Application of streptomyces rochei D74 in delaying leaf senescence

Abstract

The invention belongs to the technical field of biology, and relates to application of an existing strain streptomyces rochei D74 in delaying leaf senescence. According to the invention, the antioxidant enzyme activity in the later growth and development period of tomato leaves can be improved, the photosynthesis intensity and the redox steady state are maintained, and yellowing and aging of the tomato leaves are delayed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an application of a strain, in particular to an application of Streptomyces rocheri D74 in delaying leaf senescence. Background Art

[0002] As one of the world's most important fruit and vegetable crops, tomato fruit has high nutritional value and is an important source of nutrition in the human diet. Tomato leaf senescence is a key process in determining fruit yield and nutritional value. It is accompanied by the degradation of chloroplasts and chlorophyll, the imbalance of redox homeostasis, and the remobilization of nutrients such as nitrogen from leaves to fruit. Research has found that delaying the onset and progression of leaf senescence can significantly increase tomato fruit yield and improve fruit quality.

[0003] Current research focuses on delaying the onset and progression of leaf senescence through exogenous addition of hormones and antioxidants or endogenous molecular genetic editing of plants. Numerous studies have found that plant growth-promoting rhizobacteria play an important role in promoting plant growth through a variety of mechanisms, including the production of plant hormones, promotion of the solubility of insoluble nutrients such as phosphate, nitrogen fixation, production of siderophores, and secretion of ACC deaminase. These positive effects have also been shown to persist across multiple growth and development stages of a plant's life cycle, such as promoting crop germination and emergence, promoting root growth, increasing fruit yield and quality, and regulating flowering and leaf development. For example, Streptomyces. Streptomyces, the most extensively studied actinomycete, produces a variety of bioactive compounds, including plant hormones such as auxins, making it a key player in plant growth-promoting rhizobacteria. Previous studies have found that the actinomycete Streptomyces rochei D74, as an excellent plant rhizosphere growth-promoting bacterium, plays a positive role in regulating plant growth and improving resistance to biotic / abiotic stresses.

[0004] However, the effect of S. rochei D74 inoculation on tomato leaf senescence, a specific and important plant developmental process, remains unclear. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems existing in the background technology, the present invention provides an application of Streptomyces rocheri D74 in delaying leaf senescence, which can improve the antioxidant enzyme activity of tomato leaves in the late growth and development period, maintain photosynthesis intensity and redox homeostasis, and delay the yellowing and senescence of tomato leaves.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of Streptomyces rocheri D74 in delaying tomato leaf senescence.

[0008] Application of Streptomyces rocheri D74 in delaying tomato leaf senescence by preventing chlorophyll degradation and / or maintaining photosynthesis intensity.

[0009] Streptomyces rocheri D74 is used to delay tomato leaf senescence by increasing antioxidant enzyme activity and / or maintaining cellular redox homeostasis. The antioxidant enzymes are SOD, CAT, APX, and / or POD; and maintaining cellular redox homeostasis is achieved by reducing MDA content and / or electrolyte leakage.

[0010] The invention relates to the application of Streptomyces rocheri D74 in delaying the senescence of tomato leaves by inducing changes in metabolites in tomato leaves, especially in delaying the senescence of tomato leaves by increasing metabolites in tomato leaves.

[0011] Among them, metabolites in tomato leaves include carbohydrates, tryptophan, phenylpropanoid compounds and / or flavonoid compounds.

[0012] Sugars include D-fructose-6-phosphate, D-glucose-6-phosphate, D-glucose-1-phosphate and / or uridine 5'-diphosphate-D-glucose;

[0013] The tryptophan includes 2-amino-3-methoxybenzoic acid, 2-(formylamino)benzoic acid, L-tryptophan, methoxyindoleacetic acid, tryptamine, N-hydroxytryptamine and / or serotonin;

[0014] The phenylpropanoid compounds include L-tyrosine, cinnamic acid, caffeic acid, 5-O-p-coumarylquinic acid and / or chlorogenic acid;

[0015] The flavonoids include 5-O-p-coumaroylquinic acid, chlorogenic acid, naringenin, quercetin, isoflavone, naringenin-7-O-glucoside, hesperetin-7-O-glucoside, quercetin-3-O-glucoside, quercetin-3-O-(2"-O-xylosyl)rutinoside, luteolin-7-O-neohesperidoside, petunidin-3-O-(6"-O-p-coumaroyl)rutinoside-5-O-glucoside, malvidin-3-O-(p-coumaroyl)rutinoside-5-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-(6-O-p-coumaroyl)glucoside and / or cyanidin-3-O-(6"-O-caffeoyl)glucoside.

[0016] The dosage of Streptomyces rocheri D74 is not less than 0.5 g of Streptomyces rocheri D74 per kilogram of culture medium, and the number of effective viable bacterial units of Streptomyces rocheri D74 is not less than 2×10 9 CFU·g -1Streptomyces rocherii D74 is a culture broth, a fermentation broth or a bacterial fertilizer containing Streptomyces rocherii D74.

[0017] The advantages of the present invention are:

[0018] The present invention provides an application of Streptomyces rochei D74 for delaying tomato leaf senescence. After inoculation with Streptomyces rochei D74, tomato leaf senescence can be delayed by inhibiting chlorophyll degradation and / or maintaining photosynthesis intensity, increasing antioxidant enzyme activity and / or maintaining cellular redox homeostasis, and inducing metabolite changes within tomato leaves, particularly increasing metabolites within tomato leaves. By observing the appearance of tomato leaves and measuring physiological indicators related to photosynthesis and redox systems, the present invention investigates the effects of S. rochei D74 on the senescence process of tomato leaves, aiming to identify new bacterial strain resources and potential approaches for using rhizosphere microorganisms to manipulate leaf senescence and thereby improve fruit yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison of the effects of control treatment (C) and D74 inoculation treatment (T) on tomato leaf senescence;

[0020] Figure 2 This is a comparison of the relative chlorophyll content in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0021] Figure 3 This is a comparison of the net photosynthetic rate of tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0022] Figure 4 This is a comparison of the transpiration rate of tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0023] Figure 5 This is a comparison of the stomatal conductance of tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0024] Figure 6 This is a comparison of the intercellular carbon dioxide concentration in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0025] Figure 7 is a comparison of the fresh weight of tomato fruit between the control treatment (C) and the D74 inoculation treatment (T);

[0026] Figure 8 This is a comparison of the SOD activity in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0027] Figure 9This is a comparison of the CAT enzyme activity in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0028] Figure 10 This is a comparison of the APX enzyme activity in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0029] Figure 11 This is a comparison of the POD enzyme activity in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0030] Figure 12 This is a comparison of the malondialdehyde content in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0031] Figure 13 This is a comparison of electrolyte permeability of tomato leaves between the control treatment (C) and the D74 inoculation treatment (T);

[0032] Figure 14 This is a comparison of the differences in metabolites in tomato leaves between the control treatment (C) and the D74 inoculation treatment (T). DETAILED DESCRIPTION

[0033] In order to explain the technical solution provided by the present invention in detail, the following embodiments are used:

[0034] 1. Test materials used in the present invention

[0035] 1.1 Test strains

[0036] Streptomyces rochei D74 (NCBI accession number KJ145878) was isolated from soil in the arid region of northwest China and deposited with the China Center for Type Culture Collection on March 14, 2016, under the accession number CCTCCM2016123. The strain used in this invention was purchased from the China Center for Type Culture Collection and is referred to as D74 in the following experiments. The test actinomycetes were then fermented into an actinomycete biofertilizer, which had an effective viable bacterial count of approximately 2 × 10 9 CFU·g -1 .

[0037] 1.2 Test plants

[0038] The tomato (Solanum lycopersicum L.) variety "Micro Tom" was purchased from Xi'an Jinpeng Seedling Company. Before the experiment began, several uniformly sized tomato seeds were sterilized and then soaked for germination. The specific steps were to sterilize with 75% alcohol for 30 seconds three times, then rinse five times with sterile water. After rinsing off any residual alcohol, the seeds were placed on two layers of damp sterile gauze in a sterile Petri dish and germinated at 25°C for 72 hours.

[0039] 1.3 Test soil

[0040] The test soil was Lou soil, collected from the topsoil layer (0-20 cm) of farmland in Yangling, Shaanxi Province (34°14′N, 107°59′E). Its pH was 7.2, total nitrogen content was 1.29 g / kg, total phosphorus content was 1.01 g / kg, available potassium content was 150 mg / kg, alkaline-hydrolyzable nitrogen content was 67.75 mg / mg, and available phosphorus content was 14.94 mg / kg. The soil was sieved (<1 cm) before use.

[0041] 1.4 Test matrix

[0042] The sterile seedling medium was purchased from Jiangsu Xuefu Agriculture Co., Ltd., and its main components were peat, perlite, and vermiculite (the ratio of the three was 3:1:1).

[0043] 1.5 Other tools

[0044] Seedling trays (50 holes, 48 ​​mm × 23 mm × 40 mm) and plastic flower pots (90 mm × 130 mm × 100 mm) were purchased from Jiangsu Xuefu Agriculture Co., Ltd.

[0045] 2. Potted plant experiment

[0046] This study investigated the effects of D74 on the senescence process of tomato leaves in the later stages of growth and development through potted plant inoculation experiments. Furthermore, changes in physiological indicators characterizing photosynthesis intensity and antioxidant capacity in tomato leaves after inoculation with D74 were measured to quantify its regulatory effect on the senescence process of tomato leaves.

[0047] 2.1 Potted experiment design

[0048] From September 2024 to December 2024, a potted experiment was carried out in the artificial climate chamber of the College of Resources and Environment, South Campus, Northwest Agriculture and Forestry University. The experiment set up a non-inoculated control treatment (C) and a D74 inoculation treatment (T), with 20 replicates for each treatment. The soil and substrate were mixed in a mass ratio of 2:1, no fertilizer was applied, and 5.0 kg of mixed soil was placed in each pot. For example, the D74 inoculation treatment group will additionally receive no less than 0.5 g kg –1Actinomycete fertilizer was evenly added to the mixed soil (2.5 g per pot), and no fertilizer was added to the control group. The germinated tomato seeds were sown in all pots, 1 seed per pot. Sterile distilled water was added to make the initial water holding capacity of the potting soil 60% of the maximum water holding capacity in the field, and 50 mL of sterile water was added to each pot every 7 days. All pots were randomly placed in an artificial climate chamber, and the positions of all pots were randomly adjusted each time watering was performed. The environment of the artificial climate chamber was set to a day and night temperature of 26°C / 20°C, a light and dark cycle of 12h / 12h, a light intensity of 12000Lx, and an air relative humidity of 65%.

[0049] 2.2 Tomato plant sample collection

[0050] 100 days after inoculation, the fifth true leaf from the top of the plant was collected for physiological measurement. Five replicates were randomly selected for each treatment, and representative leaves from the same location (the fifth true leaf from the top) were collected for epigenetic observation, physiological measurement, and metabolomics analysis. Ten replicates were used for single-cell transcriptome analysis. 115 days after inoculation, fruit was collected for yield measurement.

[0051] 2.3 Determination of plant phenotypic and physiological indicators

[0052] 2.3.1 Determination of tomato photosynthesis index

[0053] The SPAD value of leaves was measured using a SPAD chlorophyll meter, and the net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration of leaves were measured using a CIRAS-3 portable photosynthetic measurement system (Lufthansa, USA).

[0054] 2.3.2 Determination of related indicators of tomato antioxidant system

[0055] The malondialdehyde (MDA) content in tomato leaves was determined by the 2-thiobarbituric acid (TBA) method, and the specific determination method can be found in Yu Xiaoli (2007); the SOD enzyme activity in leaves was determined by the NBT photoreduction method, and the specific method can be found in Gao Junfeng (2006); the POD enzyme activity in leaves was determined by the guaiacol method, and the specific method can be found in He Yanhui (2020); the methods for determining the CAT enzyme and APX enzyme activities in leaves were found in Gao Junfeng (2006) and Jahan et al. (2019), respectively.

[0056] 2.4 Data Analysis

[0057] SPSS 23.0 (IBM USA) software was used for analysis of variance and multiple comparisons between the data. The least significant difference (LSD) method was used for difference significance analysis. P < 0.05 was considered a significant difference. The measurement results were presented as mean ± standard deviation, and the graphics were drawn using Origin 2023 software.

[0058] 2.5 Results and Analysis

[0059] 2.5.1 Evaluation of the effects of inoculation on the appearance and photosynthesis of leaves during natural aging

[0060] like Figure 1 As shown in the figure, the tomato leaves in the uninoculated control showed obvious yellowing and senescence. However, the tomato leaves in the same position inoculated with D74 did not show yellowing and showed the dark green characteristic of mature leaves.

[0061] The degree of leaf senescence can be firstly judged visually by whether the leaves are yellowing and the size of the yellowing area. Further, the senescence of plant leaves can be physiologically assessed by measuring the intensity of photosynthesis and the redox state. Figure 1 In the present invention, based on the obvious apparent color difference of tomato leaves at the same position under the two treatment groups, it is preliminarily judged that the inoculation of D74 can delay the occurrence and progression of the natural senescence process of tomato leaves.

[0062] like Figures 2 to 7 As shown, the SPAD value of tomato leaves in the D74 inoculation treatment group ( Figure 2 shown) and the net photosynthetic rate ( Figure 3 The transpiration rate of tomato leaves in the D74 inoculation treatment group ( Figure 4 ), stomatal conductance ( Figure 5 ) and intercellular CO2 concentration ( Figure 6 ) four physiological indicators also increased by 11.7%, 14.4% and 12.3% respectively compared with the control group. Figure 7 As shown in the figure, the fresh weight of mature tomatoes in the inoculated group also increased significantly (p<0.05) compared with the control group, with an increase of 16.2%.

[0063] In physiological measurements of photosynthesis, the SPAD value represents the relative chlorophyll content and, along with parameters such as net photosynthetic rate, transpiration, stomatal conductance, and intracellular carbon dioxide concentration, serves as an important indicator for assessing photosynthesis. In the present invention, the significantly increased SPAD value and net photosynthetic rate in tomato leaves in the D74 inoculation group compared to the control group indicate that inoculation can delay the onset and progression of leaf senescence by preventing chlorophyll degradation and the deterioration of photosynthetic capacity.

[0064] 2.5.2 Effects of inoculation on physiological indicators related to tomato leaf senescence (MDA, antioxidant enzyme activity)

[0065] The achievement of redox homeostasis depends on a dynamic balance between the production and clearance of reactive oxygen species (ROS). Within normal leaf cells, ROS are produced in large quantities primarily as a byproduct of photosynthesis. During leaf senescence, the degradation of photosensitive chlorophyll is accompanied by rapid ROS production. ROS clearance primarily relies on the antioxidant system, comprised of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), polyoxyethylene chlorinated diol (POD), and acetylcholine (APX), as well as antioxidants such as glutathione and ascorbic acid. SOD, the first line of defense against reactive oxygen species, dismutates superoxide to hydrogen peroxide (H2O2). Reductases such as APX, POD, and CAT then degrade the generated H2O2 to water. During plant leaf senescence, the gradual decline in antioxidant enzyme activity prevents ROS from being cleared promptly, leading to excessive intracellular accumulation. Excessive ROS can disrupt the integrity of the cytoplasmic membrane, leading to increased ion leakage, membrane lipid peroxidation, and the formation of the decomposition product malondialdehyde (MDA).

[0066] like Figures 8 to 13 As shown, the SOD of tomato leaves in the D74 inoculation treatment group ( Figure 8 )、CAT( Figure 9 )、APX( Figure 10 ) and POD( Figure 11 ) The activities of the four antioxidant enzymes were significantly increased compared with the control group (p<0.05), with increases of 46.5%, 46.2%, 50.6% and 31.4% respectively. In contrast, compared with the control group, the malondialdehyde (MDA) ( Figure 12 ) content and electrolyte leakage rate ( Figure 13 ) were significantly decreased (p<0.05), with the decrease rates being 28.6% and 51.6%, respectively.

[0067] In the present invention, compared with the control group, the tomato leaves in the inoculated group had significantly increased activities of four antioxidant enzymes and significantly reduced MDA content and electrolyte leakage rate, indicating that the inoculation of D74 can maintain cellular redox homeostasis by increasing antioxidant enzyme activity and avoiding damage and destruction of the cytoplasmic membrane, thereby delaying the aging of tomato leaves.

[0068] In this study, measurements of multiple physiological indicators demonstrated that inoculation with D74 functionally delayed tomato leaf senescence. Furthermore, the significantly increased fruit fresh weight after inoculation suggests that the D74-mediated delay in leaf senescence has a positive effect on fruit yield. Combined with measurements of photosynthesis indicators, it is speculated that this is likely due to the increased nutrient accumulation in the fruit by extending the photosynthesis period and improving photosynthetic efficiency during leaf growth and development.

[0069] Clearly, inoculation with D74 significantly delayed the onset and progression of natural leaf senescence in tomato plants and significantly increased fruit yield. Uninoculated tomato plants exhibited noticeable yellowing and senescence, while leaves inoculated with D74 showed no yellowing and retained their normal dark green color. Physiologically, D74 inoculation delayed the onset of leaf senescence by inhibiting chlorophyll degradation and maintaining photosynthetic activity. Furthermore, D74 inoculation increased the activity of four antioxidant enzymes—SOD, CAT, APX, and POD—in tomato leaves, reduced malondialdehyde levels and electrolyte leakage, mitigated cellular oxidative damage and membrane lipid peroxidation, and thus delayed leaf senescence. Through its positive effects on photosynthesis and redox homeostasis, D74 functionally delayed the onset and progression of natural leaf senescence in tomato plants, ultimately significantly increasing the fresh weight of tomato fruit.

[0070] 3. Study on the mechanism of action of D74 on tomato leaf senescence

[0071] 3.1 Materials and Methods

[0072] 3.1.1 Test materials

[0073] Same as 1.1, 1.2, 1.3, and 1.4.

[0074] 3.1.2 Sampling

[0075] Same as 2.2.

[0076] 3.1.3 Broad-target metabolome sequencing

[0077] Tomato leaf samples were freeze-dried in a freeze dryer (Scientz-100F) and, after complete drying, ground using a grinder (MM400, Retsch) at 30 Hz for 1.5 min. 100 mg of the powder was weighed and dissolved in 1.2 mL of 70% methanol extract (methanol:acetonitrile:water = 2:2:1, v / v / v) (Dunn et al. 2011). The extracted samples were vortexed six times, once every 30 min for 30 s, and then refrigerated at 4°C overnight. After centrifugation at 12,000 rpm for 10 min, the samples were filtered through a 0.22 μm pore size microporous membrane. The filtered solution was stored in a vial for UPLC-MS / MS analysis. The extracts were analyzed on a 2777C ultra-high performance liquid chromatography (UPLC) system (Waters, Massachusetts, USA) coupled to a Xevo G2-XS Q-TOF mass spectrometer (Waters) at Metrowell Biotechnology Co., Ltd. (Wuhan, China). Chromatographic separation was performed on an Agilent SB-C18 column (100 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution program was as follows: 5% B at 0.00 min; 5% to 95% B at 0–9.00 min; 95% to 5% B at 10–11.10 min; and 5% B at 11.10–14 min. The flow rate was 350 μL min. -1The column temperature was 40°C, and the injection volume was 4 μL. LIT and triple quadrupole (QQQ) scanning were performed using a triple quadrupole linear ion trap mass spectrometer (Q-TRAP) and an AB4500Q TRAP UPLC / MS / MS system. Analyst 1.6.3 software (AB Sciex) controlled the ionization mode, and mass spectrometry analysis was performed using the ESI Turbo ion spray interface. The operating parameters of the ESI source were set according to the method of Huo Dongao et al. (2022), with a turbojet as the ion source, a temperature set to 550°C, and an ion spray voltage (IS) controlled to 5500 V (positive ion) / -4500 V (negative ion). Ion source gas I (GSI), gas II (GSII), and gas source gas (CUR) were set to 50, 60, and 25.0 psi, respectively, and the collision ionization parameter was set to high. The instrument was set up and mass calibrated in QQQ mode using a 10 μmol / L polypropylene glycol solution. The polypropylene glycol concentration was increased to 100 μmol / L and the instrument and mass spectrometer were calibrated in the same manner in LIT mode. The QQQ scan was performed in MRM mode with the collision gas (nitrogen) set to medium. A specific set of MRM ion pairs was monitored based on the metabolites eluting in each period, and the mass spectrometric data were matched against a self-built database, MWDB (Matwell Biotechnology Co., Ltd.).

[0078] 3.2 Data Analysis

[0079] Off-line data analysis was performed using R v4.2.0 (https: / / www.r-project.org / ) using the method of Kuhl et al. (2011). Differential metabolites were identified based on the following criteria: Fold-Change ≥ 2.0, Vip ≥ 1, and p < 0.05. Dimensionality reduction was performed using principal component analysis (PCA). Pathway enrichment analysis of differential metabolites was performed using the KEGG database. Graphs were generated using Origin v2021 and R v4.2.0.

[0080] 3.3 Results and Analysis

[0081] 3.3.1 Effects of D74 inoculation on the metabolome of tomato leaves

[0082] Metabolome analysis of tomato leaves identified a total of 1,606 metabolites, which can be divided into 13 chemical categories: phenolic acids, flavonoids, lignans and coumarins, terpenoids, quinones, alkaloids, steroids, lipids, organic acids, amino acids and their derivatives, nucleotides and their derivatives, tannins, and other compounds. Alkaloids were the most common category, comprising 278 metabolites, or 17.3%. Other major compound categories include 208 lipid compounds, accounting for 13.0%, 196 phenolic acid compounds, accounting for 12.2%, 174 flavonoids, accounting for 10.8%, 166 amino acids and their derivatives, accounting for 10.3%, and 196 other compounds, accounting for 12.2%.

[0083] To compare the differences in metabolites in tomato leaves before and after D74 inoculation, principal component analysis was first performed on the metabolite data of the two groups. The results showed that the uninoculated and inoculated treatments were effectively separated on the first principal component (PC1, 34.7%; P < 0.05; Figure 14 ), indicating that there were significant differences in metabolites between the two groups.

[0084] A total of 860 metabolites were differentially accumulated in tomato leaves before and after inoculation with D74 (fold changes, FC > 2 or < 0.5, Table 1). Compared with the control (C), 700 differential metabolites (DAMs) were upregulated in the inoculated group (T), primarily including 138 alkaloids, 109 flavonoids, 94 phenolic acids, 89 lipids, 61 amino acids and their derivatives, and 70 other substances. On the other hand, 160 DAMs were downregulated, primarily including 25 steroids, 23 phenolic acids, 21 flavonoids, 19 amino acids and their derivatives, and 18 alkaloids.

[0085] Table 1 Statistics of differential metabolites

[0086]

[0087] 3.3.2 KEGG enrichment analysis of differential metabolites inoculated with D74

[0088] Metabolic pathway enrichment analysis of DAMs in tomato leaves was performed using the KEGG database. A total of 164 DAMs were significantly enriched in the KEGG database (P < 0.05, Table 2), accounting for 19.1% of all differentially expressed metabolites. As shown in Table 2, these DAMs were primarily enriched in multiple KEGG metabolic pathways, including starch and sucrose metabolism (ko00500), tryptophan metabolism (ko00380), phenylpropanoid biosynthesis (ko00360), flavonoid biosynthesis (ko00941), flavonone and flavonol biosynthesis (ko00944), and anthocyanin biosynthesis (ko00942).

[0089] Table 2 KEGG metabolic pathway enrichment statistics of differential metabolites

[0090]

[0091] (1) The starch and sucrose metabolic pathways related to carbohydrate metabolism were significantly enriched in four DAMs (p < 0.05, Table 3), including D-fructose-6-phosphate, D-glucose-6-phosphate, D-glucose-1-phosphate, and uridine 5'-diphosphate-D-glucose. After inoculation with D74, the content changes of these substances showed a significant upregulation (p < 0.05). Among them, D-fructose-6-phosphate, D-

[0092] The contents of glucose-6-phosphate and D-glucose-1-phosphate were similar to those of the control, which were 2.07, 2.09 and 2.05-fold higher.

[0093] The content of uridine 5'-diphosphate-D-glucose was increased by 2.84 times compared with the control (p < 0.05).

[0094] Table 3 Differentially enriched metabolites in the “Starch and sucrose metabolism” pathway

[0095]

[0096] (2) The tryptophan metabolic pathway significantly enriched 9 DAMs (p < 0.05), including 2-pyridine carboxylic acid, 2-amino-3-methoxybenzoic acid, 2-(formylamino)benzoic acid, L-tryptophan, methoxyindole acetic acid, tryptamine, N-acetyl-5-hydroxytryptamine, N-hydroxytryptamine, and serotonin. After inoculation with D74, the levels of 2-amino-3-methoxybenzoic acid, 2-(formylamino)benzoic acid, L-tryptophan, methoxyindole acetic acid, tryptamine, N-hydroxytryptamine, and serum were significantly upregulated (p < 0.05, Table 4). Among them, the levels of 2-(formylamino)benzoic acid and methoxyindole acetic acid increased most significantly, increasing by 16.6 and 5.19 times compared with the control, respectively (p < 0.05). L-tryptophan, tryptamine, N-hydroxytryptamine, and serotonin showed similar increases, increasing by 4.71, 4.64, 3.32, and 3.65 times, respectively, compared to the control (p < 0.05). 2-amino-3-methoxybenzoic acid showed a smaller increase, increasing by 2.46 times (p < 0.05).

[0097] Table 4 Differentially enriched metabolites in the “Tryptophan metabolism” pathway

[0098]

[0099] (3) The phenylpropanoid biosynthesis pathway significantly enriched five DAMs (p < 0.05, Table 5), including L-tyrosine, cinnamic acid, caffeic acid, 5-O-p-coumarylquinic acid, and chlorogenic acid. After inoculation with D74, the content of these substances showed a significant increase (p < 0.05). Among them, the increase in the content of L-tyrosine, caffeic acid, and chlorogenic acid was the most significant, increasing by 6.87, 4.64, 4.82, and 4.61 times, respectively, compared with the control (p < 0.05). The content of cinnamic acid and 5-O-p-coumarylquinic acid increased by 2.71 and 2.54 times, respectively, compared with the control (p < 0.05).

[0100] Table 5 Differentially enriched metabolites in the “phenylpropanoid biosynthesis” pathway

[0101]

[0102] (4) 18 differentially enriched substances were enriched in the biosynthesis-related pathways of flavonoids (including flavonoids, flavonols, anthocyanins, etc.). Among them, 8 DAMs were significantly enriched in the flavonoid biosynthesis pathway (p < 0.05), including 5-O-p-coumarylquinic acid, chlorogenic acid, naringenin, quercetin, isobarbitalin, naringenin-7-O-glucoside, hesperetin-7-O-glucoside, and hesperetin-7-O-neohesperidoside. After inoculation with D74, the relative contents of 5-O-p-coumarylquinic acid, chlorogenic acid, naringenin, quercetin, isobarbitalin, naringenin-7-O-glucoside, and hesperetin-7-O-glucoside were significantly increased (p < 0.05, Table 6). Among them, the differential accumulation of quercetin, isosalicylate, naringenin-7-O-glucoside and naringenin in response to inoculation was the most obvious, with their contents increased by 28.4, 24.6, 11.1 and 7.44 times compared with the control, respectively (p < 0.05).

[0103] Five DAMs were significantly enriched in the flavonoid and flavonol biosynthesis pathway (p < 0.05), including quercetin-3-O-sambubioside, quercetin-3-O-sophoroside, quercetin-3-O-glucoside, quercetin-3-O-(2"-O-xylosyl)rutinoside, and luteolin-7-O-neohesperidoside. After inoculation with D74, the levels of quercetin-3-O-glucoside, quercetin-3-O-(2"-O-xylosyl)rutinoside, and luteolin-7-O-neohesperidoside increased significantly, increasing by 3.10, 3.57, and 2.40 times compared with the control, respectively (p < 0.05, Table 6).

[0104] The anthocyanin biosynthesis pathway also showed significant enrichment of five DAMs (p < 0.05), including petunidin-3-O-(6-O-p-coumaryl)rutinoside-5-O-glucoside, malvidin-3-O-(p-coumaryl)rutinoside-5-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-(6-O-p-coumaryl)glucoside, and cyanidin-3-O-(6-O-caffeoyl)glucoside. After inoculation with D74, the levels of these substances were significantly upregulated (p < 0.05, Table 6). Among them, petunidin-3-O-(6”-O-p-coumaryl)rutinoside-5-O-glucoside was upregulated by 20.7 times, delphinidin-3-O-rutinoside was upregulated by 11.5 times, malvidin-3-O-(p-coumaryl)rutinoside-5-O-glucoside and cyanidin-3-O-(6”-O-caffeoyl)glucoside were both upregulated by 5.00 times, and cyanidin-3-O-(6-O-p-coumaryl)glucoside was upregulated by 2.92 times (p<0.05, Table 6).

[0105] Table 6 Differentially enriched metabolites in the “flavonoid biosynthesis”, “flavonoid and flavonol biosynthesis”, and “anthocyanidin biosynthesis” pathways

[0106]

[0107] 3.3.3 Conclusion

[0108] In plants, carbohydrates, as energy metabolites and signaling molecules, play an important role in regulating metabolism, growth and development, and stress resistance during the plant life cycle. Studies have found that during leaf aging, a decrease in photosynthetic activity leads to a decrease in the synthesis of carbohydrates such as D-fructose-6-phosphate and glucose-6-phosphate. The continued decrease in the content of these carbohydrates will serve as an induction signal for aging to accelerate the process of leaf aging. Consistent with this rule, inoculation of D74 in the present invention significantly increased the relative content of four carbohydrates, including glucose-6-phosphate and D-fructose-6-phosphate, thereby delaying the aging of tomato leaves. Among them, the increase in glucose-6-phosphate can not only provide key precursors for the synthesis of various bioactive substances such as hormones (such as auxins) and pigments (such as flavonols, anthocyanins) through glycolysis / pyruvate cycle, but has also been shown to promote the synthesis of phenolic compounds and, together with them, clear excess ROS in cells.

[0109] In addition to carbohydrates, small molecule amino acids are also involved in key physiological processes in plants, such as growth and development regulation, intracellular pH balance, and resistance to abiotic and biotic stresses. The aging of leaves is accompanied by the transformation of a large number of substances from anabolism to catabolism, especially the decomposition of proteins and amino acids. Studies have found that the exogenous addition of various amino acids such as tryptophan can delay the post-harvest aging of broccoli. Consistent with this, the inoculation of D74 in the present invention significantly increased the relative content of tryptophan and its various derivatives, including L-tryptophan, tryptamine, N-hydroxytryptamine, serotonin, etc. The accumulation of these substances can not only provide key precursors for the synthesis of growth-promoting or antioxidant functional substances such as auxin and melatonin, but has also been shown to delay the aging process of plant leaves.

[0110] As secondary metabolites produced through the phenylalanine and tyrosine pathways, phenylpropanoid compounds such as cinnamic acid, caffeic acid, and chlorogenic acid play an important role in regulating plant aging, stress resistance, and stress responses. In the present invention, inoculation with D74 significantly increased the relative levels of various substances in tomato leaves, including L-tyrosine, cinnamic acid, caffeic acid, and chlorogenic acid. The accumulation of these substances not only provides essential precursors for the synthesis of downstream lignin monomers and flavonoids but also delays plant aging through antioxidant mechanisms.

[0111] Flavonoids, a class of secondary metabolites widely distributed throughout plants, have been extensively studied for their crucial roles in protecting plants from environmental stresses such as strong light and pests and diseases, enhancing antioxidant capacity, and promoting nodulation and pollen fertility. Flavonoids can be categorized into six major groups based on the saturation and oxidation state of their carbon rings: chalcones, flavones, flavonoids, flavonols, anthocyanidins, and proanthocyanidins. Among these, flavonoids such as luteolin-7-O-neohesperidoside have been shown to possess antimicrobial and antioxidant properties and may play a key role in delaying rice leaf senescence. Similarly, flavonoids such as naringenin have been shown to alleviate osmotic stress in bean leaves by scavenging excess ROS and maintaining redox homeostasis. As the two most extensively studied classes of flavonoids, flavonols represented by quercetin-3-O-rutinoside and quercetin-3-O-glucoside, and anthocyanins represented by cyanidin-3-O-glucoside, not only have the ability to scavenge ROS free radicals, provide antioxidant protection, and delay cellular aging, but have also been shown to play a crucial role in photoprotection by absorbing ultraviolet and visible light, protecting plant leaves from excessive radiation. In the present invention, inoculation with D74 significantly increased the relative content of various flavonoids, including flavones, dihydroflavones, flavonols, and anthocyanins. The accumulation of these substances can delay leaf aging through various means, including scavenging ROS free radicals, enhancing antioxidant capacity, and absorbing ultraviolet light. Furthermore, the final step in flavonoid biosynthesis involves various chemical modifications of the basic flavonoid skeleton, including hydroxylation and glycosylation. These modifications increase their solubility and stability and have been shown to enhance their biological activity, including antioxidant activity. In the present invention, the inoculation of D74 not only promotes the accumulation of flavonoids, but also induces the hydroxylation and glycosylation modifications of these substances, thereby improving their biological activities.

[0112] In general, the present invention, through the measurement and analysis of broad-target metabolomics, found that inoculation with D74 significantly induced changes in metabolites in tomato leaves. Among the 860 compounds whose relative contents changed significantly after inoculation with D74, the relative contents of most substances (700) increased significantly. Through KEGG metabolic enrichment analysis, it was found that inoculation with D74 promoted metabolic pathways related to the biosynthesis of carbohydrates, amino acids, phenylpropanoid compounds, and flavonoid compounds, and significantly increased the relative contents of these metabolites. The accumulation of these primary metabolism or secondary metabolism-related substances can delay the occurrence and process of leaf aging in various ways, such as inhibiting the generation of aging-inducing signals, providing synthetic precursors for antioxidants, scavenging ROS free radicals, and absorbing harmful ultraviolet radiation.

Claims

1. Application of Streptomyces rocheri D74 in delaying tomato leaf senescence.

2. Application of Streptomyces rocheri D74 in delaying tomato leaf senescence by preventing chlorophyll degradation and / or maintaining photosynthesis intensity.

3. Application of Streptomyces rocheri D74 in delaying tomato leaf senescence by increasing antioxidant enzyme activity and / or maintaining cellular redox homeostasis.

4. The use according to claim 3, characterized in that: The antioxidant enzymes are SOD, CAT, APX and / or POD; and the method of maintaining cellular redox homeostasis is to reduce MDA content and / or reduce electrolyte leakage rate.

5. Application of Streptomyces rocheri D74 in delaying tomato leaf senescence by inducing metabolite changes in tomato leaves.

6. Application of Streptomyces rocheri D74 in delaying tomato leaf senescence by increasing metabolites in tomato leaves.

7. The use according to claim 5 or 6, characterized in that: The metabolites in the tomato leaves include sugars, tryptophan, phenylpropanoid compounds and / or flavonoid compounds.

8. The use according to claim 7, characterized in that: The sugars include D-fructose-6-phosphate, D-glucose-6-phosphate, D-glucose-1-phosphate and / or uridine 5'-diphosphate-D-glucose; The tryptophan includes 2-amino-3-methoxybenzoic acid, 2-(formylamino)benzoic acid, L-tryptophan, methoxyindoleacetic acid, tryptamine, N-hydroxytryptamine and / or serotonin; The phenylpropanoid compounds include L-tyrosine, cinnamic acid, caffeic acid, 5-O-p-coumarylquinic acid and / or chlorogenic acid; The flavonoids include 5-O-p-coumaroylquinic acid, chlorogenic acid, naringenin, quercetin, isoflavone, naringenin-7-O-glucoside, hesperetin-7-O-glucoside, quercetin-3-O-glucoside, quercetin-3-O-(2"-O-xylosyl)rutinoside, luteolin-7-O-neohesperidoside, petunidin-3-O-(6"-O-p-coumaroyl)rutinoside-5-O-glucoside, malvidin-3-O-(p-coumaroyl)rutinoside-5-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-(6-O-p-coumaroyl)glucoside and / or cyanidin-3-O-(6"-O-caffeoyl)glucoside.

9. The use according to any one of claims 1 to 6, characterized in that: The dosage of the Streptomyces rocherii D74 is not less than 0.5 g of Streptomyces rocherii D74 per kilogram of the cultivation medium, and the number of effective viable bacterial units of the Streptomyces rocherii D74 is not less than 2×10 9 CFU·g -1 .

10. The use according to claim 9, characterized in that: The Streptomyces rocherii D74 is a culture solution, a fermentation solution or a bacterial fertilizer containing the Streptomyces rocherii D74.

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