A complex microbial agent for preventing and treating citrus huanglongbing, and a preparation method and application thereof

Abstract

The present application relates to a kind of complex microbial inoculant for preventing and treating citrus Huanglongbing and its preparation method and application.The complex microbial inoculant for preventing and treating citrus Huanglongbing includes 5-10 parts of Bacillus subtilis X1, 5-10 parts of Bacillus pumilus X2, 5-10 parts of Bacillus licheniformis X3, 5-10 parts of Bacillus amyloliquefaciens X4 and 5-10 parts of Pseudomonas fluorescens X5 by weight.After adding the complex microbial inoculant, the content of callose in the diseased leaf can be effectively reduced, the mechanism of self-resistance of plant is induced, the disease resistance is improved, and the Huanglongbing bacteria has obvious inhibitory effect and growth promotion effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of prevention and treatment of citrus Huanglongbing, and in particular to a compound microbial agent for preventing and treating citrus Huanglongbing, and a preparation method and application thereof. BACKGROUND

[0002] Citrus is a typical subtropical evergreen fruit tree, and it prefers warm and humid climate and has strong adaptability but weak cold resistance. Citrus Huanglongbing has caused great damage to the global citrus industry because it can infect almost all citrus plants, whether sweet orange, grapefruit, lemon or various hybrids, and has high infectivity and is difficult to eradicate. The leaves of plants infected with Huanglongbing bacteria usually show symptoms such as mottled yellowing of leaves, yellowing of leaf tips, woodiness of leaf veins, small and dull fruits, uneven distribution of green and yellow ("red-nosed fruits"), etc. Some symptoms are similar to citrus plant deficiency, such as leaf yellowing. In the later stage of Huanglongbing, the root system of the plant will rot, and the xylem layer will turn black, which seriously affects the absorption of soil nutrients by the plant.

[0003] The use of pharmaceutical preparations plays an important role in the prevention and control of citrus Huanglongbing and is an indispensable part. Chen Shiqin et al. (2014) conducted an 8-month infusion test on the phloem of citrus plants using 6 kinds of drugs, and found that DBNPA, MIT and AMP had a 90.0% control effect on Huanglongbing, proving that the use of antibiotics can effectively control the spread of the disease. Similar research results also show that antibiotics such as TET and OTC can reach the goal of preventing and controlling Huanglongbing by methods such as tree trunk injection and soaking infected branches (Han et al., 2021; Archer et al., 2022). Huang Yang (2021) explored the inhibitory effect of 2 antibiotics, SDM and AMP, on the Huanglongbing bacteria of "Newhall" navel orange and their effects on the bacterial community structure in the rhizosphere of diseased navel orange trees. The results showed that AMP antibiotics showed a certain inhibitory effect on Huanglongbing bacteria, while SDM had poor inhibitory effect on Huanglongbing bacteria, but the addition of antibiotic agents affected the structure of the bacterial community in the rhizosphere of diseased navel orange trees.

[0004] A research team isolated a strain of Bacillus subtilis, named Bacillus subtilis L1-21 (B. subtilis L1-21), which is a native endophyte isolated from healthy citrus plants. The bacteria were cultured in LB broth medium in a microbial shaker until the late growth stage, then 0.1% Tween-20 was added in proportion to mix, and the bacterial liquid was sprayed on the citrus leaves. Through indoor and field experiments, it was found that the bacteria could effectively prevent and control the citrus Huanglongbing bacteria, which was the first time to use citrus endophyte to prevent and control Huanglongbing disease, and it was also the first successful large-scale biological technology to prevent and control Huanglongbing disease in the world (Munir et al., 2021). However, due to the large differences in indigenous microbial communities and soil heterogeneity in different regions, direct application of a single beneficial strain may not be stable and obvious, and a single strain may have limitations in adapting to environmental changes and metabolic division of labor. SUMMARY

[0005] The purpose of the present application is to provide a complex bacterial agent for preventing and treating citrus Huanglongbing disease and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] A complex bacterial agent for preventing and treating citrus Huanglongbing disease, comprising, by weight: 5-10 parts of Bacillus subtilis X1, 5-10 parts of Bacillus pumilus X2, 5-10 parts of Bacillus licheniformis X3, 5-10 parts of Bacillus amyloliquefaciens X4 and 5-10 parts of Pseudomonas fluorescens X5, wherein the preservation number of Bacillus subtilis X1 is GDMCC NO:65614, the preservation number of Bacillus pumilus X2 is GDMCC NO:65272, the preservation number of Bacillus licheniformis X3 is GDMCC NO:65587, the preservation number of Bacillus amyloliquefaciens X4 is GDMCC NO:65273, and the preservation number of Pseudomonas fluorescens X5 is GDMCC NO:65274.

[0008] Bacillus subtilis X1 is preserved in Guangdong Microbial Culture Collection Center, the address of which is No. 59, Building 5, 100, Jiefang Road, Guangzhou, and the preservation time is December 12, 2024.

[0009] Bacillus pumilus X2 is preserved in Guangdong Microbial Culture Collection Center, the address of which is No. 59, Building 5, 100, Jiefang Road, Guangzhou, and the preservation time is December 06, 2024.

[0010] Bacillus licheniformis X3 is preserved in Guangdong Microbial Culture Collection Center, the address of which is No. 59, Building 5, 100, Jiefang Road, Guangzhou, and the preservation time is December 06, 2024.

[0011] Bacillus amyloliquefaciens X4 is preserved in Guangdong Microbial Culture Collection Center, the address of which is No. 59, Building 5, 100, Jiefang Road, Guangzhou, and the preservation time is October 15, 2024.

[0012] Pseudomonas fluorescens X5 is preserved in Guangdong Microbial Culture Collection Center, the address of which is No. 59, Building 5, 100, Jiefang Road, Guangzhou, and the preservation time is December 06, 2024.

[0013] In one preferred embodiment, the quantity ratio of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens and Pseudomonas fluorescens in the complex microbial agent for preventing and treating Huanglongbing of citrus is 1-2:1-3:1-2:1-2:1-3.

[0014] In one preferred embodiment, the concentration of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens and Pseudomonas fluorescens in the complex microbial agent for preventing and treating Huanglongbing of citrus is independently 5.5×10 7 -1×10 8 cfu / mL.

[0015] Based on the same inventive concept, the present application also claims the preparation method of the complex microbial agent for preventing and treating Huanglongbing of citrus, which comprises: inoculating Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens and Pseudomonas fluorescens in PDB medium respectively, and culturing under the condition of 30±5℃ and 100-200r / min for 24-48h to obtain five kinds of bacterial suspensions, and mixing the bacterial suspensions in proportion to obtain the complex microbial agent for preventing and treating Huanglongbing of citrus.

[0016] Based on the same inventive concept, the application also claims the use of the complex microbial agent for preventing and treating citrus Huanglongbing in promoting plant growth.

[0017] The addition of the complex microbial agent can effectively reduce the content of callose in the diseased leaves, improve the activities of three defense enzymes, POD, SOD and CAT, and improve the disease resistance.

[0018] Based on the same inventive concept, the application also claims the use of the complex microbial agent for preventing and treating citrus Huanglongbing in promoting plant growth.

[0019] The application of the complex microbial agent significantly increases the stem width, seedling height, fresh weight, dry weight, chlorophyll and root activity of the seedling, and has a significant growth-promoting effect.

[0020] In one preferred embodiment, the application includes directly irrigating the complex microbial agent for preventing and treating citrus Huanglongbing to the roots of the plants.

[0021] In one preferred embodiment, the application includes spraying the complex microbial agent for preventing and treating citrus Huanglongbing to the leaves of the plants after dilution.

[0022] In one preferred embodiment, the complex microbial agent for preventing and treating citrus Huanglongbing is sprayed after dilution with Tween.

[0023] In one preferred embodiment, the volume ratio of the complex microbial agent to Tween is 200-600:1.

[0024] In one preferred embodiment, the Tween is one or more of Tween 20, Tween 60 or Tween 80.

[0025] In one preferred embodiment, the application includes directly irrigating the complex microbial agent for preventing and treating citrus Huanglongbing to the roots of the plants, and spraying the complex microbial agent for preventing and treating citrus Huanglongbing to the leaves of the plants after dilution.

[0026] In one preferred embodiment, the plants are citrus.

[0027] The application is further explained as follows:

[0028] The five bacterial agents of the present application produce obvious synergies, on the one hand, because different bacterial agents have different metabolic pathways and functions, which can complement each other's functions, and together promote plant growth and inhibit diseases. On the other hand, the combination of multiple bacterial agents inhibits the growth of pathogenic bacteria through multiple mechanisms, such as producing antibacterial substances, competing for nutrients, secreting cell wall-degrading enzymes, etc. Secondly, different bacterial agents occupy different ecological niches in the soil, and through their combination, they can more comprehensively cover the ecological niches in the soil, thereby more effectively utilizing resources and space. At the same time, different bacterial agents promote plant growth through different mechanisms, such as promoting plant root development, improving plant nutrient absorption efficiency, regulating plant internal hormone levels, etc.

[0029] The complex bacterial agent of the present application can prevent and control Huanglongbing disease and promote plant growth. On the one hand, the beneficial bacteria in the bacterial agent can compete with Huanglongbing bacteria in the plant for nutrients and ecological niches in the phloem, thereby reducing the number and infection opportunities of the pathogen. On the other hand, some bacterial agents can induce plants to produce systemic resistance and activate the immune system of the plant, making the plant more resistant to Huanglongbing bacteria infection. At the same time, the complex bacterial agent of the present application can improve the soil environment and promote plant health, enhance the plant's own immunity, and also promote the circulation of nutrients such as nitrogen, phosphorus, and potassium in the soil, so that the plant can more effectively utilize these nutrients and promote healthy growth.

[0030] The present application found through experiments that the treatment of foliar spraying of bacterial agent and the treatment of simultaneous root irrigation and foliar spraying group have the best effect, both of which make the positive rate of citrus seedlings decrease by 75%, followed by the root irrigation group, after which the seedling positive rate decreases by 70%. The seedlings in the NPK fertilizer treatment group do not change in positive rate, maintaining at 100%. At the same time, the addition of the complex bacterial agent can effectively reduce the content of callose in the diseased leaves. The addition of the complex bacterial agent can improve the activities of three defense enzymes, POD, SOD, and CAT, in citrus seedlings, indicating that the addition of the complex bacterial agent can induce the plant to produce self-resistance mechanism to improve disease resistance. Therefore, the addition of the complex bacterial agent has a significant inhibitory effect on Huanglongbing bacteria.

[0031] In addition, the application of the complex microbial agent significantly increased the stem width, seedling height, fresh weight, dry weight, chlorophyll and root activity of citrus seedlings with Huanglongbing disease, and had a significant growth-promoting effect. Among them, the effect of the JS treatment (simultaneous irrigation and foliar spraying) was the best, and the growth amount of the six indicators was the highest. It is speculated that this may be due to the fact that roots and leaves are the two main parts of plant absorption and metabolism. The main function of the root system is to absorb water and nutrients, while the photosynthesis of the leaf provides energy. On this basis, by irrigating the two parts at the same time, the synergistic effect between the root system and the leaf can be effectively promoted, thereby improving the overall health status and growth efficiency of the plant. The addition of the complex microbial agent significantly reduced the accumulation of starch and soluble sugar caused by Huanglongbing disease, and promoted the transformation of starch. Among them, the PY treatment (foliar spraying) and the JS treatment (simultaneous irrigation and foliar spraying) had relatively outstanding effects. It is speculated that foliar spraying can directly and rapidly act on the disease site, and the leaf has good absorption capacity, and the comprehensive factors lead to such results. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Plate streaking of citrus rhizosphere and phyllosphere microorganisms;

[0033] Figure 2 Antagonistic test of citrus rhizosphere and phyllosphere microorganisms;

[0034] Figure 3 Morphological observation of antagonistic strains;

[0035] Figure 4 Phylogenetic tree of five antagonistic strains; wherein 4a is the phylogenetic tree of the X1 strain; 4b is the phylogenetic tree of the X1 strain; 4c is the phylogenetic tree of the X1 strain; 4d is the phylogenetic tree of the X1 strain; and 4e is the phylogenetic tree of the X1 strain;

[0036] Figure 5 Verification effect of the complex microbial agent;

[0037] Figure 6 Seedling height and stem width of citrus seedlings under different treatments;

[0038] Figure 7 Chlorophyll content of citrus seedlings under different treatments;

[0039] Figure 8 Correlation results of leaf starch and absorbance value and leaf starch content of citrus seedlings under different treatments; wherein Figure 8 a is a correlation graph of leaf starch and absorbance value, Figure 8 b is a column chart of leaf starch content of citrus seedlings under different treatments;

[0040] Figure 9 Correlation results of leaf soluble sugar content and absorbance value and leaf soluble sugar content of citrus seedlings under different treatments; whereinFigure 9 a is the correlation graph of callose content and absorbance value, Figure 9 b is the column chart of callose content of leaves of citrus seedlings of different treatments;

[0041] Figure 10 is the correlation result of callose content and absorbance value and the callose content of leaves of citrus seedlings of different treatments; wherein Figure 10 a is the correlation graph of callose content and absorbance value, Figure 10 b is the column chart of callose content of leaves of citrus seedlings of different treatments;

[0042] Figure 11 is the correlation result of TIF content and absorbance value and the root activity of citrus seedlings of different treatments; wherein Figure 11 a is the correlation graph of TIF content and absorbance value, Figure 11 b is the column chart of root activity of leaves of citrus seedlings of different treatments;

[0043] Figure 12 is the change of POD enzyme activity of leaves of citrus seedlings of different treatments with time;

[0044] Figure 13 is the change of SOD enzyme activity of leaves of citrus seedlings of different treatments with time;

[0045] Figure 14 is the change of CAT enzyme activity of leaves of citrus seedlings with time. DETAILED DESCRIPTION

[0046] The principles and features of the present application are described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art is used, or the product specification is used. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0047] The instruments and reagents required during the determination process: applicator / inoculation loop, alcohol lamp / alcohol cotton, culture dish, pipette, clean bench, high-pressure steam sterilization pot and constant temperature incubator; distilled water, beef extract peptone (LB), potato dextrose agar medium (PDA), potato dextrose liquid medium (PDB).

[0048] All data processing and statistical analysis methods of the present application are as follows:

[0049] Barcode sequence identification was used to accurately pair-end sequence assembly combined with FLASH online tools. Fastp software was used to control data quality and remove low-quality sequences. Subsequently, the reference database was compared to exclude chimeric pollution. With the help of QIIME2 software platform, deep sequence denoising, species annotation and phylogenetic tree construction were performed to generate Amplicon Sequence Variants (ASVs) set and corresponding feature abundance matrix.

[0050] By using SPSS24 software, the significant differences between the two groups of data were evaluated based on independent sample t-test. To further reveal the heterogeneity of microbial community structure, non-metric multidimensional scaling analysis (NMDS) was used to intuitively display the distance and similarity between communities. For the analysis of the correlation between soil chemical factors and protist community, redundancy analysis (RDA) and correlation heatmap were realized by using Lianchuan biological cloud platform. LEfSe analysis and correlation coefficient plot were completed by using NovoCloud platform. Mantel analysis was realized by using R software vegan package and MASS package. The completion of the rest of the charts also relies on bioinformatics cloud platform, Excel 2021, Origin 2022 software and AI (Adobe Illustrator).

[0051] Example 1

[0052] 1 Screening of citrus rhizosphere and phyllosphere strains

[0053] Dilution coating method was used to isolate and purify bacteria and fungi in soil fine roots. 5g of healthy and diseased citrus soil fine roots were weighed and cut into 0.1cm size, then ground thoroughly with a sterile mortar. After mixing, 100ml of sterile water was added to the 250ml flask, the shaking bed temperature was set to 30°C and the rotation speed was set to 180r / min. The mixed solution was shaken for 30min. 1ml of the supernatant was transferred to 9ml of sterile water, mixed evenly, and then diluted to 10 -2 to 10 -9Afterwards, 60 μL of the dilution was added to Luria Bertani (LB) medium (for culturing bacteria) and Potato Dextrose Agar medium (PDA) respectively, and evenly spread, 3 plates were spread for each gradient, if the plate microbial growth is too dense, according to the above method again after dilution and spread. Set the incubator temperature to 37°C (bacteria) and 28°C (fungi) respectively, and place the spread plates in the incubator upside down, and incubate for 1-3 days. Using the plate streak method, pick up a single colony with different colony characteristics, and culture and purify until a single colony appears on the medium. The purified strain is inoculated into Luria Bertani medium and Potato Dextrose Agar medium respectively, and stored in a 4°C refrigerator for standby.

[0054] Screening of phyllosphere strains: 5 g of healthy and diseased citrus leaves were weighed respectively, cut as much as possible and ground in a mortar, then mixed together, placed in a 250 ml flask and added with 100 ml of sterile water, and the rest of the steps were the same as the above-mentioned rhizosphere strain screening method.

[0055] The results obtained are shown in Figure 1 , wherein Figure 1 a is the colony of citrus rhizosphere microbial plate streaking and purification, Figure 1 b is the colony of phyllosphere plate streaking and purification.

[0056] 2Primary screening of functional bacteria

[0057] Using the relative bacteria of Huanglongbing bacteria, Sinorhizobium meliloti, the microorganisms with antibacterial performance screened by plate confrontation method. On the PDA plate, use a microsyringe or a seeding tool to evenly spread or seed the bacterial liquid of the candidate antibacterial microorganism on one side or the periphery of the plate, and inoculate the target relative bacteria on the opposite position of the plate, and use a puncher to make a 5mm diameter bacterial cake and place it at a certain distance from the candidate microorganism. Adjust the constant temperature incubator to 30°C, place the inoculated plate in it, and incubate for 2-3 days. Observe the growth of the pathogenic bacteria, if the candidate microorganism or its metabolites have antibacterial activity, a non-growing area, i.e. an antibacterial circle, will be formed between the target strain and the inoculation point of the microorganism to be screened. Measure the diameter of the antibacterial circle with a vernier caliper, the larger the diameter of the antibacterial circle, the better the antibacterial effect.

[0058] Using the relative bacteria of Huanglongbing bacteria, Sinorhizobium meliloti, the microorganisms with antibacterial performance screened by plate confrontation method. On the PDA plate, use a microsyringe or a seeding tool to evenly spread or seed the bacterial liquid of the candidate antibacterial microorganism on one side or the periphery of the plate, and inoculate the target relative bacteria on the opposite position of the plate, and use a puncher to make a 5mm diameter bacterial cake and place it at a certain distance from the candidate microorganism. Adjust the constant temperature incubator to 30°C, place the inoculated plate in it, and incubate for 2-3 days. Observe the growth of the pathogenic bacteria, if the candidate microorganism or its metabolites have antibacterial activity, a non-growing area, i.e. an antibacterial circle, will be formed between the target strain and the inoculation point of the microorganism to be screened. Measure the diameter of the antibacterial circle with a vernier caliper, the larger the diameter of the antibacterial circle, the better the antibacterial effect. Figure 2 a).

[0059] 3Rescreening of citrus Huanglongbing bacteria antagonistic strains

[0060] Reference Munir et al. (2021) Breeding of Huanglongbing-suppressive bacteria using the "double leaf method". (1) Biocontrol bacteria culture and preparation: First, inoculate the candidate biocontrol bacteria with the potential to suppress citrus Huanglongbing into PDB medium, set the shaking bed temperature to 30°C, and the rotation speed to 170 r / min. Under this condition, shake culture for 48 h. After the culture is completed, collect the bacterial cells by centrifugation (12000 r / min, 10 min), discard the supernatant, resuspend the bacterial cells with 0.01M PBS buffer, and adjust the concentration of biocontrol bacteria in the bacterial suspension to about 1x10 8 cfu / mL for subsequent use. (2) Disease leaf pretreatment and pathogen content detection: Collect citrus leaves with citrus Huanglongbing and cut them into two halves horizontally from the middle. Take the lower half of the leaf midrib and use qPCR technology to detect the content of citrus Huanglongbing pathogen, which is used as the control value before the experiment. (3) Biocontrol bacteria treatment and culture: Place the other half of the leaf in a sterile culture dish, lay a wet filter paper on the bottom, and spray it with potential biocontrol bacteria suspension added with Tween-20 (the ratio of bacterial suspension to Tween-20 is 500:1), ensuring that the leaf is evenly covered on both sides. Each treatment group contains at least 12 leaves, and the experiment is repeated three times. After the leaf is dried after spraying the bacterial suspension, it is placed in a constant temperature incubator at 30°C for 4 days. (4) Detection of pathogen content after treatment and calculation of control effect: After 4 days, take the leaf midrib of the treated half leaf, extract DNA using the CTAB method, and detect the content of citrus Huanglongbing pathogen again using qPCR. By comparing the changes in pathogen content before and after treatment, the control effect of biocontrol bacteria is calculated. (5) Selection of high-efficiency biocontrol bacteria: According to the control effect, high-efficiency suppressive bacteria strains are selected, and the strain with the highest control effect is considered to be a biocontrol bacteria with good control effect on citrus Huanglongbing. The control group is sprayed with the same amount of water or culture medium for comparison. The calculation formula is as follows:

[0061] Citrus Huanglongbing bacteria reduction rate (%) = (pre-treatment pathogen content - post-treatment pathogen content) / pre-treatment pathogen content x 100% (2-1)

[0062] The results are as follows: The preliminarily selected suppressive bacteria are re-screened using the "double leaf method", and 7 strains of suppressive bacteria with antagonistic effect on Huanglongbing bacteria are obtained (Table 1).

[0063] Table 1 Inhibition rate of suppressive bacteria on Huanglongbing bacteria

[0064]

[0065]

[0066] Among them, GX1 and GX3 have the best inhibition effect on Huanglongbing, reaching 79.67% and 88.3% respectively, as shown in Table 1. The obtained suppressive bacteria strains are then subjected to antagonistic test (Figure 2 b), finally 5 strains of non-antagonistic pathogen-inhibiting bacteria were obtained, Figure 3 a-e are Bacillus subtilis X1, Bacillus pumilus X2, Bacillus licheniformis X3, Bacillus amyloliquefaciens X4 and Pseudomonas fluorescens X5, respectively. Figure 3 ).

[0067] The above pathogen-inhibiting bacteria were combined, and the inhibition rate of the combined bacteria on HLB pathogen was tested. The test method was the same as above. The results are shown in the following table.

[0068] Table 2 Inhibition rate of combined bacterial agent on HLB pathogen

[0069]

[0070] Note that the combination without the proportion is combined according to the same mass and the same concentration.

[0071] It can be seen that YX1+YX2+GX1+GX2+GX3 has the best effect after being combined according to the same mass and the same concentration.

[0072] 4. Morphological and molecular identification

[0073] (1) Colony purification: the colonies obtained by re-screening were inoculated on PDA plates by multiple plate streaking for strain purification to obtain single colonies. The plates were placed in a 30°C constant temperature incubator for 2 days. After single colonies appeared, the shape, size, edge characteristics (such as smooth or rough, regular or irregular), surface texture (such as sticky, dry, shriveled, etc.), elevation shape (flat, convex, wrinkled, etc.), transparency (clear, turbid) and colony color of the colonies were observed and recorded.

[0074] (2) Identification of interaction between strains: plate confrontation experiment was used to inoculate different functional strains obtained by re-screening on PDA medium by cross streaking to observe their interaction. After incubation in a 30°C constant temperature incubator for 2-3 days, the growth of colonies at the cross points was checked, and the synergistic effect, synergistic effect or competitive stress relationship between strains was analyzed through the inhibition, promotion or no effect of colony growth.

[0075] (3) Antagonistic strain gene sequence determination and analysis: The genomic DNA of the screened antagonistic strains was extracted using bacterial and fungal DNA extraction kits (TIAN GEN). The corresponding gene fragments were amplified by PCR, and the PCR products were purified and sequenced. Then, the obtained gene sequences were compared with the reference sequences in the NCBI database for homology analysis, the sequences with high homology were selected for genetic distance calculation, and the phylogenetic tree was constructed using MEGA software, so as to determine the classification status of the strains and the relationship with other species.

[0076] The phylogenetic tree of the obtained antagonistic strains is shown in Figure 4 , wherein 4a is the phylogenetic tree of X1 strain; 4b is the phylogenetic tree of X1 strain; 4c is the phylogenetic tree of X1 strain; 4d is the phylogenetic tree of X1 strain; 4e is the phylogenetic tree of X1 strain; X1-X5 are Bacillus subtilis YX1, Bacillus pumilus GX1, Bacillus licheniformis GX2, Bacillus amyloliquefaciens YX2 and Pseudomonas fluorescens GX3 (Pseudomonas fluorescens X5) in turn. The preservation number of Bacillus subtilis X1 is GDMCC NO: 65614, the preservation number of Bacillus pumilus X2 is GDMCC NO: 65272, the preservation number of Bacillus licheniformis X3 is GDMCC NO: 65587, the preservation number of Bacillus amyloliquefaciens X4 is GDMCC NO: 65273, and the preservation number of Pseudomonas fluorescens X5 is GDMCC NO: 65274.

[0077] 5 Effect of complex microbial agent on citrus Huanglongbing disease

[0078] The strains with strong antagonistic function and not antagonistic to each other were inoculated in PDB medium respectively, and cultured at 30°C, 120r / min for 24-48h to prepare bacterial suspension. Each strain was mixed at a ratio of 1:1 to prepare culture solution, which could be used for direct root irrigation. The complex microbial agent for foliar spraying was prepared by adding 1mL Tween-20 to 500mL culture solution.

[0079] The test seedlings are one-year-old seedlings of non-nuclear Wucheng, all from the Citrus Research Institute of Guilin City, Guangxi Zhuang Autonomous Region, and were grafted with branches infected with Huanglongbing in October 2022 for experimental use. In April 2023, the seedlings were transplanted into plastic pots (specification diameter 28 cm x height 28 cm), with 1 plant per pot. The cultivation soil matrix was a mixture of local mature soil and organic fertilizer (organic fertilizer chicken manure and sugarcane residue fermented, with modified biochar and selected functional microorganisms, with N, P, and K contents of 2.91%, 3.64%, and 3.01%, respectively. The organic fertilizer used in this experiment was this fertilizer) in a 9:1 ratio. The NPK fertilizer treatment was a mixture of the same sterilized soil and NPK fertilizer (NPK fertilizer used in this study was Double Ji brand 15-15-15 compound fertilizer) in the same proportion of organic fertilizer and macronutrients. Each pot weighed about 6 kg, and the soil was from the Jingu Ice Spring Villa, with a pH of 6.5, suitable for the growth of Wucheng seedlings.

[0080] The Wucheng seedlings were previously grafted with plants determined to be positive for Huanglongbing by molecular detection. The concentration of the compound microbial agent was adjusted to about 1 x 10 8 cfu / mL, applied every 7 days for a total of 28 times. The non-microbial agent treatment group was irrigated with an equal amount of culture solution (culture solution was PDA liquid medium containing bacteria, and was sterilized by high-pressure steam before use). The organic fertilizer and NPK fertilizer were calculated for their macronutrient content before the experiment to ensure that the nutrient elements added in each treatment were consistent. The pot experiment was set up with 6 groups, with 20 pots in each treatment group, a total of 120 pots. The treatment groups are shown in Table 3.

[0081] Table 3 Different methods of treating citrus seedlings

[0082]

[0083] Note: CK: blank control group, CG: positive control group, PY: foliar spraying group, JS: simultaneous root irrigation and leaf spraying group, NPK: NPK fertilizer application group, GJ: root irrigation group.

[0084] The incidence of citrus Huanglongbing in each treatment group was measured, and the data are shown in the following table:

[0085] Table 4 Statistics of positive rate of citrus Huanglongbing

[0086]

[0087]

[0088] Note: CK: blank control group, CG: positive control group, PY: foliar spraying group, JS: simultaneous root irrigation and leaf spraying group, NPK: NPK fertilizer application group, GJ: root irrigation group. The same below.

[0089] It can be seen that after the treatment of complex microbial agent, the positive rate of citrus pot seedlings decreased. The effect of leaf spraying microbial agent treatment (PY) and simultaneous root irrigation and leaf spraying group (JS) was the best, which made the positive rate of citrus seedlings decrease by 75%, followed by root irrigation group (GJ), which made the positive rate of seedlings decrease by 70%. The seedlings treated by adding NPK fertilizer had no change in positive rate, which remained at 100%. The results showed that the addition of complex microbial agent could effectively reduce the occurrence of citrus Huanglongbing positive seedlings and inhibit the reproduction of Huanglongbing bacteria, which had obvious prevention and control effect.

[0090] 6Effect of complex microbial agent on the growth of citrus

[0091] Determination of citrus physiological indicators

[0092] After the last microbial agent treatment for 12h in the pot experiment, the 3-4 leaves at the base of the plant top were taken (Xu Yuanyuan et al., 2022), and the samples were wrapped with tin foil paper, quickly frozen in liquid nitrogen, and then quickly transported to the laboratory for storage in a -80℃ refrigerator for determination of soluble sugar, starch and chlorophyll 3 physiological indicators. After 24h of the last treatment, 3-5 leaves in the middle of the seedlings were taken for determination of callose content. At the same time, the root tips of the seedlings were collected for determination of root activity.

[0093] Instruments and reagents required during determination: enzyme marker / spectrophotometer, 96-well plate / cuvette, mortar, multichannel pipette, table centrifuge, constant temperature water bath and distilled water.

[0094] Determination of citrus leaf starch

[0095] (1) Sample treatment: The plant starch content kit (boxbio) was used for determination. 50mg of fresh sample was weighed in a mortar and ground, 1mL of eluent was added, and after homogenization, it was transferred to an EP tube. The water bath was preheated to 80℃, and the EP tube was placed in the water bath for extraction for 30min. Centrifugation for 5min (3000g, 25℃), discard the supernatant, leave the precipitate; add 0.5mL of distilled water to the precipitate, and put it in a 95℃ water bath for 15min (cover tightly to prevent water loss); after cooling, add 1mL of extraction solution, extract at 25℃ for 15min, and shake 3-5 times; after extraction, mix well, centrifuge for 10min (3000g, 25℃), and take the supernatant for determination.

[0096] (2) Absorbance measurement: The spectrophotometer was preheated for more than 30 min, the wavelength was adjusted to 620 nm, and distilled water was used for zero calibration. According to the reagent kit instructions, the appropriate amount of reagent was added into the centrifuge tube in turn. After completing the above sample pretreatment step and adding the reagent mixture, 1 mL of reaction solution was taken from the centrifuge tube into a 1 mL glass cuvette. The spectrophotometer was used to measure the absorbance of the reaction solution in the cuvette at a wavelength of 620 nm, which was recorded as "A measurement", and the absorbance of the standard solution (standard with known concentration) and the blank solution (control group without the substance to be measured) was also measured, which was recorded as "A standard" and "A blank", respectively. (3) Data analysis: In order to eliminate the influence of instrument background and other non-specific absorption, the absorbance difference between each sample and blank control was calculated, i.e. "ΔA measurement = A measurement - A blank". The same calculation was also performed for the standard, "ΔA standard = A standard - A blank", which was used to draw the standard curve. Combined with the standard curve (y = ax + b, the corresponding ΔA standard is the ordinate, which can be calculated by substituting x (mg / mL)), the content of starch in the sample can be calculated. The absorbance measurement of the blank tube only needs to be performed 1-2 times.

[0097] (3) Data analysis:

[0098] The calculation formula is as follows:

[0099] Starch content (mg / g) = M n x V1 x 0.9 x D / W = 1.35 x x D / W (2-2)

[0100] Wherein Mn: starch concentration (mg / mL); V1: total volume of sample to be measured (1.5 mL); D: dilution multiple of sample to be measured; 0.9: conversion factor of glucose to starch; W: sample mass (g).

[0101] Citrus leaf soluble sugar determination

[0102] The determination was performed using a plant soluble sugar content kit (boxbio). (1) Sample processing: 0.1 g of plant sample was weighed, 1 mL of distilled water was added, and it was ground into a uniform slurry by a grinder. The ground sample was poured into a centrifuge tube with a cover, and then placed in 95°C water for water bath treatment for 10 min, during which the centrifuge tube was tightly covered to prevent water evaporation. After water bath, the centrifuge tube was taken out and cooled, and then centrifuged at 8000 g at room temperature for 10 min. After centrifugation, the supernatant was carefully removed and transferred into a 10 mL capacity test tube, and distilled water was added to 10 mL, and then shaken well for standby use. (2) Absorbance determination: The enzyme marker was preheated for more than 30 min, the wavelength was adjusted to 620 nm, and distilled water was used for zero calibration. According to the operation instruction provided by the kit, the corresponding reagents were added to the treated sample solution and other standard tubes and blank tubes. The mixed solution was placed in a 95°C water bath for 10 min, and the test tube was tightly covered to prevent water evaporation. After cooling to room temperature, the absorbance of each tube was measured at 620 nm wavelength on the enzyme marker, and was marked as Ablank, Atest and Astandard, respectively. (3) Data analysis: The absorbance difference was calculated, i.e. “ΔAtest = Atest-Ablank”, representing the sample absorbance after removing background interference; “ΔAstandard = Astandard-Ablank”, used to draw a standard curve. Combined with the standard curve (y = ax + b, the corresponding ΔAstandard is the ordinate, which can be calculated by substitution), the content of soluble sugar in the sample can be calculated. In this process, only 1-2 tubes of blank and standard tubes are required to meet the experimental requirements. The calculation formula is as follows:

[0103] Soluble sugar content (mg / g) = x x V1 x D / W (2-3)

[0104] In the formula, V1: total volume of the sample to be tested (1 mL); D: dilution multiple of the sample to be tested (10); W: sample mass (g).

[0105] Citrus leaf chlorophyll determination

[0106] (1) Sample preparation: Fresh plant leaves or other green tissues are selected, washed with distilled water and surface water is absorbed, and the midrib is removed. About 0.1 g of sample is weighed, cut into pieces and placed in a mortar or homogenizer. 1 mL of distilled water and about 10 mg of reagent I are added, and grinding is performed under dark or weak light conditions to protect the chlorophyll from light damage. The ground sample is transferred to a 10 mL test tube, and the mortar is washed with extraction solution and the washing solution is also transferred to the same test tube. The volume in the test tube is made up to 10 mL with extraction solution, and the test tube is placed in the dark or wrapped in tin foil for 3 h of extraction, and the color of the bottom tissue residue is observed until it is close to white, indicating that the extraction is basically complete. If the color does not change white, the extraction time needs to be extended until the color of the tissue residue is close to white. (2) Absorbance measurement: The microplate reader needs to be preheated for more than 30 min. 200 μL of the upper extraction solution is injected into a 96-well plate, 200 μL of extraction solution is used as a blank, and the absorbance is measured at two wavelengths of 663 nm and 645 nm, respectively, and recorded as A 663 and A 645 . The calculation formula is as follows:

[0107] Chlorophyll a content (mg / g) = (25.43 x A 663 - 5.17 x A 645 ) x V1 x D / W / 1000

[0108] = 0.01 x (25.43 x A 663 - 5.17 x A 645 ) x D / W (2-4)

[0109] Chlorophyll b content (mg / g) = (45.76 x A 645 - 9.34 x A 663 ) x V1 x D / W / 1000

[0110] = 0.01 x (45.76 x A 645 - 9.34 x A 663 ) x D / W (2-5)

[0111] Total chlorophyll content (mg / g) = (40.59 x A 645 + 16.09 x A 663 ) x V1 x D / W / 1000

[0112] = 0.01 x (40.59 x A 645 + 16.09 x A 663 ) x D / W (2-6)

[0113] wherein V1: volume of extraction solution, (10 mL); D: dilution factor; W: sample mass, g.

[0114] Citrus leaf callose determination

[0115] Determination was carried out by using plant callose content kit (cominbio). 0.1 g sample was weighed in an EP tube and soaked in 98% alcohol solution overnight. Then, the alcohol was discarded, 1 mL extraction solution was added, and it was mixed evenly, and then it was placed in a 80°C water bath for 20 min, and centrifuged at 25°C for 10 min, and the supernatant was taken for determination. According to the kit instructions, various reagents were added to the supernatant in turn, and after uniform mixing, it was soaked in a 50°C water bath for 30 min, and placed at room temperature for 1 h until the blue color in the solution disappeared. If the blue color still does not fade, it can be continuously soaked in 50°C water until the blue color disappears. 200 μL of the solution to be measured was placed in a 96-well plate, and the fluorescence intensity was measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. Similarly, the standard solution was prepared and determined according to the kit instructions, and the standard curve was drawn. The calculation formula is as follows:

[0116] Callose content (mg / g) = c / (m / V1)

[0117] = c 上 / 0.1 (2-7)

[0118] In the formula, c: the concentration of callose in the sample supernatant (mg / mL); m: the mass of the sample (g); V1: the volume of the extraction solution (mL); c 上 : the concentration of callose in the sample supernatant (mg / mL).

[0119] Citrus root activity determination

[0120] The root activity of the seedling was determined by using plant root activity detection kit (TTC method) (solarbio). The standard solution was diluted and determined according to the kit instructions, and the absorbance of 200 μL prepared standard and 200 μL ethyl acetate (i.e. 0 μg / mL) was determined at 485 nm. The standard value of ΔA = A (20μg / mL) -A (0μg / mL) . 0.1 g of the sample to be measured was weighed into the test tube and the control tube, and various reagents were added according to the instructions. The sample should be completely soaked in the prepared solution, and the dark reaction was carried out at 37°C for 4 h, and then it was immediately placed in ice bath for 5 min, and the filtrate was removed, and the water in the roots was absorbed as much as possible with filter paper, and then it was placed in a mortar. After grinding evenly, the sample was placed in a centrifuge tube, the speed was adjusted to 12000 rpm, 4°C, and centrifuged for 10 min, and 200 μL of supernatant was taken, and the absorbance was measured at 485 nm. ΔA determination = A measurement - A control. The calculation formula is as follows:

[0121] TTC [ μg / (g·h) ] =ΔA determination × C standard / ΔA standard × V / (W×T)=5×ΔA determination / ΔA standard / W (2-8)

[0122] In the formula, W: root weight (g); Cstandard: standard solution concentration (20 μg / mL); T: reaction time (4 h); V: volume of reagent 4, i.e., homogenate volume (1 mL).

[0123] Determination of citrus defense enzyme activity

[0124] After the final inoculum treatment in the pot experiment at 0h, 1h, 12h, 24h, and 36h, the 3rd to 4th leaves from the base of the plant shoot were taken. The samples were wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and then quickly transported to the laboratory and stored at -80℃ for testing of POD, SOD, and CAT enzyme activities.

[0125] The instruments required for the assay include: microplate reader, 96-well plate, mortar and pestle, multichannel pipette, benchtop centrifuge, constant temperature water bath and distilled water.

[0126] POD enzyme activity assay

[0127] The peroxidase (POD) activity assay was performed using a BoxBio kit. Refer to the kit's instruction manual for detailed operating procedures. Immediately mix thoroughly and start timing. Measure the absorbance at 470 nm (A1) at 30 s and at 470 nm (A2) at 90 s. Calculate ΔA = A2 - A1. Unit definition: A1 = (g tissue per mL of system per minute) 470 A change of 0.01 corresponds to one unit of enzyme activity. The calculation formula is as follows:

[0128]

[0129] In the formula, Vreaction total: total volume of the reaction system (0.2 mL); Vsample: volume of crude enzyme solution added to the reaction system (0.01 mL); Vsample total: total volume of crude enzyme solution (1 mL); T: reaction time (1 min); W: sample mass (g).

[0130] SOD enzyme activity assay

[0131] Superoxide dismutase (SOD) activity was measured using a BoxBio kit. Detailed operating procedures are described in the kit's instruction manual. Absorbance measurement: The absorbance at 560 nm was measured and recorded as Ameasured, Acontrol, Ablank1, and Ablank2. ΔAmeasured = Ameasured - Acontrol, ΔAblank = Ablank1 - Ablank2. Blank groups 1 and 2 only require 1-2 measurements, and a control group should be included for each sample. The calculation formula is as follows:

[0132]

[0133] The calculation of the inhibition percentage:

[0134]

[0135] The inhibition percentage should be controlled in the range of 30-70%, the closer to 50%, the more accurate; if the inhibition percentage is less than 30% or greater than 70%, the sample size needs to be adjusted and then re-determined: if the inhibition percentage is too high, the crude enzyme solution needs to be appropriately diluted and then re-determined; if the inhibition percentage is too low, the sample size needs to be appropriately increased and then re-determined, and the calculation is modified accordingly.

[0136] In the formula, Vtotal: total volume of the reaction system (0.2 mL); Vsample: volume of the crude enzyme solution added to the reaction system (0.02 mL); Vtotal sample: total volume of the crude enzyme solution (1 mL); W: sample mass (g); D: sample dilution multiple.

[0137] CAT enzyme activity determination

[0138] Determined by hydrogen peroxidase (CAT) activity detection kit (ammonium molybdate colorimetric method) kit (boxbio). The specific operation steps are shown in the kit operation instruction manual. 200 μL of reaction solution was taken into a 96-well plate, and the absorbance at 405 nm was measured, recorded as Ameasurement, Acontrol, Astandard and Ablank, and calculated as ΔAmeasurement = Acontrol-Ameasurement, ΔAstandard = Astandard-Ablank. Note: the blank tube only needs to be determined 1-2 times, and each sample needs to be set with a control tube. With 150, 100, 80, 40, 20, 10 μmol / mL as the abscissa (x), and its corresponding ΔAstandard as the ordinate (y), a standard curve was drawn, and a standard equation y = ax + b was obtained. ΔAmeasurement was brought into the formula to obtain x (μmol / mL). The unit definition: 1 μmol H2O2 degradation catalyzed per g of tissue per minute is defined as one enzyme activity unit. The calculation formula is as follows:

[0139]

[0140] In the formula, Vsample: volume of the crude enzyme solution added to the reaction system (0.02 mL); Vtotal sample: total volume of the crude enzyme solution, (1 mL); V S2 : volume of reagent two added to the reaction system (0.02 mL); W: sample mass (g); T: reaction time (2 min).

[0141] The results are shown below:

[0142] 1. From Figure 5It can be seen that the addition of compound microbial agent has a certain effect on the growth of citrus seedlings and alleviates the symptoms of huanglongbing. The yellowing symptoms of citrus seedlings in the treatment group with added microbial agent are significantly reduced compared to other treatment groups, and the overall plant appears more vigorous. The growth of citrus seedlings in the NPK fertilizer treatment group and the positive control group is significantly poorer, and the leaves have significant yellowing symptoms.

[0143] 2、By Figure 6 It can be seen that the addition of compound microbial agent has a certain effect on the growth of citrus seedlings and alleviates the symptoms of huanglongbing. The yellowing symptoms of citrus seedlings in the treatment group with added microbial agent are significantly reduced compared to other treatment groups, and the overall plant appears more vigorous. The growth of citrus seedlings in the NPK fertilizer treatment group and the positive control group is significantly poorer, and the leaves have significant yellowing symptoms. Figure 6 b can be seen that in the various treatment groups with continuous addition of microbial compound agent, the stem width of citrus seedlings is wider than that of other treatment groups. At the same time, the stem width of citrus seedlings in the root irrigation and leaf spraying group (JS) and the positive control group is significantly different (P<0.05), and the stem height of citrus seedlings in the NPK fertilizer treatment group is very significant (P<0.001). The stem width of citrus seedlings in the root irrigation group (GJ) and the NPK fertilizer treatment group is significantly different (P<0.05). As can be seen from the above, the addition of citrus compound microbial agent has a significant effect on the growth of citrus seedlings.

[0144] 3、The results of fresh weight and dry weight of citrus seedlings are shown in Table 5:

[0145] Table 5 Fresh and dry weight of citrus seedlings under different treatments

[0146]

[0147] Note: Different lowercase letters represent significant differences (P<0.05) in fresh weight and dry weight of each treatment.

[0148] As can be seen from Table 5, the fresh weight of citrus seedlings in the root irrigation group (GJ) and the simultaneous root irrigation and leaf spraying group (JS) is greater than that of other treatment groups, and there is a significant difference (P<0.05) in fresh weight between the treatment group without added microbial agent. The dry weight of the three treatment groups with added microbial agent is greater than that of the other three treatment groups, and there is a significant difference (P<0.05). It can be seen that after applying citrus microbial compound agent, it can effectively promote the growth and development of citrus seedlings and the accumulation of biomass.

[0149] 4、By Figure 7It can be concluded that the chlorophyll content of citrus seedlings treated with the last microbial agent was significantly increased compared with the positive control and NPK fertilizer treatment groups. Among them, the chlorophyll content of the group treated with simultaneous root irrigation and leaf spraying (JS) was the highest, reaching an average of 2.15 mg / g, which was significantly different from other groups without microbial agent (P<0.05). In summary, the addition of microbial agents is beneficial to the synthesis of chlorophyll in citrus seedlings, thereby enhancing photosynthesis, promoting the growth of citrus seedlings, and improving the health of seedlings.

[0150] 5、 Figure 8 a is the correlation diagram of leaf starch and absorbance value, Figure 8 b is the column chart of leaf starch content of citrus seedlings treated with different treatments. From Figure 8 a can be concluded that the correlation between leaf starch and absorbance value is good. From Figure 8 b can be concluded that the leaf starch content of citrus seedlings treated with the last microbial agent was significantly reduced. The leaf starch content of the groups treated with leaf spraying (PY), root irrigation (GJ), and simultaneous root irrigation and leaf spraying (JS) was significantly different from the two control groups and the NPK fertilizer group (P<0.05). The leaf starch content of the groups treated with leaf spraying (PY) and simultaneous root irrigation and leaf spraying (JS) decreased the most, with starch contents of 16.87 mg / g and 16.38 mg / g, respectively. The starch content of the positive control treatment group was 40.53 mg / g, which was 2.34 and 2.47 times lower, respectively. In summary, the addition of microbial agents significantly reduced the starch accumulation caused by Huanglongbing disease and promoted the conversion of starch, which played an important role in promoting the growth of citrus seedlings.

[0151] 6、 Figure 9 a is the correlation diagram of leaf soluble sugar content and absorbance value, Figure 9 b is the column chart of leaf soluble sugar content of citrus seedlings treated with different treatments. From Figure 9 a can be concluded that the correlation between leaf soluble sugar content and absorbance value is good. From Figure 9 b can be concluded that the leaf soluble sugar content of citrus seedlings treated with the last microbial agent was significantly reduced. The leaf soluble sugar content of the groups treated with leaf spraying (PY), root irrigation (GJ), and simultaneous root irrigation and leaf spraying (JS) was significantly different from the positive control and NPK fertilizer groups (P<0.05). The leaf soluble sugar content of the groups treated with leaf spraying (PY) and simultaneous root irrigation and leaf spraying (JS) decreased the most, with soluble sugar contents of about 33.36 mg / g. The soluble sugar content of the positive control treatment group was 56.57 mg / g, which was nearly 1.7 times lower. In summary, the groups treated with leaf spraying (PY) and simultaneous root irrigation and leaf spraying (JS) had the best effect on reducing the soluble sugar content of diseased leaves.

[0152] 7、 Figure 10 a is the correlation graph of callose content and absorbance value, Figure 10 b is the column chart of leaf callose content of different treatments of citrus seedlings. From Figure 10 a can be obtained that the correlation of callose content and absorbance value is good. From Figure 10 b can be obtained that after the last application of microbial compound inoculant treatment of citrus seedlings, the leaf callose content is significantly reduced, and has significant difference (P<0.05) with the results of the other three treatment groups. The callose content of the three treatment groups of applying compound inoculant is 0.72 mg / g (GJ), 0.79 mg / g (PY) and 0.82 mg / g (JS), respectively, and the starch content of the positive control treatment group is 0.92 mg / g, which is reduced by 1.28, 1.16 and 1.12 times, respectively. It can be seen that the addition of compound inoculant can effectively reduce the content of callose in diseased leaves, among which the effect of root drenching inoculant (GJ) is the best.

[0153] 8、 Figure 11 a is the correlation graph of TIF content and absorbance value, Figure 11 b is the column chart of root activity of different treatments of citrus seedlings. From Figure 11 a can be obtained that the correlation of TIF content and absorbance value is good. From Figure 11 b can be obtained that the root activity of citrus seedlings of drenching bacteria group (GJ) and simultaneous drenching root and spraying leaves group (JS) is strong, which has significant difference (P<0.05) with the positive control group and NPK fertilizer group. The root activity of drenching bacteria group (GJ) and simultaneous drenching root and spraying leaves group (JS) is 111.34 μg / (g.h) and 115.64 μg / (g.h), respectively, and the starch content of the positive control treatment group is 92.28 μg / (g.h), which is increased by 9.36% and 11.24%, respectively. The application of NPK fertilizer does not enhance the root activity of diseased seedlings, and the root activity of the treatment group of spraying leaves with bacteria (PY) is increased by 3.21% compared with the positive control treatment group. It can be seen that the addition of compound inoculant can effectively enhance the root activity of diseased seedlings. Among them, the effect of drenching bacteria treatment (GJ) and simultaneous drenching root and spraying leaves treatment (JS) is better, which significantly enhances the root activity of citrus seedlings.

[0154] 9、From Figure 12It can be concluded that the changes of enzyme activity in each treatment group are different. Compared with the treatment group without adding bacterial agent, the three treatment groups with bacterial agent have significant differences, and the change trend of peroxidase (POD) activity in the three groups is first increased, reaching the maximum at 24 h, and then decreased. The POD enzyme activities of the three treatments with bacterial agent are 2039.46 U / g (JS), 1625.81 U / g (PY) and 1533.77 U / g (GJ) at 24 h. The POD enzyme activity of the positive control group is 451.25 U / g at 24 h, and the treatment groups with compound bacterial agent each increase by 63.77% (JS), 56.55% (PY) and 54.53% (GJ) compared with the positive control group. At the same time, the POD enzyme activity of the group with root irrigation and foliar spraying of bacterial agent (JS) increases most rapidly within 24 h after treatment. The application of NPK fertilizer has no significant effect on the POD enzyme activity of the diseased plants. In summary, the application of compound bacterial agent can effectively improve the POD enzyme activity of the leaves of the potted seedlings.

[0155] 10、from Figure 13 It can be seen that the changes of enzyme activity in each treatment group are different. The superoxide dismutase (SOD) activities of the three treatment groups with bacterial agent have significant differences with the treatment group without adding bacterial agent at 24 h and 36 h. The change trends of SOD enzyme activity in the groups with root irrigation (GJ) and simultaneous root irrigation and foliar spraying of bacterial agent (JS) are similar, and the overall trend is increasing. The SOD enzyme activity of the treatment group with foliar spraying of bacterial agent (PY) reaches the peak at 24 h, and decreases within 24 h-36 h. The SOD enzyme activities of the three treatments with bacterial agent are 725.67 U / g (JS), 651.38 U / g (PY) and 630.07 U / g (GJ) at 36 h. The SOD enzyme activity of the positive control group is 565.98 U / g at 36 h, and the treatment groups with compound bacterial agent each increase by 12.36% (JS), 7.02% (PY) and 5.36% (GJ) compared with the positive control group. The SOD enzyme activity of the group with simultaneous root irrigation and foliar spraying of bacterial agent (JS) increases most rapidly within 1 h after treatment, and also shows a rapid upward trend within 1 h-36 h. The effect of NPK fertilizer on the SOD enzyme activity of the diseased plants is the smallest, followed by the two control groups. In summary, the application of compound bacterial agent can effectively improve the SOD enzyme activity of the leaves of the potted seedlings, and the treatment effect of the group with simultaneous root irrigation and foliar spraying of bacterial agent (JS) is the best.

[0156] 11、from Figure 14It can be seen that the changes of enzyme activities in each treatment group are different. Compared with the treatment group without adding bacterial agent, the three treatment groups with bacterial agent have significant differences, and the changes of catalase (CAT) activity in the three groups are first increased, reach the maximum at 24 h, and then show a downward trend. At the same time, the CAT enzyme activity in the root irrigation and foliar spraying bacterial agent treatment (JS) and the bacterial irrigation treatment (GJ) increases most rapidly in the 12 h-24 h period; the CAT enzyme activity in the foliar spraying bacterial agent group increases most rapidly in 1 h-24 h. The CAT enzyme activities in the three treatment groups with bacterial agent are 203.41 U / g (JS), 185.97 U / g (PY) and 161.58 U / g (GJ) at 24 h, respectively. The CAT enzyme activity in the positive control group is 102.36 U / g at 24 h, and the treatment groups with compound bacterial agent increase by 33.48% (JS), 29% (PY) and 22.44% (GJ) compared with the positive control group. At the same time, the root irrigation and foliar spraying bacterial agent treatment (JS) has the best effect on the promotion of CAT enzyme activity in diseased plants, and the application of NPK fertilizer has no obvious promotion effect. In summary, the application of compound bacterial agent can effectively improve the CAT enzyme activity of potted seedling leaves.

[0157] In summary, the pot experiment of the application sets up 6 treatment groups to control huanglongbing bacteria, uses bacterial agent to treat 28 times, and then detects huanglongbing bacteria by using qPCR technology. The results show that the treatment of spraying bacterial agent on the leaves and the treatment of spraying bacterial agent on the leaves and irrigating roots at the same time have the best effect, and the positive rate of citrus seedlings is reduced by 75%. The second is the irrigation group, and the positive rate of seedlings is reduced by 70% after treatment. The seedlings of the NPK fertilizer treatment group have no change in positive rate and remain at 100%. Therefore, the addition of the compound microbial agent has obvious inhibitory effect on huanglongbing bacteria. The application of the compound microbial agent significantly increases the stem width, seedling height, fresh weight, dry weight, chlorophyll and root activity of citrus seedlings with huanglongbing, and has a certain growth-promoting effect. Among them, the effect of the treatment of spraying bacterial agent on the leaves and irrigating roots at the same time (JS) is the best, and the growth amount of the six indexes is the highest. It is speculated that the root and the leaf are the two main parts of plant absorption and metabolism. The main function of the root system is to absorb water and nutrients, and the photosynthesis of the leaf provides energy. On this basis, by irrigating the two parts at the same time, the synergistic effect between the root and the leaf can be effectively promoted, so as to improve the overall health status and growth efficiency of the plant. The addition of the compound microbial agent significantly reduces the accumulation of starch and soluble sugar caused by huanglongbing, and promotes the transformation of starch. Among them, the effect of the treatment of spraying bacterial agent on the leaves (PY) and the treatment of spraying bacterial agent on the leaves and irrigating roots at the same time (JS) is more prominent. It is speculated that spraying the leaves can directly and rapidly act on the disease part, and the leaves have good absorption capacity. The comprehensive factors lead to such results. After adding the compound microbial agent, the content of callose in the diseased leaves can be effectively reduced. After adding the compound microbial agent, the activities of three defense enzymes POD, SOD and CAT of the citrus seedlings are improved, which indicates that the addition of the compound microbial agent can induce the plant to produce self-resistance mechanism to improve the disease resistance.

[0158] The above-described embodiments only express the implementation manners of the present application, and cannot be understood as the limitation to the scope of the present application, nor as any form of limitation to the structure of the present application. It should be noted that, for those skilled in the art, some changes and improvements can be made without departing from the concept of the present application, and these changes and improvements are within the protection scope of the present application.

Claims

1. A compound microbial agent for controlling citrus Huanglongbing (HLB), characterized in that, Composed of the following components by weight: 5-10 parts Bacillus subtilis Bacillus subtilis X1, 5-10 portions of Bacillus pumilus Bacillus pumilus X2, 5-10 parts of Bacillus licheniformis Bacillus licheniformis X3, 5-10 parts of Bacillus amyloliquefaciens Bacillus amyloliquefaciens X4 and 5-10 samples of *Pseudomonas fluorescens* Pseudomonas fluorescens X5; Bacillus subtilis Bacillus subtilis X1 has the accession number GDMCC NO: 65614, and the described Bacillus pumilus... Bacillus pumilus X2 has the accession number GDMCC NO: 65272, and the Bacillus licheniformis described therein... Bacillus licheniformis X3 has the accession number GDMCC NO: 65587, and the described Bacillus amyloliquefaciens is... Bacillus amyloliquefaciens X4 has the accession number GDMCC NO: 65273, and the described fluorescent Pseudomonas bacteria Pseudomonas fluorescens The accession number for X5 is GDMCC NO: 65274; in the compound microbial agent used to control citrus Huanglongbing, the ratio of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Pseudomonas fluorescens is 1-2:1-3:1-2:1-2:1-3; in the compound microbial agent used to control citrus Huanglongbing, the concentration of each of Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Pseudomonas fluorescens is independently 5.5 × 10⁻⁶. 7 -1×10 8 cfu / mL.

2. The method for preparing the compound microbial agent according to claim 1, characterized in that, The process includes the following steps: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Pseudomonas fluorescens are inoculated into PDB medium and cultured with shaking at 30±5 ℃ and 100-200 r / min for 24-48 h to obtain five bacterial suspensions. The bacterial suspensions are then mixed in proportion to obtain the compound bacterial agent used for the prevention and control of citrus Huanglongbing.

3. The application of the compound microbial agent according to claim 1 in the prevention and control of citrus Huanglongbing.

4. The application of the compound microbial agent according to claim 1 in promoting the growth of citrus plants.

5. The application according to claim 3 or 4, characterized in that, The application includes: directly irrigating the plant roots with the compound microbial agent used to prevent and control citrus Huanglongbing.

6. The application according to claim 3 or 4, characterized in that, The application includes: diluting the compound microbial agent used to prevent and control citrus Huanglongbing and spraying it on the leaves of the plants.

7. The application according to claim 6, characterized in that, The compound microbial agent used to control citrus Huanglongbing is diluted with Tween and sprayed, with a volume ratio of compound microbial agent to Tween of 200-600:

1.

8. The application according to claim 3 or 4, characterized in that, The application includes: simultaneously applying the compound microbial agent for the prevention and control of citrus Huanglongbing directly to the plant roots and spraying the diluted compound microbial agent for the prevention and control of citrus Huanglongbing onto the plant leaves.

Images