour the liquid medium cooled to about 55°C into a culture dish and cool naturally.

[0029] The 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 and trifoliate orange [J]. Journal of Huazhong Agricultural University, 2023, 42(4): 150-157. The citrus rootstock red mandarin (Citrus reticulata Blanco, cv. Hongju) is referred to as red mandarin in the following text.

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

[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 min, let stand for 10 min, 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 Different dilutions of bacterial suspension. Take 0.1 mL of bacterial suspension from each dilution gradient and evenly spread it on LB solid medium, and culture it at 28°C for 2-3 days.

[0036] 2. After completing step 1, pick a single colony on the LB solid medium and purify it repeatedly for more than 3 times. The screened bacterial strain was named 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 homology between bacterial Rhiz_8E1 and Rhizobium tropici was the highest, reaching 99.7%.

[0040] 3. Preservation

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

[0042] Preservation of bacterial strains: A single colony of Rhiz_8E1 was inoculated into LB liquid culture medium and cultured in a shaking incubator at 28°C and 200 r / min for 8 hours to obtain bacterial culture solution. One volume of bacterial culture solution was mixed with one volume of 60% (v / v) glycerol and stored at -80°C.

[0043] (2) Preparation of bacterial 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, and culture it at 28°C and 200 r / min for 8 h. The obtained bacterial liquid is centrifuged to collect the bacterial cells, and then dissolved in sterile water and its OD 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 culture medium: Dissolve 4.46 g MS basic culture medium powder (Coolaber), 20 g sucrose and 3 g plant gel in distilled water, then dilute to 1 L with distilled water, adjust the pH to 5.8, then 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 was 12000Lx.

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

[0052] 1. Take tangerine seeds, treat them with 1 mol / L NaOH solution for 15 minutes, wash them three times with sterile water, then treat them with 2.6% (v / v) sodium hypochlorite aqueous solution for 15 minutes, and 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 place them in a 28°C constant temperature biochemical incubator for dark culture. Transplant the seeds after they grow 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, 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, and after vernalization at 4°C for two days, place them in a constant temperature incubator at 22°C, 14h light / 10h dark, and 5000Lx light intensity until the tomato seedlings grow two cotyledons before transplanting.

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

[0058] 7. Use Rhiz_8E1 strain OD 600nm The inoculum with a value of 0.2 was inoculated into 5 pots of Mic-Tom tomatoes (3 plants per pot), 5 pots of red mandarin oranges (3 plants per pot), 5 pots of fruiting Newhall navel oranges (1 plant per pot), and 10 Majia pomeloes by root irrigation. Each red mandarin and Mic-Tom tomato seedling was inoculated with 5mL of inoculum, each Newhall was inoculated with 3L of inoculum, and each Majia pomelo was inoculated with 15L of inoculum. Red mandarin and tomatoes were inoculated once every 2 weeks for a total of 3 times, and potted Newhall navel oranges and field Majia pomeloes were inoculated once a month for a total of 3 times. Another 5 pots of Mic-Tom tomatoes, 5 pots of red mandarin oranges, 5 pots of fruiting Newhall navel oranges, and 10 field Majia pomeloes were taken as controls. Red mandarin and Mic-Tom tomatoes were inoculated with 5mL of water every 2 weeks as controls, Newhall navel oranges were inoculated with 3L of water every 1 month as controls, and Majia pomeloes were inoculated with 15L of water every 1 month as controls, for a total of 3 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 ), and count the height of Mic-Tom tomatoes ( Figure 7 )、Fresh weight( Figure 8 ) and dry weight ( Fig. 9 ).

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

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

[0062] 11. By Fig.11 , Fig.13 and Fig.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 above is an example of the best embodiment of the present invention, and the parts not described in detail are common knowledge of ordinary technicians in the field. The protection scope of the present invention shall be based on the content of the claims, and any equivalent transformation based on the technical enlightenment of the present invention is also within the protection scope of the present invention.

Claims

1. A Rhizobium bacterium, characterized in that 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 the deposit number CCTCC M20242702.

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

3. The use according to claim 2, characterized in that: The application method specifically comprises treating plant roots with a bacterial agent containing the Rhizobium bacteria described in claim 1.

4. The use according to claim 3, characterized in that: OD of the inoculant containing Rhizobium spp. 600 The nm value is 0.2.

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