Bacillus subtilis S1703 and application thereof

Abstract

The invention discloses bacillus subtilis S1703 and application thereof, and belongs to the technical field of plant disease and pest control. The name of the bacterial strain is bacillus subtilis S1703, and the preservation number of the bacterial strain is GDMCC No: 66890. The bacterial strain S1703 has a wider antibacterial spectrum, and can effectively inhibit the growth of pathogenic bacteria such as fusarium oxysporum, colletotrichum gloeosporioides, alternaria alternata, aspergillus, phyllosticta, citrus reticulata, fusarium and the like; fermentation liquor of the S1703 has a good antibacterial effect on the psidium guajava wilt pathogen and the psidium guajava branch blight pathogen; the bacteriostasis rate on sigatoka sigatoka and guava leaf spot germs reaches 95% or above, and an excellent bacteriostasis effect is achieved. The strain S1703 can avoid potential adverse effects of chemical pesticides on the environment and crop safety, and has potential commercial development and application values in biological prevention and control practice of diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant disease and pest control technology, specifically involving a Bacillus subtilis strain S1703 and its application. Background Technology

[0002] Fusarium wilt is a major disease in plant production, mostly soil-borne; once it spreads, it can cause devastating damage to plant growth. Currently reported pathogens causing Fusarium wilt include *Ralstonia solanacearum* (…). Pseudomonas nightshade Klebsiella pneumoniae ( Klebsiella variicola Fusarium oxysporum ( Fusarium oxysporum Fusarium solani () F. solani ), Pythium ( Pythium ), Clostridium neothesis ( Neofusicoccumbarvum ) and Staphylococcus aureus ( Botryosphaeria dothidea These pathogens, such as wilt, often infect the same plant species simultaneously, sometimes in one or more forms. Because fruit trees are perennial plants, the lack of disease-resistant varieties and effective chemical fungicides means that once wilt occurs, the leaves of fruit trees wilt and branches wither, leading to the death of one or several trees in mild cases, and potentially causing the destruction of the entire orchard in severe cases. Currently, the main wilt diseases affecting fruit trees include: banana wilt, papaya stem base rot, guava wilt, and citrus foot rot.

[0003] Plant anthracnose is caused by fungi of the genus Anthracnose ( Colletotrichum Anthracnose is an airborne disease caused by *Colletotrichum gloeosporioides* (spp.). It has a wide host range and can occur during the growing season and post-harvest period. During the growing season, it mainly affects flowers, leaves, fruits, and stems. When affecting leaves, the fungus initially invades from the leaf tip or margin, gradually expanding into yellowish-brown, cloud-like, V-shaped lesions, which can cause significant leaf drop in severe cases. When anthracnose affects shoots, it often occurs at the shoot tip, initially appearing as light brown, oval lesions, eventually leading to the death of the entire shoot tip. Currently known common pathogens include *Colletotrichum gloeosporioides* (spp.). C. gloeosporioides ), Anthrax bacillus ( C. acutatum ), Karst anthrax bacteria ( C. karstii ), *Colletotrichum candida* ( C. fructicola ) and Siamese spirococcosis ( C. siamese )wait.

[0004] Leaf spot diseases occur throughout the entire growth period of almost all plants, including canker, brown spot, black spot, angular spot, and stripe, etc. They can be caused by fungi, bacteria, viruses, etc., affecting plant photosynthesis, leading to weakened plant growth, and in severe cases, causing yield reduction, greatly impacting plant output and economic value. Common leaf spot diseases on fruit trees are mainly caused by fungi of the genus *Fusarium* (…). Fusarium sp.), Anthrax ( Colletotrichum sp.), Alternaria ( Alternaria sp.), genus *Plasmodium* ( Pestalotiopsis sp.), Aspergillus (sp.), Aspergillus genus ( Aspergillus sp.), Neopyrium ( Neocytalidium The same bacteria can cause different symptoms on different hosts, and a single or several bacteria can also occur together on the same plant.

[0005] The pathogens that cause guava stem rot and browning include Clostridium neoformans ( Neofusicoccumbarvum ) and Fusarium oxysporum ( Fusarium oxysporum Among them, *Clostridium neoformans* can also cause guava leaf spot and fruit rot. Although guava is a rare and high-quality fruit in the Lingnan region, its unique taste and nutritional value are increasingly appreciated and accepted by the public, and its planting area is increasing year by year. Because research on new guava varieties and pest and disease control is still in its early stages, the current control of guava wilt mainly involves drenching with chemical fungicides and removing diseased trees. However, due to the complexity of the pathogen and the emergence of drug resistance, chemical fungicides have very little effect on controlling the disease, which easily damages the soil. Therefore, it is urgent to find new control methods. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a strain of Bacillus subtilis S1703 and its applications. Bacillus subtilis S1703 is a Gram-positive bacterium capable of forming highly resistant spores. It is widely distributed in soil and decaying organic matter, exhibits rapid growth and strong metabolic capacity, and can utilize various carbon and nitrogen sources. In agriculture, this bacterium is used as a biopesticide and fertilizer to inhibit pathogens; industrially, it is used in the production of enzyme preparations and antibiotics; and in the pharmaceutical field, it is used as a probiotic to regulate intestinal flora and enhance immunity. Its safety and multifunctionality make it of significant application value in biological control, green agriculture, environmental protection, and the health industry.

[0007] The objective of this invention is achieved through the following technical solution: The first objective of this invention is to provide a strain of Bacillus subtilis, named Bacillus subtilis (… Bacillus subtle S1703 was deposited on August 28, 2025 at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66890.

[0008] The second objective of this invention is to provide a reagent containing the above-mentioned Bacillus subtilis S1703 as an active ingredient.

[0009] Preferably, the reagent is a biocontrol agent, and the Bacillus subtilis S1703 is at least one of live Bacillus subtilis S1703 cells, cell fragments, fermentation broth, fermentation supernatant, and its metabolites.

[0010] The third objective of this invention is to provide the application of the above-mentioned Bacillus subtilis S1703 in the prevention and control of plant diseases or in the inhibition of plant pathogens.

[0011] Preferably, the plant pathogen is a pathogen that causes plant diseases.

[0012] Preferably, the plant disease is plant wilt, plant anthracnose, and / or plant leaf spot, etc.

[0013] Preferably, the pathogen of the plant wilt disease is *Fusarium wiltii*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, or *Fusarium oxysporum* twig blight; the pathogen of the plant anthracnose is *Colletotrichum* (*Colletotrichum*). Colletotrichum Fungi; the pathogen of the plant leaf spot disease is Alternaria. Alternaria sp., *Plasmodium spp.* Pestalotiopsis sp., Fusarium Fusarium sp., genus *Neocys* Neofusicoccus sp., Aspergillus Aspergillus sp., genus *Leaf-spot* Phyllosticta sp., Neocytophyte Neocytalidium sp. or genus *S.* Diaporthe sp.

[0014] Preferably, the banana wilt pathogen is Fusarium oxysporum race 4, Cuban specific type (…). F. oxysporum f .sp . From Cuba race 4, FOC4); the passion fruit stem base rot pathogen is Fusarium oxysporum ( Fusarium oxysporum The pathogen causing the wilt of Coptis chinensis is *Schloa crus-galli* (Cucumber spores). Plectosphaerellacucumerina The pathogen causing the foot rot of Xinhui citrus is *Fusarium oxysporum* (…). Fusarium oxysporum The pathogen causing the wilt of Polygonatum sibiricum is Fusarium solani (…). Fusarium solani The guava wilt pathogen is *Fusarium oxysporum* guava-specific strain (…). Fusarium oxysporum f. sp. psidii); the guava twig blight pathogen is Clostridium neoformans (f. sp. psidii); Neofusicoccumbarvum ); Preferably, the pathogen causing the plant anthracnose is *Prunus triloba* anthracnose, *Guava* anthracnose, *Passion fruit* anthracnose, *Dragon fruit* anthracnose, or *Lemon* anthracnose; the guava anthracnose pathogen is *Colletotrichum gloeosporioides* (…). C. gloeosporioides ) and fruit-borne thorn-like spores ( C. fructicola At least one of the following: the anthracnose fungus of the three-flowered plum is *Colletotrichum oryzae* (Fructus *Colletotrichum oryzae*). C. fructicola The passion fruit anthracnose is caused by *Colletotrichum gloeosporioides* ( ); C. gloeosporioides ) and anthrax bacteria ( C. truncatum At least one of the following: the lemon anthracnose fungus is *Colletotrichum citrinum* (Fructus *Colletotrichum citrinum*). C. fruit-growing The anthracnose pathogen affecting dragon fruit is *Colletotrichum candida* (…). C. gloeosporioides ).

[0015] Preferably, the Alternaria species Alternaria sp. is Alternaria alternifolia ( A. alternata ); the *Plasmodium spp.* Pestalotiopsis sp. is *Microsporum spp.* P. microspora ); the Fusarium genus Fusarium sp. is Fusarium canis ( F. sacchari ); the new genus *Neocystium* Neofusicoccus sp. is Clostridium neoformans ( No. small ); the Aspergillus genus Aspergillus sp. is Aspergillus niger ( A. niger ); the genus *Leafylodis* Phyllosticta sp. mold on guava leaves ( Phyllostictapsidia Tassi); the interseat shell belongs to Diaporthe sp. is a citrus metamorphosum ( D. lemon ); the new genus *Pycnopyrum* Neocytalidium sp. is *Neopterospora darkiformis* ( N. halved ).

[0016] The present invention has the following advantages and effects compared with the prior art: (1) The newly discovered Bacillus subtilis S1703 in this invention has a field control efficiency of over 85% against guava diseases.

[0017] (2) The Bacillus subtilis S1703 provided by this invention has a broader antibacterial spectrum, effectively inhibiting the growth of 17 pathogenic fungi, including Fusarium wilt pathogens on 6 plant species, anthracnose pathogens on 9 plant species, Alternaria alternata, Aspergillus, Phyllostachys, Citrus melilothorax, and Fusarium. The fermentation broth of S1703 has good antibacterial effects against Fusarium wilt and Fusarium truncatum on guava. It also has an inhibition rate of over 95% against leaf spot pathogens of banana and guava, demonstrating excellent antibacterial effects. These functions have not been reported in the prior art. Since strain S1703 was isolated from the rhizosphere soil of plants, it can be considered for use in the control of soil-borne diseases. This strain can avoid the potential adverse effects of chemical pesticides on the environment and crop safety, and has potential commercial development and application value in the biological control of diseases. Attached Figure Description

[0018] Figure 1 Based on the 16S rDNA full genome sequence, strain S1703 and Bacillus ( ) were constructed using the maximum natural method. Bacillus Phylogenetic tree of other minor species (spp.).

[0019] Figure 2 The diagram shows the morphology of strain S1703 on different culture media (30℃, 1 day); where A: LB medium; B: NA medium; C: PDA medium; D: TSA medium.

[0020] Figure 3 To determine the different biological characteristics of strain S1703.

[0021] Figure 4 The diagrams show the confrontation cultures of strain S1703 with different plant anthracnose pathogens (7 days and 20 days). A and CH: Diagrams showing strain S1703 after 7 days of confrontation culture with *Colletotrichum cirrhosa* (anthracnose pathogen of *Prunus triloba*), *Colletotrichum cirrhosa* (anthracnose pathogen of *Guava*), *Colletotrichum gloeosporioides* (anthracnose pathogen of *Guava*), *Colletotrichum gloeosporioides* (anthracnose pathogen of *Passion fruit*), *Colletotrichum gloeosporioides* (anthracnose pathogen of *Dragon fruit*), *Colletotrichum cirrhosa* (anthracnose pathogen of *Lemon*), and *Colletotrichum gloeosporioides* (anthracnose pathogen of *Passion fruit*). B: Diagram showing strain S1703 after 20 days of confrontation culture with *Colletotrichum cirrhosa* (anthracnose pathogen of *Prunus triloba*).

[0022] Figure 5 Strain S1703 is associated with leaf spot pathogens affecting bananas, black tiger fruit, dragon fruit, Buddha's hand, tangerine peel, figs, custard apples, or guava. Neofusicoccumbarvum A diagram illustrating the confrontation training process (7 days).

[0023] Figure 6 This is a schematic diagram of the confrontation culture between strain S1703 and guava spot pathogen (7 days).

[0024] Figure 7 This is a schematic diagram (7 days) of confrontation culture between strain S1703 and the wilt pathogens of passion fruit, Xinhui mandarin orange, guava, Coptis chinensis, banana, and Polygonatum sibiricum.

[0025] Figure 8 To antagonize the guava pathogen Clostridium neoformans of strain S1703 ( Neofusicoccumbarvum The antibacterial mechanism of ).

[0026] Figure 9 The diagram shows the control effect of strain S1703 on guava diseases in the field. Among them, A: Schematic diagram of diseased guava plants in the field; B: Control effect of strain S1703 on guava diseases in the field; C: Schematic diagram of diseased guava plants not inoculated with S1703. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. To better explain the present invention, the principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical solutions involved in the embodiments of the present invention are conventional solutions in the art; unless otherwise specified, the reagents or materials mentioned are all from commercial channels.

[0028] Example 1: Bacillus subtilis Bacillus subtilis Isolation, identification and biological characteristics analysis of S1703 I. Isolation of strain S1703 Rhizosphere soil from guava orchards in Nansha District, Guangzhou City, Guangdong Province was collected. A soil suspension was prepared with sterile water and uniformly spread onto PDA agar plates using a gradient dilution method. After the plates were dried, they were incubated at 26°C. Single colonies were collected daily and compared with *Fusarium wiltii* (the pathogen causing guava wilt). Fusarium oxysporum f. sp. Psidii) and guava twig blight fungus ( Neofusicoccumbarvum After confrontation culture, a strain of Bacillus subtilis that showed significant inhibitory effects on both Fusarium wilt and Fusarium truncatum was obtained after primary and secondary screening, and named S1703.

[0029] II. Morphological characteristics of strain S1703 Strain S1703 can grow on LB (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar) and NA (3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, 20 g / L agar) media at 30 ℃. Its colonies are milky white, smooth, and do not expand with a flash. Figure 2 (A and B in the text); On PDA (potato dextrose agar dry powder 39 g / L) and TSA (tryptone 15.0 g / L, soybean peptone 5.0 g / L, sodium chloride 5.0 g / L, agar 20 g / L) media, the colonies were coarse, milky white to slightly yellow, without shimmering but expanding ( Figure 2 (C and D in the text).

[0030] III. Identification of the taxonomic attributes of strain S1703 Strain S1703 was inoculated into 100 mL of PDB (potato dextrose powder 24 g / L) liquid medium and cultured overnight at 26°C and 180 rpm in a shaker. The cells were collected by centrifugation at 10,000 rpm / 7 min, ground in liquid nitrogen, and genomic DNA was extracted from strain S1703 using a standard DNA extraction kit. Using the genomic DNA of strain S1703 as a template, PCR amplification and sequencing were performed using the universal 16S rDNA primers 27F (SEQ ID NO.1, 5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (SEQ ID NO.2, 5'-GGTTACCTTGTTACGACTT-3'). The 16S rDNA sequencing results of strain S1703 (SEQ ID NO.3) were compared with BLASTn on NCBI to obtain the sequence information of closely related strains. Phylogenetic trees were constructed by downloading the 16S rDNA sequences of closely related species and related type strains. Figure 1 Strain S1703 was identified as Bacillus subtilis using molecular systematics methods. Bacillus subtilis S1703.

[0031] Bacillus subtilis ( Bacillus subtilis S1703 was deposited on August 28, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The accession number is GDMCC No. 66890. Classification and nomenclature: Bacillus subtle .

[0032] In this invention, Bacillus subtilis ( Bacillus subtilis S1703 is abbreviated as S1703.

[0033] Example 2: Cultivation and fermentation of Bacillus subtilis S1703 I. Cultivation of strain S1703 Using an inoculation loop, scrape an appropriate amount of bacterial cells from the surface of the S1703 colony, streak them on the surface of PDA medium, and then incubate them overnight at 30°C.

[0034] II. Preparation of fermentation broth for strain S1703 Using an inoculation loop, scrape an appropriate amount of bacterial cells from the surface of the S1703 colony, inoculate it into PDB liquid medium, and then incubate it overnight at 30°C and 180 rpm as a seed culture.

[0035] Using a sterile 1 mL pipette tip, 1 mL of seed culture was inoculated into a 250 mL sterile Erlenmeyer flask containing 100 mL of PDB liquid medium. After incubation at 26°C and 180 rpm for 3 days, the fermentation broth was filtered through a single layer of sterile gauze and the filtrate was collected to obtain the fermentation broth of strain S1703.

[0036] Example 3: Determination of biological characteristics of Bacillus subtilis S1703 This invention describes the determination of protease, cellulase, or β-1,3-glucanase in strain S1703. Protease detection media (12 g / L skim milk powder, 20 g / L agar), cellulase detection media (10 g / L sodium carboxymethyl cellulose, 10 g / L peptone, 5 g / L yeast extract, 1 g / L KH₂PO₄, 5 g / L NaCl, 20 g / L agar), and β-1,3-glucanase detection media (5 g / L yeast extract, 10 g / L peptone, 0.4 g / L Congo red, 5 g / L NaCl, 20 g / L agar) were prepared. Three plates were prepared for each medium, and four Oxford cups were placed on each plate. 50 μL of overnight cultured strain S1703 was added to each cup. After overnight culture, the results were observed. Figure 3 The results showed that strain S1703 possessed protease and β-1,3-glucanase activities, but not cellulase activities.

[0037] Example 4: Antagonistic effect of Bacillus subtilis S1703 against plant anthracnose pathogens This invention uses the confrontation culture method to determine the effect of strain S1703 on the genus *Colletotrichum*, which causes anthracnose on passion fruit, dragon fruit, plum, lemon, and guava. Colletotrichum The antifungal activity of the fungi was tested. The tested fungi were *Colletotrichum gloeosporioides* from passion fruit, dragon fruit, and guava. C. gloeosporioides () Figure 4 E, F, D in the text), and fruit-borne spirilla on plums, guavas, or lemons ( C. fructicola () Figure 4 A, C, G) and anthracnose on passion fruit ( C. truncated () Figure 4 (H in the text).

[0038] Five-mm diameter hyphal fragments were collected from the edge of freshly cultured *Colletotrichum* colonies (5 days prior) and inoculated onto new PDA agar plates 2 cm from the edge. S1703 cells were scraped using an inoculation loop and spread 4.5 cm from the pathogen. The uninoculated S1703 treatment served as a control. Five replicates were performed for each treatment. After incubating the plates at 26°C for 7 days, the width of the inhibition zone was measured, and the inhibition rate was calculated. Inhibition rate (%) = (4.5 - width of inhibition zone in treatment group) / 4.5 × 100.

[0039] The results are as follows Figure 4 As shown, strain S1703 exhibits strong inhibitory effects against all tested *Colletotrichum* fungi. Strain S1703 also shows strong inhibitory effects against *Colletotrichum cirrhosa*, the anthracnose causal agent of *Prunus triloba*. C. fructicola The antibacterial effect was strongest, reaching 44.58% after 7 days of confrontation culture and 63.49% after 20 days; it also showed the strongest antibacterial effect against *Colletotrichum gloeosporioides*, the causal agent of guava anthracnose. C. gloeosporioides The antibacterial effect of strain S1703 was the weakest, at 20%. These results indicate that the antibacterial activity of S1703 against anthracnose has a certain persistence, demonstrating that strain S1703 has good potential for controlling plant anthracnose.

[0040] Example 5: Antagonistic effect of Bacillus subtilis S1703 against Fusarium wilt pathogen. This experiment selected the fungus causing stem rot of passion fruit (… Fusarium oxysporum ), Xinhui citrus foot rot fungus ( F. oxysporum ), banana wilt pathogen ( F. oxysporum f .sp . From Cuba race 4, FOC4), Fusarium wilt pathogen ( Plectosphaerellacucumerina ), Polygonatum wilt pathogen ( F. solani Guava twig blight fungus ( Neofusicoccumbarvum ) and guava wilt fungus ( Fusarium oxysporum f. sp. psidii) was cultured in confrontation with S1703, and the antibacterial activity of S1703 against the plant wilt pathogen was determined. Figure 7 ).

[0041] Passion fruit stem rot fungus, *Xinhui citrus foot rot fungus*, *Polygonatum sibiricum*, *Flavorum sibiricum*, *Coptis chinensis*, *Guava* branch blight fungus, and *Guava* wilt fungus were inoculated onto PDA agar plates and incubated at 26℃ for 5 days. Mycelial blocks with a diameter of 5 mm were collected from the edge of the colonies and inoculated onto new PDA agar plates 2 cm from the edge. S1703 cells were scraped using an inoculation loop and spread at a distance of 4.5 cm from the pathogen. The treatment without S1703 inoculation served as a control. Five replicates were performed for each treatment. After incubating the plates at 26℃ for 7 days, the width of the inhibition zone was measured, and the inhibition rate was calculated. Inhibition rate (%) = (4.5 - width of inhibition zone in treatment group) / 4.5 × 100.

[0042] The results are as follows Figure 7 As shown, strain S1703 exhibits strong inhibitory effects against all tested Fusarium wilt pathogens, and also against *Fusarium solani* (the causal agent of guava branch blight). Neofusicoccumbarvum The antibacterial rate was 38.53% ( Figure 5 H in the text), against guava wilt pathogen ( Fusarium oxysporum The inhibition rate of *Fusarium oxysporum f. sp. psidii* was 43.67%; the inhibition rate of *Fusarium oxysporum* stem rot pathogen was 35.42%, that of *Fusarium oxysporum* wilt pathogen was 4.79%, that of *Fusarium oxysporum* wilt pathogen was 47.96%, that of *Fusarium oxysporum* wilt pathogen was 24.26%, and that of *Fusarium oxysporum* foot rot pathogen was 39.04%. In conclusion, strain S1703 has good biocontrol potential against *Fusarium oxysporum* wilt pathogens.

[0043] Example 6: Antagonistic effect of Bacillus subtilis S1703 against fungi causing plant leaf spot disease This experiment used a confrontation culture method to determine the antifungal activity of strain S1703 against six pathogenic fungi that cause leaf spot disease in plants. These pathogenic fungi include Alternaria alternata (…). A. alternata (Cherlenchus custard apple, Citrus reticulata peel), Microsporum simsii ( Pestalotiopsis microspora (Guava, Black Tiger), Neodatura darkens ( Neoscytalidium dimidiatum )(pitaya) 、 Clostridium neothesis ( Neofusicoccumbarvum (Guava), Guava Leaf Spotting ( Phyllostictapsidia Tassi (guava), Aspergillus niger ( A. niger (fig), genus *Cercis* ( Diaporthecitri (Buddha's Hand) and Fusarium ( ) F. sacchari (Banana). The determination method is the same as in Example 5.

[0044] The results are as follows Figure 5 and Figure 6As shown, strain S1703 exhibits strong inhibitory effects against all eight tested plant pathogenic fungi, including those causing banana leaf spot disease. F. sacchari It had the highest antibacterial rate, at 95.91%; against the leaf spot pathogen of custard apple. A. alternate It had the lowest inhibition rate, at 3%, while its inhibition rates against other pathogens were all above 35%; it also showed the best inhibition rate against the pathogen causing guava spot disease. Phyllostictapsidia Tassi showed an inhibition rate of 95.3%. After 30 days of confrontational culture, S1703 showed resistance to the banana leaf spot pathogen. F. sacchari Guava Spot Disease P. psidii Tassi maintained an inhibition rate of over 95%. This indicates that strain S1703 not only has a broad antibacterial spectrum but also exhibits persistent activity. Therefore, it has significant development and application value in the biological control of plant diseases.

[0045] Example 7: Bacillus subtilis S1703 against Clostridium neoformans, the pathogen of guava ( Neofusicoccumbarvum A Preliminary Study on Antibacterial Mechanism I. Preparation of fermentation broth plates for strain S1703 Add 100 μL of the fermentation broth of strain S1703 obtained in Example 2 to 100 mL of melted PDA medium (cooled to 40°C), mix well, and pour into plates, 25 mL of medium per plate. Add 100 μL of PDB medium to the control group.

[0046] II. The effect of fermentation broth of strain S1703 on guava branch blight pathogen Neofusicoccumbarvum Inhibition effect on mycelial growth In this invention, *Guava twig blight* pathogens that have been freshly cultured for 5 days are selected. Neofusicoccumbarvum Five-mm diameter mycelial blocks were taken from the edge of the plate and inoculated into the center of the fermentation broth plate from step one above. The plates were incubated at 26 °C, with five replicates per treatment. The mycelial blocks were also inoculated into blank PDA medium as a control group. From day 3 of incubation, the colony diameter was observed and measured daily until day 7. The mycelial growth inhibition rate of the sterile fermentation broth of strain S1703 against the measured pathogen was calculated. Mycelial growth inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100.

[0047] Select strain S1703 and guava twig blight pathogen Neofusicoccumbarvum Confrontation culture of bacterial colonies for 7 days and guava twig blight pathogen on PDA Neofusicoccumbarvum Colonies cultured without confrontation for 7 days were examined under an electron microscope, and mycelia at the colony edges were selected. The presence of *Guava twig blight* fungus treated with strain S1703 was observed. NeofusicoccumbarvumMelanin deposition in the hyphae indicates accelerated hyphal senescence. It is speculated that strain S1703 may inhibit pathogens by accelerating hyphal senescence. Figure 8 ).

[0048] Example 8: Field control efficacy of Bacillus subtilis S1703 against guava diseases This experiment used the field drenching inoculation method on two-year-old guava trees to determine the disease control effect of the fermentation broth of strain S1703 on guava diseases.

[0049] Identification of Guava Diseases: (1) Identification of Pathogens in the Field: Using the conventional tissue separation method, select the root of the infected guava and use a scalpel to remove a 0.2×0.2 cm tissue sample from the junction of diseased and healthy tissue. 2 Small pieces were placed in 70% alcohol for 30 seconds, 2% NaClO for 2-3 minutes, rinsed with sterile water 3-5 times, placed on sterile filter paper and dried in a clean bench, and finally placed on a PDA plate for dark culture at 25 ℃. After 3 days, white colonies grew on the isolate. Single spores were picked and inoculated on a new PDA plate and cultured for another 7 days. The morphology and size of the hyphae and spores of the isolated strain were examined under a microscope, and at least 15-20 fields of view were observed. (2) Pathogenicity test: The isolated strain and 7 healthy guava plants cultured on PDA plates for 7 days were selected and inoculated using the non-invasive method and the needle puncture method respectively (one small hole was punctured at each inoculation point with a sterile needle). First, 0.5 mm of the isolated strain and blank PDA medium were taken with a punch. 2 The mycelial block was placed with the mycelial side attached to the healthy guava stem, and blank PDA medium was used as a negative control. On the 5th and 10th day after inoculation, the plantlets were observed and recorded; (3) Molecular biological identification: the whole genome DNA of the fungal strain that can cause rot and browning of guava stem was extracted and amplified by PCR using primers for the internal transcribed spaces of fungal DNA ribosomes (ITS, ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' / ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). The amplified PCR products were sequenced and compared by BLAST in NCBI. Finally, it was confirmed that the pathogen causing the above symptoms in guava was Guava twig blight fungus ( Neofusicoccumbarvum ) and guava wilt fungus ( Fusarium oxysporum f. sp.Psidii).

[0050] Inoculum preparation: Prepare inoculum solutions with concentrations of OD0.05 and OD0.05. 600 Fermentation broth of strain S1703 with a coefficient of 2.0.

[0051] Inoculation: Ten guava plants already infected with the same degree of guava disease were selected from the field for the experiment. Figure 9 (A) Among them, 5 diseased plants were irrigated with 500 mL of strain S1703 fermentation liquid every month starting from April 2025, and the other 5 plants were irrigated with 500 mL of PDB culture medium.

[0052] Field control efficiency calculation method: Guava diseases are classified into different levels according to their severity: Level 0 (healthy), Level 1 (leaf on 1-2 branches is yellow / purple; slight cracking symptoms appear on the stem), Level 2 (leaf on half of the branches of the entire tree is yellow / purple; cracking symptoms appear on the stem), and Level 3 (the entire tree is weak, most leaves are yellow; bark peeling symptoms appear on the stem); Disease index = 100 × ∑(number of diseased leaves at each level × representative value of each level) / (total number of leaves surveyed × highest level representative value); Control efficiency (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100.

[0053] Disease survey: In October 2025, a field survey was conducted on the control of guava diseases using strain S1703. Results showed that most of the five guava trees treated with root drenching recovered. Figure 9 (B in the text), and the fruit load is comparable to that of healthy guava plants, with a control efficiency of over 85%; untreated guava trees with diseased fruit exhibit the phenomenon of complete necrosis ( Figure 9 (C in the text). This indicates that strain S1703 shows promising biocontrol potential in the field against guava diseases.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis, characterized by: The name is Bacillus subtilis ( Bacillus subtilis S1703 was deposited on August 28, 2025 at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 66890.

2. A reagent, characterized in that, It contains Bacillus subtilis S1703 as the active ingredient as described in claim 1.

3. The reagent according to claim 2, characterized in that, The reagent is a biocontrol agent, and the Bacillus subtilis S1703 is at least one of the following: live Bacillus subtilis S1703 cells, cell fragments, fermentation broth, fermentation supernatant, and its metabolites.

4. The application of Bacillus subtilis S1703 according to claim 1 or the reagent according to any one of claims 2 to 3 in the prevention and control of plant diseases, characterized in that: The plant diseases mentioned are plant wilt, plant anthracnose, and / or plant leaf spot.

5. The application of Bacillus subtilis S1703 according to claim 1 or the reagent according to any one of claims 2 to 3 in inhibiting plant pathogens, characterized in that: The plant pathogens are pathogens that cause plant diseases; the plant diseases are plant wilt, plant anthracnose, and / or plant leaf spot.

6. The application according to claim 4 or 5, characterized in that: The pathogens of plant wilt are *Fusarium wiltii*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, or *Fusarium oxysporum* twig blight; the pathogens of plant anthracnose are *Colletotrichum* (*Colletotrichum*). Colletotrichum Fungi; the pathogen of the plant leaf spot disease is Alternaria. Alternaria sp., *Plasmodium spp.* Pestalotiopsis sp., Fusarium Fusarium sp., genus *Neocys* Neofusicoccum sp., Aspergillus Aspergillus sp., genus *Leaf-spot* Phyllosticta sp., Neocytophyte Neoscytalidium sp. or genus *S.* Diaporthe sp.

7. The application according to claim 6, characterized in that: The fungus causing banana wilt is Fusarium oxysporum, specifically race 4 of the Cuban variant. F. oxysporum f .sp. Cubense race 4); the passion fruit stem base rot pathogen is Fusarium oxysporum ( Fusarium oxysporum The pathogen causing the wilt of Coptis chinensis is *Schloa crus-galli* (Cucumber spores). Plectosphaerella cucumerina The pathogen causing the foot rot of Xinhui citrus is *Fusarium oxysporum* (…). Fusarium oxysporum The pathogen causing the wilt of Polygonatum sibiricum is Fusarium solani (…). Fusarium solani The guava wilt pathogen is *Fusarium oxysporum* guava-specific strain (…). Fusarium oxysporium f. sp. psidii); the guava twig blight pathogen is Clostridium neoformans (f. sp. psidii); Neofusicoccum parvum ).

8. The application according to claim 6, characterized in that: The pathogens causing plant anthracnose are anthracnose of plum, guava, passion fruit, dragon fruit, or lemon.

9. The application according to claim 8, characterized in that: The guava anthrax bacterium is *Colletotrichum gloeosporioides* (… C. gloeosporioides ) and fruit-borne thorn-like spores ( C. fructicola At least one of the following: the anthracnose fungus of the three-flowered plum is *Colletotrichum oryzae* (Fructus *Colletotrichum oryzae*). C. fructicola The passion fruit anthracnose is caused by *Colletotrichum gloeosporioides* ( ); C. gloeosporioides ) and anthrax bacteria ( C. truncatum At least one of the following: the lemon anthracnose fungus is *Colletotrichum citrinum* (Fructus *Colletotrichum citrinum*). C. fructicola The anthracnose pathogen affecting dragon fruit is *Colletotrichum candida* (…). C. gloeosporioides ).

10. The application according to claim 6, characterized in that: Alternaria Alternaria sp. is Alternaria alternifolia ( A. alternata ); the *Plasmodium spp.* Pestalotiopsis sp. is *Microsporum spp.* P. microspora ); the Fusarium genus Fusarium sp. is Fusarium canis ( F. sacchari ); the new genus *Neocystium* Neofusicoccum sp. is Clostridium neoformans ( N. parvum ); the Aspergillus genus Aspergillus sp. is Aspergillus niger ( A. niger ); the genus *Leafylodis* Phyllosticta sp. mold on guava leaves ( Phyllosticta psidii Tassi); the interseat shell belongs to Diaporthe sp. is a citrus metamorphosum ( D. citri ); the new genus *Pycnopyrum* Neoscytalidium sp. is *Neopterospora darkiformis* ( N. dimidiatum ).

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