Symbiotic bacteria of genus Mycosphaeria and application thereof to tomato growth

Abstract

The invention relates to the field of microorganisms, and in particular relates to three symbiotic bacteria, namely, Acrocentrum sp.E00677, Acrocentrum sp.E01299A and Acrocentrum sp.E01299B, of the malachium, and an application of the symbiotic bacteria to the growth of tomatoes, of the symbiotic bacteria, namely, Acrocentrum sp.E00677, Acrocentrum sp.E01299A and Acrocentrum sp.E01299B. The strain provided by the invention can promote tomato growth, improve tomato leaf quality, and enhance oxidation resistance and stress resistance of tomatoes.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to three symbiotic bacteria of the genus Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B and applications thereof to tomato growth. Background Art

[0002] Most plants in natural ecosystems can coexist with endophytic fungi, which can effectively promote plant growth and ecological adaptation to adversity. Plant roots (including the root and rhizosphere) have diverse fungal communities, which can affect the adaptability, phenotype and environmental tolerance of plants. In addition to the widely studied mycorrhizal fungi, there is also a large group of non-mycorrhizal endophytic fungi in plant roots, such as dark septate endophytes (DSE) that are localized to the roots of living plants and have melanized septate hyphae or small sclerotia. DSE fungi have a wide range of hosts and ecological distribution characteristics, and are widely distributed in different ecological environments such as deserts, alpine polar regions, temperate and tropical regions. The roots of more than 600 plant species from more than 110 families and 320 genera are colonized by DSE. Both DSE and mycorrhizal fungi colonize plant roots specifically, but compared with mycorrhizal fungi, DSE has stronger saprophytic ability and can degrade organic matter in the soil, and can improve plant nutrition through mineralization of organic matter in the soil; and compared with saprophytic fungi, DSE colonizes plant roots, which is more beneficial to host plants. Laburnicola rhizohalophila DSE plays an important role in improving plant adaptability. For example, Laburnicola rhizohalophila DSE isolated from Suaeda salsa, a halophyte of Amaranthaceae, is a core member of the root fungal community of Suaeda salsa and plays an important role in plant resistance to salt stress; Paraphoma chrysanthemicola DSE isolated from Artemisia ordosica, a desert Asteraceae, has good application potential in improving the drought resistance of Artemisia ordosica. Under the long-term symbiotic evolution, DSE and its host have established a good mutually beneficial relationship. On the one hand, DSE obtains nutrients needed for life from the plant body, and on the other hand, endophytes also have beneficial effects on the physiological activities of the host. DSE can antagonize plant pathogens through nutrient and space competition, production of anti-pathogen secondary metabolites, and induction of host defense-related gene expression, thereby reducing the incidence of plant diseases. In addition, DSE has the advantage of not causing infection or disease in the host, so endophytes are potential candidate strain sources for biological control applications.

[0003] The invention of CN108192832A "A new endophytic fungus QTY from Acrocalymma sp. and its application in biological control" discloses an endophytic fungus (Acrocalymma sp.) QTY from Acrocalymma sp., whose preservation number is CCTCC M 2018058; it also discloses the application of the endophytic fungus QTY from Acrocalymma sp. in the field of biological control. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide three strains of symbiotic bacteria of the genus Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B and uses thereof.

[0005] In order to solve the above technical problems, the present invention provides three strains of Acrocalymma symbiotic bacteria, namely Acrocalymma sp.E00677 with a preservation number of GDMCC.No: 63140, Acrocalymmasp.E01299A with a preservation number of GDMCC.No: 63143, and Acrocalymma sp.E01299B with a preservation number of GDMCC.No: 63153.

[0006] The deposit information of Acrocalymma sp.E00677 is as follows:

[0007] Deposit name: Acrocalymma sp., deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: January 18, 2023, deposit number: GDMCC No: 63140.

[0008] The deposit information of Acrocalymma sp.E01299A is as follows:

[0009] Deposit name: Acrocalymma sp., deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: January 18, 2023, deposit number: GDMCC No: 63143.

[0010] The deposit information of Acrocalymma sp.E01299B is as follows:

[0011] Deposit name: Acrocalymma sp., deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: January 18, 2023, deposit number: GDMCC No: 63153.

[0012] The present invention also provides the use of the above strain, which is at least any of the following:

[0013] Promote tomato growth: increase the number of lateral roots, optimize the root-crown ratio, and increase the average root weight, thereby enhancing the stability of the plant and its ability to absorb nutrients;

[0014] Improve the quality of tomato leaves: Specifically, it increases the chlorophyll content in tomato leaves, which helps to improve photosynthesis efficiency; significantly increases the anthocyanin content, thereby enhancing antioxidant capacity;

[0015] As well as improving root vitality, providing tomato plants with stronger nutrient absorption and stress resistance support.

[0016] The strains involved in the present invention have significant technical advantages and beneficial effects:

[0017] 1. After application on tomato plants, it can effectively increase the average root weight, significantly increase the number of lateral roots, and improve the root-crown ratio, indicating that it has a wide range of application potential in promoting plant growth;

[0018] 2. Significantly increase the chlorophyll and anthocyanin content of tomatoes, while enhancing root activity, which not only helps to improve tomato yield and quality, but also enhances the plant's tolerance to environmental stress, bringing significant economic and ecological benefits to agricultural production.

[0019] In summary, the strain of the present invention can promote tomato growth, improve the quality of tomato leaves, and enhance the antioxidant and stress resistance of tomatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0021] Figure 1 This is the result of phylogenetic tree analysis of Acrocalymma, indicating that the symbiotic bacteria of Acrocalymma sp.E00677, Acrocalymma sp.E01299A and Acrocalymma sp.E01299B all belong to the genus Acrocalymma.

[0022] Figure 2 This is the result of the co-cultivation experiment between Acrocalymma sp.E00677 and tomato. Compared with the control CK (left), the Acrocalymma sp.E00677 (right) strain promoted the growth and improved the stress resistance of tomatoes on 1 / 2MS medium.

[0023] Figure 3The experimental results of promoting the growth and improving the stress resistance of Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma;

[0024] Figure 3 middle:

[0025] A is the number of lateral roots of tomatoes treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0026] B is the comparison of the root-to-shoot ratio of tomatoes after being treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0027] C is the average root weight of tomatoes treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0028] D is the root activity of tomatoes treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0029] E is the chlorophyll a content of tomatoes treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0030] F is the chlorophyll b content of tomatoes treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0031] G is the total chlorophyll content of tomatoes treated with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0032] H is the relative content of anthocyanins in tomatoes after treatment with Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, compared with the control.

[0033] Figure 4 The experimental results of promoting growth and improving stress resistance of Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma;

[0034] Figure 4 middle:

[0035] A is the number of lateral roots of tomatoes treated with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0036] B is the comparison of the root-to-shoot ratio of tomatoes after being treated with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0037] C is the average root weight of tomatoes treated with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0038] D is the comparison of tomato root activity after treatment with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, with the control;

[0039] E is the chlorophyll a content of tomatoes treated with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0040] F is the chlorophyll b content of tomatoes treated with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0041] G is the total chlorophyll content of tomatoes treated with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0042] H is the relative content of anthocyanins in tomatoes after treatment with Acrocalymma sp.E01299A, a symbiotic bacterium of the genus Acrocalymma, compared with the control.

[0043] Figure 5 The experimental results of promoting growth and improving stress resistance of Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma;

[0044] Figure 5 middle:

[0045] A is the number of lateral roots of tomatoes treated with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0046] B is the comparison of the root-to-shoot ratio of tomatoes after being treated with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0047] C is the average root weight of tomatoes treated with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0048] D is the comparison of tomato root activity after treatment with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, with the control;

[0049] E is the chlorophyll a content of tomatoes treated with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0050] F is the chlorophyll b content of tomatoes treated with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0051] G is the total chlorophyll content of tomatoes treated with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control;

[0052] H is the relative content of anthocyanins in tomatoes after treatment with Acrocalymma sp.E01299B, a symbiotic bacterium of the genus Acrocalymma, compared with the control. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0054] Example 1: Symbiotic bacteria Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B of the genus Acrocalymma were isolated and obtained.

[0055] Potato dextrose agar (PDA): The formula includes 200g of potatoes (peeled), 20g of glucose, 20g of agar, and distilled water to 1000mL; the method is as follows: wash and peel the potatoes, weigh 200g of potatoes, cut them into small pieces, add water and cook until they are soft (boil for 20 to 30 minutes, until they can be pierced by a glass rod), filter with eight layers of gauze, heat, and then add 20g of agar according to actual experimental needs, continue heating and stirring to mix, and after the agar is dissolved, add glucose and stir evenly. After cooling slightly, add water to 1000mL, divide into test tubes or conical flasks, add stoppers, bandage, (1.1 atmospheres, 121°C sterilization for 20 minutes) take out after sterilization, cool and store for later use.

[0056] Water agar medium (WA): 20 g agar powder, 1000 mL distilled water, and conventional high-temperature sterilization (1.1 atmospheres, 121°C for 20 min).

[0057] The strain of the present invention can be obtained by isolating and culturing in the laboratory under the following conditions.

[0058] The symbiotic bacteria of the genus Acrocalymma sp.E00677, Acrocalymma sp.E01299A and Acrocalymma sp.E01299B were isolated from Oryza verruca in the Naban River Basin National Nature Reserve, Xishuangbanna, Yunnan Province (30.39°N, 119.88°E). The collected plant samples were collected and brought back to the laboratory and rinsed under tap water to remove surface impurities. After rinsing, the surface was disinfected.

[0059] After surface disinfection, the wild rice was cut into 0.5 cm × 0.5 cm size, placed on PDA medium and cultured in an incubator until fungi grew out. After three times of purification culture, pure culture was obtained.

[0060] The morphological characteristics of Acrocalymma sp.E00677, Acrocalymma sp.E01299A and Acrocalymma sp.E01299B strains of the symbiotic bacteria of the genus Acrocalymma are as follows: on the PDA plate, the center of the back of the colony is grayish white to grayish black, with radial cracks, and the colony is circular with annular patterns; the hyphae contain dark septate hyphae and many branches, and form irregular thick-walled spores distributed in chains.

[0061] Example 2: Identification of Symbiotic Bacteria of the Genus Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B

[0062] 1. Molecular identification:

[0063] (1) PCR amplification of fungal DNA genes

[0064] PCR amplification was performed using a 50 μL reaction system containing: 2 μM each of upstream and downstream primers, 200 μM dNTPs, Mg 2+ 1.5mM, 10×PCR buffer 5μL, bacterial solution 2μL, Taq enzyme 2U, ddH 2 The volume of 4% HO was added to 50 μL. PCR amplification reaction was carried out on a BIORAD S1000 PCR instrument.

[0065] The reaction conditions, primers and amplification procedures were as follows:

[0066]

[0067]

[0068] (2) Recovery and purification of PCR products:

[0069] After the PCR reaction was completed, the PCR product was detected by 1% agarose gel electrophoresis and then purified using a DNA gel purification kit from Axygen Biotechnology Company according to the steps in the kit manual.

[0070] Here are the steps:

[0071] ① After electrophoresis, cut the gel containing the target DNA fragment with a blade under ultraviolet light, place it in a 2mL centrifuge tube, and weigh it.

[0072] ② Add 3 times the volume of DE-A buffer and keep warm at 75℃ for 10 min, shaking several times during the process, until it is completely melted.

[0073] ③Add 0.5 times volume of DE-B buffer and mix well.

[0074] ④ Place the DNA preparation tube into a 2 mL centrifuge tube, transfer the mixed solution into the DNA preparation tube, centrifuge at 12000 rpm for 1 min, and discard the supernatant.

[0075] ⑤Put the DNA preparation tube back into the 2 mL centrifuge tube, add 500 μL buffer W1, and centrifuge at 12000 rpm for 30 seconds.

[0076] ⑥Put the DNA preparation tube back into the 2 mL centrifuge tube, add 700 μL buffer W2, and centrifuge at 12000 rpm for 30 seconds.

[0077] ⑦ Repeat step (6) once.

[0078] ⑧Put the DNA preparation tube back into a 2 mL centrifuge tube and centrifuge at 12000 rpm for 2 min to drain the washing solution on the membrane.

[0079] ⑨Put the DNA preparation tube back into the 2 mL centrifuge tube and add 50 μL of ddH 2 O, centrifuge at 10000rpm for 1min, and store the eluted DNA at -20℃.

[0080] (3) Gene sequencing and sequence analysis

[0081] The purified and recovered target DNA fragments were sent to Hangzhou Shangya for sequencing using an ABIPRISMA377 automatic sequencer after electrophoresis detection. After strict verification of the sequencing results, the corresponding DNA fragment sequences were obtained, specifically as SEQ ID NO: 1-12 in the sequence table (ITS of E00677, E01299A, and E01299B are SEQ ID NO: 1 to SEQ ID NO: 3, LSU are SEQ ID NO: 4 to SEQ ID NO: 6, SSU are SEQ ID NO: 7 to SEQ ID NO: 9, and TEF1 are SEQ ID NO: 10 to SEQ ID NO: 12). The measured nucleotide sequences were searched and compared with homologous or similar nucleotide sequences in GenBank using BLAST.

[0082] Phylogenetic analysis was performed using the PhyloSuite platform. MAFFT was used to align the sequences of the ITS, LSU, SSU, and tef1α regions. The aligned sequences were trimmed and connected. ModelFinder was used to select the most appropriate partitioning model using the Akaike information criterion (AIC). IQ-TREE was used to perform 5000 ultra-fast bootstrap inferences on the aligned sequences to obtain the maximum likelihood phylogeny. FigTree v1.4.3 was used to visualize the phylogenetic tree, and the maximum likelihood score (MLBS) was greater than 75% in Figure 1 Displayed in.

[0083] Combined with morphological and phylogenetic analysis, Acrocalymma sp.E00677, Acrocalymmasp.E01299A and Acrocalymma sp.E01299B were identified as fungi of the genus Acrocalymma.

[0084] The deposit information of the three obtained symbiotic bacteria of the genus Chisana is as follows:

[0085] Acrocalymma sp.E00677, deposit name: Acrocalymma sp., deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: January 18, 2023, deposit number: GDMCC.No: 63140.

[0086] Acrocalymma sp.E01299A, deposit name: Acrocalymma sp., deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: January 18, 2023, deposit number: GDMCC.No: 63143.

[0087] Acrocalymma sp.E01299B, deposit name: Acrocalymma sp., deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: January 18, 2023, deposit number: GDMCC.No: 63153.

[0088] The three strains of symbiotic bacteria of the genus Acrocalymma, Acrocalymma sp.E00677, Acrocalymmasp.E01299A and Acrocalymma sp.E01299B, in the present invention have the following classification status: Fungi, Ascomycota, Dothideomycetes, Pleosporales, Morosphaeriaceae, Acrocalymma.

[0089] Example 3: Growth-promoting effect of Acrocalymma sp. E00677 on tomatoes

[0090] The specific process is as follows:

[0091] 1. Prepare MS culture medium (conventional culture medium). The MS salts used are shown in Table 1:

[0092] Table 1. MS salts

[0093]

[0094]

[0095] 1 / 2MS medium (conventional medium): 2.2g MS salt, 5.0g sucrose, 0.5g MES, add water to make up to 1L, sterilize at 121℃ at 1.1 atmospheres for 20 minutes, then cool and store for later use; PDA medium: weigh 43g PDA powder (Hangzhou Microbiological Reagent Co., Ltd.) and dissolve in 1000mL purified water, boil until dissolved, divide into portions, sterilize at 121℃ for 20 minutes, then cool and store for later use.

[0096] 2. Co-cultivation of Acrocalymma sp.E00677 and sterile tomato seedlings:

[0097] ① Select whole tomato (Diana tomato) seeds, soak them in 75% ethanol for 30 seconds, then soak them in 0.5% sodium hypochlorite for 15 minutes, and wash them with sterile water 4 times, 2 minutes each time. Place the seeds on sterilized dry filter paper and air dry the surface moisture in a clean bench.

[0098] ②Put the sterilized 1 / 2MS culture medium into sterile 13 cm square culture dishes on the clean bench. Place the sterilized seeds on the 1 / 2MS culture medium (25°C, 16 h light, 8 h dark, fluorescent lamp: 120-150 mol m -2 s -1 ), 5 mm E00677 cake was inoculated to 2 cm below the seed, 5 cakes per dish, repeated 5 times, and 5 mm blank sterile PDA cake was placed in the control dish; cultured for about 15 days; Figure 2 .

[0099] The preparation method of E00677 cake is as follows: firstly, a fungal PDA colony containing E00677 is cultured, and a sterile puncher with a diameter of 5 mm is used to press the fungal block from top to bottom on the cultured fungal PDA plate to obtain a round cake.

[0100] The preparation method of blank sterile PDA cake is as follows: a round blank sterile PDA cake is obtained by pressing from top to bottom on a PDA plate using a sterile hole puncher with a diameter of 5 mm.

[0101] 3. Collect tomato plants, wash them with tap water to remove the agar adhering to the tomato roots, wipe off the surface moisture with absorbent paper, cut the above-ground parts and roots of the seedlings, and measure agronomic traits such as average root weight and average above-ground weight.

[0102] The above experiment was repeated three times and the average value was taken to obtain the co-culture results of Acrocalymma sp.E00677 and tomato. The experimental results showed that Acrocalymma sp.E00677 significantly promoted the growth of Diana tomatoes, mainly manifested as follows:

[0103] The number of lateral roots increased significantly: The strain promoted the branching and lateral root development of tomato roots, providing the plant with a larger surface area for nutrient absorption; compared with the control group, the number of lateral roots increased by an average of 53.77%, which is 1.54 times that of the control group ( Figure 3 A).

[0104] The average root weight was slightly reduced: compared with the control group, the average root weight was reduced by 1.34% ( Figure 3 C).

[0105] The root-to-crown ratio was significantly improved: The strain enhanced the relative strength of the tomato's root growth and optimized the growth coordination between the roots and the aboveground parts, thereby improving the stability and absorption efficiency of the plant. Compared with the control group, the root-to-crown ratio increased by an average of 53.90%, which is 1.54 times that of the control group ( Figure 3 B).

[0106] The above experimental results clearly demonstrate the outstanding advantages of Acrocalymma sp.E00677, a symbiotic bacterium of the genus Acrocalymma, in promoting tomato growth and demonstrate its wide application potential as an agricultural microbial resource.

[0107] 4. Effect of Acrocalymma sp.E00677 on stress resistance of tomatoes

[0108] The above-ground parts and roots of the seedlings (obtained after cultivation for about 15 days) were taken to measure the chlorophyll content, anthocyanin content and root activity.

[0109] ① Determination of chlorophyll content: Remove the main vein of tomato leaves. Weigh about 0.5g of leaves on a 0.0001 balance and place in a 50mL centrifuge tube. Add 25mL of 95% ethanol and seal. Extract at room temperature in the dark for 24-36h. Dilute the extract to a certain multiple (depending on the specific situation). Then, perform colorimetry using 95% ethanol as a blank control at wavelengths of 665, 652, 649, and 470nm on an ultraviolet spectrophotometer.

[0110] ②Anthocyanin content determination: Take about 100 mg of tomato leaves and place them in a 1.5 mL centrifuge tube, record the sample weight, add 1 mL of 2% hydrochloric acid methanol solution (concentrated hydrochloric acid: 99% methanol = 2:98), and place at 4°C for 24 hours. Take 1 mL and pour it into a colorimetric dish, use 2% hydrochloric acid methanol solution as a negative control to adjust to zero, and use a UV spectrophotometer to measure the absorbance at wavelengths of 530 nm and 637 nm. Formula:

[0111] Gree formula to calculate OD value, OD = 530nm absorbance - 0.25 × 637nm absorbance

[0112] Anthocyanin content = (OD × 1) / (4.62 × weight);

[0113] ③ Root activity determination: Weigh 50 mg of freshly harvested root tissue, add 5 mL of 0.4% TTC solution (w / v) and 5 mL of 0.067 M phosphate buffer (pH 7.4), and mix well. Incubate the mixture at 40°C for 3 hours, then add 2 mL of 2M H 2 SO 4 . Add 10 mL of ethyl acetate solution, grind, and extract red triphenylformazan from the root. Determine its concentration by spectrophotometry at 485 nm and use A485 g -1 h -1 express.

[0114] The above experiment was repeated three times and the average value was taken to obtain the co-culture results of Acrocalymma sp.E00677 and tomato. The experimental results showed that the strain showed significant effects in promoting tomato growth, improving quality and enhancing resistance to adverse stress, including:

[0115] Significant increase in chlorophyll content: Co-cultivation significantly increased the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of tomato 'Diana', indicating that the strain can enhance photosynthesis efficiency and provide plants with higher energy reserves; compared with the control group, the chlorophyll a content increased by an average of 9.28% ( Figure 3 E), the chlorophyll b content increased by an average of 18.18% ( Figure 3 F), the total chlorophyll content increased by an average of 7.76% ( Figure 3 G).

[0116] Increased anthocyanin content: The experimental results showed that the strain was able to increase the accumulation level of anthocyanins in tomato plants, which not only improved the antioxidant capacity of tomatoes, but also had a potential contribution to improving fruit quality; compared with the control group, the anthocyanin content increased by an average of 37.52% ( Figure 3 H).

[0117] Root activity determination: The root activity of the strain-treated cells was equivalent to that of the control group ( Figure 3 D).

[0118] Promote growth and enhance stress resistance: Overall, the experimental data showed that the symbiotic bacteria Acrocalymmasp.E00677 of the genus Acrocalyx significantly promoted the growth and development of tomato 'Diana', and at the same time enhanced the plant's survival and recovery ability under adverse stress.

[0119] Example 4: Growth-promoting effect of Acrocalymma sp. E01299A on tomatoes

[0120] The method is as in Example 3.

[0121] The experimental results show that:

[0122] When the symbiotic bacteria Acrocalymma sp.E01299A of the genus Acrocalymma was co-cultured with tomato 'Diana', it showed a similar promoting effect as Acrocalymma sp.E00677, and showed significant effects on multiple growth and quality indicators:

[0123] 1. Enhance root development

[0124] Co-cultivation experiments showed that the symbiotic bacteria Acrocalymma sp. E01299A of the genus Acrocalymma can significantly increase the number of lateral roots of tomatoes and optimize the root-crown ratio. The increased number of lateral roots provides the plant with a larger absorption surface area, while the increased root-crown ratio indicates that the plant root system dominates the growth, making it more efficient in absorbing nutrients and water. Compared with the control group, the number of lateral roots increased by an average of 36.66%, which is 1.37 times that of the control group ( Figure 4 A), the root-shoot ratio increased by an average of 125.04%, which is 2.25 times that of the control group ( Figure 4 B), the average root weight increased by 13.09% ( Figure 4 C).

[0125] 2. Increase the content of chlorophyll and anthocyanin

[0126] The experimental results showed that under the action of the Acrocalymma sp. E01299A strain, the chlorophyll a, chlorophyll b and total chlorophyll content of tomatoes were significantly increased, enhancing the efficiency of photosynthesis. In addition, the increase in anthocyanin content not only improved the antioxidant capacity of tomatoes, but also may have a positive effect on the improvement of their fruit quality. Compared with the control group, the chlorophyll a content of tomatoes increased by an average of 32.12% ( Figure 4 E), the chlorophyll b content increased by an average of 50.85%, which is 1.51 times that of the control group ( Figure 4 F), the total chlorophyll content increased by an average of 34.39% ( Figure 4 G), the relative content of anthocyanins increased by an average of 87.18%, which is 1.87 times that of the control group ( Figure 4 H).

[0127] 3. Enhance root vitality and resistance

[0128] The symbiotic bacteria of the genus Acrocalymma sp.E01299A also significantly improved the vitality of tomato roots, which was manifested by enhanced root metabolic activity and improved absorption capacity. This effect enables tomatoes to survive and recover better under adverse conditions (such as drought, salinity or disease pressure), providing them with higher environmental adaptability. Compared with the control group, the root vitality increased by an average of 5.77% ( Figure 4 D).

[0129] In summary, the symbiotic bacteria of the genus Acrocalymma sp.E01299A have broad application potential in promoting tomato growth and improving stress resistance. They can not only significantly improve the growth performance of crops, but also provide important support for improving agricultural production efficiency and crop quality.

[0130] Example 5: Growth-promoting effect of Acrocalymma sp. E01299B on tomato

[0131] The method is the same as Example 3.

[0132] The experimental results showed that the symbiotic bacteria Acrocalymma sp.E01299B of the genus Acrocalymma showed significant effects on plant growth and quality improvement when co-cultivated with tomato 'Diana'. The strain promoted the development of tomato plants through multiple mechanisms, which are specifically reflected in the following aspects:

[0133] 1. Promote root development

[0134] Experimental data showed that the symbiotic bacteria of the genus Acrocalymma sp.E01299B can significantly increase the number of lateral roots of tomatoes and significantly improve the root-crown ratio. This shows that the strain can not only promote the development of root branches, but also optimize the coordination between the roots and the aboveground parts, thereby providing strong support for the plant's absorption of water and mineral nutrients. Compared with the control group, the number of lateral roots increased by an average of 53.77%, which is 1.54 times that of the control group ( Figure 5 A), the root-shoot ratio increased by an average of 90.17%, which is 1.9 times that of the control group ( Figure 5 B), the average root weight increased by 13.29% ( Figure 5 C).

[0135] 2. Improve leaf quality indicators

[0136] Under the promotion of Acrocalymma sp.E01299B, the chlorophyll a, chlorophyll b and total chlorophyll contents in tomato leaves increased, especially chlorophyll b, indicating that the strain enhanced the photosynthesis capacity of the plant and provided more energy for the growth and development of tomatoes. At the same time, the increase in anthocyanin levels not only enhanced the antioxidant properties of tomatoes, but also may further improve the quality of the fruit, which has important economic value. Compared with the control group, the chlorophyll a content increased by an average of 23.64% ( Figure 5 E), the chlorophyll b content increased by an average of 31.50%, which is 1.31 times that of the control group ( Figure 5 F), the total chlorophyll content increased by an average of 23.51% ( Figure 5 G), the relative content of anthocyanins increased by an average of 33.75% ( Figure 5 H).

[0137] 3. Enhance root vitality and environmental adaptability

[0138] The symbiotic bacteria of the genus Acrocalymma sp.E01299B enhanced the activity of tomato roots, which was manifested by a significant improvement in root metabolism and absorption. This enhancement enabled tomatoes to show stronger resistance to environmental stresses (such as drought or salt damage), providing key physiological support for the plants. Compared with the control group, the root activity increased by an average of 5.77% ( Figure 5 D).

[0139] In summary, the symbiotic bacteria of the genus Acrocalymma sp.E00677, Acrocalymma sp.E01299A and Acrocalymma sp.E01299B of the genus Acrocalymma promote tomato growth in many aspects, showing broad prospects in agricultural applications. By improving plant growth performance, improving leaf quality indicators and enhancing antioxidant and stress resistance, the value of the symbiotic bacteria of the genus Acrocalymma sp.E00677, Acrocalymma sp.E01299A and Acrocalymma sp.E01299B in modern agriculture cannot be ignored.

[0140] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Three strains of the genus Trichoderma that are beneficial to tomato growth, characterized in that They are: Acrocalymma sp. E00677 with a deposit number of GDMCC. No: 63140, Acrocalymma sp.E01299A with a deposit number of GDMCC.No:63143, Acrocalymma sp. E01299B with the deposit number of GDMCC.No:63153.

2. The use of three strains of the symbiotic bacteria of the genus Zeaxanthin as claimed in claim 1, characterized in that: Promotes tomato growth.

3. The use of three strains of the symbiotic bacteria of the genus Zeaxanthin according to claim 2, characterized in that: Enhance the stability and nutrient absorption capacity of tomatoes.

4. The use of three strains of the symbiotic bacteria of the genus Zea mays according to claim 2 or 3, characterized in that At least one of the following: Increase the number of tomato lateral roots; Improve the root-to-shoot ratio of tomatoes; Increase the average weight of tomato roots; Improve the quality of tomato leaves, increase chlorophyll content, and thus improve photosynthesis efficiency; Increase the anthocyanin content of tomato leaves and enhance antioxidant capacity; Improve the vitality of tomato root system and provide stronger nutrient absorption and stress resistance support for tomato plants.

5. The use of three strains of symbiotic bacteria of the genus Zea mays according to any one of claims 2 to 4, characterized in that: The tomato varieties include 'Diana'.

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