Method for constructing synthetic bacterial community in tea-leaf oil tree with disease prevention effect, constructed bacterial community and application thereof

Abstract

The present invention discloses a method for constructing a synthetic bacterial community with disease prevention effect in tea leaves, the constructed bacterial community and its application, which comprises: (1) collecting tea leaves, analyzing the bacterial community structure in the tea leaves, and obtaining the relative abundance ranking of the bacterial community; (2) analyzing the interaction network of the bacterial community in the tea leaves, and obtaining the centrality ranking of microbial species; (3) screening bacteria with antibacterial and siderophore producing abilities from the microorganisms in the tea leaves, and selecting tea leaves bacteria with both antagonistic and siderophore producing abilities to construct a community based on the relative abundance and centrality ranking of the community; (4) determining the leaf colonization time and anthracnose prevention effect of the synthetic bacterial community, and selecting the optimal synthetic bacterial community. The colonization time and prevention effect of the optimal synthetic bacterial community are significantly better than those of a single bacterium, and the systemic resistance of tea leaves can be induced. The method of the present invention can obtain a synthetic bacterial community with long-term colonization in the tea leaves, and has the effects of disease prevention and resistance induction.

Description

Technical Field

[0001] The invention belongs to the biotechnology field of constructing composite functional bacterial communities using microbial strains, and specifically relates to a method for constructing disease-resistant composite bacterial communities, composite functional bacterial communities and applications of the composite functional bacterial communities in preventing and controlling plant diseases. Background Art

[0002] The microbial communities of plants, also known as the plant microbiome (or plant microbiota), exist in the rhizosphere, phyllosphere, and within the plant. These plant microbiomes play an important role in improving plant immunity, inhibiting disease occurrence, providing nutrients required for survival, and protecting against biotic and abiotic environmental stresses.

[0003] The artificially created microbial community of two or more species co-cultured under known culture conditions is called a synthetic functional flora. The biological control of plant diseases is often based on the application of a single strain, but a single strain is difficult to adapt to the complex environment of the field, and some strains that work in the laboratory do not achieve the desired effect in the field. With the continuous development of research, the use of microbial communities or synthetic flora for control has gradually become a trend, with the goal of higher effectiveness, versatility and stability in the environment.

[0004] Phyllosphere microorganisms live in the special environment of the host plant and co-evolve with the host. On the one hand, the plant provides them with the energy and nutrients necessary for growth; on the other hand, phyllosphere microorganisms can affect the plant body through their own metabolites or through signal transduction. Phyllosphere microorganisms are closely related to the healthy growth of the host plant. Some leaf pathogens can cause plant diseases, while the diversity of non-pathogenic microorganisms on the leaves can protect the plants.

[0005] Camellia oleifera anthracnose is one of the main diseases of Camellia oleifera. The pathogen can infect the flower buds, leaf buds, fruits, branches and leaves of Camellia oleifera, usually causing the phenomenon of flower and fruit drop and branch withering of Camellia oleifera. In severe cases, it can cause the death of the whole plant. It occurs in all Camellia oleifera producing areas in my country. Each province (region) suffers from a 10% to 30% reduction in seed yield due to the disease each year, and 40% to 50% in severely affected areas. In typical forest stands, diseased buds account for 26% to 45% of the total number of buds dropped. Although diseased fruits can be harvested in the late stage, the oil content of the seeds is only half of that of healthy seeds, or even lower. At present, the prevention and control of anthracnose is mainly based on chemical control, but it is easy to cause environmental pollution and drug resistance, so it is urgent to develop biological control methods. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a method for constructing a synthetic flora of tea leaves with disease prevention effects, the flora thus constructed, and applications of the flora.

[0007] The present invention is based on the characteristics of the microbial community structure of the tea leaves and the disease resistance of the strains, and utilizes bacteria separated from the microorganisms of the tea leaves to construct a composite bacterial community with good colonization effect and disease resistance, thereby avoiding the ecological imbalance problem caused by the long-term use of a single bacterial agent.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The method for constructing a synthetic bacterial community in tea leaves with disease prevention effect comprises the following steps:

[0010] (1) Collect tea leaves, analyze the microbial community structure of the tea leaves, and obtain the relative abundance ranking of the bacterial community; (2) Analyze the interaction network of the bacterial community of the tea leaves, and obtain the centrality ranking of microbial species; (3) Screen bacteria with antibacterial and siderophore-producing abilities from the microorganisms of the tea leaves, and select tea leaves with the ability to antagonize tea pathogens and produce siderophores based on the relative abundance and centrality ranking of the community to construct a community; (4) Determine the leaf colonization time and anthracnose prevention effect of the synthetic bacterial community, and select the optimal synthetic bacterial community.

[0011] The construction method involves collecting healthy leaves of Camellia oleifera from at least three regions and three varieties for relative abundance and centrality ranking analysis and screening.

[0012] The construction method is to collect the main varieties of Hunan oil-tea camellia: common oil-tea camellia 'Huashuo', common oil-tea camellia 'Xianglin 210', and Youxian oil-tea camellia.

[0013] The present invention also provides a synthetic bacterial community in tea oil leaves with disease prevention effect, comprising:

[0014] At least two of Bacillus sp. H12 with a deposit number of CCTCC NO: M 20231284, Pseudomonas sp. H28 with a deposit number of CCTCC NO: M20231283, Bacillus sp. H6-9 with a deposit number of CCTCC NO: M 20231285, and Bacillus sp. H6-19 with a deposit number of CCTCC NO: M 20231286.

[0015] The synthetic flora of tea leaves with disease prevention effects preferably comprises any of the following combinations:

[0016] Bacillus sp.H12 and Bacillus sp.H6-9;

[0017] Bacillus sp.H12 and Bacillus sp.H6-19;

[0018] Pseudomonas sp.H28 and Bacillus sp.H6-19;

[0019] Bacillus sp.H12, Pseudomonas sp.H28 and Bacillus sp.H6-19;

[0020] Bacillus sp.H12, Bacillus sp.H6-9 and Bacillus sp.H6-19;

[0021] Pseudomonas sp.H28, Bacillus sp.H6-9 and Bacillus sp.H6-19;

[0022] Bacillus sp.H12, Pseudomonas sp.H28, Bacillus sp.H6-9 and Bacillus sp.H6-19.

[0023] The present invention also provides the application of the synthetic bacterial flora in tea oil leaves with disease prevention effect, namely, application in the prevention and treatment of anthracnose, especially application in the prevention and treatment of anthracnose in tea oil leaves.

[0024] The present invention also provides the application of the synthetic bacterial flora in the oil tea leaf with disease prevention effect, which is used to improve the activities of defense enzymes such as POD, SOD, PPO and PAL in the oil tea leaves.

[0025] The present invention also provides the application of the synthetic bacterial flora in the tea-leaf oleifera with disease prevention effect, which is used for inducing the up-regulation of the expression of resistance genes such as POD, SOD1 and PR1B1 of the tea-leaf oleifera.

[0026] The deposit information of the four strains of bacteria of the present invention is as follows:

[0027] Classification and nomenclature: Bacillus sp.H12, deposit number: CCTCC NO:M 20231284;

[0028] Classification and nomenclature: Pseudomonas sp.H28, deposit number: CCTCC NO:M 20231283;

[0029] Classification and nomenclature: Bacillus sp.H6-9, deposit number: CCTCC NO:M 20231285;

[0030] Classification name: Bacillus sp.H6-19, preservation number is CCTCC NO:M 20231286; preservation time is July 12, 2023.

[0031] Name of the collection center: China Center for Type Culture Collection

[0032] Deposit location: Wuhan University, Wuhan, Hubei Province.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The leaf colonization time of the synthetic bacterial community of the present invention is significantly higher than that of a single bacterium. Under indoor conditions, the number of viable bacteria of the synthetic bacterial community after inoculation for 30 days is 3.35 times that of a single bacterium. Under outdoor conditions, the number of viable bacteria of the synthetic bacterial community after inoculation for 30 days is 2.96 times that of a single bacterium. The synthetic bacterial community has a better control effect on anthracnose of oil tea than a single bacterium. The control effect of the synthetic bacterial community is as high as 100%, such as: the control effect of a single strain Bacillus sp. H6-9 is only 16.21%. The synthetic bacterial community of the present invention can significantly increase the activity of the defense enzyme of oil tea leaves, and can also significantly induce the up-regulation of the expression of oil tea resistance genes; it has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 :Composition and relative abundance of phyllospheric bacterial communities with genera greater than 5% in different cultivars and regions of Camellia oleifera;

[0036] Figure 2 :Interaction network of bacterial communities in tea oleifera leaves;

[0037] Figure 3 : Changes in defensive enzyme activities of Camellia oleifera leaves induced by synthetic microbiota;

[0038] Figure 4 : Expression of defense-related disease resistance genes in Camellia oleifera leaves induced by synthetic microbiota.

[0039] Figure 3 and Figure 4 CK: sterile water (control group); CF: pathogens; BA: synthetic flora; BF: pathogens were inoculated with synthetic flora 12 hours later. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described in detail below through specific embodiments, but the present invention is not limited to these embodiments.

[0041] Example 1

[0042] 1 Analysis of tea-leaf bacterial communities and interaction networks

[0043] 1.1 In May, June and late July 2021, oil-tea camellia samples were collected from three areas: Tianxin District (N 41°29.454′, W07′30.398°, C), You County (N 41°32.593′, W 07′07.445°, A) and Wangcheng (N 41°32.756′, W07′07.590°, B) in Changsha City, Hunan Province. The samples collected were Youxian oil-tea camellia, common oil-tea camellia ‘Huashuo’ and common oil-tea camellia ‘Xianglin 210’.

[0044] 1.2 Randomly select 3 healthy tea oil plants of each cultivar in each plot. Use sterilized scissors and gloves to randomly collect leaves from the selected tea oil plants, put them in sterile ziplock bags, mark the sample information and number them. Change gloves and sterilize scissors in time when taking different samples to prevent cross contamination.

[0045] 1.3 All samples were frozen in ice packs and sent back to the laboratory. Three strains of each cultivar were taken from each plot, for a total of 27 samples. Some samples were stored in a -80℃ refrigerator for microbial community analysis. Another part of the samples was temporarily stored in a 4℃ refrigerator for the isolation of leaf bacteria.

[0046] 1.4 Weigh 10g of Camellia oleifera leaves, cut them into pieces and put them into a sterile conical flask, add 200ml of sterile TE buffer (10mmol / L Tris-HCl, 1mmol / L EDTA, pH8.0). Place the conical flask containing the sample on a shaker at a speed of 200r / min for 30min, then place it in a 40kHz ultrasonic cleaner for 15min, and use a vacuum filtration device in a sterile environment to collect the microorganisms in the shaking liquid onto a 0.22μm filter membrane. The total genomic DNA of the microbial community was extracted according to the DNA Isolation Kit instructions, the quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop 2000.

[0047] 1.5 Sequencing was completed by Shanghai Meiji Biotechnology Co., Ltd. Using the above extracted DNA as a template, the upstream primer 515F (5'-GTGCCAGCMGCCGCGGTAA-3', see SEQ NO.1) carrying the Barcode sequence and the downstream primer 907R (5'-CCGTCAATTCMTTTRAGTTT-3', see SEQ NO.2) were used to perform PCR amplification of the V4-V5 variable region of the 16S rRNA gene, with 3 replicates for each sample.

[0048] 1.6 Use the R language vegan package to draw a Heatmap diagram to cluster samples according to the similarity of abundance, and use color changes to reflect the similarities and differences in community composition at the sample genus taxonomy level.

[0049] 1.7 The composition and relative abundance of tea-leaf bacterial communities were analyzed at the genus level, and the bacterial communities with a relative abundance of >5.00% were selected as the dominant bacterial communities. Figure 1 .

[0050] Networkx software was used to analyze and construct the network between microorganisms. Based on the spearman correlation, |r|>0.6p<0.05, species were selected for correlation network drawing. The results are shown in Figure 2 .

[0051] 1.8 In the leaf-sphere bacterial interaction network, the top five bacteria in terms of centrality are Exiguobacterium, Methylobacterium, Paenibacillus, Pseudonocardia, and Ochrobactrum. These species have high centrality and correlation. The community construction strains were selected from the genus ranked 30 in terms of centrality. The results are shown in Figure 2 .

[0052] 2 Screening and identification of antagonistic bacteria in tea oil leaves

[0053] 2.1 Screening of biocontrol bacteria for anthracnose of oil tea by confrontation culture method. First, the pathogen cake (5 mm in diameter) was inoculated in the center of the PDA solid culture medium plate, and then the leaf bacteria were inoculated in three triangular symmetrical positions 15 mm away from the center of the plate. Each treatment was repeated three times, and the plate inoculated with only pathogens in the middle was used as the control. Then the plate was placed in a 28°C constant temperature incubator and cultured for 7 days to observe whether there was an inhibition zone. A total of 214 strains of bacteria with antagonistic effects on C. fructicola were screened.

[0054] 2.2 Take 1 μL of the antagonistic bacterial solution of tea oil leaves as a PCR amplification template, and perform colony PCR on it with the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', see SEQ NO.3) and 1492R (5'-TACGACTTAACCCCAATCGC-3', see SEQ NO.4) of bacterial 16S rDNA. The amplified PCR stock solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the obtained DNA sequence was compared and analyzed on the NCBI website. Molecular biological identification showed that they mainly came from 6 genera, namely 139 strains of Bacillus, 42 strains of Pseudomonas, 19 strains of Paenibacillus, 7 strains of Stenotrophomonas, 5 strains of Microbacterium and 2 strains of Acinetobacter.

[0055] 3 Screening of siderophore-producing phyllospheric bacteria

[0056] 3.1 Qualitative screening of siderophore-producing phyllospheric bacteria After inoculating antagonistic bacteria from oil tea leaves on LB plates for 1 day, the colonies were inoculated on CAS solid test plates using a sterilized toothpick using the spot inoculation method. Three replicates were set for each strain and cultured in a 28°C incubator for 3 to 7 days. Observe whether a yellow halo, i.e., a siderophore, is formed around the colonies.

[0057] 3.2 Quantitative detection of iron carrier production capacity Use a toothpick to take the iron carrier-producing leaf bacteria for qualitative detection and place them in a conical flask containing 20 mL of MKB liquid culture medium, and shake and culture them at 200 r / min and 28°C for 2 days. Take 2 ml of the bacterial solution and centrifuge it in a 10,000 r / min high-speed refrigerated centrifuge for 15 minutes. Take 1 mL of the supernatant and add it to a centrifuge tube containing the same volume of CAS detection solution. Mix it thoroughly, keep it away from light for 1 hour, and then use an ultraviolet spectrophotometer to measure the absorbance of the reaction solution at a wavelength of 630 nm. 32 iron-producing strains were screened from 214 strains of antagonistic bacteria in the tea oleifera. (Table 1)

[0058] Table 1 Siderophore production capacity of antagonistic bacteria in tea oil leaves

[0059]

[0060] 4 Construction of synthetic bacterial community in tea oleifera

[0061] 4.1 Combining the relative abundance and centrality ranking of the community, 5 strains were selected as test strains from the 32 antagonistic bacteria against Colletotrichum oleifera that have the ability to produce siderophores (Table 2).

[0062] Table 2 Characteristics of strains used to construct synthetic bacterial communities

[0063]

[0064] 5. Colonization of synthetic flora on tea leaves

[0065] 5.1 Inoculation of leaves with synthetic flora Spread the disinfected leaves evenly on absorbent paper in a sterile operating table and spray 1 ml of 10% 10 CFU / mL of a single bacterial or combined bacterial suspension, in which various bacteria in the combined bacterial population are mixed in equal volumes (the concentration of each bacteria before mixing is 10 8 CFU / mL), set up 3 replicates, and after the leaves were dried, placed in a 150mm culture dish and cultured outdoors and indoors for moisturizing. (Table 3)

[0066] Table 3 Synthetic bacterial community members

[0067]

[0068]

[0069] 5.2 Observation of the colonization of synthetic flora The colonization was observed on five days: 1d, 3d, 7d, 15d, and 30d. The leaves were placed in a sterile triangular flask and 10mL of sterile water was added. The leaves were cleaned with ultrasound (40Hz) for 5min, and then placed in a constant temperature shaking incubator at 200r / min and 28℃ for 20min. Then the oil tea leaves were taken out and the liquid was divided into 5mL sterile centrifuge tubes. After centrifugation at 8000r / min for 5min, the supernatant was discarded and the precipitate was left. The volume was then made up to 5mL with sterile water. The suspension was diluted in different multiples according to the number of days. Take 100μL of each dilution and evenly spread it on the LB solid plate, set up 3 replicates, and place it in a constant temperature incubator at 28℃ for 2d, and count the number of colonies of the synthetic flora.

[0070] 5.3 The viable count of synthetic bacterial flora H12 / H28 / H6-9 / H6-19 on day 30 under indoor conditions was 9.4893×10 4 CFU / mL, the number of live bacteria of single bacteria H28 is 2.8294×10 4 CFU / mL, the synthetic flora was 3.35 times that of the single bacteria; the number of viable bacteria of the synthetic flora H12 / H28 / H6-9 / H6-19 on the 30th day under outdoor conditions was 5.4333×10 2 CFU / mL, the number of live bacteria of single bacteria H28 is 1.8314×10 2 CFU / mL, the synthetic flora was 2.96 times that of a single bacterium, and the leaf colonization effect of the synthetic flora was better than that of a single bacterium (Table 4, Table 5).

[0071] Determination of the efficacy of synthetic bacterial flora on the control of anthracnose in tea oil

[0072] 6.1 After disinfection of the leaf surface, inoculate with antagonistic bacteria. The inoculation method of antagonistic bacteria single strain and synthetic flora is shown in 5.1. Spraying with sterile water is used as a control. After the leaves are dried, use a sterilized toothpick to treat the wounds in a small area, and inoculate the fruit spiny disc spore cake with a diameter of 5mm on the surface of the oil tea leaves. Set up 10 replicates for each treatment. Put the treated leaves in a culture dish, put them in a 25℃ artificial climate box to moisturize and culture for 5 days, observe the infection status, calculate the disease index and control effect. Grade standard for anthracnose of oil tea: Grade 0: no lesions; Grade 1: lesion area <20%; Grade 2: 20% < lesion area <50%; Grade 3: 50% < lesion area <75%; Grade 4: lesion area >75%.

[0073] Disease index = (disease level × number of cases at that level) / (total number × highest disease level) × 100

[0074] Preventive effect (%) = (control disease index - treatment disease index) / control disease index × 100%

[0075] 6.2 The results of in vitro control test of tea leaves under different treatments are shown in Table 6. Table 6 shows that the control effect of the synthetic bacterial communities H12 / H28 / H6-9 / H6-19 and C2 / H12 / H6-9 / H28 is 100%, and the control effect of the single strain H6-9 is 16.21%. Based on this, the control effect of the synthetic bacterial community is significantly higher than that of the single strain. Therefore, the in vitro control effect of the synthetic bacterial community is better than that of the single strain.

[0076] Table 4 Colonization of single bacteria and synthetic bacteria in tea-leaf oleifera (indoor)

[0077]

[0078]

[0079] Table 5 Colonization of single bacteria and synthetic bacteria in tea-leaf oleifera (outdoor)

[0080]

[0081]

[0082] Table 6 The protective effect of synthetic bacterial flora on detached leaves

[0083]

[0084]

[0085] 7 Synthetic bacterial flora induces changes in defensive enzyme activities in tea leaves

[0086] 7.1 This experiment has 4 treatments, numbered CF, BA, BF and CK. CF is for spraying pathogens only, BA is for spraying H12 / H28 / H6-9 / H6-19 synthetic flora only, BF is for spraying synthetic flora 12 hours before spraying pathogens, CK is for sterile water (control group), and each treatment has 5 replicates. Take 200mL of sterile water for each treatment, 10 10 CFU / mL bacterial suspension (synthetic flora) or 10 8 CFU / mL spore suspension (pathogen) was evenly sprayed on individual tea plants and cultured under natural light. The various bacteria in the combined flora were mixed in equal volumes (the concentration of each bacteria before mixing was 10 8 CFU / mL),

[0087] 7.2 Extraction of crude enzyme solution A total of 10 time points were set for leaf collection, namely 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h and 72h after inoculation. Weigh 0.5g of leaves and put them into a mortar, add 10mL phosphate buffer (pH 7.8) and a small amount of quartz sand for grinding and homogenization, transfer to a 10mL centrifuge tube, centrifuge at 8000r / min for 15min, and the supernatant is the crude enzyme solution required for SOD, POD and PPO activity determination. Weigh 0.5g of leaves and put them into a precooled mortar, add 10ml boric acid buffer (pH 8.8) containing 5mmol / L mercaptoethanol, 0.5g polyvinyl pyrrolidone (PVP) and a small amount of quartz sand for grinding and homogenization, transfer to a 10mL centrifuge tube, centrifuge at 8000r / min for 15min, and the supernatant is the crude enzyme solution required for PAL activity determination.

[0088] 7.3 Enzyme activity determination The SOD, POD, PPO and PAL enzyme activities were determined using the Solebol kit.

[0089] 8 Synthetic bacterial flora induces the expression of defense-related disease resistance genes in tea leaves

[0090] 8.1 Extraction of total RNA Weigh 0.1 g of sample and grind thoroughly in liquid nitrogen, place in a 1.5 mL centrifuge tube, and then The total RNA of Camellia oleifera leaves was extracted according to the instructions of RNAprep Pure Plant Plus Kit. After extraction, the RNA samples that met the requirements were directly reverse transcribed, and the remaining RNA was stored in a -80℃ refrigerator for later use.

[0091] 8.2 cDNA synthesis and use cDNA was synthesized using ⅢRT SuperMix for qPCR (+gDNA Remover) kit and the synthesized cDNA was diluted 5 times before use.

[0092] 8.3 Fluorescence quantitative PCR (qRT-PCR) using TSINGKE TSE203 The Master qPCRMix kit was used to detect the expression of POD, SOD1, and PR1B1 genes in Camellia oleifera using cDNA as template and EF1-α gene as internal reference. The amplification primers are shown in Table 7. Primer synthesis was completed by Beijing Qingke Biotechnology Co., Ltd. (Changsha).

[0093] Table 7 Primers used in qRT-PCR

[0094]

[0095] The primers in Table 7 are shown in SEQ NO. 5-12 respectively.

[0096] 8.4 Inoculation of C. fructicola on the surface of tea leaves can significantly increase the activities of defense enzymes such as POD, SOD, PPO and PAL in tea leaves. Figure 3 .

[0097] 8.5 Quantitative analysis of resistance genes in Camellia oleifera showed that the synthetic bacterial community induced the upregulation of the expression of resistance genes such as POD, SOD1 and PR1B1 in Camellia oleifera. Figure 4 .

Claims

1. A synthetic flora of tea-leaf oil leaves with disease prevention effects, characterized in that: Contains: CCTCC NO:M20231284 Bacillus sp. H12 、 The deposit number is CCTCC NO:M 20231283 Pseudomonas sp. H28, with a deposit number of CCTCC NO: M 20231285 Bacillus sp. H6-9, CCTCC NO:M20231286 Bacillus sp. At least two of H6-19.

2. The synthetic flora of tea-leaf oil leaf having disease prevention effect according to claim 1, characterized in that: Contains any combination of the following: Bacillus sp. H12 and Bacillus sp. H6-9; Bacillus sp. H12 and Bacillus sp. H6-19; Pseudomonas sp. H28 and Bacillus sp. H6-19; Bacillus sp. H12 、Pseudomonas sp. H28 and Bacillus sp. H6-19; Bacillus sp. H12 、Bacillus sp. H6-9 and Bacillus sp. H6-19; Pseudomonas sp. H28, Bacillus sp. H6-9 and Bacillus sp. H6-19; Bacillus sp. H12 、Pseudomonas sp. H28, Bacillus sp. H6-9 and Bacillus sp. H6-19.

3. The use of the synthetic bacterial flora of tea leaves with disease prevention effect as claimed in claim 1 or 2, characterized in that: Used for the prevention and control of anthracnose of tea oil plants.

4. The use of the synthetic flora of tea leaves with disease prevention effect according to claim 3, characterized in that: Used to increase the activity of POD, SOD, PPO and PAL defense enzymes in tea leaves.

5. The use of the synthetic flora of tea-leaf oil leaf having disease prevention effect according to claim 3, characterized in that: Used to induce tea oil POD , SOD1 and PR1B1 The expression of resistance genes was upregulated.

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