Highly biocompatible biogenic silver nanoparticles, method for their preparation, microcapsules comprising them and use thereof

Abstract

The application provides a kind of high biocompatibility biological silver nanoparticles, its preparation method, compound microcapsule and application, the biological silver nanoparticles are based on the fermentation broth or fermentation filtrate of biocontrol bacillus B268, the metabolite of B268 is combined on the surface of particle, the antagonistic activity to plant pathogenic bacteria is enhanced, and show high biocompatibility to biocontrol bacteria, biocontrol fungi, plant, model nematode, can be directly embedded with bacteria liquid, the microcapsule prepared fuses the compatible interaction relationship between bacteria, bacterial metabolite, silver nanoparticles, shows synergistic antibacterial effect, and the bacteriostatic activity is higher than that of microcapsule embedded by bacteria liquid or silver nanoparticles alone, and under low temperature preservation condition, microcapsule can keep the stability of bacteria, silver nanoparticles and its synergistic antibacterial activity, provides preparation raw material and preparation method for developing nano biological pesticide.

Description

Technical Field

[0001] This invention belongs to the field of microbial strains and their application technology, and relates to a highly biocompatible biological silver nanoparticle, its preparation method, compound microcapsules and applications. Background Technology

[0002] Nanoparticle-based pesticides are novel pesticides combining nanotechnology and bioactive ingredients. Their core forms are nanocarriers and nanoencapsulation. The former utilizes nanoparticles to carry natural bioactive substances (such as proteins, peptides, and oligosaccharides), while the latter encapsulates nanoparticles and active substances or functional bacteria in a bio-based matrix. The aim is to achieve more efficient and precise pest and disease control with lower dosages. Highly selective and low-toxicity nanoparticle-based pesticides are a key research focus. Using biocontrol bacteria with good interactions with plants to synthesize silver nanoparticles with inherent antibacterial properties and further preparing corresponding nanoparticle-based pesticides is an effective approach.

[0003] Bacillus is one of the most successfully applied biocontrol bacteria, possessing both disease resistance and health-promoting functions as well as growth-promoting and yield-increasing properties. It has broad ecological adaptability and high compatibility with various formulations, making it an excellent strain for the design and creation of nano-biological pesticides. Bacillus belye belongs to the Bacillus amyloliquefaciens clade genus and is one of the representative strains of plant growth-promoting bacteria. However, research on its use in the biosynthesis of nanoparticles is limited, and there are even fewer reports on the development of nano-biological pesticides with high selectivity and biocompatibility.

[0004] Based on this, the present invention proposes a bio-silver nanoparticle with both broad-spectrum and selective anti-life activity. It is synthesized from biocontrol Bacillus B268 and has excellent biocompatibility. It is compatible with B268 cells and its active metabolites. The three can form a compound microcapsule through composite encapsulation technology, which shows a stable synergistic effect in antagonizing bacterial wilt. Summary of the Invention

[0005] This invention first provides a superior biocontrol Bacillus B268 strain possessing antibacterial activity, growth-promoting activity, and extracellular silver nanoparticle synthesis activity, as well as silver nanoparticles synthesized from B268 exhibiting both selective and broad-spectrum antagonistic activities. This provides materials for the synergistic application of bacterial cells and nanoparticles in plant disease resistance and growth promotion. The metabolites of the strain B268 involved in this invention possess anti-plant pathogenic bacteria and fungal activity, and exhibit stress-resistance and growth-promoting effects on Arabidopsis thaliana. Screening further revealed its ability to synthesize extracellular silver nanoparticles. The silver nanoparticles synthesized by B268 exhibit anti-plant pathogenic bacteria, fungi, oomycetes, and nematodes activity, while showing no inhibitory effect on various biocontrol bacteria, plants, and model nematodes, including B268. Secondly, this invention provides a method for synthesizing silver nanoparticles from B268, and a method for preparing a compound microcapsule that combines silver nanoparticles and B268. By synergistically combining the biological activities of the strain and the nanoparticles, the purpose of synergistic effect is achieved. Its features are that it combines the following properties: (1) The surface of the synthesized silver nanoparticles is bound to the metabolites of B268, which has both broad-spectrum antimicrobial activity against plant pathogens and biocompatibility with plants and non-pathogens; (2) The synthesized silver nanoparticles can adhere to the surface of B268 cells and synergistically fight bacteria with B268. The compound microcapsule prepared in this way utilizes the natural compatibility between B268 cells, active substances and nanoparticles, avoids mutual antagonism between the two, and forms a stable synergistic effect.

[0006] The specific technical solution adopted is as follows:

[0007] A method for preparing highly biocompatible bio-silver nanoparticles includes the following:

[0008] Bacillus velezensis B268 (the preservation information of which has been disclosed in patent CN106635922A) with accession number CGMCC No. 13225 was cultured in liquid culture medium to obtain a fermentation broth. The cell-free fermentation filtrate or the fermentation broth was directly mixed with AgNO3 solution and incubated. After centrifugation and rinsing, the highly biocompatible bio-silver nanoparticles were obtained, which are silver nanoparticles with proteins / peptides bound to their surface.

[0009] In the above technical solution, the high biocompatibility further includes: the bio-silver nanoparticles, at concentrations up to 2 mg / mL, have no antagonistic effect on various biocontrol Bacillus species, including B268, but the half-lethal concentration against bacterial wilt (i.e., *Ralstonia somnifera*) is only 5.35 μg / mL; at concentrations up to 28 mg / mL, they have no antagonistic activity against biocontrol Trichoderma, but the half-lethal concentration against apple tree rot fungus (i.e., *Trichoderma oleracea*), chestnut blight fungus (i.e., *Cryptococcus parasiticus*), *Fusarium graminearum*, *Botrytis cinerea*, etc., is only 28.75-570 μg / mL; at 250 μg / mL, they have no adverse effect on seed germination and seedling development of rice, wheat, cucumber, sorghum, and casuarina; at a concentration of 5 μg / mL, the killing rate against pine wilt nematode is 85.7%, and there is no antagonistic effect against *C. elegans*.

[0010] Furthermore, the culture is carried out for 24-48 hours.

[0011] Furthermore, the optimal final concentration of AgNO3 after mixing the AgNO3 solution with the cell-free fermentation filtrate is 5-7 mM, and the optimal final concentration of AgNO3 after mixing the AgNO3 solution with the fermentation broth is 3-5 mM.

[0012] Furthermore, the incubation is carried out at 37 ℃, 200 rpm, and in the dark for 24-48 h.

[0013] A highly biocompatible bio-silver nanoparticle is prepared by the method described in any of the preceding methods.

[0014] A method for preparing a compound microcapsule with synergistic antibacterial effect includes the following:

[0015] The above-mentioned bio-silver nanoparticles were dispersed in deionized water, sodium alginate was added, and then fermentation broth of B268 was added and mixed. The mixture was then pressed into calcium chloride solution through a needle to solidify, thus obtaining compound microcapsules.

[0016] In the above technical solution, the synergistic antibacterial effect further refers to the fact that microcapsules prepared based on B268 or silver nanoparticles alone have antibacterial activity against Raulella tobaccois, while the antibacterial activity is enhanced in the composite microcapsules that combine the two.

[0017] Furthermore, the silver nanoparticles are dispersed in deionized water at a concentration of 100 μg / mL.

[0018] Furthermore, the sodium alginate is added at a concentration of 15 g / L and dissolved under high temperature and high pressure.

[0019] Furthermore, the bacterial concentration of the B268 fermentation broth is OD. 600 =2.0, and the volume ratio when mixed is 1:1.

[0020] A compound microcapsule with synergistic antibacterial effect is prepared by the method described in any of the preceding methods.

[0021] Application of B268 fermentation filtrate in the preparation of pesticides antagonizing plant pathogens, wherein the plant pathogens are plant pathogenic fungi or plant pathogenic bacteria, including *Cryptospira macrantha*, *Cryptospira macrantha*, *Fusarium graminearum*, and *Botrytis cinerea*, and plant pathogenic bacteria including *Raulella vesicae*.

[0022] The application of the above-mentioned bio-silver nanoparticles in the preparation of pesticides antagonizing plant pathogens, wherein the plant pathogens are plant pathogenic fungi, bacteria, oomycetes, and nematodes, wherein the fungi include *Cryptospira macrantha*, *Cryptospira macrantha*, *Fusarium graminearum*, and *Botrytis cinerea*, the bacteria include *Raulella vesicae*, the oomycetes include *Phytophthora camphorata*, and the nematodes include *Pinus wood nematode*.

[0023] The application of the compound microcapsules described above in the preparation of pesticides antagonizing Rochertis tobacco.

[0024] Specifically:

[0025] (1) Preservation and taxonomic revision of strain B268

[0026] Strain B268 was deposited on November 1, 2016, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 13225. Previously, based on phylogenetic analysis of its 16S rRNA gene sequence, it was identified as *Bacillus siamensis*. However, subsequent genomic phylogenetic analysis revealed that it belongs to the conspecific *Bacillus velezensis*. Therefore, this invention revises its classification to *Bacillus velezensis*.

[0027] (2) Plant growth-promoting activity of strain B268

[0028] Using Arabidopsis thaliana as the research subject, the plant growth-promoting activity of B268 was detected through plate inoculation experiments. Under non-stress conditions, B268 significantly improved the stem, leaf, and lateral root growth of Arabidopsis thaliana. Under NaCl stress conditions, B268 improved the salt tolerance of Arabidopsis thaliana, mainly through root protection, including stabilizing the number of lateral roots and root biomass.

[0029] (3) Anti-viability of fermentation filtrate of strain B268

[0030] The fermentation filtrate of strain B268 exhibited antagonistic activity against Ralstonia nicotianae, Valsa mali var. mali, Cryphonectria parasitica, Fusarium graminearum, and Botryosphaeria dothidea, but showed no antagonistic activity against Phytophthora cinnamomi and Bursaphelenchus xylophilus.

[0031] (4) Silver nanoparticle synthesis activity of strain B268

[0032] Fermentation broth, fermentation filtrate, and sterile aqueous suspension of B268 cells were prepared separately. After incubation with an equal concentration of AgNO3, the color of the fermentation broth and filtrate changed from light brown to dark brown, and a characteristic absorption peak between 400-450 nm was detected by surface plasmon resonance (SPR). Similar characteristic absorption peaks were also detected after collecting cells from the fermentation broth and dysplasticizing them with ultrasound, indicating that silver nanoparticles can be synthesized both inside and outside the cells. However, the sterile aqueous suspension of cells did not exhibit the above phenomena, indicating that no silver nanoparticles were generated inside or outside the cells. These results demonstrate that B268 synthesizes silver nanoparticles through specific metabolites inside and outside the cells under normal growth conditions.

[0033] (5) Characterization of silver nanoparticles

[0034] Transmission electron microscopy revealed spherical silver nanoparticles that were dispersed in the fermentation filtrate without significant aggregation. These nanoparticles were uniformly dispersed on the surface of B268 cells or remained free extracellularly (with aggregation). Fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS) analyses indicated that B268 protein / peptide metabolites, acting as reducing agents and capping agents, participated in the synthesis of silver nanoparticles and surface modification.

[0035] (6) Anti-aging properties of silver nanoparticles

[0036] The purified silver nanoparticles (cell-free) showed antagonistic activity against *Rauvolfia tobaccois*, *Heterophytes macrantha*, *Cryptococcus parasiticus*, *Fusarium graminearum*, *Botrytis cinerea*, *Phytophthora camphorata*, and *Wood nematode*.

[0037] (7) Biocompatibility of silver nanoparticles

[0038] The purified silver nanoparticles showed good compatibility with biocontrol Bacillus species, including B268, at concentrations up to 2 mg / mL. They exhibited no inhibitory effect on biocontrol Trichoderma harzianum at concentrations up to 28 mg / mL. At a concentration of 250 μg / mL, they had no adverse effects on seed germination and seedling development in rice, wheat, cucumber, sorghum, and casuarina, and even showed a promoting effect. At a concentration of 5 μg / mL, they achieved a kill rate of 85.7% against pine wilt nematode and showed no antagonistic effect against Caenorhabditis elegans.

[0039] (8) Microcapsules of B268 and silver nanoparticles

[0040] It exhibits high cell encapsulation efficiency, high cell viability, and synergistic antibacterial activity. Its inhibitory activity against *Rabotomyces tobaccoeris* is higher than that of microcapsules encapsulated with B268 or silver nanoparticles alone. Furthermore, after storage at 4 °C for 5 months, the number of viable cells and the synergistic antibacterial activity remain stable. Compared with existing technologies, the advantages of this invention are:

[0041] (1) Explore the ability of biocontrol Bacillus to synthesize metal nanoparticles, and use the synthesized silver nanoparticles to enhance the antibacterial activity of the strain through composite encapsulation technology.

[0042] (2) Silver nanoparticles were prepared by using the extracellular products of plant growth-promoting Bacillus as reducing agents and capping agents, which improved the compatibility between nanoparticles and biocontrol bacteria and plants.

[0043] (3) The microcapsule preparation process makes full use of the Bacillus fermentation broth (cells, active substances, and nutrients). The active substances are used to synthesize and modify silver nanoparticles. After composite encapsulation, the cells, active substances, and silver nanoparticles are the antibacterial components of the microcapsule, while the nutrients are the nutrient reserves of the microcapsule. After the microcapsule dissolves, it can be used as the initial nutrient matrix for the resuscitation of B268.

[0044] (4) Sodium alginate is a biological polysaccharide that is green and non-toxic. Using it to prepare microcapsules ensures the survival rate of bacteria, the stability of silver nanoparticles, and the synergistic effect of their activity. Attached Figure Description

[0045] Figure 1 A phylogenetic tree (compressed tree) of the lineage of Bacillus amyloliquefaciens.

[0046] Figure 2The growth-promoting effect of Bacillus velezensis B268 on Arabidopsis thaliana under non-stress conditions, where CK was MS culture plates inoculated with blank YTGB medium, and B268 was MS culture plates inoculated with B268 seed culture.

[0047] Figure 3 The salt tolerance and growth-promoting effect of Bacillus velezensis B268 on Arabidopsis thaliana under NaCl stress was investigated. The control (CK-80 mM NaCl) involved inoculating MS culture plates with blank YTGB medium containing 80 mM NaCl. The B268-80 mM NaCl control involved inoculating MS culture plates with B268 seed culture containing 80 mM NaCl. The control (CK-100 mM NaCl) involved inoculating MS culture plates with blank YTGB medium containing 100 mM NaCl. The B268-100 mM NaCl control involved inoculating MS culture plates with B268 seed culture containing 100 mM NaCl.

[0048] Figure 4 Effects of Bacillus velezensis B268 on fresh weight of Arabidopsis thaliana under NaCl stress.

[0049] Figure 5 Effects of Bacillus velezensis B268 on root development in Arabidopsis thaliana under NaCl stress.

[0050] Figure 6 The antagonistic effects of the fermentation filtrate of Bacillus velezensis B268 on plant pathogenic fungi were determined. CK-Bd was the control plate for Botryosphaeria dothidea, and B268-Bd was the antagonistic plate for Botryosphaeria dothidea. CK-Vm was the control plate for Valsa mali var. mali, and B268-Vm was the antagonistic plate for Valsa mali var. mali. CK-Cp was the control plate for Cryphonectria parasitica, and B268-Cp was the antagonistic plate for Cryphonectria parasitica. CK-Fg was the control plate for Fusarium graminearum, and B268-Fg was the antagonistic plate for Fusarium graminearum.

[0051] Figure 7 The antagonistic effect of fermentation filtrate of Bacillus velezensis B268 at different culture times on Bacillus velezensis. The time indicated in the lower left corner represents the amount of filter paper dropped onto the fermentation filtrate prepared at the corresponding fermentation time.

[0052] Figure 8 Silver nanoparticle synthesis activity of Bacillus velezensis B268 fermentation broth, fermentation filtrate, and cell resuspension. A represents the silver nanoparticle synthesis solution. From left to right, the images show B268 fermentation broth, fermentation filtrate, and cell resuspension after incubation with AgNO3. B is the scanning spectrum of the silver nanoparticle synthesis solution. C is the scanning spectrum of B268 cells before and after cell lysis.

[0053] Figure 9 Morphology and distribution characteristics of silver nanoparticles synthesized from Bacillus velezensis B268 fermentation broth and fermentation filtrate, where A represents silver nanoparticles synthesized from fermentation broth (× 40K) and B represents silver nanoparticles synthesized from fermentation filtrate (× 40K).

[0054] Figure 10 Chemical characterization of silver nanoparticles synthesized from fermentation filtrate of Bacillus velezensis B268, where A is the FTIR spectrum and B is the EDS spectrum.

[0055] Figure 11 Compatibility of silver nanoparticles synthesized from Bacillus velezensis B268 fermentation filtrate with biocontrol Bacillus velezensis.

[0056] Figure 12 The compatibility of silver nanoparticles synthesized from the fermentation filtrate of Bacillus velezensis B268 with biocontrol Trichoderma was studied. CK was cultured after treating the mycelium cake with sterile water, while AgNPs was cultured after treating the mycelium cake with a 28 mg / mL silver nanoparticle solution.

[0057] Figure 13 The effects of silver nanoparticles synthesized from Bacillus velezensis B268 on Caenorhabditis elegans and pine wood nematode.

[0058] Figure 14 The effects of silver nanoparticles synthesized from the fermentation filtrate of Bacillus velezensis B268 on seed radicle development are shown in Figure A, which shows the taproot length of five plants under different concentrations of silver nanoparticles, and Figure B shows the lateral root length of three plants under different concentrations of silver nanoparticles (cucumber and casuarina had not yet formed lateral roots at the same time).

[0059] Figure 15 Microcapsules with different encapsulation targets: empty microcapsules refer to those without encapsulation of B268 fermentation broth and silver nanoparticles; B268 microcapsules refer to those with only encapsulation of B268 fermentation broth; AgNPs microcapsules refer to those with only encapsulation of silver nanoparticles; and B268-AgNPs microcapsules refer to those with encapsulation of B268 fermentation broth and silver nanoparticles.

[0060] Figure 16 Scanning chromatograms of four microcapsule solutions before (A) and after (B) storage at 4 ℃.

[0061] Figure 17 Antagonistic activity of four microcapsule solutions against Raulella tobaccois before (A) and after (B) storage at 4 ℃. Detailed Implementation

[0062] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0063] Example 1: Re-identification of strain B268

[0064] The complete genome sequence (CP053764) of strain B268 was obtained using SMRT single-molecule sequencing on the PacBio RS II sequencing platform. Genomes of 230 strains from the Bacillus amyloliquefaciens phylum (including B. amyloliquefaciens, B. velezensis, B. siamensis, and B. methyltrophicus) were downloaded from the NCBI genome database (https: / / www.ncbi.nlm.nih.gov / ) and identified as B. cereus NCTC2599. T As outgroups, single-copy orthologous genes were extracted, pruned and assembled into a core genome after multiple sequence alignment, and a genome phylogenetic tree was constructed using IQ-TREE with maximum likelihood. Figure 1 As shown, the 174 genomes form three branches, constituting the Bacillus amyloliquefaciens phylogenetic lineage: the B. amyloliquefaciens branch, the B. siamensis branch, and the allospecific B. velezensis branch. The allospecific B. velezensis branch consists of a cluster of strains from the original B. velezensis, the original B. methyltrophicus, and the original B. amyloliquefaciens subsp. plantarum, with a topological structure consistent with previous reports. B268 and the remaining 55 genomes form a separate branch, located between the B. siamensis branch and the allospecific B. velezensis branch, and are more closely related to the latter. Therefore, B268 was re-identified as B. velezensis instead of B. siamensis.

[0065] Example 2: Plant growth-promoting activity of strain B268

[0066] After surface sterilization, Arabidopsis thaliana Columbia seeds were spot-inoculated onto MS agar plates and vernalized at 4 ℃ in the dark for 2 days. The incubator was then transferred to a 22 ℃ light incubator with a day-night ratio of 16:8 (h) for 6 days. The -80 ℃ glycerol culture solution of strain B268 was inoculated at 1% (v / v) into YTGB liquid medium in test tubes (4 mL / tube) and cultured at 28 ℃ and 140 rpm for 24 h to prepare the seed culture.

[0067] Arabidopsis thaliana seedlings with uniform growth were selected and randomly transplanted onto MS plates with NaCl concentrations of 0 mM, 80 mM, and 100 mM (5 seedlings / plate), with 3 replicates for each concentration. Simultaneously, B268 seed culture was prepared and the inoculum concentration was adjusted to OD0.05. 600 =0.5, and 10 µL / plate was inoculated onto the above MS plates at a position 4.5 cm below the Arabidopsis seedlings. Blank YTGB medium was used as a control instead of the seed culture. After the bacterial culture was absorbed by the solid medium, the plates were sealed and placed upright on the culture rack, and randomly placed in a 22 ℃ light incubator with a day-night ratio of 16:8 (h) for 24 days.

[0068] After cultivation, the roots of the Arabidopsis thaliana were photographed, and the root images were analyzed using EZ-Rhizo 2.0 to obtain data on the taproot and lateral roots. Simultaneously, the stems, leaves, and roots were weighed (fresh weight) using a 0.01% balance.

[0069] like Figure 2 As shown in Table 1, under non-stress conditions (0 mM NaCl), compared with the control group, B268 significantly increased the fresh weight of stems and leaves of Arabidopsis thaliana, while regulating the root structure, which was manifested as limiting the length of the taproot and promoting the elongation of lateral roots.

[0070] Table 1. Effects of B268 on various growth parameters of Arabidopsis thaliana under non-stress conditions.

[0071]

[0072] like Figure 3 , 4As shown in Figures 5, under 80 mM NaCl stress, B268 significantly increased the fresh weight of stems, leaves, and roots of Arabidopsis thaliana compared to the control group. Meanwhile, compared to growth indicators under non-stress conditions, 80 mM NaCl inhibited lateral root development in the control group, manifested as a significant decrease in the number of lateral roots and root fresh weight, while B268 inoculation maintained lateral root development and root fresh weight. Under 100 mM NaCl stress, compared to growth indicators under non-stress conditions, the fresh weight of stems and leaves in both the control and inoculated groups significantly decreased, showing obvious salt damage. However, the fresh weight of stems and leaves in the B268-inoculated Arabidopsis thaliana was still higher than that of the uninoculated Arabidopsis thaliana, and the number of lateral roots was still maintained.

[0073] MS medium: MS pre-prepared medium (Haibo Biotechnology HB8469-5) 2 g, sucrose 10 g, agar powder 8 g, distilled water 1000 mL, pH 6.0.

[0074] YTGB medium: 3 g beef extract powder, 5 g Tryptone, 10 g glucose, 0.5 g yeast extract, 1000 mL distilled water, pH 7.2-7.4.

[0075] Example 3: Antifungal activity of fermentation filtrate of strain B268

[0076] The B268 seed culture was prepared in the same manner as in Example 2, and transferred to YTGB liquid culture medium (100 mL / 250 mL) in a shake flask at 1% (v / v). The culture was carried out at 28 °C and 140 rpm for 24 h to prepare the fermentation broth. The fermentation broth was then subjected to conventional centrifugation and filtration to obtain cell-free fermentation filtrate.

[0077] The fermentation filtrate was mixed with sterile PDA fungal culture medium or PC oomycete culture medium at a ratio of 1:10 (v / v) to prepare antibacterial plates. Blank YTGB culture medium was used to prepare control plates instead of fermentation filtrate. Different plant pathogenic fungi or oomycetes were inoculated and incubated at 25 °C. When the fungal colonies of the control plate covered 2 / 3 of the plate area, the colony diameter was measured and the inhibition rate was calculated. The calculation formula is: [(control plate colony diameter - antibacterial plate colony diameter) / (control plate colony diameter - inoculated mycelium diameter)] × 100%.

[0078] like Figure 6 As shown, the fermentation filtrate of B268, after being diluted 10 times, still exhibited antagonistic activity against plant pathogenic fungi such as Cryptosporidium parasiticum, Botrytis cinerea, Black Rot Fungus of Apple, and Fusarium graminearum, with inhibition rates of 100±0.76%, 92.98±0.76%, 86.84±5.18%, and 35.56±4.07%, respectively. However, it showed no antagonistic activity against Phytophthora camphorata.

[0079] PDA fungal culture medium: 200 g potato, 10 g glucose, 1000 mL distilled water, pH 7.0.

[0080] PC Oomyces culture medium: 400 mL V8 fruit and vegetable juice, 1 g yeast extract, 5 g Tryptone, 3 g calcium carbonate, 600 mL distilled water, pH 7.0.

[0081] Example 4: Antibacterial activity of fermentation filtrate of strain B268

[0082] The B268 fermentation broth was prepared in the same manner as in Example 3, but the fermentation time was set to 8, 12, 24, 48, 72, and 96 h, respectively. The fermentation broth was then subjected to conventional centrifugation and filtration to obtain the corresponding cell-free fermentation filtrate.

[0083] Ralstonia nicotianae cultured in -80℃ glycerol tubes was inoculated at 1% (v / v) into YTGB liquid medium in test tubes (4 mL / tube) and cultured at 28℃, 140 rpm for 36 h. The resulting seed culture was then transferred at 1% (v / v) to YTGB liquid medium in shake flasks (100 mL / 250 mL) and cultured at 28℃, 140 rpm for 36 h. The bacterial concentration in the shake flasks was adjusted to OD. 600 =1, transfer to sterile YTGB medium at 5% (v / v) and prepare double-layer plates together with blank YTGB medium (bottom layer is 10 mL / plate of blank YTGB, top layer is 20 mL / plate of inoculated YTGB).

[0084] Take a sterile filter paper disc, add 30 μL of fermentation filtrate for different fermentation times to each disc, and use a disc with 30 μL of blank YTGB added as a control. Place the discs on the surface of the above double-layer plate and incubate at 28 ℃ for 72 h. Observe the inhibition zone.

[0085] like Figure 7 As shown, when B268 was cultured for 24 hours, the fermentation filtrate began to show antibacterial activity. The inhibition zone diameter was the largest when cultured for 24-48 hours, which means that the fermentation filtrate had the highest antagonistic activity against Raulella tobaccois at this time.

[0086] Example 5: Silver nanoparticle synthesis activity of strain B268

[0087] The 24-hour fermentation broth of B268 was prepared as in Example 4 and divided into two equal portions. One portion was reserved (Liquid I), and the other portion was centrifuged to collect cells and fermentation supernatant. The fermentation supernatant was filtered to prepare cell-free fermentation filtrate (Liquid II). The cells were washed with sterile water to remove residual culture medium and then resuspended in an equal volume of sterile water (Liquid III). Liquids I, II, and III were dispensed into sterile test tubes (5 mL / tube), and 1 mL of 30 mM AgNO3 stock solution was added to each tube and mixed well (final concentration 5 mM). The reaction was carried out at 37 ℃, 200 rpm, and in the dark for 24 h. The reaction solution was then scanned and analyzed between 300 and 700 nm.

[0088] like Figure 8 As shown in Figures A and B, the fermentation broth (Liquid I) and fermentation filtrate (Liquid II) of B268 turned dark brown after the reaction, and absorption peaks were detected at 425 nm and 419 nm, respectively, indicating that the synthesis of silver nanoparticles was positive. However, the cell resuspension (Liquid III) did not change color after the reaction, and no absorption peak was detected, indicating that no silver nanoparticles were synthesized.

[0089] The fermentation broth and cell resuspension after the reaction were centrifuged at 12,000 rpm for 20 min. The collected precipitate was rinsed with sterile water and centrifuged at 12,000 rpm for 5 min. This rinsing process was repeated multiple times to remove free silver nanoparticles. Samples were then scanned and analyzed in the range of 300-700 nm. At the same time, the remaining samples were subjected to ultrasonic cell disruption (power ratio 30%, 2 s / 3 s, 20 min). The intracellular extract obtained was also scanned and analyzed in the range of 300-700 nm.

[0090] like Figure 8 As shown in Figure C, the cell suspension without extracellular silver nanoparticle synthesis did not show any absorption peak after rinsing and cell disruption treatment, indicating that no intracellular silver nanoparticles were synthesized. However, the fermentation broth treated with the same method showed absorption peaks before and after cell disruption, with the absorption peak after cell disruption being higher than that before. This suggests that extracellularly synthesized silver nanoparticles may have adsorbed onto the cell surface and could not be completely washed away, while intracellular silver nanoparticles were also synthesized, hence the increased absorbance after cell disruption.

[0091] Figure 9 Image A is a transmission electron microscope image of the product synthesized from the fermentation broth, which shows that uniform and fine nanoparticles (12.6-25 nm in diameter, with an average of 16.4 nm) are evenly distributed on the cell surface, while spherical nanoparticles of varying sizes (4.5-111.7 nm in diameter, with an eccentric distribution and an average of 29.2 nm) are also free in the extracellular space, showing aggregation. Figure 9Image B is a transmission electron microscope image of the product synthesized from the fermentation filtrate. The spherical nanoparticles are dispersed, and their average size is similar to that of the free nanoparticles in the fermentation broth (particle size 3.2-64 nm, near normal distribution, average 29.2 nm).

[0092] Example 6: Preparation of silver nanoparticles

[0093] The B268 seed culture was prepared in the same manner as in Example 2, and transferred to a shake flask of YTGB liquid medium (100 mL / 250 mL) at 1% (v / v). The culture was incubated at 28 °C and 140 rpm for 48 h to prepare the fermentation broth. The broth was then sterilized by filtration through a 0.22 μm filter membrane to prepare the fermentation filtrate. 20 mL of 18 mM AgNO3 stock solution (final concentration 3 mM) was added to every 100 mL of fermentation broth, or 20 mL of 30 mM AgNO3 stock solution (final concentration 5 mM) was added to every 100 mL of fermentation filtrate. The mixture was reacted at 37 °C and 200 rpm under light-protected conditions for 24 h to obtain the silver nanoparticle synthesis solution.

[0094] The synthesis solution was centrifuged at 12000 rpm for 20 min to collect the precipitate, which was then washed twice with 5 mM NaCl solution to remove residual Ag. + The mixture was then rinsed three times with sterile water to remove residual culture medium. Finally, it was resuspended in a small amount of sterile water and freeze-dried into powder, which is pure silver nanoparticles (the silver nanoparticles synthesized from the fermentation broth contain a small number of cells). It was stored at 4 ℃ for subsequent experiments (Examples 7-13).

[0095] Example 7: Chemical Characterization Analysis of Silver Nanoparticles

[0096] Pure silver nanoparticles synthesized from fermentation filtrate were used as samples for surface analysis and elemental composition analysis, respectively, using Fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS).

[0097] like Figure 10 As shown in Figure A, compared with two references—blank YTGB medium and pure fermentation filtrate without AgNO3—the silver nanoparticle spectra synthesized from the fermentation filtrate (3440, 2921, 1630, 1520, 1381, 1049, 602 cm⁻¹) are as follows: -1 It has the following changes: ① 3440 cm -1The peak represents the OH / NH stretching vibration, originating from proteins (peptone, beef extract) and carbohydrates (glucose) in the culture medium. The peak position continuously shifts, while the peak width increases and the peak intensity decreases, indicating that the corresponding substances are absorbed and utilized by B268 during fermentation, and transformed into new proteins / peptides / organic acids, etc. These then coordinate with the surface of nanoparticles through amino / hydrogen bonds during synthesis, and may undergo conformational changes; ② 1630 cm⁻¹ -1 The peak belongs to the amide I band, dominated by the C=O group of amides, and also exhibits in-plane bending vibration of NH4+, originating from proteins in the culture medium. The peak position continuously shifts, while the peak intensity decreases, indicating the aforementioned changes; ③ 2921 cm -1 The peak represents the aliphatic CH stretching vibration, detected only in silver nanoparticles, indicating that the redox reaction during synthesis produced specific oxidation products along with elemental silver; ④ 1520 cm⁻¹ -1 1381 cm -1 Peak and 1406 cm of fermentation filtrate -1 All peaks belong to the amide II band, indicating CN vibration, along with in-plane NH bending vibration, representing the presence of protein / peptide. These peaks were not detected in the culture medium, indicating a protein / peptide concentration at 1406 cm⁻¹. -1 The peak originates from a metabolite of B268, and its position differs significantly from the two peaks of the nanoparticles, suggesting that it may act as a reducing agent, undergoing oxidation during synthesis, or as a capping agent, undergoing conformational changes during coordination binding; ⑤ 1049 cm⁻¹ -1 The peak represents the OH vibration that forms hydrogen bonds, which may be coordination bonds on the surface of nanoparticles; ⑥ 602 cm⁻¹ -1 The peaks represent lattice vibrations or bending vibrations of Ag-O, and may also be coordination bonds on the nanoparticle surface. In summary, the FTIR spectra demonstrate that B268 protein / peptide metabolites are involved in the synthesis of silver nanoparticles and bind to the particle surface.

[0098] like Figure 10 As shown in Figure B, the main element in the nanoparticles synthesized from the filtrate is Ag, proving that they are silver nanoparticles. C, O, S and other elements were also detected, indicating that the protein / peptide is a potential capping agent that binds to the particle surface.

[0099] Example 8: Antagonistic activity of silver nanoparticles against plant pathogens

[0100] The half-lethal concentration (LC50) of pure silver nanoparticles synthesized from fermentation filtrate against different plant pathogens was determined.

[0101] (1) Pathogenic bacteria: Prepared the *Rahuella tumefaciens* bacterial suspension as in Example 4, and adjusted the OD value. 600=1. Add 200 μL of YTGB medium, 10 μL of bacterial suspension, and 20 μL of silver nanoparticles (setting an appropriate concentration gradient) sequentially to a 96-well plate, with 6 parallel wells for each concentration. Two control groups were set up: one group used 10 μL of sterile water instead of the bacterial suspension, and the other group used 20 μL of sterile water instead of the silver nanoparticles. Incubate at 28 ℃ and 140 rpm for 24 h. OD of each well was measured using a microplate reader. 600 The values ​​were obtained by performing nonlinear fitting on the data using Origin 2022 to obtain LC. 50 The results showed that silver nanoparticles had a significant LC50 effect on different strains of *Rahuella tobaccois*. 50 The value was 5.35 μg / mL.

[0102] (2) Pathogenic fungi and oomycetes:

[0103] ① Casting plate method (used when nanoparticle concentration ≤ 250 μg / mL): Prepare PDA medium, add different amounts of lyophilized nanoparticles to a gradient concentration, sterilize at 121 ℃ for 20 min, shake thoroughly, pour into plates, cool and solidify, and inoculate with pathogenic fungal cakes. Use inoculated plates without nanoparticles as a control. Incubate at 25 ℃, calculate the inhibition rate as in Example 3, and obtain LC as in Example 8 (1). 50 .

[0104] ② Dropping method for fungal cakes (used when nanoparticle concentration > 250 μg / mL): Pathogenic fungi and oomycetes fungal cakes were inoculated onto PDA and PC plates, respectively. 30 μL of silver nanoparticles at gradient concentrations were evenly dropped onto the fungal cakes, with three fungal cakes treated at each concentration. Fungal cakes with an equal volume of sterile water were used as a control. The plates were incubated at 25 °C. The inhibition rate was calculated as in Example 3, and the LC ratio was obtained as in Example 8(1). 50 .

[0105] The results showed that silver nanoparticles exhibited LC-resistance against *Cryptococcus parasiticus*, *Black rot of apple*, *Fusarium graminearum*, *Botrytis cinerea*, and *Phytophthora camphorata*. 50 The values ​​were 28.7 μg / mL, 148.4 μg / mL, 184.6 μg / mL, 0.57 mg / mL, and 1.59 mg / mL, respectively.

[0106] (3) Pine wood nematode: Add 50 μL of fresh mixed nematode suspension of each instar and 100 μL of silver nanoparticles of varying concentrations to a 96-well plate, mix well, and make 6 parallel wells for each concentration. Use sterile water instead of silver nanoparticles as a control. Incubate at 25℃ for 4 h. Take an appropriate amount of treatment solution (not less than 100 nematodes) on a glass slide, and revive at room temperature for 30 min. Count the nematodes under a microscope and calculate the mortality rate. The calculation formula is: (number of dead nematodes / total number of counted nematodes) * 100%. Same as (1) to obtain LC50 The results showed that silver nanoparticles exhibited LC50 activity against pine wood nematodes. 50 The value was 3.75 μg / mL.

[0107] Example 9: Biocompatibility of silver nanoparticles with its synthetic strain

[0108] Seed cultures were prepared for Bacillus strains exhibiting silver nanoparticle synthesis activity, similar to those in Example 2, and the bacterial concentrations were adjusted to OD values. 600 =1.0, and transferred to sterile YTGB medium at 4% (v / v), and prepared bilayer plates together with blank YTGB medium (both the bottom layer of blank YTGB and the top layer of inoculated YTGB were 10 mL / plate), and wells (6 mm) were punched in the top plate. Silver nanoparticles synthesized by the above strains were prepared as in Example 6. Silver nanoparticles synthesized by the same strain (2 mg / mL) were added to the bilayer plates of each strain at 20 μL / well, with an equal volume of sterile water added as a control, and incubated at 28 ℃. The formation of inhibition zones was recorded.

[0109] The results are shown in Table 2. Different strains showed different tolerances to the silver nanoparticles they synthesized. Four strains, represented by B268, did not show inhibition zones, while the other six strains were inhibited to varying degrees, indicating that B268 has good compatibility with the silver nanoparticles it synthesized and modified.

[0110] Table 2. Antagonistic effect of silver nanoparticles on its synthetic strains

[0111]

[0112] Example 10: Biocompatibility of silver nanoparticles with biocontrol bacteria and model nematodes

[0113] (1) Biocontrol bacteria

[0114] The same biocontrol bacterial seed culture was prepared as in Example 2 (with B268 as a negative control), and the same *Raulella tobaccoeri* seed culture was prepared as in Example 4 (as a positive control). The bacterial concentration was adjusted to OD. 600 =0.6, the same as in Example 9, a double-layer perforated plate was prepared, and 20 μL of silver nanoparticles synthesized from LB268 fermentation filtrate (2 mg / mL) was added into the well. An equal volume of sterile water was added as a control. The plate was incubated at 28 °C, the inhibition zone was counted, and the antagonism index was calculated. The calculation formula is: inhibition zone diameter / well diameter.

[0115] The results are as follows Figure 11 As shown, among the 12 biocontrol-active Bacillus strains targeted, only 2 strains showed inhibition zones, and their inhibition indices were significantly lower than those of Raulella tobaccois, indicating that the silver nanoparticles synthesized by B268 are compatible with most biocontrol bacteria.

[0116] (2) Biocontrol fungi

[0117] The antagonistic activity of silver nanoparticles against Trichoderma was detected using the same method as in Example 8 (drop-on mycelium cake method), and the results are as follows: Figure 12 As shown, no antagonistic effect was observed at the highest concentration (28 mg / mL), indicating that the silver nanoparticles synthesized by B268 are also compatible with biocontrol fungi.

[0118] (3) Model nematode

[0119] Similar to Example 8, using pine wood nematode as a control, the toxicity of silver nanoparticles synthesized by B268 to Caenorhabditis elegans was tested. The results are as follows: Figure 13 As shown, at a concentration of 4-5 μg / mL, the mortality rate of pine wood nematode reached 64.4%-85.7%, while the mortality rate of Caenorhabditis elegans was below 7%. Within the range of counting error and natural mortality, this indicates that the nanoparticles are compatible with Caenorhabditis elegans.

[0120] Example 11: Biocompatibility of silver nanoparticles with plant seeds

[0121] Wheat, cucumber, rice, sorghum, and casuarina seeds were soaked in sterile water. Damaged and empty seeds on the top layer were discarded, and the seeds on the bottom layer were collected. The seeds were soaked in silver nanoparticles of varying concentrations for 2 hours. The treated seeds were then evenly spread in petri dishes (each dish containing no less than 50 seeds) lined with three layers of sterile gauze and one layer of sterile filter paper. The petri dishes were moistened with an appropriate amount of sterile water and incubated at 25 ℃ in the dark for 4-7 days. Germination was observed. When the number of germinated seeds no longer changed, the seeds were removed, and the germination rate and radicle length were recorded.

[0122] The germination rate results are shown in Table 3. Silver nanoparticles promoted the germination of sorghum and casuarina seeds, with the highest germination rates at 250 μg / mL and 150 μg / mL, respectively. Within the concentration range of 0–250 μg / mL, there was no significant difference in the germination of cucumber and rice seeds, and within the concentration range of 0–200 μg / mL, there was no significant difference in the germination of wheat seeds.

[0123] Table 3. Effects of different concentrations of silver nanoparticles on seed germination

[0124]

[0125] Statistical results of radicle length are as follows Figure 14As shown, silver nanoparticles did not significantly inhibit the growth of the primary root of wheat, but promoted the growth of lateral roots in the concentration range of 150-250 μg / mL; they did not significantly inhibit the growth of lateral roots of sorghum, but tended to stimulate the growth of primary roots at a concentration of 200 μg / mL; and they had no significant effect on the development of the embryonic root of rice, casuarina, and cucumber.

[0126] Example 12: Preparation of B268-AgNPs compound microcapsules

[0127] The B268 fermentation broth was prepared in the same manner as in Example 6. Part of it was used to prepare filtrate and synthesize silver nanoparticles, while the remainder was used for encapsulation, as detailed below:

[0128] (1) B268-AgNPs composite encapsulation: Take an appropriate amount of silver nanoparticles synthesized from B268 fermentation filtrate, add water to a concentration of 100 μg / mL, add sodium alginate powder at a concentration of 15 g / L, stir and dissolve appropriately, then sterilize at 121 ℃ for 20 min (completely dissolved); add B268 fermentation broth (OD 600 =2) Mix with an equal volume of the above sodium alginate-AgNPs mixture, add to a syringe pump, squeeze and drip into a 20 g / L calcium chloride solution through a 30 G needle, solidify for 30 min, collect the formed microcapsules, wash twice with 9 g / L sodium chloride solution, drain and freeze dry, and store at 4 ℃.

[0129] (2) Separate encapsulation of B268: Prepare a sodium alginate solution with a concentration of 15 g / L, stir to dissolve appropriately, sterilize at 121 ℃ for 20 min (completely dissolved), and add an equal volume of B268 fermentation broth (OD). 600 =2) Mix, and the subsequent operations are the same as above.

[0130] (3) Individual encapsulation of AgNPs: Take an appropriate amount of silver nanoparticles synthesized from B268 fermentation filtrate, add water to a concentration of 50 μg / mL, add sodium alginate powder at a concentration of 7.5 g / L, stir and dissolve appropriately, sterilize at 121 ℃ for 20 min (completely dissolved), let stand and cool, and follow the same procedures as above.

[0131] (4) Empty embedding: Prepare a sodium alginate solution with a concentration of 7.5 g / L, stir and dissolve it appropriately, sterilize it at 121 ℃ for 20 min (completely dissolved), let it stand and cool, and then follow the same procedure as above.

[0132] The microcapsules obtained from the above four groups of encapsulation are as follows: Figure 15 As shown, B268 microcapsules, AgNPs microcapsules, and empty microcapsules were used as controls for B268-AgNPs microcapsules.

[0133] Example 13: Property evaluation of B268-AgNPs compound microcapsules

[0134] The properties of the four microcapsules described in Example 12 were evaluated as follows:

[0135] (1) Packaging ratio

[0136] Take 1 g of lyophilized B268 microcapsules and B268-AgNPs microcapsules, add them to 9 mL of 0.1 M sterile sodium citrate solution and dissolve them thoroughly. Simultaneously, take the B268 fermentation broth (OD) used for encapsulation. 600 = 2.0), respectively according to 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 Perform serial dilutions, spread on YTGB plates, incubate at 28 ℃, count colonies, and calculate the viable cell encapsulation rate using the formula: EE (%) = (N / N o ) × 100%, where N is the number of live cells released from 1 g of lyophilized microcapsules upon dissolution. o The actual number of cells added to prepare 1g of microcapsules.

[0137] Take 1 g of lyophilized AgNPs microcapsules and empty capsules as controls, add 9 mL of 0.1 M sterile sodium citrate solution and dissolve them thoroughly. Take samples together with the above two microcapsule solutions and scan them between 30-700 nm to detect the encapsulation of silver nanoparticles.

[0138] (2) Moisture content

[0139] Weigh out 1 g of each of the four types of microcapsules after draining and label them W. wet After freeze-drying, weigh again and record the weight as W. dry To calculate the moisture content, use the formula: MC (%) = [(W) wet -W dry ) / W wet ] ×100%.

[0140] (3) Expansion rate

[0141] Weigh out 1 g of each of the four types of freeze-dried microcapsules, denoted as W. dry Add sufficient 9 g / L sterile NaCl solution, soak at room temperature for 24 h, remove, drain, and weigh, denoted as W. wet Calculate the expansion rate using the formula: Expansion rate (%) = [(W)] wet -W dry ) / W wet ]×100%.

[0142] (4) Antibacterial activity

[0143] Take 0.1 g of each of the four lyophilized microcapsules and add 0.9 mL of 0.1 M sterile sodium citrate solution until the microcapsules are completely dissolved. Prepare the seed culture of *Rahuella tumefaciens* as in Example 4. Take 70 μL of the seed culture and streak it onto a YTGB plate. Then take 10 μL of the microcapsule solution and spot it onto the plate. Make three replicates per plate and incubate at 28 °C to observe the inhibition zone.

[0144] As shown in Table 4, there were no significant differences in the live cell encapsulation rate, water content, and expansion rate among the four types of microcapsules.

[0145] Table 4. Live cell encapsulation efficiency, water content, and swelling rate of four types of microcapsules

[0146]

[0147] Scanning chromatograms of four microcapsule solutions are as follows: Figure 16 As shown in Figure A, no absorption peak was observed in the 400-450 nm range for either the unloaded microcapsule or the B268 microcapsule, while absorption peaks were detected in both the AgNPs microcapsule and the B268-AgNPs composite microcapsule, proving the presence of silver nanoparticles.

[0148] The antagonistic activity of four microcapsule solutions against Raulella tobaccois, such as Figure 17 As shown in Figure A, the empty microcapsules showed no inhibition zone, indicating that neither the encapsulating agent sodium alginate nor the sol agent sodium citrate had antibacterial activity. The other three types of microcapsules all showed inhibition zones. The inhibition zone of the B268-AgNPs composite microcapsules was significantly larger than that of the B268 microcapsules and the AgNPs microcapsules, indicating a synergistic antibacterial effect between the bacteria and the silver nanoparticles. Furthermore, B268 growth was observed at the seeding sites on both the B268 microcapsule and the B268-AgNPs composite microcapsule plates, further demonstrating the compatibility between B268 and the silver nanoparticles.

[0149] Example 14: Storage stability assessment of B268-AgNPs compound microcapsules

[0150] After the four types of lyophilized microcapsules described in Example 12 were stored at 4 °C for 5 months, the encapsulation efficiency, expansion rate, and antibacterial activity were tested again according to the method in Example 13. The results are as follows: Figure 16 Table B, Table 5 and Figure 17As shown in Figure B, characteristic absorption peaks of silver nanoparticles were detected in both AgNPs microcapsules and B268-AgNPs composite microcapsules. The absorption peak of AgNPs microcapsules remained unchanged, while the absorption peak of B268-AgNPs composite microcapsules showed a red shift (412 nm → 467 nm), indicating an increase in particle size and suggesting potential aggregation of particles towards cells. The expansion rates of the four microcapsules did not change significantly. The viable cell encapsulation rate of B268-AgNPs composite microcapsules increased slightly, while that of B268 microcapsules decreased slightly, which is considered to be mainly due to experimental error. Meanwhile, except for the empty microcapsules, the other three types of microcapsules maintained antibacterial activity. The inhibition zone of B268-AgNPs composite microcapsules was still significantly larger than that of the other two microcapsules, indicating stable synergistic antibacterial activity.

[0151] Table 5. Live cell encapsulation rate and expansion rate of the four microcapsules after low-temperature storage.

[0152]

[0153] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A highly biocompatible bio-silver nanoparticle, characterized in that, Its preparation methods include the following: Bacillus B268 with accession number CGMCC No. 13225 was cultured in liquid medium to obtain fermentation broth. The cell-free fermentation filtrate or the fermentation broth was directly mixed with AgNO3 solution and incubated. After centrifugation and washing, the highly biocompatible bio-silver nanoparticles were obtained. These are silver nanoparticles with protein / peptide bound to their surface. The high biocompatibility includes: the bio-silver nanoparticles have no antagonistic effect on various biocontrol Bacillus species, including B268, at concentrations up to 2 mg / mL, but the half-lethal concentration (LD50) against bacterial wilt is only 5.35 μg / mL; at concentrations up to 28 mg / mL, they have no antagonistic activity against biocontrol Trichoderma, but the LD50 against *Rhizoctonia solani*, *Cryptococcus parasiticus*, *Fusarium graminearum*, and *Botrytis cinerea* is only 28.75-570 μg / mL; at 250 °C... At a concentration of 5 μg / mL, it had no adverse effects on seed germination and seedling development of rice, wheat, cucumber, sorghum, and casuarina; at a concentration of 5 μg / mL, it achieved a kill rate of 85.7% against pine wilt nematode and had no antagonistic effect against Caenorhabditis elegans.

2. The bio-silver nanoparticles according to claim 1, characterized in that, The culture period is 24-48 hours.

3. The bio-silver nanoparticles according to claim 1, characterized in that, The optimal final concentration of AgNO3 after mixing the AgNO3 solution with the cell-free fermentation filtrate is 5-7 mM, and the optimal final concentration of AgNO3 after mixing the AgNO3 solution with the fermentation broth is 3-5 mM.

4. The bio-silver nanoparticles according to claim 1, characterized in that, The incubation period was 24-48 h at 37 ℃, 200 rpm, and in the dark.

5. A compound microcapsule, characterized in that, The compound microcapsules were prepared by dispersing the bio-silver nanoparticles as described in claim 1 in deionized water, adding sodium alginate, then adding the fermentation broth of B268 and mixing, and then pressing them into a calcium chloride solution with a needle to solidify them.

6. The application of the bio-silver nanoparticles as described in claim 1 in the preparation of pesticides antagonizing plant pathogens, characterized in that, The plant pathogens were selected from *Cryptospira macrantha*, *Cryptospira macrantha*, *Fusarium graminearum*, *Botrytis cinerea*, *Raulella vesicae*, *Phytophthora camphorata*, and *Wood nematode*.

Images