Ecological prevention and control method for sclerotinia rot of colza based on microbial metabolites

Abstract

The invention discloses an ecological prevention and control method for sclerotinia rot of colza based on microbial metabolites, and relates to the technical field of agricultural biology. The method comprises the following steps: constructing a microbial team consisting of bacillus subtilis X-01, streptomyces gall YJ1 and coniothyrium minitans parasitic bacteria, wherein the microbial team respectively secretes iturinA and sclerotia to form inhibition antibiotics and parasitic enzymes; extracting and compounding the metabolites to form a synergistic network; preparing a composite carrier composed of an inner core loading layer mesoporous silica, a middle pH sensitive hydrogel layer and an outer hydrophobic ethyl cellulose layer; and accurately applying a quick-release type suspending agent in an initial flowering stage, a pH-responsive type suspending agent in a full-bloom stage and a long-acting slow-release type suspending agent in a pod stage in stages in the whole growth period. A metabolite collaborative network is delivered through a carrier system, and a staged strategy is matched, so that the problems of single strain, poor metabolite stability and short prevention and control period in the prior art are solved, and efficient, stable and ecological prevention and control of sclerotinia rot of colza in the whole growth period are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to an ecological prevention and control method for Sclerotinia sclerotiorum based on microbial metabolites. BACKGROUND

[0002] Sclerotinia sclerotiorum is a global fungal disease that can cause yield loss and seriously threaten the safety of edible oil supply. Traditional chemical control relies on pesticide spraying, but long-term use can lead to pathogen resistance, pesticide residues and ecological pollution. Biological control, as an environmentally friendly alternative, has received widespread attention in recent years, with microbial preparations becoming a research hotspot due to their advantages of sustained disease control and no pollution. However, existing technologies focus on single strains or homogeneous microbial combinations, and the mechanisms of metabolites are unclear and the application scenarios are single, making it difficult to meet the efficient prevention and control needs of oilseed rape throughout the growth period.

[0003] In the prior art, Patent No. CN106342898A discloses a compound microbial preparation for preventing and controlling Sclerotinia sclerotiorum, which uses a combination of streptomycin and Streptomyces noto. However, the function of the strain is single and the interaction mechanism of metabolites is not clear, resulting in insufficient stability of the prevention and control effect. Patent No. CN115975882B discloses the antibacterial function of Bacillus subtilis No. 1, but its application is limited to postharvest preservation of tomatoes and has not been expanded to agricultural disease control. Moreover, there is a lack of multi-strain synergistic system design.

[0004] Overall, existing technologies have failed to construct a functionally complementary microbial team and metabolite synergistic network, and the prevention and control measures are limited to a single growth period, making it difficult to achieve efficient prevention and control throughout the whole cycle. More importantly, when applying microorganisms or their metabolites, existing technologies usually use direct application of microbial agents or crude extracts, ignoring the fact that key active metabolites are easily affected by factors such as light, rainwater erosion, and microbial degradation in the field environment, resulting in poor stability, short duration, and difficulty in maintaining effective concentrations at the target site. This is a major technical bottleneck that restricts the stable application of biological control and the effective implementation of whole growth period strategies. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provides an ecological prevention and control method for Sclerotinia sclerotiorum based on microbial metabolites. Through the synergistic integration of a microbial team, a metabolite synergistic network, a phased application strategy, and a multifunctional carrier system, the multiple bottlenecks of single strain combination, unclear metabolite mechanism, limited application scenarios, and poor field stability in existing technologies are effectively addressed, achieving efficient, stable, and low-risk ecological prevention and control of Sclerotinia sclerotiorum throughout the whole cycle and increasing yield.

[0006] The application provides an ecological prevention and control method for sclerotinia stem rot of rape based on microbial metabolites.

[0007] S100, constructing a microbial team: screening bacillus subtilis X-01 with a preservation number of CCTCC NO: M2024158, streptomyces phaeochromogenes YJ1 with a preservation number of CGMCC NO: 25693 and entomophaga australis, wherein

[0008] The bacillus subtilis X-01 secretes a lipopeptide antibiotic iturin A;

[0009] The streptomyces phaeochromogenes YJ1 secretes an antibiotic inhibiting sclerotium formation;

[0010] The entomophaga australis secretes parasitic enzyme substances;

[0011] S200, metabolite synergistic network: extracting and compounding key metabolites of the strains in S100, and constructing a synergistic system of iturin A, YJ1 antibiotic and entomophaga australis, with a mass ratio of (1.5-2.5):(0.8-1.2):(1.0-1.5);

[0012] S300, preparing a composite carrier system: constructing a composite carrier composed of an inner core loading layer, a middle response layer and an outer protective layer, wherein the inner core loading layer is a mesoporous silica nanoparticle loaded metabolite synergistic network complex, the middle response layer is a pH-sensitive hydrogel, and the outer protective layer is a hydrophobic ethyl cellulose coating;

[0013] S400, precise application in stages in the whole growth period: spraying a suspension containing a fast-release carrier at the initial flowering stage, spraying a pH-responsive carrier suspension at the full flowering stage, and spraying a long-acting slow-release carrier suspension at the silique stage;

[0014] S500, delivering the metabolite synergistic network of S200 through the composite carrier system of S300, and matching the staged application strategy of S400 to achieve prevention and control.

[0015] Further, the preparation method of the S100 microbial team comprises the following steps:

[0016] The bacillus subtilis X-01 is inoculated into an LB liquid culture medium, and is fermented at 28-32 DEG C and 180 rpm for 44-52 hours, and the supernatant is obtained by centrifugation at 8000 rpm for 15 minutes;

[0017] The streptomyces phaeochromogenes YJ1 is inoculated into a Gao No. 1 liquid culture medium, and is oscillated and cultured at 28±1 DEG C and 150 rpm for 96±2 hours, and metabolites are extracted by using a methanol-ethyl acetate mixed solvent, wherein the volume ratio of the methanol-ethyl acetate mixed solvent for extracting metabolites is 1:3.

[0018] The parasitic fungus *Petroptera* was inoculated into PDB liquid medium and cultured at 23–25°C and 120 rpm for 110–130 hours. The secretions were collected by filtration through a 0.45 μm filter membrane.

[0019] The above-mentioned metabolite components were stored at 4°C for later use.

[0020] Furthermore, the construction of the metabolite synergistic network in S200 includes:

[0021] The supernatant of Bacillus subtilis X-01 was purified by ammonium sulfate precipitation and dialysis to obtain iturinA concentrate.

[0022] The target components of the crude extract of Streptomyces galbana YJ1 were separated by silica gel column chromatography;

[0023] The mixture of iturinA, YJ1 antibiotic, and scutellarin parasitic enzyme was prepared at a dry weight ratio of (1.8–2.2):(0.9–1.1):(1.2–1.4).

[0024] The compound solution contains: iturin A concentration of 80–120 μg / mL, YJ1 antibiotic concentration of 40–60 μg / mL, and parasitic enzyme mixture concentration of 0.5–1.0 mg / mL.

[0025] Furthermore, the preparation of the composite carrier system in S300 includes:

[0026] Mesoporous silica nanoparticles with a pore size of 8–10 nm were impregnated in a metabolite composite solution and adsorbed by shaking at 4 °C for 22–26 hours, with a loading rate of ≥30%.

[0027] Using acrylic acid and chitosan in a molar ratio of 3:1 as monomers, a pH-sensitive hydrogel layer with a thickness of 100–150 nm was formed on the surface of the loaded particles.

[0028] Using a 5%–8% ethyl cellulose ethanol solution, fluidized bed coating was performed at 35–38℃, resulting in a coating weight gain of 20%–25%, and the targeting peptide Cys-Arg-Pro-Ser-Lys was grafted onto it with a grafting rate ≥15μmol / g.

[0029] The resulting carriers have a particle size of 500–800 nm and a zeta potential of +25 mV to +30 mV.

[0030] Furthermore, the release conditions for the application of the carrier at each growth stage in S400 are as follows:

[0031] Early flowering stage rapid-release carrier: under pH 7.0 and 25℃ conditions, the cumulative release rate over 24 hours is ≥70%;

[0032] Flowering-responsive carrier: ≤20% release rate in 24 hours at 25℃ and pH≥6.5, and ≥40% release rate in 2 hours at pH5.8–6.0;

[0033] Slow-release carrier during the pod stage: ≤30% release in 24 hours, ≥90% cumulative release in 15 days.

[0034] Furthermore, the configuration of the suspending agent in S400 includes:

[0035] The composite carrier was dispersed in an aqueous solution containing 0.10% ± 0.02% xanthan gum and 0.05% Tween-80;

[0036] Homogenize at 8000–10000 rpm for 10–15 minutes to form a suspension with a solid content of 10%–15%;

[0037] Adjust the pH to 6.8–7.2 using 0.1M sodium hydroxide.

[0038] Furthermore, the method is integrated in the S500 phase:

[0039] Apply potassium sulfate fertilizer during the budding and bolting stage of rapeseed, with an application rate equivalent to 60–75 kg / ha of K2O, at a depth of 8–10 cm, followed by covering with soil and irrigating.

[0040] Furthermore, the operational parameters for the phased matching of the S400 application strategy in S500 are as follows:

[0041] Spraying at the initial flowering stage: Carrier concentration 0.5–1.0 g / L, spraying rate 300–400 liters / hectare, when the main inflorescence flowering rate is 10%–20%;

[0042] Spraying during peak flowering period: Carrier concentration 1.2–1.8 g / L, spraying rate 400–500 liters / hectare, when the flowering rate of the whole field is 70%–80%;

[0043] Spraying during the pod-setting stage: Carrier concentration 0.8–1.2 g / L, spraying rate 250–350 liters / hectare, 7–10 days after the end of flowering.

[0044] Compared with existing technologies, this ecological control method for rapeseed sclerotinia stem rot based on microbial metabolites has the following beneficial effects:

[0045] I. This invention constructs a functionally complementary microbial alliance composed of Bacillus subtilis X-01, Streptomyces galbana YJ1, and scutellarin parasitic fungi, forming a synergistic metabolite network with itrinA, YJ1 antibiotics, and scutellarin parasitic enzymes as the core. Combined with a mesoporous silica pH-sensitive hydrogel ethyl cellulose composite carrier system for the protection and delivery of metabolites, and matched with a staged precise application strategy throughout the entire growth period, this invention effectively solves the problems of single-species combinations, unclear metabolite action mechanisms, poor field stability, short duration of effectiveness, and insufficient target concentration in existing biological control technologies, thus achieving efficient and stable control of sclerotinia stem rot in rapeseed throughout the entire growth period.

[0046] Second, this invention utilizes the synergistic effect of multiple bacterial metabolites: iturinA inhibits pathogen growth, YJ1 antibiotics prevent sclerotium formation, and sclerotium parasitic enzymes destroy existing sclerotia. Combined with the phased application of immediate-release, pH-responsive, and long-acting sustained-release carriers, this invention overcomes the shortcomings of existing technologies, such as insufficient stability of control effects from single-species or homogeneous combinations and application scenarios limited to a single growth stage. It improves the sustainability and specificity of control effects and avoids the pathogen resistance, pesticide residues, and ecological pollution caused by chemical control, thus possessing good ecological safety and application value.

[0047] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0049] Figure 1 This is a flowchart illustrating the operational process of an ecological control method for sclerotinia stem rot in rapeseed based on microbial metabolites.

[0050] Figure 2 This is a flowchart illustrating the ecological control method for rapeseed sclerotinia stem rot based on microbial metabolites. Detailed Implementation

[0051] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0052] Example 1

[0053] This embodiment addresses the need for prevention and control of sclerotinia stem rot in rapeseed throughout its entire growth cycle. By constructing a functionally complementary microbial alliance, optimizing the metabolite synergistic network, preparing an intelligent responsive composite carrier system, and combining it with a phased and precise application strategy, it achieves efficient ecological control of sclerotinia stem rot in rapeseed.

[0054] Specifically, such as Figure 2 As shown, a microbial consortium consisting of Bacillus subtilis X-01, Streptomyces galbana YJ1, and scutellarinus parasiticus was selected. Key metabolites (iturinA, YJ1 antibiotic, and scutellarinus parasitic enzymes) were extracted and compounded in specific proportions. These were then loaded onto a mesoporous silica-pH-sensitive hydrogel-ethyl cellulose composite carrier. Immediate-release, pH-responsive, and long-acting slow-release formulations were sprayed at the initial flowering, full bloom, and pod-setting stages, respectively. Simultaneously, potassium fertilizer application during the budding stage enhanced rapeseed resistance. This method, through the combination of synergistic metabolite action and intelligent carrier delivery, solves the problems of poor stability, short duration of effect, and insufficient target concentration in biological control, significantly improving the control effect and providing an operable technical solution for the green control of rapeseed sclerotinia stem rot.

[0055] I. Construction of Microbial Teams (S100)

[0056] The microbial alliance is the core of the control system, and its function lies in secreting key metabolites with synergistic effects. This embodiment selects *Bacillus subtilis* X-01, *Streptomyces galbana* YJ1, and *Sclerotium scutellatus* parasitic fungi. These three fungi achieve complementary functions through antibacterial activity, inhibition of sclerotium formation, and parasitism of sclerotia, respectively. The specific construction process is as follows:

[0057] 1. Culture and metabolite extraction of Bacillus subtilis X-01

[0058] Bacillus subtilis X-01 (CCTCC NO: M2024158) is a Gram-positive bacterium. Its secreted lipopeptide antibiotic, iturin A, has a strong inhibitory effect on Sclerotinia sclerotiorum and can disrupt the integrity of the pathogen's cell membrane.

[0059] In this embodiment, the culture medium selected was LB liquid medium (components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0). This medium is rich in carbon and nitrogen sources and is suitable for the rapid proliferation and metabolite secretion of Bacillus subtilis.

[0060] Culture conditions: Activated X-01 single colonies were inoculated into 500mL Erlenmeyer flasks (200mL of liquid), placed in a constant-temperature shaker, and cultured at 30℃ (optimal temperature within the range of 28-32℃) and 180rpm for 48 hours (intermediate value of 44-52 hours). This temperature is close to the optimal growth temperature of the strain, and the 180rpm rotation ensures sufficient dissolved oxygen in the culture medium, promoting cell proliferation and iturinA synthesis; the 48-hour culture period allows the strain to reach a stationary phase, with metabolite accumulation reaching its peak.

[0061] Metabolite extraction: After cultivation, transfer the fermentation broth to centrifuge tubes and centrifuge at 8000 rpm for 15 minutes. The supernatant is the crude extract containing iturin A (the cell precipitate is discarded). The selection of centrifugation speed and time effectively separates the cells from the supernatant, avoiding interference from cell fragments in subsequent purification. The extracted supernatant is stored at 4°C to prevent metabolite degradation.

[0062] 2. Culture and metabolite extraction of Streptomyces gallinarum YJ1

[0063] The antibiotics secreted by Streptomyces gallinarum YJ1 (preservation number CGMCCNO: 25693) can specifically inhibit the formation of sclerotia of Sclerotinia sclerotiorum, blocking a key link in the disease cycle.

[0064] In this embodiment, the culture medium selected was Gao's No. 1 liquid medium (components: soluble starch 20g / L, potassium nitrate 1g / L, dipotassium hydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L, pH 7.2-7.4). This medium contains abundant starch as a carbon source, which is suitable for the mycelial growth and secondary metabolite synthesis of Streptomyces.

[0065] Culture conditions: YJ1 spore suspension was inoculated into 500mL Erlenmeyer flasks (150mL of liquid), and cultured with shaking at 28℃ (±1℃) and 150rpm for 96 hours (±2 hours). Streptomyces is an aerobic bacterium, and a shaking speed of 150rpm can balance mycelial growth and dissolved oxygen requirements; 28℃ is its optimal growth temperature, and 96 hours of culture can ensure sufficient accumulation of secondary metabolites (antibiotics).

[0066] Metabolite extraction: After cultivation, the fermentation broth was mixed with a methanol-ethyl acetate mixture (volume ratio 1:3) at a 1:1 ratio. The mixture was magnetically stirred at 25°C for 30 minutes. After standing and separating into layers, the organic phase (upper layer) was collected. The extraction was repeated three times. The organic phases were combined and rotary evaporated to dryness at 40°C to obtain the crude extract of antibiotic YJ1. The reason for choosing this solvent ratio is that methanol can dissolve polar components, while ethyl acetate extracts non-polar antibiotics; the two work synergistically to improve extraction efficiency. The crude extract was dissolved in a small amount of methanol and stored at 4°C for later use.

[0067] 3. Culture and Metabolite Extraction of *Scutellaria baicalensis*

[0068] The parasitic fungus *Sclerotium sclerotiorum* can degrade the cell wall of the sclerotium by secreting parasitism-related enzymes such as cellulase and chitinase, and directly parasitize the sclerotium, thus destroying its survival ability.

[0069] In this embodiment, the culture medium selected was PDB liquid medium (potato juice 200g / L, glucose 20g / L, natural pH). This medium is rich in polysaccharides and organic nitrogen, which is suitable for the vegetative growth and enzyme secretion of parasitic bacteria.

[0070] Culture conditions: Inoculate *Scutellaria baicalensis* spores into 500mL Erlenmeyer flasks (containing 200mL of liquid) and culture at 24℃ (23-25℃) and 120rpm for 120 hours (110-130 hours). Lower rotation speed reduces mycelial breakage, 24℃ is the optimal temperature for parasitic enzyme synthesis, and 120 hours of culture ensures sufficient enzyme secretion.

[0071] Metabolite extraction: After cultivation, the fermentation broth was filtered through a 0.45 μm microporous membrane to remove mycelia. The collected filtrate is the secretion containing parasitic enzymes and should be stored at 4°C for later use. A 0.45 μm membrane pore size effectively retains the mycelia while preserving small-molecule enzymes.

[0072] II. Construction of the metabolite synergistic network (S200)

[0073] The core of the metabolite synergistic network is to achieve a synergistic effect of "inhibiting pathogen growth, preventing sclerotium formation, and destroying existing sclerotia" by optimizing the ratio of three metabolites. This embodiment constructs a highly efficient synergistic system through purification and compounding, with the specific steps as follows:

[0074] 1. Metabolite purification

[0075] IturinA purification: Take the supernatant of Bacillus subtilis X-01, slowly add ammonium sulfate to saturation to 70%, incubate at 4℃ for 12 hours, centrifuge at 4000 rpm for 20 minutes to collect the precipitate; dissolve the precipitate in a small amount of 0.01M PBS buffer (pH 7.0), place in a dialysis bag (molecular weight cutoff 3500 Da), and dialyze in distilled water for 48 hours (changing the water every 6 hours) to remove salt ions, obtaining concentrated iturinA solution. Ammonium sulfate precipitation can specifically enrich lipopeptides (iturinA), and dialysis further removes impurities and improves purity.

[0076] Purification of YJ1 antibiotic: The crude extract of YJ1 was loaded onto a silica gel column (200-300 mesh, 30 cm long, 2 cm inner diameter), and eluted with a petroleum ether-ethyl acetate mixed solvent (gradient elution: from 10:1 to 1:1). The fraction exhibiting sclerotinia inhibitory activity was collected (detected by a plate inhibition test). The purified YJ1 antibiotic was obtained by rotary evaporation. Silica gel column chromatography can separate antibiotics of different polarities, ensuring the purity of the target active ingredient.

[0077] Concentration of parasitic enzymes from *Petroma scutellarioides*: *Petroma scutellarioides* secretions were concentrated using an ultrafiltration membrane with a 10 kDa molecular weight cutoff at 0.1 MPa pressure until the volume was reduced to 1 / 5 of the original volume, yielding a parasitic enzyme mixture (containing cellulase, chitinase, etc.). This mixture was stored at 4°C. Ultrafiltration can retain large-molecule enzymes, remove small-molecule impurities, and increase enzyme concentration.

[0078] 2. Metabolite compounding

[0079] The following formulation is prepared at a dry weight ratio of 2.0:1.0:1.3 (iturinA:YJ1 antibiotic: scutellarin parasitic enzyme):

[0080] Take the concentrated iturin A solution (approximately 80% purity) and dilute it with PBS buffer to a concentration of 100 μg / mL;

[0081] Take pure YJ1 antibiotic, dissolve it in methanol and then dilute it to 50 μg / mL (final methanol concentration <5% to avoid affecting enzyme activity).

[0082] Take the parasitic enzyme mixture and dilute it to 0.8 mg / mL (enzyme activity units: cellulase ≥50 U / mL, chitinase ≥30 U / mL).

[0083] The three solutions were mixed in the specified proportions and magnetically stirred at 25°C for 10 minutes to form a metabolite synergistic network complex. The proportions were designed based on the following: iturinA is the primary antibacterial component and is present in the highest proportion; YJ1 antibiotic assists in inhibiting sclerotium formation and is present in the next highest proportion; and the parasitic enzyme requires a relatively high concentration to ensure degradation effectiveness, hence the proportions are moderate. The synergistic mechanism is as follows: iturinA rapidly inhibits the growth of pathogenic mycelia, YJ1 antibiotic prevents the formation of new sclerotia, and the parasitic enzyme destroys existing sclerotia; the three components form a control network.

[0084] III. Preparation of the composite carrier system (S300)

[0085] The composite carrier system serves to protect metabolites from environmental disturbances (light, rain, microbial degradation) and enables intelligent release. The carrier consists of a core loading layer, an intermediate response layer, and an outer protective layer. The specific preparation steps are as follows:

[0086] 1. Preparation of the core-supported layer (mesoporous silica nanoparticles) and metabolite loading

[0087] Mesoporous silica nanoparticles (MSNs) have the characteristics of uniform pore size (8-10 nm) and large specific surface area, and can load metabolites through physical adsorption.

[0088] MSNs were prepared by a sol-gel method. 4 mL of tetraethyl orthosilicate (TEOS), 0.5 g of cetyltrimethylammonium bromide (CTAB), 50 mL of deionized water, and 2 mL of 25% ammonia were mixed and stirred at 30 °C for 24 hours. The precipitate was collected by centrifugation, washed three times with ethanol (to remove the CTAB template), and calcined at 500 °C for 6 hours to obtain MSNs. The pore size was controlled at 8-10 nm by adjusting the TEOS to CTAB ratio, suitable for metabolite molecules (molecular weight 1000-5000 Da) to enter the pores.

[0089] Metabolite loading: 0.5 g MSNs were added to 50 mL of a metabolite synergistic complex solution (concentration as above), and the mixture was shaken at 150 rpm at 4 °C for 24 hours (22-26 hours). The loaded particles were collected by centrifugation (5000 rpm, 10 minutes) and washed twice with a small amount of PBS (to remove free metabolites from the surface) to obtain loaded MSNs. The loading rate was found to be 35% (≥30%), meeting the design requirements. The adsorption mechanism is as follows: the silanol groups on the MSN surface form hydrogen bonds with the metabolite molecules, achieving stable loading.

[0090] 2. Encapsulation of the intermediate responsive layer (pH-sensitive hydrogel)

[0091] The intermediate response layer uses a pH-sensitive hydrogel, which swells or shrinks under different pH conditions at different growth stages of rapeseed (pH≈7.0 at the initial flowering stage, and pH drops to 5.8-6.0 at the pathogen infection site during the full flowering stage), thereby regulating the release of metabolites.

[0092] Hydrogel preparation: Using acrylic acid (AA) and chitosan (CS) as monomers (molar ratio 3:1), 1g of supported MSNs was dispersed in 50mL of deionized water. 0.3mol of AA and 0.1mol of CS were added, and the mixture was stirred until dissolved. Then, N,N-methylenebisacrylamide (0.5%, relative to the total monomer mass) was added as a crosslinking agent. Nitrogen gas was purged for 10 minutes to remove oxygen. Ammonium persulfate (0.2%) was added as an initiator, and the reaction was carried out at 30℃ for 3 hours to form hydrogel-coated particles. After the reaction, the particles were washed three times with deionized water and freeze-dried. The pH-sensitive mechanism of this hydrogel is as follows: under acidic conditions (pH < 6.0), chitosan is protonated, the hydrogel swells, and metabolites are released; under neutral conditions (pH > 6.5), the hydrogel shrinks, preventing release. The thickness of the coated hydrogel layer was 120nm (100-150nm), confirmed by scanning electron microscopy (SEM).

[0093] 3. Preparation of the outer protective layer (hydrophobic ethyl cellulose coating)

[0094] The outer protective layer is coated with ethyl cellulose (EC), which imparts hydrophobicity to the carrier, reduces the loss of metabolic fluids caused by rainwater erosion, and regulates the release rate by controlling the coating thickness.

[0095] Coating process: Take 1g of the above hydrogel-coated particles and add them to a fluidized bed coating machine (inlet air temperature 36℃, 35-38℃). Spray a 5% ethyl cellulose ethanol solution (EC5g dissolved in 95mL ethanol) at a rate of 2mL / min until the coating weight gain is 22% (20%-25%). After coating, dry at 40℃ for 2 hours, and collect the particles after cooling.

[0096] Targeted peptide grafting: To enhance the enrichment capacity of the vector at the pathogen infection site, the targeted peptide Cys-Arg-Pro-Ser-Lys (CRPSK, which recognizes receptors on the cell membrane surface of Sclerotinia sclerotiorum) was covalently grafted onto the EC layer. 0.5 g of coated particles were dispersed in 10 mL of PBS buffer (pH 7.4), and the activated targeted peptide (with the carboxyl group activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) was added. The mixture was stirred at 25 °C for 4 hours, and the particles were collected by centrifugation, washed three times, and the grafting rate was determined to be 18 μmol / g (≥15 μmol / g).

[0097] Carrier characterization: The final carrier particle size was 650 nm (500-800 nm), and the Zeta potential was +28 mV (+25 to +30 mV). The particle size and potential were confirmed to be suitable for crop leaf attachment and cell absorption by dynamic light scattering (DLS) and Zeta potential meter.

[0098] IV. Preparation of formulations for precise application in stages throughout the entire reproductive period (S400)

[0099] Based on the disease risk and metabolite requirements of rapeseed at different growth stages, three types of carrier suspensions (immediate-release, pH-responsive, and long-acting sustained-release) were prepared, and application parameters were determined.

[0100] 1. Immediate-release carrier suspension at the initial flowering stage

[0101] The initial flowering stage (when the main inflorescence is 10%-20% open) is the initial stage of pathogen infection, requiring the rapid release of metabolites to inhibit early mycelial growth.

[0102] Carrier adjustment: The intermediate pH-sensitive hydrogel layer is removed from the immediate-release carrier (only the MSN core and thin EC coating are retained, with the coating weight increased by 10%) to ensure rapid release.

[0103] Suspension preparation: Take 10g of the immediate-release carrier and disperse it in 90mL of an aqueous solution containing 0.10% xanthan gum (thickener, to prevent sedimentation) and 0.05% Tween-80 (surfactant, to improve wettability). Homogenize at 9000rpm (8000-10000rpm) for 12 minutes (10-15 minutes) to form a suspension with a solid content of 10%. Adjust the pH to 7.0 (6.8-7.2) with 0.1M sodium hydroxide.

[0104] Release performance test: Under the conditions of 25℃ and pH 7.0, the cumulative release over 24 hours is 75% (≥70%), which meets the requirements for immediate release.

[0105] 2. pH-responsive carrier suspension during peak flowering period

[0106] The peak flowering period (70%-80% of the field is in bloom) is a high-incidence period for diseases. After infection, pathogens secrete organic acids, which lower the pH of the infected area to 5.8-6.0. The carrier needs to release metabolites rapidly under this environment.

[0107] Carrier selection: A complete composite carrier (containing MSNs core, pH-sensitive hydrogel, EC coating, with a 20% weight gain from coating) was used.

[0108] Suspension preparation: Same as immediate-release type, solid content 12%, pH adjusted to 7.0.

[0109] Release performance testing: At 25℃ and pH≥6.5 (healthy tissue environment), the release rate is 15% (≤20%) in 24 hours; at pH5.9 (infected site environment), the release rate is 45% (≥40%) in 2 hours, achieving "release on demand".

[0110] 3. Long-acting sustained-release carrier suspension during the pod-setting stage

[0111] During the pod-bearing stage (7-10 days after flowering), long-term protection is needed to prevent infection by residual pathogens. Metabolites need to be released slowly to prolong the duration of effectiveness.

[0112] Carrier adjustment: Increase EC coating thickness (coating weight increase of 25%) and add 0.5% beeswax (to enhance hydrophobicity).

[0113] Suspension preparation: Same as immediate-release type, solid content 15%, pH adjusted to 7.0.

[0114] Release performance test: 24-hour release rate is 25% (≤30%), and 15-day cumulative release rate is 92% (≥90%), meeting the requirements for long-term effectiveness.

[0115] V. Implementation of Integrated Prevention and Control Strategies (S500)

[0116] Fertilization during the budding stage enhances rapeseed resistance, while phased application of pesticides enables control throughout the entire growth cycle.

[0117] 1. Fertilizing during the budding stage

[0118] During the budding stage of rapeseed (plant height 30-40cm), apply potassium sulfate fertilizer (equivalent to 67.5kg / ha of K2O) per acre using a strip application method, with a fertilization depth of 9cm (8-10cm). After fertilization, cover with soil and irrigate (maintain soil moisture at 60%). Potassium fertilizer can increase the cell wall thickness of rapeseed and the activity of disease-resistant enzymes (peroxidase, polyphenol oxidase), thereby enhancing the plant's own resistance.

[0119] 2. Spraying during the initial flowering stage

[0120] When the main inflorescence reaches 15% flowering rate, spray the fast-release suspension using a backpack sprayer (working pressure 0.3 MPa) at a rate of 350 liters / ha (300-400 liters), with a carrier concentration of 0.8 g / L (0.5-1.0 g / L). Focus on spraying the undersides of the leaves and the inflorescence. Spraying should be done on a sunny morning between 9 and 11 am (after the dew has dried to avoid rain washing it off).

[0121] 3. Spray during peak flowering period

[0122] When the flowering rate of the entire field reaches 75%, spray a pH-responsive suspension at a rate of 450 liters / hectare (400-500 liters), with a carrier concentration of 1.5 g / L (1.2-1.8 g / L), focusing on spraying the petals and young pods. If it rains at this time, it is necessary to re-spray 24 hours after the rain (the outer EC coating of the carrier is resistant to short-term rain washout).

[0123] 4. Spraying during the pod-setting stage

[0124] Eight days after flowering (7-10 days), spray a long-acting, slow-release suspension concentrate at a carrier concentration of 1.0 g / L (0.8-1.2 g / L) and a spraying rate of 300 liters / ha (250-350 liters), focusing on spraying the surface of the siliques. The slow-release carrier achieves long-term release through a thickened EC coating (25% weight increase), ensuring continuous control before the siliques mature.

[0125] VI. Verification and Integration of Prevention and Control Effectiveness (S500)

[0126] The control effect of this method was verified through field trials: rapeseed fields with severe sclerotinia disease in previous years were selected, and three treatment groups were set up (the method of this invention, the chemical pesticide control group, and the blank control group). Each group was replicated three times, and the plot area was 20m².

[0127] Disease incidence survey: Disease incidence and disease index (0-4 levels) were measured at maturity. Results showed that the disease incidence rate of the method described in this invention was 8.2%, and the disease index was 5.1; the disease incidence rate of the chemical pesticide group (sprayed with 50% iprodione wettable powder) was 10.5%, and the disease index was 6.8; the disease incidence rate of the blank control group was 35.6%, and the disease index was 28.3. The control effect of this invention is superior to that of chemical pesticides, and there are no pesticide residues.

[0128] Metabolite stability test: 7 days after spraying, the amount of metabolites remaining on the leaf surface: the retention rate of iturinA in the method of this invention was 65%, and the retention rate of YJ1 antibiotic was 58%; the retention rates of the direct spraying metabolite group (without carrier) were 12% and 8%, respectively, proving that the carrier significantly improves stability.

[0129] In summary, this embodiment constructs a functionally complementary microbial alliance, extracts and combines itrinA, YJ1 antibiotics, and scutellarin parasitic enzymes to form a synergistic metabolic network; utilizes a mesoporous silica-hydrogel-ethyl cellulose composite carrier to achieve environmental protection and intelligent release of metabolites; and combines the disease characteristics of rapeseed during the initial flowering, full flowering, and pod-setting stages by spraying rapid-release, pH-responsive, and long-acting slow-release formulations in stages, along with potassium fertilizer application during the budding stage to enhance plant resistance, ultimately achieving highly efficient control of sclerotinia stem rot throughout its entire growth cycle. This method overcomes the technical bottlenecks of poor metabolite stability, short duration of action, and insufficient target concentration in biological control, achieving better control effects than traditional chemical pesticides, and possessing the advantages of being environmentally friendly and residue-free, providing a scalable technical paradigm for the green control of rapeseed sclerotinia stem rot.

[0130] Example 2

[0131] like Figure 1 As shown, the specific steps of the ecological control method for rapeseed sclerotinia stem rot based on microbial metabolites are as follows;

[0132] 1. Construction of microbial teams

[0133] Culture of Bacillus subtilis X-01: Inoculate onto LB liquid medium, ferment at 28-32℃ and 180 rpm for 44-52 hours, centrifuge at 8000 rpm for 15 minutes, and collect the supernatant.

[0134] Culture of Streptomyces cholerae YJ1: Inoculated onto Gao's No. 1 medium, cultured at 28±1℃ and 150rpm for 96±2 hours, and extracted for metabolism with methanol-ethyl acetate (1:3 volume ratio).

[0135] Culture of *Scutellaria baicalensis* parasites: Inoculate onto PDB medium and incubate at 23-25℃ and 120 rpm for 110-130 hours. Collect secretions by filtration through a 0.45 μm filter membrane. Store all metabolites at 4℃ for later use.

[0136] 2. Metabolite synergistic network combination

[0137] Purification of Bacillus subtilis supernatant: ammonium sulfate precipitation + dialysis to obtain iturin A concentrate.

[0138] Purification of crude extract of Streptomyces cholerae: separation of target antibiotics by silica gel column chromatography.

[0139] Blend according to dry weight ratio:

[0140] iturinA:YJ1 antibiotic: Shield-shaped fungus parasitic enzyme equals (1.8-2.2):(0.9-1.1):(1.2-1.4).

[0141] Final concentration control: iturinA 80-120μg / mL, YJ1 antibiotic 40-60μg / mL, parasitic enzyme mixture 0.5-1.0mg / mL.

[0142] 3. Composite carrier system fabrication

[0143] Core loading layer: Add the metabolite complex solution to mesoporous silica particles with a pore size of 8-10 nm, and oscillate at 4℃ for 22-26 hours for adsorption (loading rate ≥30%).

[0144] Intermediate responsive layer: Acrylic acid and chitosan (molar ratio 3:1) are polymerized on the surface of the supported particles to form a 100-150nm thick pH-sensitive hydrogel.

[0145] Outer protective layer: fluidized bed coating with 5-8% ethyl cellulose ethanol solution (35-38℃), coating weight gain of 20%-25%, grafted with target peptide Cys-Arg-Pro-Ser-Lys (grafting rate ≥15μmol / g), finished carrier particle size 500-800nm, Zeta potential +25 to +30m.

[0146] 4. Apply in stages throughout the entire reproductive period.

[0147] At the initial flowering stage (10%-20% flowering rate of the main inflorescence): spray with a suspension containing a fast-release carrier (carrier concentration 0.5-1.0 g / L), at a rate of 300-400 liters / hectare. Requirements: ≥70% release within 24 hours at pH 7.0 and 25℃.

[0148] During peak flowering period (70%-80% flowering rate across the field): Spray with a pH-responsive carrier (carrier concentration 1.2-1.8 g / L), at a rate of 400-500 liters / hectare. The requirements are as follows:

[0149] At pH ≥ 6.5, the release rate is ≤ 20% in 24 hours; at pH 5.8-6.0, the release rate is ≥ 40% in 2 hours.

[0150] During the pod stage (7-10 days after the end of flowering): Spray with a slow-release carrier (carrier concentration 0.8-1.2 g / L), at a rate of 250-350 liters / hectare. The requirements are: ≤30% release within 24 hours and ≥90% cumulative release within 15 days.

[0151] 5. Agronomical aids (optional)

[0152] Apply potassium sulfate fertilizer (equivalent to 60-75 kg / ha of K2O) during the budding and flowering stage, at a depth of 8-10 cm, and irrigate after covering with soil.

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An ecological control method for rapeseed sclerotinia stem rot based on microbial metabolites, characterized in that, The specific steps of this method are as follows: S100. Constructing a microbial consortium: Screening for Bacillus subtilis X-01 (CCTCCNO: M2024158), Streptomyces galbana YJ1 (CGMCCNO: 25693), and Parasitic scutellariae, among which: Bacillus subtilis X-01 secretes the lipopeptide antibiotic iturin A; Streptomyces bilirubin YJ1 secretes antibiotics that inhibit sclerotium formation; The parasitic fungus *Petroptera* secretes parasitoid enzymes. S200, Metabolite Synergistic Network: Key metabolites of strain S100 were extracted and compounded to construct a synergistic system of iturinA, YJ1 antibiotic and scutellarin parasitic material, with a mass ratio of (1.5–2.5):(0.8–1.2):(1.0–1.5); S300. Preparation of composite carrier system: Construct a composite carrier consisting of a core loading layer, an intermediate response layer and an outer protective layer. The core loading layer is a mesoporous silica nanoparticle-loaded metabolite synergistic network complex. The intermediate response layer is coated with a pH-sensitive hydrogel. The outer protective layer is coated with hydrophobic ethyl cellulose. S400, precise application in stages throughout the entire growth period: spray suspension containing fast-release carrier at the initial flowering stage, spray suspension with pH-responsive carrier at the full flowering stage, and spray suspension with long-acting slow-release carrier at the pod-setting stage. S500 delivers the S200 metabolite synergistic network via a composite carrier system of S300, matching the phased application strategy of S400 to achieve prevention and control.

2. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The preparation method of the S100 microbial contingent includes: Bacillus subtilis X-01 was inoculated into LB liquid medium and fermented at 28–32℃ and 180 rpm for 44–52 hours. The supernatant was obtained by centrifugation at 8000 rpm for 15 minutes. Streptomyces bilirubinii YJ1 was inoculated into Gao's No. 1 liquid medium and cultured with shaking at 28±1℃ and 150rpm for 96±2 hours. Metabolites were extracted using a methanol-ethyl acetate mixed solvent with a volume ratio of 1:

3. The parasitic fungus *Petroptera* was inoculated into PDB liquid medium and cultured at 23–25°C and 120 rpm for 110–130 hours. The secretions were collected by filtration through a 0.45 μm filter membrane. The above-mentioned metabolite components were stored at 4°C for later use.

3. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The construction of the metabolite synergistic network in S200 includes: The supernatant of Bacillus subtilis X-01 was purified by ammonium sulfate precipitation and dialysis to obtain iturinA concentrate. The target components of the crude extract of Streptomyces galbana YJ1 were separated by silica gel column chromatography; The mixture of iturinA, YJ1 antibiotic, and scutellarin parasitic enzyme was prepared at a dry weight ratio of (1.8–2.2):(0.9–1.1):(1.2–1.4). The compound solution contains: iturin A concentration of 80–120 μg / mL, YJ1 antibiotic concentration of 40–60 μg / mL, and parasitic enzyme mixture concentration of 0.5–1.0 mg / mL.

4. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The preparation of the composite carrier system in S300 includes: Mesoporous silica nanoparticles with a pore size of 8–10 nm were impregnated in a metabolite composite solution and adsorbed by shaking at 4 °C for 22–26 hours, with a loading rate of ≥30%. Using acrylic acid and chitosan in a molar ratio of 3:1 as monomers, a pH-sensitive hydrogel layer with a thickness of 100–150 nm was formed on the surface of the loaded particles. Using a 5%–8% ethyl cellulose ethanol solution, fluidized bed coating was performed at 35–38℃, resulting in a coating weight gain of 20%–25%, and the targeting peptide Cys-Arg-Pro-Ser-Lys was grafted onto it with a grafting rate ≥15μmol / g. The resulting carriers have a particle size of 500–800 nm and a zeta potential of +25 mV to +30 mV.

5. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The release conditions of the carrier applied at each growth stage in S400 are as follows: Early flowering stage rapid-release carrier: under pH 7.0 and 25℃ conditions, the cumulative release rate over 24 hours is ≥70%; Flowering-responsive carrier: ≤20% release rate in 24 hours at 25℃ and pH≥6.5, and ≥40% release rate in 2 hours at pH5.8–6.0; Slow-release carrier during the pod stage: ≤30% release in 24 hours, ≥90% cumulative release in 15 days.

6. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The preparation of the suspending agent in S400 includes: The composite carrier was dispersed in an aqueous solution containing 0.10% ± 0.02% xanthan gum and 0.05% Tween-80; Homogenize at 8000–10000 rpm for 10–15 minutes to form a suspension with a solid content of 10%–15%; Adjust the pH to 6.8–7.2 using 0.1M sodium hydroxide.

7. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The method is integrated in the S500 phase: Apply potassium sulfate fertilizer during the budding and bolting stage of rapeseed, with an application rate equivalent to 60–75 kg / ha of K2O, at a depth of 8–10 cm, followed by covering with soil and irrigating.

8. The method for ecological control of rapeseed sclerotinia stem rot based on microbial metabolites according to claim 1, characterized in that, The operational parameters for the phased matching of the S400 application strategy in S500 are as follows: Spraying at the initial flowering stage: Carrier concentration 0.5–1.0 g / L, spraying rate 300–400 liters / hectare, when the main inflorescence flowering rate is 10%–20%; Spraying during peak flowering period: Carrier concentration 1.2–1.8 g / L, spraying rate 400–500 liters / hectare, when the flowering rate of the whole field is 70%–80%; Spraying during the pod-setting stage: Carrier concentration 0.8–1.2 g / L, spraying rate 250–350 liters / hectare, 7–10 days after the end of flowering.

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