Bacillus velezensis JR1A and application thereof in inhibition of garlic pathogenic fungi

Abstract

The invention relates to the technical field of biocontrol strains for crops, in particular to bacillus velezensis JR1A and application thereof to inhibition of garlic pathogenic fungi. The bacillus velezensis JR1A is preserved in the China General Microbiological Culture Collection Center on June 30, 2025, and the preservation number of the bacillus velezensis JR1A is CGMCC No.35059. The bacillus velezensis JR1A is named as bacillus velezensis JR1A. The JR1A inhibits mycelial growth and spore production of pathogenic fungi of common garlic diseases, has an excellent biocontrol effect and a certain growth promoting function, can reduce the relative abundance of pathogenic fungi in garlic crop rotation soil, and reduces the occurrence of related garlic diseases.

Description

Technical Field

[0001] The invention relates to the technical field of crop biocontrol strains, in particular to a Bacillus velezensis JR1A and application thereof in inhibiting garlic pathogenic fungi. Background Art

[0002] Intensive agriculture is an agricultural model that maximizes land output and agricultural production efficiency through high inputs (such as labor, capital, technology, fertilizers, and pesticides). Modern intensive agriculture typically relies on monoculture, low-diversity cropping, and short rotations, which can easily trigger negative plant-soil feedback. Continuous cropping obstacles are a typical manifestation of this phenomenon. Continuous cropping obstacles refer to the phenomenon in which continuous planting of the same crop type or family on the same plot of land results in suppressed crop growth, reduced yields, and increased pests and diseases. Continuous cropping obstacles have become a major constraint on the sustainable development of agriculture in China.

[0003] The complexity of the mechanism of continuous cropping disorder stems from the interaction between "soil-plant-microorganism". At present, a large number of studies have shown that the imbalance of soil microbial communities is the most critical factor leading to continuous cropping disorder. Zeng et al. found that continuous cropping increased the diversity and relative abundance of harmful microorganisms in the soil, while the indicators related to beneficial bacteria showed a downward trend. The balance of soil microbial communities was broken, which seriously affected the ecological functions of the soil and restricted plant growth and development. Diseases caused by soil-borne pathogens are the most direct and main manifestations of continuous cropping disorder, such as soft rot, root rot, ear rot, etc. More than 70% of the related damages caused by continuous cropping disorder can be attributed to the infection and spread of soil-borne pathogens.

[0004] Garlic is highly susceptible to soil-borne diseases, especially during continuous cropping. Large-scale infestations of pathogenic fungi (such as Fusarium and Stemphylium) can cause garlic root rot, leaf blight, and purple spot, resulting in significant economic losses. Inhibiting the growth of harmful microorganisms is an effective way to alleviate continuous cropping problems. Summary of the Invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provide a strain of Bacillus velez JR1A and its use in inhibiting garlic pathogenic fungi. JR1A inhibits the mycelial growth and spore production of pathogenic fungi that cause common garlic diseases, has excellent biocontrol effects and certain growth-promoting functions, can reduce the relative abundance of pathogenic fungi in garlic rotation soil, and reduce the occurrence of garlic-related diseases.

[0006] The object of the present invention is achieved through the following technical solution: a strain of Bacillus velezensis JR1A, characterized in that the Bacillus velezensis JR1A is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of June 30, 2025, and a deposit number of CGMCC No.35059.

[0007] The present invention also provides a microbial agent comprising at least one of the bacterial cells, spores and fermentation liquid of the above-mentioned Bacillus Velez JR1A.

[0008] The present invention also provides a use of Bacillus velezensis JR1A for inhibiting pathogenic fungi of Liliaceae plants.

[0009] Furthermore, the lily family plant is garlic.

[0010] Furthermore, the pathogenic fungi include at least one of Fusarium odoratum, Chaetomium globosum and Sclerotium zeae.

[0011] The present invention also provides a method for preventing and controlling garlic diseases, comprising the following steps: applying the above-mentioned Bacillus Velezii or the above-mentioned microbial agent to pathogens, the above-ground parts of plants or the growth environment of plant roots.

[0012] Furthermore, the garlic diseases include at least one of root rot, leaf blight, rust and soft rot.

[0013] The present invention has the following beneficial effects: 150 strains were isolated from garlic rhizosphere soil, and Bacillus Velez JR1A was screened out as having the strongest inhibitory effect on pathogens of common garlic diseases. Among them, the present invention has an inhibitory effect far exceeding that of other microorganisms on major infecting pathogens that can cause garlic root rot, leaf blight, root diseases in the early growth stage of garlic (root rot, soft rot), and foliar diseases in the late growth stage of garlic (leaf blight, rust, and soft rot), with an inhibitory effect as high as 84% ​​to 100%.

[0014] Patent CN117286050A discloses that Bacillus velezensis JSBV63 has an inhibition rate of 78% against the garlic leaf blight pathogen Stemphylium; Bacillus velezensis LXS-N2 has an inhibition efficiency of 76.02% against the ginseng pathogen Pythium ginseng (a member of the same genus as Stemphylium) in a plate confrontation experiment (Liu Ziyao et al., 2025), which is the highest inhibition rate that Bacillus velezensis can achieve against Stemphylium pathogens reported in the current research literature; similarly, in a related patent, Bacillus velezensis ZF438 has an inhibition rate of 70.51% against tomato Stemphylium in a plate confrontation experiment (Li Lei et al., 2023); CN114032182B A fungus that has both antagonistic effects on garlic root rot pathogens and growth-promoting functions, with an inhibition rate of 66-78% against common garlic pathogens; CN116286492A a strain of Paenibacillus polymyxa C2 and its application, with an inhibition rate of mainly 70-75%.

[0015] By comparing the inhibition rates, it can be seen that the inhibitory effect of the Bacillus Velez JR1A of the present invention on garlic disease bacteria is not only far superior to that of the same type of Bacillus Velez, but also far exceeds the inhibitory effect of all garlic disease inhibitory bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To construct phylogenetic trees of culturable bacteria (a) and fungi (b) based on 16S rRNA / ITS gene sequences; Figure 2 To isolate the strains and plate confrontation with garlic pathogenic fungi; Figure 3 is the mycelial inhibition rate of the rhizosphere isolated strains against garlic pathogenic fungi (%); Figure 4 The fermentation supernatant inhibited the mycelial growth of Fusarium T9; Figure 5 The inhibition rates (%) of fermentation supernatant on mycelium (a) and spore production (b) of Fusarium T9. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0018] The main instruments and equipment involved in the present invention are as follows: Table 1 name model factory Clean bench SW-CJ-2FD-11 Suzhou Antai Air Technology Co., Ltd. High-speed refrigerated centrifuge OSE-HC-02 Tiangen Biochemical Technology (Beijing) Co., Ltd. Shaking incubator ZQZY-980V Shanghai Zhichu Instrument Co., Ltd. Vertical laminar flow clean bench HCB-1300V Qingdao Haier Special Electrical Appliance Co., Ltd. Constant temperature incubator EM7KCGW3-NR Shanghai Yiheng Scientific Instrument Co., Ltd. Gel electrophoresis system Sub-Cell Bio-Rad Gel imaging system HVA-85 Sanyo Corporation Gradient PCR instrument K960 Beckman Coulter pH meter FE28 Methole-Toledo Instruments (Shanghai) Co., Ltd. Vertical high pressure steam sterilizer LDZX Shanghai Shen'an Medical Equipment Factory Intelligent inverted fluorescence microscope Leica DMI4000B Leica Micro-wave oven EM7KCGW3-NR Guangdong Midea Kitchen Appliance Manufacturing Co., Ltd. The culture medium formula involved in the present invention is as follows: (1) Bacterial screening culture medium formula R2A medium: 0.5 g / L soluble starch, 0.5 g / L casein hydrolysate, 0.3 g / L potassium hydrogen phosphate, 0.5 g / L yeast extract powder, 0.5 g / L peptone, 0.5 g / L glucose, 0.024 g / L anhydrous magnesium sulfate, 0.3 g / L sodium pyruvate, and 20 g / L agar powder.

[0019] LB medium: yeast extract 5.0 g / L, agar powder 20 g / L, sodium chloride 10.0 g / L, tryptone 10.0 g / L.

[0020] (2) Fungal screening culture medium formula PDA medium: potato extract powder 12.0 g / L, glucose 20.0 g / L, agar powder 20 g / L.

[0021] (3) Antagonistic culture medium formula Potato extract powder 12.0 g / L, sodium chloride 10.0 g / L, agar powder 20 g / L, tryptone 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L.

[0022] Example 1 Bacterial source and screening The bacterial strains involved in this invention were primarily screened from rhizosphere soil samples of fresh garlic stored at 4°C for one and ten years. The fungal strains were primarily screened from rhizosphere soil samples of fresh garlic stored at 4°C for ten years, as well as from plant tissue at the diseased-healthy junction of diseased garlic. (The soil and garlic were sourced from the Wenjiang Rice-Garlic Rotation Base in Chengdu, Sichuan Province.) (1) Isolation, purification, identification and preservation of bacterial strains Weigh 10 g of fresh soil sample and dissolve it in 90 mL of sterile water. Shake at 4°C and 150 rpm for 30 min to prepare a soil suspension. Aspirate 1 mL of the soil suspension and serially dilute it with sterile water to a concentration of 10⁻¹. Spread 100 μL of the serial dilutions evenly onto each culture plate. Repeat each serial dilution three times. Incubate the plates upside down at 30°C for 3–5 days. Select individual colonies with distinct morphological characteristics and re-streak them until a purified strain is obtained.

[0023] Single colonies from the purified plates were picked for PCR identification. Universal primers 27F (5'-AGAGTTTGATCCTGGCTCACAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACT-3') were used for bacterial amplification. The PCR reaction system and protocol settings are shown in Tables 2 and 3. The PCR stock solution was sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The resulting sequences were uploaded to the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for species identification by homology comparison. Purified single colonies were inoculated and cultured in the corresponding liquid medium until logarithmic phase. The mixture was mixed with 60% glycerol solution at a ratio of 1:2 by volume, aliquoted into cryovials, and stored at -80°C in an ultra-low temperature freezer until further use.

[0024] Table 2 Bacterial PCR reaction system Components Volume (μL) KOD One™ PCR Master Mix 10.0 <![CDATA[ddH2O]]> 8.0 Primer 1 0.5 Primer 2 0.5 DNA template 1.0 Total volume 20.0 Table 3 Bacterial PCR reaction procedures temperature time Number of cycles 95℃ 5 min 35 94℃ 30 s 35 53℃ 30 s 35 72℃ 1 kb / min 35 72℃ 5 min 35 4℃ ∞ 35 (2) Isolation, purification, identification and preservation of fungal strains Remove the diseased garlic leaf from the junction of the diseased and healthy parts and cut the diseased tissue into 5 mm x 5 mm pieces using a sterile blade. Soak the diseased tissue in 75% ethanol and 5% NaClO solution for 30 seconds, respectively. Finally, rinse three times with sterile water and transfer to sterile filter paper to dry. Place the diseased tissue on polydimethylsiloxane (PDA) medium supplemented with streptomycin (30 mg / mL), placing four pieces per dish. After inverted culture for 7 days, select the hyphae that have grown colonies and transfer them to PDA medium for purification. Soil dilution isolation is performed as described above for bacterial dilution isolation.

[0025] Mycelia were collected from the purified plates and identified by PCR using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system and protocol settings are shown in Tables 4 and 5. The PCR stock solution was sent to Beijing Qingke Biotechnology Co., Ltd. for ITS gene sequencing. The resulting sequences were uploaded to the NCBI website, and species information was determined by homologous sequence alignment. The color and morphology of the fungal colonies were observed and recorded. Mycelia were collected for slide preparation and microscopic examination of the morphological characteristics of the mycelia and spores. Mycelia from the purified fungal colonies were inoculated into slant test tubes and cultured in a 30°C incubator for 7 days before being stored in a 4°C refrigerator until further use.

[0026] Table 4 Fungal PCR reaction system Components Volume (μL) KOD One™ PCR Master Mix 10.0 <![CDATA[ddH2O]]> 8.0 Primer 1 0.5 Primer 2 0.5 DNA template 1.0 Total volume 20.0 Table 5 Fungal PCR reaction procedures temperature time Number of cycles 95℃ 5 min 35 94℃ 30 s 35 55℃ 30 s 35 72℃ 1 kb / min 35 72℃ 5 min 35 4℃ ∞ 35 (3) Screening results Using R2A and LB culture media, 150 bacterial strains were isolated and identified from the rhizosphere soil of 1-year-old and 10-year-old garlic, covering 6 phyla and 57 genera ( Figure 1 a). Among them, the most isolated strains were Bacillus, Pseudomonas, Pseudoxanthomonas, Lysobacter, Flavobacterium, Stenotrophomonas, Chryseobacterium, and Microbacterium. Bacillus was the most isolated in the rhizosphere soil, with a total of 13 strains of different species. A total of 13 fungal strains were isolated and identified, covering 2 phyla and 9 genera ( Figure 1 b) Fusarium and Stemphylium were the most commonly isolated strains. Fusarium was most abundant in the rhizosphere soil, while Stemphylium, Chaetomium, and Pleospora were most abundant in diseased tissues.

[0027] The isolated fungal strains were morphologically observed using culture plates and a microscope. Four common garlic pathogens, Fusarium foetens T9, Chaetomium globosum C1, Stemphylium eturmiunum C3, and Pleospora rosae-caninae C2, were isolated, along with a common beneficial fungus, Trichodermahamatum T4. Fusarium, Stemphylium, Chaetomium, and Pleospora are known garlic pathogens (CHEN et al., 2023; DEWI et al., 2025; LEE et al., 2024; REYES-TENA et al., 2024). Large-scale field infestations can cause garlic root rot and leaf blight. Fusarium T9 is the primary pathogen causing root diseases in the early growth phase of garlic, while Stemphylium C3 is the primary pathogen causing foliar diseases in the later growth phase.

[0028] Example 2 Inhibitory Effect of Microbial Cells on Garlic Pathogenic Fungi (1) Experimental methods Antagonism test between microorganisms and garlic pathogenic fungi: Isolated strains were inoculated into 5 mL of LB liquid medium and cultured at 30°C and 180 rpm to an OD600 of 1.00. A bacterial seed solution was obtained for later use. An 8 mm cake was taken from the purified fungus PDA plate using a hole punch and inoculated onto a PDA plate. The plate was then incubated upside down at 30°C for 7 days. For isolated bacteria, an 8 mm cake was taken from the pathogenic fungus plate and placed in the center of the antagonistic medium. Using sterile tweezers, filter paper discs were placed 2 cm from either side of the cake. Using a pipette, 4 μL of the bacterial seed solution was gently dripped onto the filter paper discs. A drop of ddH2O was used as a control (CK) control. This experiment was repeated three times for each group. For isolated fungi, an 8 mm cake was taken from the pathogenic fungus plate and placed on one side of the antagonistic medium. An 8 mm cake of the isolated fungus was placed 3 cm from the other side. A drop of ddH2O was used as a CK control. This experiment was repeated three times for each group. The opposing plates were incubated at 30°C for 7 days, and the mycelial growth inhibition rate was calculated. Mycelial growth inhibition rate (%) = (pathogenic fungal colony diameter in the control - pathogenic fungal colony diameter after confrontation) / pathogenic fungal colony diameter in the control × 100% (ZHOU et al., 2022b; ZHOU et al., 2023d).

[0029] Antagonism test between fermentation supernatants and garlic pathogenic fungi: The isolated bacterial seed liquid was inoculated into 100 mL of liquid LB medium and cultured at 30°C and 180 rpm until the logarithmic phase. After centrifugation at 4°C and 4000 rpm for 10 minutes, the supernatant was filtered through a 0.22 μm filter in a clean bench to obtain a sterile bacterial fermentation supernatant. The isolated fungal cake was inoculated into 100 mL of PDB medium and cultured at 30°C and 180 rpm for 7 days. After centrifugation at 4°C and 10,000 rpm for 20 minutes, the supernatant was filtered through a 0.22 μm filter in a clean bench to obtain a sterile fungal fermentation supernatant. The inhibitory effect of the fermentation supernatant on the mycelial growth of the pathogenic fungus was evaluated using the Oxford cup method. An 8 mm cake of the pathogenic fungus was placed in the center of the antagonist plate. 100 μL of fermentation supernatant was pipetted into each well of an Oxford cup 2 cm away. ddH₂O was added as a CK control. The plates were incubated at 30°C for 7 days, and the mycelial growth inhibition rate was calculated.

[0030] Add more than 20 mL of fermentation supernatant to a 250 mL Erlenmeyer flask containing 30 mL of PDB liquid medium. Next, place an 8 mm piece of bacterial cake from the pathogen culture plate into the culture medium. ddH2O was added as a control. All treatments were incubated at 30°C and 180 rpm for 7 days. After incubation, appropriate amounts of culture medium were collected to prepare slides. The pathogen spore concentration and sporulation inhibition rate were calculated using a hemocytometer under a microscope. Sporulation inhibition rate (%) = (pathogen spore concentration in control - pathogen spore concentration after co-culture) / pathogen spore concentration in control × 100%.

[0031] (2) Inhibition results The 150 microorganisms isolated in Example 1 were subjected to a plate confrontation experiment with garlic pathogenic fungi. The inhibitory effects of the microorganisms with antagonistic functions on garlic pathogenic fungi were as follows: Figure 2 The microbial groups that showed inhibitory effects were mainly concentrated in Bacillus, Streptomyces, Paenibacillus and Trichoderma. Among them, Bacillus velezensis JR1A, Paenibacillus peoriae BR40, Streptomyces hydrogenans BR52, Streptomyces vinaceus BR3 and Trichoderma hamatumT4 all showed general inhibitory effects on three garlic pathogens, Fusarium T9, Chaetomium C1 and Stemphylium C3. Most of the other rhizosphere microorganisms and key microbial groups did not show obvious inhibitory effects on garlic pathogens.

[0032] like Figure 3JR1A exhibited the best pathogen inhibition. Its inhibition rates against Fusarium T9 were 84.41%, against Chaetomium C1 were 90.87%, and against Stemphylium C3, reaching almost 100%, significantly higher than those of the other isolates (P < 0.05). Paenibacillus BR40 exhibited inhibition rates of 48.52%, 55.08%, and 60.83% against the three pathogen genera, respectively. Streptomyces BR52 and BR3 exhibited inhibition rates of 43.63%, 35.22%, 49.25%, 50.75%, and 65.29%, and 48.7%, respectively. Trichoderma T4 (a common beneficial fungus) exhibited inhibition rates of 48.24%, 66.44%, and 61.70%, respectively, against the three pathogen genera.

[0033] Although the plate confrontation can directly show the inhibitory effect of the strains on pathogenic fungi, there is an impact that the strains compete for living space and nutrients. In order to eliminate this effect, the present invention continues to explore the inhibitory effect of the sterile fermentation supernatant of the above strains JR1A, BR40, BR52 and T4 on garlic pathogenic fungi. Figure 4 The fermentation supernatant of JR1A inhibited the mycelial growth of Fusarium T9 by 34.06%. However, the fermentation supernatants of BR40, BR52 and T4 strains had almost no inhibitory effect on the mycelial growth of Fusarium T9 ( Figure 5 a). Next, we will continue to explore the effect of fermentation supernatant on the production of pathogenic fungal spores. Figure 5 b. Similar to the results of hyphae inhibition, the fermentation supernatant of JR1A inhibited Fusarium T9 spore production by 74.06%. However, the fermentation supernatants of BR40, BR52, and T4 strains showed little inhibition on Fusarium T9 spore production.

[0034] In addition, the present invention found through phosphate solubilization function assay and siderophore production function assay that JR1A has the ability to solubilize phosphate and produce siderophore, and can promote plant growth.

[0035] The foregoing description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Rather, the present invention can be used in various other combinations, modifications, and environments and can be modified within the scope of the concept described herein through the above teachings or techniques or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention are intended to be protected by the appended claims.

Claims

1. A strain of Bacillus velezensis JR1A, characterized in that: The Bacillus velezensis JR1A was deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of June 30, 2025, and a deposit number of CGMCC No. 35059.

2. A microbial agent, characterized in that: The method comprises at least one of the bacterial cells, spores and fermentation liquid of the Bacillus velezensis JR1A according to claim 1.

3. Use of the Bacillus Velez JR1A according to claim 1 for inhibiting pathogenic fungi of Liliaceae plants.

4. The use according to claim 3, characterized in that The lily family plant is garlic.

5. The use according to claim 4, characterized in that The pathogenic fungi include at least one of Fusarium odoratum, Chaetomium globosum and Sclerotium zeae.

6. A method for preventing and controlling garlic diseases, characterized in that: The method comprises the following steps: applying the Bacillus Velez subtilis according to claim 1 or the microbial agent according to claim 2 to pathogenic bacteria, the above-ground parts of plants or the growth environment of plant roots.

7. A method for preventing and controlling garlic diseases according to claim 6, characterized in that: The garlic diseases include at least one of root rot, leaf blight, rust and soft rot.

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