Microbial organic fertilizer and application thereof in prevention and control of citrus huanglongbing and recovery of tree vigor

Abstract

The invention relates to the technical field of biology, in particular to a microbial organic fertilizer and application thereof in prevention and control of Citrus Huanglongbing and recovery of tree vigor. The method comprises the following steps: converting chicken manure by hermetia illucens larvae to obtain a fecula organic fertilizer, carrying out solid fermentation on bacillus TD1-2, and mixing the fecula organic fertilizer and the bacillus TD1-2 to form the microbial organic fertilizer rich in effective microbial communities. The organic fertilizer is continuously applied to citrus seedlings infected with the Huanglongbing, the result shows that the pathogen titer is greatly reduced, and the disease-resistant effect is remarkable. Meanwhile, the organic fertilizer with a proper dosage can significantly promote the growth of citrus seedlings and improve the conditions of leaves and roots. Metabolic component analysis shows that the organic fertilizer is rich in metabolites closely related to plant disease resistance and normal physiological and biochemical metabolism, and theoretical support is provided for the disease resistance effect. In addition, the safe use range of the microbial organic fertilizer is defined, the application effect of the microbial organic fertilizer under different air temperature conditions is ensured, and a new strategy is provided for biological prevention and control of the citrus liberobacter asiaticum.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a microbial organic fertilizer and its application in preventing and controlling citrus huanglongbing and restoring tree vigor. Background Art

[0002] Citrus huanglongbing (HLB), a quarantine disease caused by unculturable phloem bacteria (Candidatus Liberibacter spp.), poses a severe challenge to the global citrus industry due to its long incubation period, fast transmission speed, and wide damage range. In China, this disease has affected approximately 300 counties in 11 provinces, causing a large number of citrus plants to be damaged, significantly reducing the yield and quality of citrus, and severely restricting the healthy development of the industry.

[0003] The main prevention and control measures for citrus huanglongbing mainly include spraying pesticides to kill the vector insect psyllids, cutting down diseased trees, and cultivating virus-free seedlings, etc. However, these methods have significant limitations. Although the insect-proof net technology can effectively isolate psyllids, its high cost limits its wide application and is not conducive to mechanized and intelligent management. Chemical control is effective in the short term, but it is easy to cause the enhancement of psyllid drug resistance, and at the same time cause pesticide residues and ecological environment damage. Antibiotic control may cause phytotoxicity, affect the fruit appearance, and enhance the drug resistance of pathogens after long-term use, and the disease is prone to relapse after stopping the drug.

[0004] In view of the limitations of traditional prevention and control strategies, biocontrol bacteria show great potential in the prevention and control of citrus huanglongbing. Biocontrol bacteria act alone or synergistically through various mechanisms, such as antagonism, competition, hyperparasitism, induction of plant resistance, and promotion of plant growth, etc., to play specific effects at different development stages or parts of the plant, effectively inhibiting or killing pathogenic microorganisms and ensuring plant health. Among them, inducing plant resistance is the key way to enhance plant defense ability, including two ways: systemic acquired resistance (SAR) and induced systemic resistance (ISR). SAR means that after the plant is infected by a pathogen, resistance can also be generated in the non-inoculated parts; while ISR means that after the plant is stimulated by a non-pathogen (such as biocontrol bacteria), it stimulates the plant to produce physical or chemical barriers to enhance the overall resistance. In addition, biocontrol bacteria promote plant growth by improving plant nutrient absorption, regulating hormone levels, or enhancing metabolic activity, indirectly improving the disease resistance of plants.

[0005] The cultivation of citrus is mainly concentrated in the acidic soil areas in the south. As an important biocontrol microorganism, Bacillus is restricted in survival and reproduction in acidic soil. Therefore, it is of great significance to screen out Bacillus that has good control effects on citrus diseases and tolerance to acidic environments. In view of the lack of control measures for citrus huanglongbing, developing new, efficient, and environmentally friendly control strategies and providing virus-free seedlings have become urgent needs in current research. Summary of the Invention

[0006] In view of this, the present invention provides a microbial organic fertilizer and its application in controlling citrus huanglongbing and restoring tree vigor. The present invention combines the excrement of black soldier fly larvae and the fermentation product of Bacillus sp. TD1-2, showing significant effects on the control of citrus huanglongbing and the continuous restoration of citrus tree vigor.

[0007] The technical solution of the present invention is realized as follows:

[0008] In the first aspect, the present invention provides a preparation method of a microbial organic fertilizer, including the following steps:

[0009] S1. Inoculate black soldier fly larvae into chicken manure. After 6-10 days of transformation, screen out the larval bodies, retain the excrement of the larvae and the remaining residues to obtain the excrement of black soldier fly larvae;

[0010] S2. Inoculate a single colony of Bacillus sp. TD1-2 into LB liquid medium for seed culture to obtain a seed solution;

[0011] S3. Ferment the seed solution in a solid medium at 28-32 °C for 7-15 days, dry the culture at 60 °C, and then pulverize it to obtain a solid fermentation product;

[0012] S4. Mix the excrement of black soldier fly larvae and the solid fermentation product according to a mass ratio of 1000:8 to obtain the microbial organic fertilizer;

[0013] The Bacillus sp. TD1-2 was deposited at the China Center for Type Culture Collection on October 30, 2019, with the deposit number CCTCC NO: M 2019873 and the deposit address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0014] In some specific embodiments, in step S1, the black soldier fly larvae are 6-day-old, and 100 larvae are inoculated into every 200 g of chicken manure. The culture amount can be increased proportionally according to experimental needs; control the water content of the chicken manure to be maintained at 65%-70%.

[0015] In some specific embodiments, in step S2, the LB liquid medium comprises: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride; pH 7.4;

[0016] Further, in some specific embodiments, the seed culture is carried out at 180 - 220 rpm and 28 - 32 °C until the OD of the seed liquid 600 reaches 0.8 - 1.2.

[0017] In some specific embodiments, in step S3, 1 kg of rice bran, 1 kg of wheat bran, 0.5 kg of starch, 0.5 kg of soybean meal, and 15 g of brown sugar are mixed to prepare a culture medium matrix; it is configured into a solid medium according to the ratio of the culture medium matrix to water of 1:1.3, and then 20 mL of the seed liquid is added for solid fermentation.

[0018] In some specific embodiments, in steps S3 and S4, the viable bacteria count in the solid fermented product is ≥ 20 billion per gram.

[0019] In a second aspect, the present invention provides a microbial organic fertilizer prepared according to the above preparation method.

[0020] In a second aspect, the present invention provides the application of the microbial organic fertilizer in preventing and controlling citrus huanglongbing and restoring tree vigor.

[0021] In some specific embodiments, the microbial organic fertilizer is applied to the roots or soil of citrus seedlings at a dosage of 10 - 20 g / plant / pot each time; it is applied once a week for 20 consecutive times; after 4 - 6 months, the microbial organic fertilizer is additionally applied once a week for a total of 10 times.

[0022] In some specific embodiments, the microbial organic fertilizer is used for the following purposes:

[0023] A1. Reducing the pathogen titer of Candidatus Liberibacter;

[0024] A2. Preventing and controlling the citrus huanglongbing pathogen;

[0025] A3. Alleviating leaf yellowing or mottling symptoms;

[0026] A4. Improving the poor root development and browning phenomenon;

[0027] A5. Cultivating virus - free citrus seedlings.

[0028] The beneficial effects of the present invention at least include the following:

[0029] First, after treating citrus seedlings with the microbial organic fertilizer provided by the present invention, the pathogen titer of the main pathogen (phloem bacillus) of huanglongbing in the vein samples of the treatment group decreased significantly. When the microbial organic fertilizer was applied 30 times, the percentage decrease in pathogen titer was extremely significant compared to the samples of the control CK group. Comparing the results of applying the microbial organic fertilizer of the present application 20 times and 30 times, it was found that the percentage decrease in pathogen titer when applied 30 times was also extremely significant compared to when applied 20 times. However, when comparing the results of treating with black soldier fly manure alone 20 times and 30 times, it was found that there was no significant difference in the percentage decrease in pathogen titer between the two.

[0030] Second, at an appropriate treatment dose (10 - 20 g / plant / pot), the microbial organic fertilizer provided by the present invention significantly promoted the normal growth of citrus seedlings, manifested as the leaf color turning green, the growth vigor significantly enhanced, and its growth state was significantly better than that of the control group without using organic fertilizer. The microbial organic fertilizer provided by the present invention has a positive effect on improving the condition of citrus leaves. The leaves of the seedlings in the treatment group were lush, the roots were developed, and there were abundant new roots, showing a good growth trend; while the roots of the seedlings in the control group were poorly developed, accompanied by browning, and the growth was inhibited.

[0031] In addition, in terms of metabolite component analysis, black soldier fly manure contains rich metabolites, among which lipids and lipid-like molecules, organic acids and their derivatives, phenyl-like compounds, organic heterocyclic compounds, etc. account for a significant proportion; Bacillus TD1-2 contains rich secondary metabolite gene clusters, including: multifunctional enzyme complexes, aromatic PKS, chalcone-type PKS. These metabolites are closely related to plant disease resistance and normal physiological and biochemical metabolism. In particular, the active ingredients with plant disease resistance-related functions account for a certain proportion, which is closely related to the decrease in pathogen titer in the control of huanglongbing on potted seedlings, providing a solid theoretical support for the study of the microbial organic fertilizer against citrus huanglongbing.

[0032] Finally, the present invention also defines the safe use range of the microbial organic fertilizer. Under the condition that the average monthly temperature is above 17 °C, the application amount of the organic fertilizer should not exceed 20 g / plant / pot to avoid adverse effects such as leaf withering and shedding caused by excessive application. From November to January, when the application amount of the microbial organic fertilizer reaches 1000 g / plant for each adult tree, it is also safe and will not cause root burning. Subsequent tests also proved that in January 2024, when the amount is 10 kg / plant for each adult tree, it is also safe.

[0033] Generally, the symptoms of huanglongbing are more prominent in autumn and winter than in spring and summer, and the pathogen concentration shows an increase. However, this application shows that even when pathogen detection is carried out under low-temperature conditions such as in autumn and winter, it is found that the microbial organic fertilizer can also withstand the test, effectively control the condition of huanglongbing, and ensure the healthy growth of citrus seedlings. At the same time, it should be noted that when using the said microbial organic fertilizer, special attention should be paid to supplementing medium and trace elements to prevent nutrient competition and ensure the actual operation and application effect of the microbial organic fertilizer.

[0034] Biological preservation

[0035] In this application, Bacillus sp. TD1-2 was obtained by isolation and purification from the vegetable garden soil of the Agricultural Microbiology Engineering Center of Huazhong Agricultural University in Wuhan, Hubei Province. The said Bacillus sp. TD1-2 was preserved in the China Center for Type Culture Collection on October 30, 2019, with the preservation number CCTCC NO: M 2019873 and the preservation address at No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province. Brief description of the drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 It is the standard curve of the RNR+ / RNR- primers in the embodiments of the present invention;

[0038] Figure 2 It is the influence of the microbial organic fertilizer treatment on the morphological characteristics of citrus seedlings in the embodiments of the present invention; Figures A, B, C, and G represent the microbial organic fertilizer treatment group; Figures D, E, F, and H represent the untreated control group; specifically as follows:

[0039] Figures A and D (taken on March 17, 2022): Show the initial states of the treatment group and the control group. The leaves of both groups show yellowing or mottling symptoms, serving as the test baseline. Figures B and E (taken on October 12, 2022): Figure B shows the shoots after being treated with microbial organic fertilizer 12 times. The leaves turn green and present a healthy state. Figure E, as the control group, is taken at the same time point, and the leaves are still yellowing. Figures C and F (taken on December 13, 2022): Figure C shows the shoots after being treated 20 times. The leaves are dark green and have a waxy luster. Figure F of the control group at the same time point shows continuous yellowing of the leaves. Figures G and H (taken on February 15, 2023): Figure G shows the treated seedlings with lush leaves, well-developed roots, and abundant new roots. Figure H of the control group shows poor root development with browning.

[0040] Figure 3 Comparison of the effects of different application amounts of microbial organic fertilizer on seedlings in the embodiments of the present invention.

[0041] Figures A - F: Responses of seedlings 2 days after treatment with different application doses in August 2022. The difference in application amount significantly affects the growth state of seedlings. Specifically as follows:

[0042] Figure A: At the application amount of 10 g / plant, the plants perform normally. Figure B: At the application amount of 20 g / plant, the plants also perform normally. Figure C: At the application amount of 30 g / plant, the plants are overall normal, but Figure D (Figure D is a partial magnification of Figure C) shows that a small amount of top leaves present a withered state. Figure E: At the application amount of 50 g / plant, the leaves show water loss and wilting, and the fruits turn yellow in advance. Figure F: At the application amount of 200 g / plant, all the plants wither and shed leaves severely.

[0043] Figure G: The plant state at 17 December 2024 (average temperature 10 °C) with an application amount up to 1000 g / plant. At this time, the plants maintain normal growth with dark green leaves, indicating that the use of the microbial organic fertilizer at this dose is safe under these conditions.

[0044] Figure 4 Types and percentages of the top 10 metabolites contained in the black soldier fly manure in the embodiments of the present invention.

[0045] Figure 5 Identification results of metabolites in Bacillus amyloliquefaciens strain MG - 2.

[0046] Figure 6 Statistical results of gene clusters and corresponding gene numbers of Bacillus TD1 - 2 samples in the embodiments of the present invention. Detailed implementation manners

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0048] The citrus seedlings involved in this application are the Nanhong tangerine seedlings obtained by this application group from Jiangxi Province. First, through qPCR detection of the Huanglongbing pathogen, the positive seedlings are identified. Then, trifoliate orange is used as the rootstock. When the thickness of the root neck of trifoliate orange reaches 0.8 - 1 cm, in August - September of summer, budding grafting is carried out using positive scions to obtain uniform positive seedlings. When the seedlings grow for 2 years, samples are taken from each seedling for detection, and the positive seedlings are used for experiments. Each treatment has at least 6 biological replicates.

[0049] Example 1 Preparation and Application of Microbial Organic Fertilizer

[0050] 1. Preparation Method of Microbial Organic Fertilizer

[0051] (1) Preparation of black soldier fly manure:

[0052] The black soldier fly manure described in this application is prepared on behalf by Wuhan Kewei Biotechnology Co., Ltd. Its preparation method includes the following steps:

[0053] Chicken manure is used as the raw material, and 6 - day - old black soldier fly larvae are inoculated, with an inoculation ratio of adding 100 larvae to every 200 grams of chicken manure. During the preparation process, the water content of the chicken manure is strictly controlled within the range of 65% to 70% (mass percentage) to ensure suitable conversion conditions. Subsequently, after an 8 - day biological conversion cycle, during which the black soldier fly larvae ingest the chicken manure and complete the processes of digestion, absorption, and metabolism in their bodies.

[0054] After the conversion is completed, the larvae are separated from the manure and its residues through screening means. The larvae are screened out, and the remaining part is the black soldier fly manure product rich in various beneficial microorganisms.

[0055] On October 12, 2023, Wuhan Kewi Microbial Technology Co., Ltd. submitted samples to the Quality Monitoring and Testing Center for Microbial Products of the Ministry of Agriculture and Rural Affairs for quality inspection, and the inspection results met the NY / T 525-2021 standard. The center issued an official inspection report (Report No.: NO2023-05712), confirming the accuracy and validity of the inspection data. Instruments and equipment including a Kjeldahl nitrogen analyzer (model WZJ-43), a visible spectrophotometer (model WZJ076), a biochemical incubator (model WZJ061), an inductively coupled plasma spectrometer (model WZJ070), and an atomic fluorescence photometer (model WZJ-059) were used during the inspection process.

[0056] The inspection report shows that the microbial flora in this black soldier fly manure product covers various beneficial bacterial species such as Lactobacillus, Actinobacteria, and Bacillus, which is consistent with the descriptions in the literature (Zhang et al., 2023; Zhao et al., 2023; Shao et al., 2023). The total number of probiotics in the product exceeds 200 million CFU / g, the total nutrient content (N + P 2 O 5 + K 2 O) is not less than 10%, the organic matter content is not less than 40%, and the pH value is maintained within the suitable range of 5.5 to 8.5.

[0057] (2) Preparation of Bacillus seed liquid and solid fermented product:

[0058] Preparation of seed liquid: Pick a single colony of Bacillus sp. TD1-2 (hereinafter abbreviated as Bacillus TD1-2), and culture it in LB liquid medium (formula: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of deionized water; pH 7.4) at 200 rpm and 30 °C until OD 600nm = 1.0.

[0059] Preparation of solid fermented product: Take 20 mL of the Bacillus TD1-2 bacterial liquid cultured to OD 600nm = 1.0 and add it to the sterilized medium. The formula ratio of the medium is: 1 kg of rice bran, 1 kg of wheat bran, 0.5 kg of starch, 0.5 kg of soybean meal, 15 g of brown sugar, and mix evenly according to the ratio of medium matrix: water = 1:1.3 (mass ratio), and scale up according to actual needs. Ferment at 30 °C for 8 days, stirring once a day. After fermentation is completed, dry the culture at 60 °C, crush it with a pulverizer, and set aside. The viable bacteria count of the fermented product is detected to be about 20 billion per gram or more.

[0060] (3) Preparation of microbial organic fertilizer

[0061] The prepared black soldier fly feces and Bacillus solid fermentation product are fully mixed in a mass ratio of 1000:8 to form microbial organic fertilizer for standby use.

[0062] 2. Application method of microbial organic fertilizer

[0063] (1) Application method and dosage of microbial organic fertilizer in treatment groups

[0064] The microbial organic fertilizer treatment group was applied at a dosage of 10g / plant / pot in the plastic pots for planting seedlings (the diameter of the pot top was 32cm and the height was 28cm), and was evenly applied into the soil about 10cm-15cm away from the main trunk. The soil was loosened appropriately to facilitate the contact between the microbial organic fertilizer and the soil.

[0065] (2) Processing time:

[0066] In September 2021, the seedlings will be tested first and the seedlings for the experiment will be prepared.

[0067] From July to December 2022, microbial organic fertilizer was applied once a week at a dosage of 10 g / plant for 20 consecutive times. From May to July 2023, microbial organic fertilizer was applied 10 times at 10 g / plant / pot. Therefore, a total of 30 applications were made in one year (from July 2022 to July 2023). In daily management, potted seedlings were watered and fertilized in the usual way.

[0068] (3) Control group setting

[0069] The control group potted seedlings were not treated with microbial organic fertilizer (referred to as BDB2). The control group was also watered and fertilized in the same way as the treatment group. This application also set up a black soldier fly feces treatment alone (referred to as BDB1) test to explore a method to minimize the pathogen titer.

[0070] Example 2 qPCR detection of citrus Huanglongbing pathogen

[0071] 1. Sampling time:

[0072] (1) The sampling time before the plants were experimentally treated was September 4, 2021.

[0073] (2) Between July and December 2022, after 20 treatments with microbial organic fertilizer, samples were collected for pathogen detection on December 6, 2022;

[0074] (3) Between May and July 2023, process 10 more times. After a total of 30 processes, collect samples on July 1, 2023 for pathogen detection.

[0075] 2. Sampling method:

[0076] Each time, strictly disinfect the scissors with 4% (volume ratio: v / v) sodium hypochlorite, wipe them clean with a wet tissue before use. After collecting samples in the greenhouse, wipe the leaf surface clean with a wet tissue containing 70% alcohol, cut off the leaf veins, immediately put them into a 2 mL centrifuge tube sterilized by autoclaving, then put them into an ice bath together. After bringing them to the laboratory, immediately freeze the samples with liquid nitrogen, and then store them in a -70°C ultra-low temperature freezer for detection.

[0077] 3. qPCR (Quantitative Polymerase Chain Reaction) detection

[0078] The sampling method on the test plants is to mix-sample the 3rd to 5th leaves on the branches at the east, south, west, and north directions of each citrus plant, and cut off the middle veins of the leaves for extracting total DNA of the leaf veins. Use the CTAB method (Chen Kunsong et al., 2004) suitable for extracting citrus leaf vein DNA to extract DNA. The main instruments used are shown in Table 1. Perform qPCR detection on the extracted DNA, and the reaction system and conditions are shown in Table 2.

[0079] The internal reference gene is the plant cytochrome oxidase gene COX, and the primers are COX+ / COX- (Li et al., 2006). The target gene is the RNR gene of the Asian type pathogen of citrus huanglongbing with 5 copies, that is, the ribonucleotide reductase gene, and the primers are RNR+ / RNR-. The sequence information of the primers is shown in Table 3.

[0080] qPCR detection method:

[0081] Use the sample DNA diluted to 100 ng / μL as the template for real-time fluorescence quantitative PCR detection, and set 3 biological replicates for each sample. The callus DNA of healthy 'Valencia orange' is used as the negative control, and the leaf vein DNA of 'Shatangju' detected as a positive plant in the laboratory is used as the qPCR positive control.

[0082] Table 1

[0083]

[0084]

[0085] Table 2

[0086]

[0087] Table 3

[0088]

[0089] 4. Preparation Method of Standard Curve for qPCR Primers for Detection of Huanglongbing in Citrus

[0090] (1) Amplification of Target Fragment and Ligation and Transformation of Recombinant Plasmid

[0091] Using the DNA extracted from the leaf veins of Huanglongbing-positive plants as a template, ordinary PCR amplifications were performed using the primers COX+ / COX- (Li et al 2006) and RNR+ / RNR- (Zheng et al 2016) respectively. The reaction parameters were set as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s for 35 cycles; and finally extension at 72°C for 5 min. The PCR amplification products were detected by 1% agarose gel electrophoresis. And the amplified PCR products were recovered using the gel extraction kit of ComWin Biotech Co., Ltd. The PCR products were ligated with the pCloneEZ-NRS vector using the pCloneEZ-NRS-Omni-Kan HC cloning kit of Clone smarter company, and then the ligated recombinant plasmid was transformed into JM-109 Escherichia coli competent cells.

[0092] (2) Screening and Sequencing Identification of Recombinant Plasmid

[0093] Picking colonies with a sterile needle and placing them into 1 mL of sterile LB liquid medium containing 100 mg / mL kana, culturing overnight at 37°C with shaking at 150 rpm. The recombinant plasmid was extracted using a plasmid extraction kit. And PCR identification was performed using M13 primers, and the PCR amplification products were detected by 1% agarose gel electrophoresis. And the samples were sent to Quintiles (Wuhan) Biotechnology Co., Ltd. for sequencing identification.

[0094] (3) Purification and Dilution of Recombinant Plasmid

[0095] The recombinant plasmid was extracted from the propagated bacterial solution using the plasmid extraction kit of ComWin Biotech Co., Ltd. The concentration was measured using a UV spectrophotometer, and the standard plasmid was diluted according to the concentration to 1×10 7 ,1×10 6 ,1×10 5 ,1×10 4 ,1×10 3 ,1×10 2 ,1×10 1Plasmids at a concentration of [[COPY_NUMBER]] copy / μL. Real-time fluorescence quantitative PCR was performed on the corresponding plasmids using the primers COX+ / COX- and RNR+ / RNR- respectively. Reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 20 s for 40 cycles, and finally extension at 72°C for 5 min. A standard curve was plotted and the primer efficiency and detection limit were calculated. Using the diluted recombinant plasmids at different concentrations as templates, primers at a concentration of 100 pM were used to perform real-time quantitative PCR reactions under the same conditions, with three replicates set for each concentration, and the effect of different primer concentrations on the fluorescence signal was observed.

[0096] (4) Calculation of primer efficiency and detection limit

[0097] The amplification efficiency E of each gene = (10^(-1 / slope) - 1) × 100%, which was calculated from the slope of the corresponding standard curve. The lowest detection limit was calculated based on the CT value corresponding to the aggregation of the amplification curves of the plasmids at lower concentrations.

[0098] 4. Results

[0099] (1) Preparation of the standard curve for specific primers

[0100] Research has shown that the standard curve of the RNR gene of Candidatus Liberibacter asiaticus Figure 1 ). Through the primer concentration gradient experiment, it was found that when the primer concentration was 100 pM, the linear regression equation of the RNR gene was y = -3.2484x + 37.727, and the regression coefficient was R2 = 0.9907. The amplification efficiency of the primers RNR+ / RNR- reached 103.2%, and the lowest detection limit was 3.419 copies. This indicates that when performing qPCR detection, RNR+ / RNR- has high repeatability and sensitivity. According to this standard curve, the CT value of the sample was converted into the number of copies / vein tissue, that is, the pathogen titer. This quantitative detection result can better reflect the effects of microbial organic fertilizer (BDB2) and black soldier fly manure (BDB1) treatments on controlling the pathogen of huanglongbing.

[0101] (2) Comparison of the effects of microbial organic fertilizer and black soldier fly manure on controlling huanglongbing

[0102] Through comparative experiments with microbial organic fertilizer and black soldier fly manure respectively, the results showed that: when black soldier fly manure (BDB1) was treated alone 20 times and 30 times. The pathogen titers of Candidatus Liberibacter asiaticus decreased by 52.61% and 61.23% respectively. When microbial organic fertilizer (BDB2) was treated 20 times and 30 times, the pathogen titers of Candidatus Liberibacter asiaticus decreased by 55.04% and 73.97% respectively.

[0103] Table 4 Effects of microbial organic fertilizer and black soldier fly manure on the pathogen titer of huanglongbing

[0104]

[0105]

[0106] In order to compare the significance of the difference between two sample percentages, the following two-sample percentage hypothesis testing statistical method is adopted in this application:

[0107] Taking the percentage of the decrease in pathogen titer when the microbial organic fertilizer is treated 20 times and comparing it with the control sample as an example, the calculation process of the hypothesis test for comparing two sample percentages is described.

[0108] A. Sample and Data Acquisition

[0109] Obtain the DBD2 sample treated with the microbial organic fertilizer and the control (CK) sample. Record the titer data of the DBD2 sample treated with the microbial organic fertilizer. Before treatment, it was 5234.57, and after 20 treatments, it was 2353.48. The titer change value Titer_change was 2881.09; the titer of the CK sample before treatment was 6433.19, and the titer corresponding to the time point when treated 20 times was 7014.20. The titer change value Titer_change was -581.01.

[0110] B. Calculation Steps of Hypothesis Testing

[0111] (B1) Calculate the proportion-related values: Calculate P1 = 2881.09 / 5234.57 = 0.5504; calculate P2 = -0.0903; calculate P = 0.19713, and then obtain q = 0.8028; calculate σ(P1 - P2) = 0.007405;

[0112] (B2) Calculate the statistic u value = 62.13;

[0113] (B2) Significance judgment: Set the significance level α = 0.01, and the corresponding critical value ∣u 0.01 ∣ = 2.5758, ∣u 0.05 ∣ = 1.959; Since ∣u∣ > 2.5758 and > 1.959), the null hypothesis can be rejected, and it is concluded that P1 ≠ P2.

[0114] The results show that when the microbial organic fertilizer BDB2 is treated 20 times, the percentage of the decrease in its pathogen titer is extremely significantly different (α = 0.01) compared with the control sample.

[0115] Further analysis using the same statistical method found that:

[0116] When the microbial organic fertilizer BDB2 was treated 30 times (α = 0.01), the percentage decrease in the pathogen titer was also extremely significantly different from that of the control sample. Comparing the results of treating with the microbial organic fertilizer BDB2 20 times and 30 times (α = 0.01), it was found that the percentage decrease in the pathogen titer when treated 30 times was extremely significantly different from that when treated 20 times.

[0117] When the black soldier fly larva manure BDB1 was treated 20 times (α = 0.01), the percentage decrease in the pathogen titer was also extremely significantly different from that of the control sample. When the black soldier fly larva manure BDB1 was treated 30 times (α = 0.01), the percentage decrease in the pathogen titer was also extremely significantly different from that of the control sample.

[0118] However, comparing the results of treating the black soldier fly larva manure BDB1 20 times and 30 times (α = 0.01), it was found that there was no significant difference in the percentage decrease in the pathogen titer between the two.

[0119] When comparing the results of treating with the microbial organic fertilizer BDB2 30 times and the black soldier fly larva manure BDB1 30 times (α = 0.01), it was found that the microbial organic fertilizer BDB2 showed a more significant effect in terms of the percentage decrease in the pathogen titer, and the difference was extremely significant.

[0120] In summary, when the microbial organic fertilizer BDB2 of the present application was treated 30 times, the effect on controlling the pathogen titer was the best.

[0121] In addition, after the temperature dropped in autumn and winter, the concentration of the pathogen of Liberibacter asiaticus in the phloem of the midribs of the top shoots of the diseased seedlings was usually at a relatively high level, and the yellowing and mottling symptoms of the leaves were the most prominent. After the treatment with the microbial organic fertilizer of the present application, sampling and testing were carried out in December 2022, and the results showed that the treatment with the microbial organic fertilizer could withstand the test of autumn and winter and achieve a good control effect.

[0122] Example 3 Effects of microbial organic fertilizer treatment on the growth status of citrus

[0123] 1. Effects of microbial organic fertilizer treatment on the morphological characteristics of citrus

[0124] After treating with the microbial organic fertilizer provided by the present application (prepared and applied as in Example 1), obvious changes occurred in the phenotypes of citrus leaves. It was observed that: before treatment, the positive seedlings in the treatment group showed typical yellowing and mottling symptoms ( Figure 2 A). Starting from July 2022, the seedlings were treated with the microbial organic fertilizer once a week. When treated 12 times, the yellowing and mottling symptoms on the leaves were alleviated or the mottling tended to disappear ( Figure 2 B). When the treatment continued to 20 times, the leaf color turned green, the surface was smooth, with wax, and the appearance was healthy.Figure 2 C). However, for the Huanglongbing positive control plants, different degrees of chlorosis or mottling symptoms could always be observed on the leaves of their branches and shoots. As the growth cycle lengthened, the leaves became more mature, but the top leaves still showed the characteristics of chlorosis or mottling. Figure 2 D, 2E, and 2F). This indicates that the microbial organic fertilizer plays an obvious role in the greening of the leaves.

[0125] From the perspective of the growth changes of the whole nursery stock, the roots of the nursery stock treated with the microbial organic fertilizer are well-developed, the branches and shoots grow vigorously, there are many branches, the leaf color is dark green, and there are many new roots. Figure 2 G), while for the nursery stock in the control group, the roots are sparse and there is browning. Figure 2 H). Thus, it can be seen that the microbial organic fertilizer shows significant effects on the restoration and rejuvenation of citrus trees.

[0126] 2. Effects of different application rates of the microbial organic fertilizer on nursery stock

[0127] As Figure 3 (Figures A - F) shows the performance of the nursery stock 2 days after treatment with different doses of the microbial organic fertilizer in August 2022 (summer, average monthly temperature 30°C); the results show that the difference in the application rate significantly affects the growth state of the nursery stock.

[0128] The microbial organic fertilizer was applied to the roots or soil of citrus nursery stock at different treatment doses each time; when the treatment dose was 10 g - 20 g / plant / pot, the nursery stock showed normal performance, the leaf color turned green, and the growth vigor increased. Figure 3 A, 3B); in mid-August, when 30 g / plant / pot was applied, a small number of leaves on the top shoots withered. Figure 3 C, 3D); when 50 g / plant / pot was applied, a large number of leaves withered, the branches and shoots drooped, and the fruits turned yellow prematurely. Figure 3 E); when 200 g / plant / pot was applied, all the leaves withered and fell off, and the main trunk and side branches gradually dried up. Figure 3 F).

[0129] In addition, during the period from July to December 2022, the average monthly temperatures were 33°C, 33°C, 28°C, 24°C, 17°C, and 10°C respectively; between May and July 2023, the average monthly temperatures were 26°C, 30°C, and 33°C respectively. When the application rate of the microbial organic fertilizer was 10 - 20 g / plant / pot, it was all safe, the nursery stock did not show the symptom of withered leaves, and the growth state was normal and significantly better than that of the control.

[0130] In December 2022 (winter, monthly average temperature 10℃), the microbial organic fertilizer was used as a base fertilizer once, with a treatment amount of up to 1000g / plant / pot; at this time, the plants maintained normal growth and the leaves were dark green, indicating that it was safe to use this dose of microbial organic fertilizer under this condition ( Figure 3 G).

[0131] It can be seen that when the monthly average temperature is above 17℃, the amount of microbial organic fertilizer used should not exceed 20g / plant / pot, because excessive use will cause some leaves to wither and fall off. In the low temperature season (when the monthly average temperature is below 10℃), the high-dose one-time application of microbial organic fertilizer as basal fertilizer (1000g / plant / pot) can still ensure the safety of seedlings. At the same time, when applying high doses, attention should be paid to the balance of trace elements, and attention should be paid to the coordinated use of trace elements and organic fertilizers in microbial organic fertilizers to prevent nutritional competition between multiple microorganisms and citrus plants. The above results provide a reference for the actual operation and application of microbial organic fertilizers.

[0132] Example 4 Non-targeted metabolomics analysis

[0133] In the non-targeted metabolite analysis method of this example, 100 mg of liquid nitrogen-ground tissue samples were collected for each sample, with 6 biological replicates. LC-MS analysis was performed (Sellick et al., 2011; Sellick et al., 2012). Mass spectrometer QExactive TM HF / Q Exactive TM HF-X Thermo Fisher Germany, chromatograph Vanquish UHPLC Thermo Fisher Germany, chromatographic column Hypesil Gold column (100×2.1 mm, 1.9 μm) Thermo Fisher USA. Non-targeted metabolomics analysis and identification were performed on 24 samples (black soldier fly feces prepared according to the method provided in Example 1) at Beijing Novogene Technology Co., Ltd., and a total of 1301 metabolites in positive ion mode (ESI+) and 742 metabolites in negative ion mode (ESI-) were identified. Differential metabolites were screened according to the standards of VIP>1.0, FC>1.2 or FC<0.833 and P-value<0.05.

[0134] The results are as follows Figure 4As shown in the figure, the excrement of Hermetia illucens has rich metabolic components. Among them, the metabolites and percentages obtained by anion analysis are as follows: lipids and lipid-like molecules (39.01%), organic acids and their derivatives (16.83%), benzene-like compounds (12.06%), organic heterocyclic compounds (10.69%), nucleosides, nucleotides and their analogues (7.92%), organic oxygen compounds (7.72%), phenylpropanoids and polyketides (5.15%), alkaloids and their derivatives (0.20%), organic nitrogen compounds (0.2%), hydrocarbons (0.2%).

[0135] Taking the Bacillus amyloliquefaciens MG-2 strain [1] (hereinafter referred to as the MG-2 strain) studied in our laboratory as a reference, Shanghai Applied Protein Technology Co., Ltd. carried out a detailed identification of the metabolites of the MG-2 strain provided by Wuhan Keno Biotechnology Co., Ltd. The results ( Figure 5 ) show that: lipids and lipid-like molecules (22.164%), organic acids and their derivatives (6.62%), benzene-like compounds (10.03%), organic heterocyclic compounds (9.37%), nucleosides, nucleotides and their analogues (1.574%), organic oxygen compounds (7.41%), phenylpropanoids and polyketides (22.492%), alkaloids and their derivatives (2.492%), organic nitrogen compounds (1.246%), hydrocarbons (0.131%).

[0136] The comparison of the metabolite composition of the excrement of Hermetia illucens (BDB1) in this application with that of the MG-2 strain [1] shows that the percentages of the above components contained in the BDB1 metabolites are significantly higher than those of the main components in the single MG-2 strain. After treating the Huanglongbing-infected positive seedlings with the bacterial powder fermented from the MG-2 strain, the pathogen positive rate decreased from 100% to 50%. The above metabolites are closely related to plant disease resistance and normal physiological and biochemical metabolism of plants, and have plant disease resistance-related functions [2-5] . These data are highly consistent with the field control effect of Huanglongbing (the pathogen titer decreased by 52.16 - 73.97%), providing strong theoretical support for the research on the resistance of microbial organic fertilizers to citrus Huanglongbing. It shows that the use of the microbial organic fertilizer in this application is of great significance for improving plant disease resistance.

[0137] Example 5 Analysis of the secondary metabolite gene cluster of Bacillus sp. TD1-2

[0138] In this example, a framework analysis of the secondary metabolite gene cluster of Bacillus sp. TD1-2 was performed. First, the construction of the Bacillus sp. TD1-2 library and the sequencing on the machine were carried out: After the DNA sample was detected to be qualified, it was randomly fragmented using a Covaris ultrasonic disruptor, and then the whole library preparation work was completed through steps such as end repair, adding A tails, adding sequencing adapters, PCR amplification, fragment screening, and purification. The final DNA library was obtained. Sequencing was performed on the Illumina platform PE150.

[0139] Analysis results of the secondary metabolite gene cluster of Bacillus TD1-2( Figure 6 ) showed that PKS can be divided into three types: Type I is also called modular PKS, which is a multi-functional enzyme complex composed of multiple domains. Type II is also called aromatic PKS, which mainly synthesizes aromatic compounds. Type III is also called chalcone-type PKS. These products are important components for improving plant disease resistance and inducing immunity. Among them, polyketide compounds and non-ribosomal peptide compounds are important raw materials for the synthesis of antibacterial drugs.

[0140] The above Examples 4 and 5 respectively explained the material basis of the microbial organic fertilizer of this application for resisting Huanglong disease from two aspects: black soldier fly manure and Bacillus TD1-2. The contents of lipids / lipoids (39.01%) and organic acids (16.83%) in black soldier fly manure were significantly higher than those of the single-strain fermentation product of Bacillus MG-2 strain (such as lipids only 22.16%), and it contained a higher proportion of plant disease resistance-related substances such as nucleotide analogs (7.92% vs 1.57%); these metabolites may synergistically enhance their effects through the following pathways: lipid substances enhance stress resistance as components of the plant cell membrane structure; organic acids regulate the rhizosphere pH value to inhibit pathogenic bacteria; and phenyl compounds induce systemic acquired resistance (SAR), etc. At the same time, the manure generated by black soldier flies treating organic waste itself has the physical effect of improving soil structure and increasing the organic matter content, which may create a more suitable microenvironment for the colonization of Bacillus TD1-2 and indirectly enhance its functional expression. The type III PKS (chalcone-type) and NRPS gene clusters contained in Bacillus TD1-2 can synthesize compounds such as flavonoids and antibacterial peptides, and these substances have dual functions in plant-microbe interactions (possibly directly inhibiting the pathogen of Huanglong disease and activating defense signaling pathways such as jasmonic acid). The hydrocarbons (0.2%) in the manure may serve as substrates for TD1-2 to synthesize polyketide substances, and the antibacterial substances (such as non-ribosomal peptides) produced by the strain can enhance the stability of the existing metabolites in the manure, forming a positive feedback loop.

[0141]

References

[0142] [1]Ding Z,Liu Y,Zhang S,Wang F,Zong Q,Yang Y,Du A,Zheng Y,Zhu J andJiang L(2024)Investigation of the anti-Huanglongbing effects usingantimicrobial lipopeptide and phytohormone complex powder prepared fromBacillus amyloliquefaciens MG-2 fermentation.Front.Microbiol.15:1458051.doi:10.3389 / fmicb.2024.1458051

[0143] [2]Kim PI,Ryu J,Kim YH,Chi YT.Production of biosurfactantlipopeptides iturin A,fengycin and surfactin A from Bacillus subtilis CMB32for control of Colletotrichum gloeosporioides.Journal of Microbiology&Biotechnology,2010,20(1):138

[0144] [3]Pathak KV,Keharia H.Identification of surfactins and iturinsproduced by potent fungal antagonist,Bacillus subtilis K1 isolated fromaerial roots of banyan(Ficus benghalensis)tree using massspectrometry.Biotech,2014,4(3):283-295

[0145] [4] Dame ZT, Rahman M. 2021, Islam T. Bacilli as sources of agrobiotechnology: recent advances and future directions[J]. Green Chemistry Letters and Reviews, 14(2): 246-71

[0146] [5] Chowdhury SP, Uhl J, Grosch R, et al. 2015, Cyclic lipopeptides of Bacillus amyloliquefaciens subsp. plantarum colonizing the lettuce rhizosphere enhance plant defense responses toward the bottom rot pathogen Rhizoctonia solani. Molecular Plant-Microbe Interactions, 28(9): 984-95

[0147] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a microbial organic fertilizer, characterized in that: The steps include: S1. Inoculating black soldier fly larvae into chicken manure, sieving out the larvae after 6-10 days of transformation, retaining the excrement and other residues to obtain black soldier fly excrement; S2, inoculating a single colony of Bacillus sp. TD1-2 into LB liquid medium for seed culture to obtain seed liquid; S3, fermenting the seed liquid in a solid culture medium at 28-32° C. for 7-15 days, drying the culture at 60° C., and then crushing to obtain a solid fermentation product; S4, mixing the black soldier fly feces with the solid fermentation product in a mass ratio of 1000:8 to obtain the microbial organic fertilizer; The Bacillus sp. TD1-2 was deposited in the China Center for Type Culture Collection on October 30, 2019, with a deposit number of CCTCC NO: M 2019873, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The preparation method according to claim 1, characterized in that: In step S1, the black soldier fly larvae are 6 days old; 100 larvae are inoculated per 200g of chicken manure; and the moisture content of the chicken manure is controlled to be maintained at 65%-70%.

3. The preparation method according to claim 1, characterized in that: In step S2, the LB liquid culture medium includes: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride; pH 7.

4.

4. The preparation method according to claim 3, characterized in that: The seed culture was carried out at 180-220 rpm and 28-32°C until the OD of the seed solution reached 600 Reach 0.8-1.

2.

5. The preparation method according to claim 1, characterized in that: In step S3, 1 kg of rice bran, 1 kg of bran, 0.5 kg of starch, 0.5 kg of soybean meal and 15 g of brown sugar are mixed to prepare a culture medium matrix; a solid culture medium is configured according to a mass ratio of the culture medium matrix to water of 1:1.3, and then 20 mL of seed liquid is added for solid fermentation.

6. The preparation method according to claim 1, characterized in that: In steps S3 and S4, the number of viable bacteria in the solid fermentation product is ≥ 20 billion / g.

7. A microbial organic fertilizer, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the microbial organic fertilizer according to claim 7 in preventing and controlling citrus Huanglongbing disease and restoring tree vigor.

9. The use according to claim 8, characterized in that: The microbial organic fertilizer is applied to the roots of citrus seedlings or in the soil at a dosage of 10-20 g / plant / pot each time; it is applied once a week for 20 consecutive times; after 4-6 months, additional microbial organic fertilizer is applied once a week for a total of 10 times.

10. The use according to claim 8 or 9, characterized in that: The microbial organic fertilizer is used for the following purposes: A1. Reduce the pathogen titer of Bacillus subtilis; A2. Prevent and control the pathogen of Huanglongbing; A3. Reduce the symptoms of yellowing or mottled leaves; A4. Improve root stunting and browning; A5. Cultivate virus-free citrus seedlings.

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