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

Abstract

The present application relates to the field of biotechnology, and particularly relates to a microbial organic fertilizer and application thereof in prevention and control of citrus Huanglongbing and recovery of tree vigor. The present application obtains worm manure organic fertilizer by converting chicken manure through black soldier fly larvae, and carries out solid fermentation on bacillus TD1-2, and the two are mixed to form microbial organic fertilizer rich in beneficial bacteria. The organic fertilizer is continuously applied to citrus seedlings infected with Huanglongbing, and the result shows that the pathogen titer is greatly reduced, and the disease resistance effect is remarkable. At the same time, the organic fertilizer under a suitable dose significantly promotes the growth of citrus seedlings, and improves the leaf and root system conditions. Metabolic component analysis shows that the organic fertilizer is rich in metabolites closely related to plant disease resistance and normal physiological and biochemical metabolism, which provides theoretical support for the disease resistance effect. In addition, the present application defines the safe use range of the microbial organic fertilizer, ensures the application effect under different temperature conditions, and provides a new strategy for biological prevention and control of citrus Huanglongbing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a microbial organic fertilizer and its application in the prevention and control of citrus Huanglongbing (HLB) and the restoration of tree vigor. Background Technology

[0002] Huanglongbing (HLB), a quarantine disease caused by the difficult-to-culture Candidatus Liberibacter spp., poses a serious challenge to the global citrus industry due to its long incubation period, rapid spread, and wide range of damage. In my country, the disease has affected approximately 300 counties in 11 provinces, resulting in the loss of a large number of citrus trees, a significant decline in citrus yield and quality, and severely hindering the healthy development of the industry.

[0003] Control measures for citrus Huanglongbing (HLB) mainly include spraying pesticides to kill the vector insect psyllid, cutting down diseased trees, and cultivating virus-free seedlings. However, these methods have significant limitations. While insect-proof netting can effectively isolate psyllids, its high cost limits its widespread application and is not conducive to mechanized and intelligent management. Chemical control is effective in the short term, but it easily leads to increased pesticide resistance in psyllids, while also causing pesticide residues and ecological damage. Antibiotic control may cause phytotoxicity, affecting fruit appearance, and long-term use can enhance pathogen resistance, making the disease prone to recurrence after discontinuation of treatment.

[0004] Given the limitations of traditional control strategies, biocontrol bacteria have shown great potential in the control of citrus Huanglongbing (HLB). Biocontrol bacteria exert specific effects at different developmental stages or parts of the plant through various mechanisms, such as antagonism, competition, hyperparasitism, induction of plant resistance, and promotion of plant growth, effectively inhibiting or killing pathogenic microorganisms and ensuring plant health. Inducing plant resistance is a key way to enhance plant defense capabilities, including two pathways: systemically acquired resistance (SAR) and induced systemic resistance (ISR). SAR refers to the development of resistance in uninoculated parts of a plant after infection with a pathogen; while ISR refers to the stimulation of the plant by non-pathogens (such as biocontrol bacteria) to induce the formation of physical or chemical barriers, enhancing overall resistance. Furthermore, biocontrol bacteria promote plant growth by improving nutrient absorption, regulating hormone levels, or enhancing metabolic activity, indirectly improving plant disease resistance.

[0005] Citrus cultivation is mainly concentrated in acidic soil areas of southern China, and Bacillus, as an important biocontrol microorganism, has limited survival and reproduction in acidic soils. Therefore, screening out Bacillus species that have good control effects on citrus diseases and are tolerant of acidic environments is of great significance. Given the current lack of effective control methods for citrus Huanglongbing (HLB), developing novel, efficient, and environmentally friendly control strategies and providing virus-free seedlings has become an urgent research need. Summary of the Invention

[0006] In view of this, the present invention provides a microbial organic fertilizer and its application in the control of citrus Huanglongbing (HLB) and the restoration of tree vigor. The present invention combines black soldier fly larvae manure with Bacillus sp. TD1-2 fermentation product, demonstrating significant effects on the control of citrus HLB and the sustained restoration of citrus tree vigor.

[0007] The technical solution of this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for preparing microbial organic fertilizer, comprising the following steps:

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

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

[0011] S3. After fermenting the seed culture in a solid culture medium at 28-32℃ for 7-15 days, the culture is dried at 60℃ and then pulverized to obtain a solid fermentation product.

[0012] S4. The black soldier fly larvae excrement and solid fermentation material are mixed at 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 accession number CCTCC NO: M 2019873, and the deposit address is 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 days old, and 100 larvae are inoculated per 200g of chicken manure. The culture quantity can be increased proportionally according to experimental needs; the moisture content of the chicken manure should be controlled at 65%-70%.

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

[0016] Furthermore, in some specific embodiments, the seed culture is carried out at 180-220 rpm and 28-32°C until the OD of the seed culture reaches a certain level. 600 It 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 substrate; a solid culture medium is prepared according to the mass ratio of culture medium substrate to water of 1:1.3, and then 20 mL of seed liquid is added for solid fermentation.

[0018] In some specific embodiments, in steps S3 and S4, the number of viable bacteria in the solid fermentation product is ≥20 billion / g.

[0019] Secondly, the present invention provides a microbial organic fertilizer, which is prepared according to the preparation method described above.

[0020] Secondly, the present invention provides the application of the microbial organic fertilizer in the prevention and control of citrus Huanglongbing and the restoration of tree vigor.

[0021] In some specific embodiments, the microbial organic fertilizer is applied to the roots of citrus seedlings or the soil at a dosage of 10-20g / plant / pot each time; applied once a week for 20 consecutive weeks; and then applied again after 4-6 months, 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. Reduce the titer of Bacillus phloem pathogens;

[0024] A2. Control of Huanglongbing pathogen;

[0025] A3. Reduce symptoms of yellowing or mottled leaves;

[0026] A4. Improves poor root development and browning;

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

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

[0029] First, after treating citrus seedlings with the microbial organic fertilizer provided by this invention, the pathogen titer of the main pathogen of Huanglongbing (HLB) in the leaf vein samples of the treated group decreased significantly. When treated with the microbial organic fertilizer 30 times, the percentage decrease in pathogen titer was highly significant compared to the control group (CK). Comparing the results of 20 and 30 treatments with the microbial organic fertilizer of this application, it was found that the percentage decrease in pathogen titer after 30 treatments was also highly significant compared to after 20 treatments. However, comparing the results of 20 and 30 treatments with black soldier fly larvae manure alone, no significant difference was found in the percentage decrease in pathogen titer between the two methods.

[0030] Secondly, under appropriate treatment dosages (10-20g / plant / pot), the microbial organic fertilizer provided by this invention significantly promoted the normal growth of citrus seedlings, manifested as greening of leaves and significantly enhanced growth vitality, with their growth status being significantly better than the control group that did not use organic fertilizer. The microbial organic fertilizer provided by this invention has a positive effect on improving the condition of citrus leaves. The treated group seedlings had lush leaves, well-developed root systems, and abundant new roots, exhibiting good growth; while the control group seedlings had poor root development, accompanied by browning, and their growth was stunted.

[0031] Furthermore, in terms of metabolic component analysis, black soldier fly frass contains abundant metabolites, with lipids and lipid molecules, organic acids and their derivatives, benzene-like compounds, and organic heterocyclic compounds accounting for a significant proportion. Bacillus TD1-2 contains abundant secondary metabolic gene clusters, including multifunctional enzyme complexes, aromatic PKS, and chalcone-type PKS. These metabolites are closely related to plant disease resistance and normal physiological and biochemical metabolism. In particular, the proportion of active ingredients with plant disease resistance-related functions is quite high, which is closely related to the reduced pathogen titer in potted seedlings controlling Huanglongbing (HLB), providing solid theoretical support for research on the resistance of microbial organic fertilizers to citrus HLB.

[0032] Finally, this invention also defines the safe application range of microbial organic fertilizer. Under conditions where the average monthly temperature is above 17℃, the amount of organic fertilizer used should not exceed 20g / plant / pot to avoid adverse effects such as leaf wilting and shedding caused by excessive application. From November to January, it is also safe to use the microbial organic fertilizer at a rate of 1000g / mature tree without causing root burn. Subsequent experiments also proved that it is safe to use at a rate of 10kg / mature tree in January 2024.

[0033] Typically, Huanglongbing (HLB) symptoms are more pronounced in autumn and winter than in spring and summer, with increased pathogen concentrations. However, this application demonstrates that even under low-temperature conditions in autumn and winter, pathogen detection using microbial organic fertilizer shows it can withstand the test, effectively controlling HLB and ensuring the healthy growth of citrus seedlings. It is important to note that when using the aforementioned microbial organic fertilizer, special attention should be paid to supplementing micronutrients to prevent nutrient competition and ensure the effective operation and application of the microbial organic fertilizer.

[0034] Biological Preservation

[0035] The Bacillus sp. TD1-2 used in this application was obtained through isolation and purification from the vegetable garden soil of the Agricultural Microbiology Engineering Center of Huazhong Agricultural University in Wuhan, Hubei Province. The Bacillus sp. TD1-2 was deposited at the China Center for Type Culture Collection (CCTCC) on October 30, 2019, with accession number CCTCC NO: M 2019873, located at No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0038] Figure 2 This invention illustrates the effect of microbial organic fertilizer treatment on the morphological characteristics of citrus seedlings. Figures A, B, C, and G represent the microbial organic fertilizer treatment groups; Figures D, E, F, and H represent the untreated control group. Details are as follows:

[0039] Images A and D (taken March 17, 2022): Show the initial state of the treatment and control groups, with leaves in both groups showing yellowing or mottling symptoms, serving as the baseline for the experiment; Images B and E (taken October 12, 2022): Image B shows the shoots after 12 treatments with microbial organic fertilizer, with leaves turning green and showing a healthy state; Image E, as the control group, was taken at the same time point, with leaves still showing yellowing; Images C and F (taken December 13, 2022): Image C shows the shoots after 20 treatments, with dark green leaves and a waxy sheen; Image F, the control group, showed leaves continuing to yellow at the same time point; Images G and H (taken February 15, 2023): Image G shows the treated seedlings with lush foliage, well-developed root systems, and abundant new roots; Image H shows the control group seedlings with poor root development and browning;

[0040] Figure 3 This is a comparison of the effects of different treatment amounts of microbial organic fertilizer on seedlings in the embodiments of the present invention;

[0041] AF plot: Seedling response two days after treatment with different dosages in August 2022; differences in treatment dosage significantly affected seedling growth status; details are as follows:

[0042] Image A: 10g / plant treatment, plants show normal performance; Image B: 20g / plant treatment, plants also show normal performance; Image C: 30g / plant treatment, plants are generally normal, but Image D (Image D is a magnified section of Image C) shows a small number of top leaves withered; Image E: 50g / plant treatment, leaves show signs of water loss and wilting, and fruits turn yellow prematurely; Image F: 200g / plant treatment, all plants wither and suffer severe leaf drop.

[0043] Figure G: Plant condition on December 17, 2024 (average temperature 10℃), with a treatment amount as high as 1000g / plant; at this time, the plants maintained normal growth and the leaves were dark green, indicating that the use of this dose of microbial organic fertilizer was safe under these conditions;

[0044] Figure 4 The types and percentages of the top 10 metabolites contained in the black soldier fly frass in this embodiment of the invention;

[0045] Figure 5 The results of the identification of metabolites in Bacillus amyloliquefaciens strain MG-2;

[0046] Figure 6 The above are the statistical results of gene clusters and corresponding gene quantities of Bacillus TD1-2 samples in the embodiments of the present invention. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0048] The citrus seedlings involved in this application were obtained by our research group from Nanfeng mandarin oranges in Jiangxi Province. First, positive seedlings were identified through qPCR detection of Huanglongbing (HLB) pathogen. Then, using trifoliate orange as rootstock, when the root collar diameter of the trifoliate orange reached 0.8-1 cm, bud grafting was performed using positive scions in August-September to obtain uniform positive seedlings. After two years of growth, samples were taken from each seedling for testing, and the positive seedlings were used for experiments. Each treatment had at least six 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 frass:

[0052] The black soldier fly frass described in this application was prepared by Wuhan Kewei Microbial Technology Co., Ltd., and its preparation method includes the following steps:

[0053] Chicken manure was used as the raw material, and 6-day-old black soldier fly larvae were inoculated at a ratio of 100 larvae per 200 grams of chicken manure. During the preparation process, the moisture content of the chicken manure was strictly controlled within the range of 65% to 70% (by weight) to ensure suitable transformation conditions. Subsequently, an 8-day biotransformation cycle was performed, during which the black soldier fly larvae ingested the chicken manure and completed the digestion, absorption, and metabolism processes within their bodies.

[0054] After the conversion is completed, the larvae are separated from the excrement and residues by screening. The larvae are screened out, while the remaining part is the black soldier fly excrement product rich in a variety of beneficial microorganisms.

[0055] On October 12, 2023, Wuhan Kewei Microbial Technology Co., Ltd. submitted samples to the Microbial Product Quality Monitoring and Testing Center of the Ministry of Agriculture and Rural Affairs for quality testing. The test results met the NY / T 525-2021 standard. The center issued a formal test report (report number: NO2023-05712), confirming the accuracy and validity of the test data. The testing process utilized 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 spectrophotometer (model WZJ-059).

[0056] The test report shows that the microbial community in this black soldier fly larvae excrement product includes various beneficial bacteria 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+P2O5+K2O) is not less than 10%, the organic matter content is not less than 40%, and the pH value is maintained within a suitable range of 5.5 to 8.5.

[0057] (2) Preparation of Bacillus seed culture and solid fermentation product:

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

[0059] Preparation of solid-state fermentation product: Take 20 mL of the above culture until OD 600nm A 1.0 g / L Bacillus TD1-2 bacterial suspension was added to a sterilized culture medium. The culture medium formula was: 1 kg rice bran, 1 kg wheat bran, 0.5 kg starch, 0.5 kg soybean meal, and 15 g brown sugar, mixed evenly at a substrate-to-water ratio of 1:1.3 (mass ratio), and scaled up according to actual needs. Fermentation was carried out at 30℃ for 8 days, stirring once a day. After fermentation, the culture was dried at 60℃, pulverized, and set aside for later use. The viable cell count of the fermentation product was found to be over 20 billion CFU / g.

[0060] (3) Preparation of microbial organic fertilizer

[0061] The prepared black soldier fly larvae excrement and Bacillus solid fermentation material are thoroughly mixed at a mass ratio of 1000:8 to form microbial organic fertilizer for later use.

[0062] 2. Application methods of microbial organic fertilizer

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

[0064] For each treatment of the microbial organic fertilizer group, apply 10g / plant / pot to the plastic pot for planting (pot top diameter is 32cm, height is 28cm), about 10cm-15cm away from the main stem, and evenly apply it into the soil. Loosen the soil appropriately to facilitate contact between the microbial organic fertilizer and the soil.

[0065] (2) Processing time:

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

[0067] From July to December 2022, apply microbial organic fertilizer once a week at a rate of 10g per plant, for a total of 20 applications. From May to July 2023, apply microbial organic fertilizer 10 more times at a rate of 10g per plant per pot. Therefore, a total of 30 applications are required within one year (from July 2022 to July 2023). In routine management, water and fertilize potted seedlings using conventional methods.

[0068] (3) Control group setup

[0069] The potted seedlings in the control group were not treated with microbial organic fertilizer (BDB2). Other routine management practices in the control group, including watering and fertilization, were consistent with the treatment group. This application also included a black soldier fly frass treatment alone (BDB1) to explore methods for maximally reducing pathogen titers.

[0070] Example 2: qPCR detection of citrus Huanglongbing pathogen

[0071] 1. Sampling time:

[0072] (1) The sampling time for the plants before the experimental treatment was September 4, 2021.

[0073] (2) Between July and December 2022, samples were collected on December 6, 2022, after being treated 20 times with microbial organic fertilizer;

[0074] (3) Between May and July 2023, the samples were processed 10 more times, for a total of 30 times. On July 1, 2023, samples were collected for pathogen detection.

[0075] 2. Sampling method:

[0076] Each time, the scissors were strictly sterilized with 4% (v / v) sodium hypochlorite, and then wiped clean with a damp paper towel before use. After collecting samples in the greenhouse, the leaf surface was wiped clean with a damp cloth containing 70% alcohol, the leaf veins were cut off, and the samples were immediately placed in autoclaved 2mL centrifuge tubes. The tubes were then placed in an ice bath and brought to the laboratory. The samples were immediately frozen with liquid nitrogen and then stored in an ultra-low temperature freezer at -70℃ for later testing.

[0077] 3. qPCR (quantitative polymerase chain reaction) detection

[0078] Sampling on the tested plants involved sampling the 3rd to 5th leaves from the branches on the east, south, west, and north sides of each citrus plant, and collecting midribs for total vein DNA extraction. DNA was extracted using the CTAB method (Chen Kunsong et al., 2004), which is suitable for citrus leaf vein DNA extraction. The main instruments used are shown in Table 1. The extracted DNA was then analyzed by qPCR; the reaction system and conditions are shown in Table 2.

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

[0080] qPCR detection method:

[0081] Sample DNA diluted to 100 ng / μL was used as a template for real-time quantitative PCR detection, with three biological replicates for each sample. DNA from callus tissue of healthy 'Village Orange' plants served as a negative control, while DNA from leaf veins of 'Satsuma Mandarin' plants that tested positive in the laboratory served as a positive control for qPCR.

[0082] Table 1

[0083]

[0084]

[0085] Table 2

[0086]

[0087] Table 3

[0088]

[0089] 4. Preparation method of qPCR primer standard curve for citrus Huanglongbing detection

[0090] (1) Amplification of target fragments and ligation and transformation of recombinant plasmids

[0091] DNA extracted from leaf veins of Huanglongbing-positive plants was used as a template for conventional PCR amplification using primers COX+ / COX- (Li et al 2006) and RNR+ / RNR- (Zheng et al 2016), respectively. The reaction parameters were set as follows: 94℃ pre-denaturation for 5 min; 35 cycles of 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s; and a final extension at 72℃ for 5 min. PCR products were detected by 1% agarose gel electrophoresis. The amplified PCR products were recovered using a gel recovery kit from Kangwei Century. The PCR products were ligated to the pCloneEZ-NRS vector using the Clone Smarter pCloneEZ-NRS-Omni-Kan HC cloning kit, and the ligated recombinant plasmid was then transformed into JM-109 E. coli competent cells.

[0092] (2) Screening and sequencing identification of recombinant plasmids

[0093] Plaques were picked up with a sterile needle and placed in 1 mL of sterile LB broth containing 100 mg / mL kanamycin. The culture was incubated overnight at 37°C with shaking at 150 rpm. Recombinant plasmids were extracted using a plasmid extraction kit. PCR identification was performed using M13 primers, and the PCR amplification products were detected by 1% agarose gel electrophoresis. The samples were then sent to Quintiles (Wuhan) Biotechnology Co., Ltd. for sequencing identification.

[0094] (3) Purification and dilution of recombinant plasmids

[0095] Recombinant plasmids were extracted from the amplified bacterial culture using a plasmid extraction kit from Kangwei Century Company. The concentration was determined using a UV spectrophotometer. Standard plasmids were diluted to 1×10⁻⁶ concentrations. 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1Plasmids were prepared at a concentration of 1 copy / μL. Real-time quantitative PCR was performed on the corresponding plasmids using primers COX+ / COX- and RNR+ / RNR-, respectively. The reaction program was: 94℃ pre-denaturation for 5 min; 40 cycles of 94℃ denaturation for 20 s, 60℃ annealing for 20 s, and 72℃ extension for 20 s, followed by a final extension at 72℃ for 5 min. A standard curve was plotted, and the efficiency and limit of detection (LOD) of each primer were calculated. Using recombinant plasmids diluted to different concentrations as templates, real-time quantitative PCR was performed under the same conditions with primers at a concentration of 100 pM. Three replicates were set for each concentration to observe the effect of different primer concentrations on the fluorescence signal.

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

[0097] The amplification efficiency of each gene, E = (10(-1 / slope)-1) × 100%, is determined by the slope of the corresponding standard curve. The limit of detection is calculated based on the CT value corresponding to the aggregation of amplification curves of lower concentration plasmids.

[0098] 4. Results

[0099] (1) Preparation of specific primer standard curve

[0100] Studies have shown that the standard curve of the RNR gene in Citrus phloem bacillus (Citrus thalassemia) Figure 1 Through primer concentration gradient experiments, it was found that when the primer concentration was 100 pM, the linear regression equation for the RNR gene was y = -3.2484x + 37.727, with a regression coefficient of R² = 0.9907. The amplification efficiency of primers RNR+ / RNR- reached 103.2%, with a minimum detection limit of 3.419 copies. This indicates that RNR+ / RNR- has high repeatability and sensitivity in qPCR detection. Based on this standard curve, the CT value of the sample was converted into copy number / leaf vein tissue, i.e., pathogen titer. This quantitative detection result can better reflect the effect of microbial organic fertilizer (BDB2) and black soldier fly dung (BDB1) treatment on controlling Huanglongbing pathogen.

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

[0102] Comparative experiments were conducted using microbial organic fertilizer and black soldier fly larvae excrement. The results showed that when black soldier fly larvae excrement (BDB1) was used alone for 20 and 30 treatments, the pathogen titer of *Bacillus phloem* decreased by 52.61% and 61.23%, respectively. When microbial organic fertilizer (BDB2) was used for 20 and 30 treatments, the pathogen titer of *Bacillus phloem* decreased by 55.04% and 73.97%, respectively.

[0103] Table 4. Effects of microbial organic fertilizer and black soldier fly larvae excrement on Huanglongbing pathogen titer.

[0104]

[0105]

[0106] To compare the significance of the difference between two sample percentages, this application uses the following statistical method for hypothesis testing of two sample percentages:

[0107] Taking the percentage decrease in pathogen titer after 20 treatments with microbial organic fertilizer as an example, this paper illustrates the hypothesis testing calculation process for comparing the percentages of the two samples.

[0108] A. Sample and data acquisition

[0109] DBD2 samples and control (CK) samples were obtained from the microbial organic fertilizer treatment. The titer data of the DBD2 samples were recorded: 5234.57 before treatment and 2353.48 after 20 treatments, with a titer change value of 2881.09. The titer of the CK samples was 6433.19 before treatment and 7014.20 at the time point corresponding to 20 treatments, with a titer change value of -581.01.

[0110] B. Hypothesis testing calculation steps

[0111] (B1) Calculate the proportional correlation value: 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 assessment: 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, we can reject the null hypothesis and conclude that P1 ≠ P2.

[0114] The results showed that when treated with microbial organic fertilizer BDB2 20 times, the percentage decrease in pathogen titer was statistically significant compared with the control sample (α=0.01).

[0115] Further analysis using the same statistical methods revealed:

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

[0117] When treated with black soldier fly larvae feces BDB1 20 times (α=0.01), the percentage decrease in pathogen titer was also highly significant compared with the control sample. When treated with black soldier fly larvae feces BDB1 30 times (α=0.01), the percentage decrease in pathogen titer was also highly significant compared with the control sample.

[0118] However, comparing the results of 20 treatments with black soldier fly larvae feces BDB1 versus 30 treatments (α = 0.01), it was found that there was no significant difference in the percentage decrease in pathogen titer between the two treatments.

[0119] When comparing the results of 30 treatments with microbial organic fertilizer BDB2 and 30 treatments with black soldier fly larvae manure BDB1 (α=0.01), it was found that microbial organic fertilizer BDB2 showed a more significant effect in reducing the percentage of pathogen titers, with the difference reaching a highly significant level.

[0120] In conclusion, the best effect on controlling pathogen titers was achieved when the microbial organic fertilizer BDB2 of this application was applied for 30 times.

[0121] Furthermore, after the temperature drops in autumn and winter, the concentration of Bacillus phloem pathogens in the leaf veins of infected seedlings is usually at a high level, and the yellowing and mottling symptoms of the leaves are most prominent. In December 2022, samples were collected and tested after treatment with microbial organic fertilizer. The results showed that the microbial organic fertilizer treatment could withstand the test of autumn and winter and achieve good control effects.

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

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

[0124] After treatment with the microbial organic fertilizer provided in this application (preparation and application as in Example 1), the phenotype of citrus leaves underwent significant changes. Observations revealed that the positive seedlings in the treatment group exhibited typical yellowing and mottling symptoms before treatment. Figure 2 A) Starting in July 2022, seedlings were treated with microbial organic fertilizer once a week. After 12 treatments, the yellowing and mottling symptoms on the leaves were reduced or the mottling tended to disappear. Figure 2 B), after 20 treatments, the leaves turn green, become smooth, waxy, and appear robust. Figure 2 C). However, in the positive control plants for Huanglongbing, varying degrees of yellowing or mottling symptoms could be observed on the leaves at the branch tips. As the growth cycle lengthened, the leaves became more mature, but the terminal leaves still showed yellowing or mottling characteristics. Figure 2 (D, 2E, and 2F). This indicates that microbial organic fertilizer plays a significant role in the greening of leaves.

[0125] From the changes in the overall growth of the seedlings, the seedlings treated with microbial organic fertilizer had well-developed root systems, vigorous branch growth, more branches, dark green leaves, and a large number of new roots. Figure 2 G), while the seedlings in the control group had sparse root systems and showed signs of browning. Figure 2 H). This shows that microbial organic fertilizer has a significant effect on the recovery and rejuvenation of citrus trees.

[0126] 2. The effects of different treatment amounts of microbial organic fertilizer on seedlings

[0127] like Figure 3 Figure AF shows the performance of seedlings treated with different doses of the aforementioned microbial organic fertilizer in August 2022 (summer, average monthly temperature 30℃) 2 days after treatment; the results show that the difference in treatment amount significantly affects the growth status of seedlings.

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

[0129] Furthermore, from July to December 2022, the average monthly temperatures were 33℃, 33℃, 28℃, 24℃, 17℃, and 10℃, respectively; and from May to July 2023, the average monthly temperatures were 26℃, 30℃, and 33℃, respectively. Under these conditions, the application rate of microbial organic fertilizer was 10-20g / plant / pot, and the seedlings did not show symptoms of leaf withering. Their growth was normal and significantly better than the control.

[0130] In December 2022 (winter, average monthly temperature 10℃), microbial organic fertilizer was applied as a base fertilizer in a single application at a rate of up to 1000g / plant / pot. At this time, the plants maintained normal growth with dark green leaves, indicating that using this dosage of microbial organic fertilizer under these conditions was safe. Figure 3 G).

[0131] Therefore, when the average monthly temperature is above 17℃, the application rate of microbial organic fertilizer should not exceed 20g / plant / pot, as excessive application can cause some leaves to wither and fall off. However, during the low-temperature season (when the average monthly temperature is below 10℃), a high-dose, one-time application of microbial organic fertilizer as a base 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 micronutrients and trace elements, and care should be taken to use the micronutrients in the microbial organic fertilizer in combination with organic fertilizer to prevent nutrient competition between various microorganisms and citrus plants. The above results provide a reference for the practical operation and application of microbial organic fertilizer.

[0132] Example 4: Non-targeted metabolomics analysis

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

[0134] The results are as follows Figure 4As shown, the excrement of the black soldier fly is rich in metabolic components. The metabolites and percentages obtained by anion analysis are as follows: lipids and lipid 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%), and hydrocarbons (0.2%).

[0135] The Bacillus amyloliquefaciens MG-2 strain studied in our laboratory [1] Using strain MG-2 (hereinafter referred to as MG-2 strain) as a reference, Zhongke New Life Biotechnology Co., Ltd. conducted a detailed identification of the metabolites of strain MG-2 provided by Wuhan Kenuo Biotechnology Co., Ltd., and the results were as follows: Figure 5 The composition of the compounds was as follows: lipids and lipid 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%), and hydrocarbons (0.131%).

[0136] The metabolite composition of black soldier fly frass (BDB1) in this application is similar to that of strain MG-2. [1] The comparison showed that the percentage of the aforementioned components in the BDB1 metabolites was significantly higher than that in the MG-2 single bacterium. Treatment of Huanglongbing-positive seedlings with the fermented powder from the MG-2 strain reduced the pathogen positivity rate from 100% to 50%. These metabolites are closely related to plant disease resistance and normal physiological and biochemical metabolism, and possess plant disease resistance-related functions. [2-5] These data are highly consistent with the field control effects of Huanglongbing (pathogen titer reduction of 52.16-73.97%), providing strong theoretical support for research on the resistance of citrus Huanglongbing to microbial organic fertilizer. This demonstrates that the use of the microbial organic fertilizer described in this application is of great significance in improving plant disease resistance.

[0137] Example 5: Analysis of secondary metabolic gene clusters of Bacillus sp. TD1-2

[0138] This embodiment provides a framework analysis of the secondary metabolic gene clusters of Bacillus sp. TD1-2. First, a Bacillus sp. TD1-2 library was constructed and sequenced: After the DNA samples passed quality checks, they were randomly fragmented using a Covaris ultrasonic disruptor. The library preparation then involved end repair, A-tailing, sequencing adapter addition, PCR amplification, fragment selection, and purification. The final DNA library was then obtained and sequenced using an Illumina PE150 platform.

[0139] Results of secondary metabolic gene cluster analysis of Bacillus TD1-2 ( Figure 6 This indicates that PKS can be divided into three types: Type I, also known as modular PKS, is a multifunctional enzyme complex composed of multiple domains. Type II, also known as aromatic PKS, mainly synthesizes aromatic compounds. Type III, also known as chalcone-type PKS. These products are important components for improving plant disease resistance and inducing immunity. Among them, polyketides and non-ribosomal peptides are important raw materials for the synthesis of antibacterial drugs.

[0140] Examples 4 and 5 above explain the material basis of the microbial organic fertilizer's resistance to Huanglongbing from the perspectives of black soldier fly frass and Bacillus TD1-2, respectively. The components of black soldier fly frass, such as lipids / lipids (39.01%) and organic acids (16.83%), are significantly higher than those in the fermentation products of Bacillus MG-2 strain (e.g., lipids are only 22.16%), and it contains a higher proportion of plant disease resistance-related substances such as nucleotide analogs (7.92% vs 1.57%). These metabolites may synergistically enhance the effects through the following pathways: lipids enhance stress resistance as structural components of plant cell membranes; organic acids regulate rhizosphere pH to inhibit pathogens; and benzene-like compounds induce systemic resistance (SAR). Simultaneously, the frass generated by black soldier fly frass processing organic waste has the physical effect of improving soil structure and increasing organic matter content, which may create a more suitable microenvironment for Bacillus TD1-2 colonization, indirectly enhancing 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 antimicrobial peptides. These substances have a dual function in plant-microbe interactions (potentially directly inhibiting the pathogen of Huanglongbing and activating defense signaling pathways such as jasmonic acid). The hydrocarbons (0.2%) in insect excrement may serve as substrates for the synthesis of polyketides by TD1-2, while the antimicrobial substances produced by the strain (such as non-ribosomal peptides) can enhance the stability of existing metabolites in insect excrement, 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 ChemistryLetters 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 rhizosphereenhance plant defense responses toward the bottom rot pathogen Rhizoctoniasolani. Molecular Plant-Microbe Interactions, 28(9):984-95

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of a microbial organic fertilizer in the control of citrus Huanglongbing (HLB) and restoration of tree vigor. The method for preparing the microbial organic fertilizer includes the following steps: S1. Inoculate 6-day-old black soldier fly larvae into chicken manure, with 100 larvae inoculated per 200g of chicken manure; control the moisture content of the chicken manure to be maintained at 65%-70% by mass; after 6-10 days of transformation, sieve out the larval bodies, retain the larval excrement and other residues to obtain black soldier fly larval excrement. S2. Inoculate a single colony of Bacillus sp. TD1-2 into LB liquid medium for seed culture to obtain seed solution; S3. Mix 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 to prepare the culture medium substrate; prepare a solid culture medium with a substrate-to-water ratio of 1:1.3, and then add 20 mL of seed culture to the solid culture medium. After fermentation at 28-32℃ for 7-15 days, the culture is dried at 60℃ and then pulverized to obtain a solid fermentation product with a viable count of ≥20 billion / g. S4. The black soldier fly larvae excrement and solid fermentation material are mixed at a mass ratio of 1000:8 to obtain the microbial organic fertilizer; The Bacillus sp. TD1-2 was deposited at the China Center for Type Culture Collection on October 30, 2019, with accession number CCTCC NO: M2019873 and address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The steps of the application include: Apply the microbial organic fertilizer at a rate of 10-20g / plant / pot to the roots or soil of citrus seedlings each time. Apply once a week for 20 consecutive weeks; after an interval of 4-6 months, apply additional microbial organic fertilizer once a week for a total of 10 times.

2. The application according to claim 1, characterized in that, The microbial organic fertilizer is used for the following purposes: It reduces the titer of Bacillus phloem pathogens; alleviates leaf yellowing or mottling symptoms; and improves poor root development and browning.

Images