mycosphaerella symbiotica and its use for tomato growth

Abstract

The present application relates to the field of microorganism, specifically to three strains of Acrocalymma sp. symbiotic bacteria E00677, E01299A and E01299B and their use for tomato growth. The strains of the present application can promote tomato growth, improve tomato leaf quality, and enhance the antioxidant and stress resistance of tomato.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to three strains of Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B and their use for tomato growth. BACKGROUND

[0002] Most plants in natural ecosystems can form symbiosis with endophytic fungi, which can effectively promote plant growth and ecological adaptation to adversity. Plant roots (including roots and rhizosphere) have diverse fungal communities, which can affect plant adaptability, phenotype and environmental tolerance. In addition to mycorrhizal fungi, which have been extensively studied, there is a large group of non-mycorrhizal endophytic fungi in plant roots, such as dark septate endophytes (DSE) that are localized in 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, high mountains, polar regions, temperate and tropical regions. More than 600 species of plants belonging to more than 320 genera of 110 families are colonized by DSE. Both DSE and mycorrhizal fungi specifically colonize plant roots, but compared with mycorrhizal fungi, DSE has stronger saprophytic ability and can degrade soil organic matter, thereby improving plant nutrition through soil organic matter mineralization. Compared with saprophytic fungi, DSE colonizes plant roots, which is more beneficial to host plants. DSE in the order Pleosporales plays an important role in improving plant adaptability, such as Laburnicola rhizohalophila, a DSE in the order Pleosporales isolated from the plant Suaeda salsa of the family Chenopodiaceae, which is a core member of the fungal community in the roots of Suaeda salsa and plays an important role in plant salt stress resistance. Paraphoma chrysanthemicola, a DSE in the order Pleosporales isolated from the plant Artemisia ordosica of the family Asteraceae, has good application potential in improving the drought resistance of Artemisia ordosica. Through long-term co-evolution, DSE and the host establish a good mutually beneficial and win-win relationship. On the one hand, DSE obtains the nutrients needed for life from the plant body, and on the other hand, the endophyte also has beneficial effects on the physiological activities of the host. DSE can produce antagonism with plant pathogens through nutrient and space competition, production of secondary metabolites against pathogens and induction of expression of host defense-related genes, thereby reducing the incidence of plant diseases. In addition, DSE has the advantage of not causing infection or disease in the host body, so endophytic fungi are a potential source of candidate strains for biological control applications.

[0003] The application of CN108192832A, "A new Acrocalymma sp. QTY and its application in biological control", discloses a Acrocalymma sp. QTY with a preservation number of CCTCC M 2018058, and the application of the Acrocalymma sp. QTY in the field of biological control. SUMMARY

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

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

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

[0007] Preservation name: Acrocalymma sp., preservation unit: Guangdong Microbial Culture Collection Center, preservation address: 5th floor, No. 59 building, Guangzhou Xianlie Road 100, preservation date: January 18, 2023, preservation number: GDMCC No:63140.

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

[0009] Preservation name: Acrocalymma sp., preservation unit: Guangdong Microbial Culture Collection Center, preservation address: 5th floor, No. 59 building, Guangzhou Xianlie Road 100, preservation date: January 18, 2023, preservation number: GDMCC No:63143.

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

[0011] Preservation name: Acrocalymma sp., preservation unit: Guangdong Microbial Culture Collection Center, preservation address: 5th floor, No. 59 building, Guangzhou Xianlie Road 100, preservation date: January 18, 2023, preservation number: GDMCC No:63153.

[0012] The application also simultaneously provides the use of the above-mentioned strains, at least any one of the following:

[0013] Promoting the growth of tomatoes: manifested as increasing the number of lateral roots, optimizing the root-shoot ratio, increasing the root average weight, thereby enhancing the stability and nutrient absorption capacity of the plants;

[0014] Improving the quality of tomato leaves: specifically including increasing the chlorophyll content in tomato leaves, which helps to improve the photosynthetic efficiency; significantly increasing the anthocyanin content, thereby enhancing the antioxidant capacity;

[0015] And increasing the root activity, providing stronger nutrient absorption and stress resistance support for tomato plants.

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

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

[0018] 2. Significantly increasing the chlorophyll and anthocyanin content of tomatoes, while enhancing the root activity, not only helping to improve the yield and quality of tomatoes, but also enhancing the tolerance of plants to environmental stress, bringing significant economic and ecological benefits to agricultural production.

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

[0020] The specific embodiments of the application will be further described in detail below in conjunction with the accompanying drawings.

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

[0022] Figure 2 is the result figure of the co-culture experiment of the symbiotic bacteria Acrocalymma sp. E00677 of the genus Acrocalymma and tomatoes, compared with the control CK (left), the strain of the symbiotic bacteria Acrocalymma sp. E00677 (right) of the genus Acrocalymma promotes the growth and improves the stress resistance of tomatoes on 1 / 2MS medium;

[0023] Figure 3The results of promoting growth and improving stress resistance of Acrocalymma sp. E00677 symbiotic fungus;

[0024] Figure 3 In:

[0025] A is the number of lateral roots of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0026] B is the root crown ratio of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0027] C is the average root weight of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0028] D is the root activity of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0029] E is the chlorophyll a content of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0030] F is the chlorophyll b content of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0031] G is the total chlorophyll content of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control;

[0032] H is the relative content of anthocyanin of tomato treated with Acrocalymma sp. E00677 symbiotic fungus compared with the control.

[0033] Figure 4 The results of promoting growth and improving stress resistance of Acrocalymma sp. E01299A symbiotic fungus;

[0034] Figure 4 In:

[0035] A is the number of lateral roots of tomato treated with Acrocalymma sp. E01299A symbiotic fungus compared with the control;

[0036] B is the root crown ratio of tomato treated with Acrocalymma sp. E01299A symbiotic fungus compared with the control;

[0037] C is the average root weight of tomato treated by Acrocalymma sp. E01299A compared with the control;

[0038] D is the root activity of tomato treated by Acrocalymma sp. E01299A compared with the control;

[0039] E is the chlorophyll a content of tomato treated by Acrocalymma sp. E01299A compared with the control;

[0040] F is the chlorophyll b content of tomato treated by Acrocalymma sp. E01299A compared with the control;

[0041] G is the total chlorophyll content of tomato treated by Acrocalymma sp. E01299A compared with the control;

[0042] H is the relative content of anthocyanin of tomato treated by Acrocalymma sp. E01299A compared with the control.

[0043] Figure 5 is the growth promotion and stress resistance improvement experiment result of Acrocalymma sp. E01299B;

[0044] Figure 5 in the middle:

[0045] A is the lateral root number of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0046] B is the root crown ratio of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0047] C is the average root weight of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0048] D is the root activity of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0049] E is the chlorophyll a content of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0050] F is the chlorophyll b content of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0051] G is the total chlorophyll content of tomato treated by Acrocalymma sp. E01299B compared with the control;

[0052] H is the relative anthocyanin content of tomato treated by Acrocalymma sp. E01299B compared with the control. DETAILED DESCRIPTION

[0053] The application will be further described in conjunction with specific examples, but the protection scope of the application is not limited to the following:

[0054] Example 1, Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B were isolated.

[0055] Potato dextrose agar (PDA) : the formula includes potato 200g (peeled), glucose 20g, agar 20g, distilled water to 1000mL; the method is that the potato is washed and peeled, 200g of potato is weighed and cut into small pieces, boiled and mashed (boiled for 20-30 minutes, which can be poked by a glass rod), filtered with eight layers of gauze, heated, and 20g of agar is added according to the actual experimental needs, continue to heat and stir to mix, after the agar is dissolved, add glucose, stir evenly, slightly cool, then add water to 1000mL, distribute test tubes or conical bottles, plug, bandage, sterilize (1.1 atm, 121℃ for 20min) after sterilization, take out and store after cooling.

[0056] Water agar (WA) : agar powder 20g, distilled water 1000mL, routine high temperature sterilization (1.1 atm, 121℃ for 20min).

[0057] The strains described in the application can be obtained by culturing in the laboratory under the following conditions.

[0058] Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B strains of Acrocalymma symbiont were isolated from Oryza pachystachys in Xishuangbanna Nature Reserve (30.39 °N, 119.88 °E) in Yunnan Province. The plant samples were collected and washed under tap water to remove surface impurities. After washing, surface sterilization was performed.

[0059] After surface sterilization, Oryza pachystachys was cut into 0.5 cm x 0.5 cm size and placed on PDA medium and placed in an incubator for culture until fungi grew out. After three times of transfer and purification culture, pure culture was obtained.

[0060] The morphological characteristics of Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B strains of Acrocalymma symbiont are as follows: on PDA plate, the back of the colony is grayish white to gray black with radial cracks, and the colony has ringed round shape; the mycelium has dark septate hyphae and multiple branches, and forms irregular thick-walled spores in chain distribution.

[0061] Example 2, identification of Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B strains of Acrocalymma symbiont

[0062] 1. Molecular identification:

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

[0064] PCR amplification was performed using a 50 μL reaction system, which contained: 2 μM of each upstream and downstream primer, 200 μM of dNTPs, 1.5 mM of Mg 2+ 5 μL of 10 x PCR buffer, 2 μL of bacterial solution, 2 U of Taq enzyme, and ddH2O to make up to 50 μL. PCR amplification was performed on a BIORAD S1000 PCR instrument.

[0065] The reaction conditions, primers and amplification program are as follows:

[0066]

[0067]

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

[0069] After the PCR reaction, the PCR products were detected by 1% agarose gel electrophoresis, and then purified by a DNA gel purification kit from Axygen Biotechnology Co., Ltd. according to the steps of the kit instructions.

[0070] The steps are as follows:

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

[0072] ② Add 3 times the volume of DE-A buffer, incubate at 75°C for 10 min, and shake several times during the incubation until completely melted.

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

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

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

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

[0077] ⑦ Repeat step (6) once.

[0078] ⑧ Put the DNA preparation tube back into the 2 mL centrifuge tube, centrifuge at 12000 rpm for 2 min, and drain the membrane wash.

[0079] ⑨ Put the DNA preparation tube back into the 2 mL centrifuge tube, add 50 μL of ddH2O, centrifuge at 10000 rpm for 1 min, and elute the DNA and store at -20°C.

[0080] (3) Sequencing and sequence analysis of the gene

[0081] The purified recovered target DNA fragments were sent to Hangzhou Shangya for sequencing by electrophoresis detection using ABI PRISMA 377 type automatic sequencer. After strict checking of the sequencing results, the corresponding DNA fragment sequences were obtained, as shown in the sequence table of SEQ ID NO: 1-12 (the ITS of E00677, E01299A and E01299B is SEQ ID NO: 1-3, respectively, the LSU is SEQ ID NO: 4-6, respectively, the SSU is SEQ ID NO: 7-9, respectively, and the TEF1 is SEQ ID NO: 10-12, respectively). The measured nucleotide sequences were searched and compared with homologous or similar nucleotide sequences in GenBank using BLAST.

[0082] PhyloSuite platform was used for phylogenetic analysis. First, MAFFT was used to align the sequences of ITS, LSU, SSU and tef1a regions. The aligned sequences were trimmed and linked. ModelFinder was used to select the most appropriate partition model using Akaike information criterion (AIC). IQ-TREE was used to infer the maximum likelihood phylogeny of the aligned sequences with 5000 ultrafast bootstrap. FigTree v1.4.3 was used to visualize the phylogenetic tree, and the maximum likelihood value (MLBS) greater than 75% was shown in the Figure 1

[0083] Based on the morphological and phylogenetic analysis, Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B were determined to be fungi of the genus Acrocalymma.

[0084] The preservation information of the three strains of Acrocalymma symbiotic fungi obtained is as follows:

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

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

[0087] Acrocalymma sp. E01299B, the deposit name: Acrocalymma sp., the deposit unit: Guangdong Microbial Culture Collection Center, the deposit address: 59, Building 5, Guangzhou Institute of Military Personnel, Guangzhou, the deposit date: January 18, 2023, and the deposit number: GDMCC. No: 63153.

[0088] The three strains of Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B in the application are classified as follows: Fungi, Ascomycota, Dothideomycetes, Pleosporales, Morosphaeriaceae, and Acrocalymma.

[0089] Example 3, the promotion of Acrocalymma sp. E00677 to tomato

[0090] The specific process is as follows:

[0091] 1. Prepare MS medium (a conventional medium), and the MS salt used is shown in Table 1:

[0092] Table 1, MS salt

[0093]

[0094]

[0095] 1 / 2MS medium (conventional medium): MS salt 2.2 g, sucrose 5.0 g, MES 0.5 g, add water to 1 L, sterilize at 1.1 atm and 121℃ for 20 min, then take out and store after cooling; PDA medium: weigh 43 g of PDA powder (Hangzhou Microbial Reagent Co., Ltd.) into 1000 mL of purified water, boil until dissolved, then sterilize at 121℃ for 20 min, then take out and store after cooling.

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

[0097] ① Select intact tomato (Diane tomato) seeds, soak in 75% ethanol for 30 s, then soak in 0.5% sodium hypochlorite for 15 min, and wash with sterile water 4 times, each time for 2 min. The seeds are placed on sterilized dry filter paper in a clean bench and the surface moisture is dried.

[0098] ② Dispense the sterilized 1 / 2 MS medium into sterile 13cm square petri dishes on a laminar flow hood. Place the sterilized seeds on the 1 / 2 MS medium (25℃, 16h light, 8h dark incubation, daylight lamp: 120-150 mol m). -2 s -1 Inoculate 5mm E00677 mycelium cakes 2cm below the seed culture, using 5 cakes per dish, with 5 replicates. Place 5mm blank sterile PDA mycelium cakes in the control dish. Incubate for approximately 15 days. Figure 2 .

[0099] The preparation method of E00677 fungal cake is as follows: First, cultivate fungal PDA colonies containing E00677. Then, use a sterile punch with a diameter of 5 mm to press the fungal PDA plate from top to bottom to obtain round fungal cakes.

[0100] The method for preparing blank sterile PDA seed cakes is as follows: use a sterile punch with a diameter of 5 mm to press out circular blank sterile PDA seed cakes from top to bottom on a PDA plate.

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

[0102] The above experiment was repeated three times and the average was taken to obtain the results of co-culturing Acrocalymma sp. E00677 with tomatoes. The experimental results showed that Acrocalymma sp. E00677 significantly promoted the growth of Diana tomatoes, mainly in the following ways:

[0103] The number of lateral roots increased significantly: the strain promoted branching and lateral root development in tomato roots, providing 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 was 1.54 times that of the control group. Figure 3 A).

[0104] The average root weight decreased slightly: compared with the control group, the average root weight decreased by an average of 1.34%. Figure 3 C).

[0105] The root-to-shoot ratio was significantly improved: the strain enhanced the relative strength of tomato root growth and optimized the growth coordination between roots and above-ground parts, thereby improving plant stability and absorption efficiency. Compared with the control group, the root-to-shoot ratio increased by an average of 53.90%, which was 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 in promoting tomato growth, and show its broad application potential as an agricultural microbial resource.

[0107] 4. Stress resistance of Acrocalymma sp. E00677 to tomato

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

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

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

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

[0112] Anthocyanin content = (OD x 1) / (4.62 x 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 H2SO4. Add 10 mL of ethyl acetate solution and grind to extract the red triphenylformazan from the roots. Measure the concentration of triphenylformazan at 485 nm using spectrophotometry, and express the result as A485 g -1 h -1 .

[0114] The above experiment was repeated three times and the average was taken to obtain the results of co-culturing Acrocalymma sp. E00677 with tomatoes. The experimental results show that this strain has significant effects on promoting tomato growth, improving quality, and enhancing resistance to abiotic stress, specifically including:

[0115] Significantly increased chlorophyll content: Co-culture significantly increased the content of chlorophyll a, chlorophyll b, and total chlorophyll in the leaves of tomato 'Diana', indicating that the strain can enhance photosynthetic efficiency and provide higher energy reserves for the plant; 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% (E). Figure 3 F), the total chlorophyll content increased by an average of 7.76% (F). Figure 3 G).

[0116] Increased anthocyanin content: Experimental results showed that this strain could increase the accumulation level of anthocyanins in tomato plants, which not only improved the antioxidant capacity of tomatoes but also had the potential to contribute 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 assay: The root activity of the strain treated was comparable to that of the control group. Figure 3 D).

[0118] Promotes growth and enhances stress resistance: Overall, experimental data show that the symbiotic fungus Acrocalymmasp.E00677 has a significant promoting effect on the growth and development of tomato 'Diana', while enhancing the plant's survival and recovery ability under adverse stress.

[0119] Example 4: The promoting effect of the symbiotic bacterium Acrocalymma sp. E01299A on tomatoes.

[0120] The method is the same as in Example 3.

[0121] Experimental results show that:

[0122] When co-cultured with tomato 'Diana', the symbiotic bacterium *Acrocalymma* sp. E01299A exhibited a similar promoting effect to *Acrocalymma* sp. E00677, showing significant benefits across multiple growth and quality indicators.

[0123] 1. Enhance root development

[0124] Co-culture experiments showed that the symbiotic fungus *Acrocalymma* sp. E01299A significantly increased the number of lateral roots in tomatoes and optimized the root-to-shoot ratio. The increased number of lateral roots provided the plant with a larger absorption surface area, while the improved root-to-shoot ratio indicated that the root system dominated growth, making it more efficient in nutrient and water absorption. Compared with the control group, the number of lateral roots increased by an average of 36.66%, which was 1.37 times that of the control group. Figure 4 A) The root-to-shoot ratio increased by an average of 125.04%, which was 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 chlorophyll and anthocyanin content

[0126] Experimental results showed that under the action of the symbiotic strain *Acrocalymma* sp. E01299A, the contents of chlorophyll a, chlorophyll b, and total chlorophyll in tomatoes were significantly increased, enhancing photosynthetic efficiency. Furthermore, the increase in anthocyanin content not only improved the antioxidant capacity of tomatoes but may also have a positive impact on 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% (F). Figure 4 G), the relative anthocyanin content 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 stress resistance

[0128] The symbiotic fungus *Acrocalymma* sp. E01299A significantly improved tomato root activity, manifested as enhanced root metabolic activity and increased absorption capacity. This effect allows tomatoes to better survive and recover under adverse conditions (such as drought, salinity, or disease stress), providing them with greater environmental adaptability. Compared with the control group, root activity increased by an average of 5.77% (…). Figure 4 D).

[0129] In summary, the symbiotic fungus *Acrocalymma* sp. E01299A has broad application potential in promoting tomato growth and enhancing stress resistance. It not only significantly improves crop growth performance but also provides important support for improving agricultural productivity and crop quality.

[0130] Example 5, Promoting effect of Acrocalymma sp. E01299B on tomato

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

[0132] The experimental results show that Acrocalymma sp. E01299B has a significant effect on the growth and quality improvement of tomato 'Diane' when co-cultured with the plant. This strain promotes the development of tomato plants through multiple mechanisms, which are reflected in the following aspects:

[0133] 1. Promoting root development

[0134] The experimental data show that Acrocalymma sp. E01299B helps to significantly increase the number of lateral roots of tomato, and significantly improves the root-shoot ratio. This indicates that the strain not only promotes the development of root branching, but also optimizes the coordination between the root and the aboveground part, thereby providing strong support for the plant's water and mineral nutrient uptake. Compared with the control group, the average number of lateral roots is increased by 53.77% ( Figure 5 A), which is 1.54 times that of the control group, the average root-shoot ratio is increased by 90.17% ( Figure 5 B), which is 1.9 times that of the control group, and the average root weight is increased by 13.29% ( Figure 5 C).

[0135] 2. Improving leaf quality indicators

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

[0137] 3. Enhancing root activity and environmental adaptability

[0138] Acrocalymma sp. E01299B enhances the activity of tomato root system, which is manifested as significant improvement in root system metabolism and absorption function. This enhancement enables tomato to exhibit stronger stress resistance when facing environmental stress (such as drought or salt damage), and provides key physiological support for the plant. Compared with the control group, the average root activity is increased by 5.77% Figure 5 D).

[0139] In summary, Acrocalymma sp. E00677, Acrocalymma sp. E01299A and Acrocalymma sp. E01299B play a promoting role in many aspects of tomato growth, showing broad prospects in agricultural applications. By improving plant growth performance, improving leaf quality indicators and enhancing antioxidant and stress resistance, the value of 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 only lists several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived or inferred from the disclosed content by those of ordinary skill in the art should be considered within the scope of the present application.

Claims

1. A Mycosphaerella sydowiana symbiotic bacteria beneficial to tomato growth, characterized in that For any of the following: Deposition No: GDMCC. No: 63140 Acrocalymma sp. E00677, Deposition No: GDMCC. No: 63143 Acrocalymma sp. E01299A, Deposition No: GDMCC. No: 63153 Acrocalymma sp. E01299B.

2. Use of a strain of Mycosphaerella graminicola symbiote according to claim 1, characterized in that For any of the following: Increasing the number of lateral roots of tomatoes; Increasing the root-shoot ratio of tomatoes; Increasing the average root weight of tomatoes; Improving the quality of tomato leaves, increasing chlorophyll content, and thus improving photosynthetic efficiency; Increasing the anthocyanin content of tomato leaves and enhancing antioxidant capacity; Increasing the root activity of tomatoes, providing stronger nutrient absorption and stress resistance support for tomato plants.

3. Use of a strain of Mycosphaerella spp. according to claim 2, characterized in that: The tomato variety includes 'Diana'.

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