Bacillus velezensis yndh2404 and application thereof

Abstract

The present application belongs to the technical field of microorganism and plant disease control, and discloses a bacillus velezensis YNDH2404 (Bacillus velezensis YNDH2404) Bacillus velezensis ), with a preservation number of CCTCC NO: M 20242825; and the application of the bacillus velezensis or its fermentation liquor is also disclosed. Experiments prove that the bacillus velezensis and fermentation products thereof isolated and identified by the present application have a significant inhibitory effect on coffee variegation caused by the short hair paint spot fungus, konjak variegation, coffee anthracnose caused by the anthracnose fungus, and coffee stem rot caused by the corchiferous fusarium. The biocontrol agent prepared by using the present application does not cause pollution to the ecological environment in the use process, is non-polluting, can reduce the use amount of other chemical pesticides, and reduces the prevention and control cost of coffee and konjak diseases, thereby providing protection for creating a coffee and konjak "green food brand", and has good popularization value.

Description

Technical Field

[0001] This invention belongs to the field of microbial and plant disease control technology, and discloses a strain of Bacillus belye YNDH2404 and its application. Background Technology

[0002] Bacillus belesiensis ( Bacillus velezensis *Bacillus belyssae* is a new species of Bacillus whose metabolites possess broad-spectrum antibacterial activity. It plays an important role in feed, plant protection, medicine, food industry, forestry, and wastewater treatment. It can inhibit the growth of bacteria, fungi, viruses, nematodes, and other pathogens, and can also increase crop yields, making it a research hotspot in recent years. Studies have shown that *Bacillus belyssae* is effective against various plant fungal diseases and promotes plant growth, including in tomatoes, peppers, rapeseed, tobacco, peanuts, and grapes. Existing research indicates that *Bacillus belyssae* can inhibit plant diseases such as pepper blight, peanut fruit rot, gray mold and sclerotinia rot in leafy vegetables, powdery mildew in red mustard and Chinese mustard, damping-off in ginseng, brown rot in peach trees, verticillium wilt in tomatoes, root rot in poplar trees, anthracnose in loquat, leaf spot in mung beans, anthracnose fungus in coffee, *Pseudomonas stolonifera* leaf spot fungus in coffee, and brown spot fungus in coffee.

[0003] coffee( Coffea L. Coffee is an important tropical crop, belonging to the genus Coffea in the Rubiaceae family. There are approximately 124 species, but only three are truly commercially viable: Arabica coffee (Coffea argyrophylla). Coffea arabica ), medium-sized coffee ( C. canephora ) and large-bean coffee ( C. liberica In 2024, the national coffee planting area was 81,300 hectares. 2 With a total output of 152,300 tons, Yunnan ranks 13th among global coffee-producing countries, with a total agricultural output value of 6.095 billion yuan; of which, Yunnan has a total planting area of ​​79,500 hectares. 2Yunnan Province, accounting for 97.79% of the national total and ranking first in the country, boasts a total coffee agricultural output value of 6.01 billion yuan, making it a pillar industry in the tropical region of Yunnan. The main variety cultivated is Arabica coffee. Konjac, belonging to the Araceae family and the Amorphophallus genus, is rich in a high-molecular-weight polysaccharide—glucomannan—which possesses functions such as weight loss, lipid reduction, blood sugar reduction, insulin resistance reduction, and immune enhancement. It has high development and utilization value in the fields of medicine, food, and cosmetics. In particular, the concept of "low-carbon and green" makes it a sustainable plant resource. Dehong Prefecture in Yunnan Province has a subtropical monsoon climate with abundant annual rainfall, suitable for konjac cultivation. In 2022, the planting area of ​​bulbils konjac in the region reached 666.7 hectares, making it one of the pillar industries for rural revitalization in border areas. Global climate change-induced biological and abiotic stresses, along with large-scale monoculture of crops, have reduced crop resistance. These adverse environmental factors have exacerbated the occurrence and spread of crop diseases, with foliar fungal pathogens seriously affecting the yield and health of economic crops. In recent years, the types of coffee diseases have been on the rise, with some minor diseases becoming major ones, such as coffee lacquer spot, which has become a major disease in nurseries.

[0004] *Myrothecium* is a new genus established by Lombard et al. in 2016. Morphologically, *Myrothecium* is distinguished from *Myrothecium* and other *Myrothecium-like* species by the presence of thin-walled bristles with 1-3 septa around the conidiomata. *Myrothecium* commonly causes leaf spot, wilt, stem canker, and fruit rot in various vegetables, ornamental plants, and economic crops. In recent years, *Myrothecium brevifolium* has also been observed. Paramyrothecium breviseta The resulting lacquer spot disease has been reported in coffee and konjac growing areas. It has occurred in nurseries across various coffee-producing regions of Yunnan, with an incidence rate as high as 40%. Symptoms include: initially, water-soaked spots appear on the leaf surface. Under high temperature and humidity, the spots expand rapidly. After 3-5 days, nearly circular lesions appear with a grayish-white to brown center and a dark brown edge, exhibiting typical concentric rings of alternating light and dark shades. The lesions are similar on both the upper and lower surfaces, with no obvious difference. The lesions are sunken and easily ruptured. A distinct yellow halo appears at the boundary between diseased and healthy tissue on the affected leaves. After spore formation, small granules of white mycelium, which are conidiophores, are found at the concentric rings. In later stages, the lesions spread to the entire leaf and even the stem, causing leaf drop. In severe cases, the entire coffee plant dies. This fungus can infect not only coffee leaves and young stems but also fresh coffee berries. The disease can also damage the leaves and petioles of konjac. Statistics show that in April 2022, the incidence rate of *Amorphophallus titanum* seedlings in greenhouses in Ruili City was 5-8%, and the incidence rate after transplanting to the field was 10%. Therefore, research on the prevention and control of lacquer spot disease in coffee and konjac has significant economic value and social benefits.

[0005] The results of the existing researches show that the inhibition rate of Bacillus velezensis MC2-1 on Xanthomonas campestris (Xbd1) is 55.8%, and the results of the researches of Liang Yanqiong et al. show that the inhibition rates of Bacillus velezensis BS2C on Xanthomonas campestris (CCG3), Botryodiplodia theobromae (BEC48) and Phomopsis sp. (FS4-1) are 65.08%, 78.3% and 55.60% respectively, the preventive effects of the Bacillus velezensis BS2C on the detached leaves are that the spot inhibition rates of "BS2C+CCG3", "BS2C+FS4-1" and "BS2C+BEC48" are 70.40%, 66.86% and 67.39% respectively, and the treatment effects of the Bacillus velezensis BS2C on the detached leaves are that the spot inhibition rates of "CCG3+BS2C", "FS4-1+BS2C" and "BEC48+BS2C" are 55.68%, 53.03% and 56.52% respectively. According to the re-search, the Bacillus velezensis has not been reported to prevent and treat the coffee leaf spot disease and the taro leaf spot disease. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a Bacillus velezensis and an application thereof.

[0007] The technical scheme of the present application is as follows:

[0008] A Bacillus velezensis YNDH2404 ( Bacillus velezensis The strain YNDH2404 is preserved in the China Center for Type Culture Collection on December 16, 2024, and the preservation number is CCTCC NO: M20242825.

[0009] The present application also protects the application of the Bacillus velezensis or the fermentation liquor thereof in preparing any one of the following products:

[0010] 1) inhibiting the short-hair Phomopsis sp. and the conidial spore germination thereof;

[0011] 2) preventing and treating the disease caused by the short-hair Phomopsis sp.;

[0012] 3) inhibiting the anthracnose pathogen and the conidial spore germination thereof;

[0013] 4) preventing and treating the disease caused by the anthracnose pathogen;

[0014] 5) inhibiting the Fusarium solani and the conidial spore germination thereof;

[0015] 6) preventing and treating the disease caused by the Fusarium solani.

[0016] Further, the disease caused by the short-hair Phomopsis sp. is the coffee leaf spot disease or the taro leaf spot disease, the disease caused by the cocoa anthracnose fungus or the anthracnose fungus is the coffee anthracnose disease, and the disease caused by the Fusarium solani is the coffee stem rot disease.

[0017] Further, the Bacillus velezensis fermentation liquid is obtained by the following steps: inoculating the preserved Bacillus velezensis strain YNDH2404 on a solid NA culture medium plate, culturing by streaking at 30 DEG C in a constant temperature incubator for 1 day, inoculating the activated biocontrol bacterial cake into a liquid NA culture solution, and culturing at 30 DEG C and 180 r / min in a shaking incubator for 3 days, and filtering the culture solution with 4 layers of sterile gauze.

[0018] The application also protects a biocontrol bacterium, which has the active ingredient of the Bacillus velezensis YNDH2404 or the fermentation liquid of the Bacillus velezensis YNDH2404.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] The Bacillus velezensis YNDH2404 provided by the application can prevent and control various diseases caused by Leptographium brevifilum, such as coffee variegation and konjak variegation, can reduce the dependence on pesticides, improve the yield and quality of crops, and reduce the negative impact on the environment, and has wide application potential in the fields of agriculture and environment. Experiments prove that the treatment effect of the Bacillus velezensis YNDH2404 provided by the application on coffee variegation and konjak variegation reaches 95.12% and 82.22% respectively, and the prevention effect reaches 96.96% and 87.85% respectively. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a colony morphology diagram of the strain YNDH2404 cultured on the NA culture medium;

[0022] Figure 2 It is a scanning electron microscope diagram of the strain YNDH2404;

[0023] Figure 3 It is a phylogenetic tree of the strain YNDH2404 constructed based on the 16s rRNA gene sequence;

[0024] Figure 4 It is a comparison diagram of the inhibition effect of the strain YNDH2404 on coffee Leptographium brevifilum strain MB-5, wherein Figure 4 A is an inhibition effect diagram of YNDH2404 on coffee Leptographium brevifilum strain MB-5, Figure 4 B is a control group;

[0025] Figure 5 It is a comparison diagram of the inhibition effect of the strain YNDH2404 on konjak Leptographium brevifilum strain M1, wherein Figure 5 A is an inhibition effect diagram of YNDH2404 on konjak Leptographium brevifilum strain M1, Figure 5 B is a control group;

[0026] Figure 6 Figure 4 is a comparison chart of the inhibitory effect of strain YNDH2404 on Colletotrichum coffeicola strain YB-6, wherein Figure 6 A is a graph of the inhibitory effect of YNDH2404 on Colletotrichum coffeicola strain YB-6, Figure 6 B is a control group;

[0027] Figure 7 Figure 5 is a comparison chart of the inhibitory effect of strain YNDH2404 on Colletotrichum gloeosporioides strain YD-4, wherein Figure 7 A is a graph of the inhibitory effect of YNDH2404 on Colletotrichum gloeosporioides strain YD-4, Figure 7 B is a control group;

[0028] Figure 8 Figure 6 is a comparison chart of the inhibitory effect of strain YNDH2404 on Fusarium solani f. sp. coffeae strain YM-4, wherein Figure 8 A is a graph of the inhibitory effect of YNDH2404 on Fusarium solani f. sp. coffeae strain YM-4, Figure 8 B is a control group;

[0029] Figure 9 Figure 7 is a comparison chart of the inhibitory effect of strain YNDH2404 on conidial germination (conidial NA liquid suspension, conidial YNDH2404 fermentation liquid suspension, conidial pure water suspension at 0h, 3h, 6h, 12h, 24h) of Mycosphaerella tashiroi strain MB-5;

[0030] Figure 10 Figure 8 is a comparison chart of the inhibitory effect of strain YNDH2404 on conidial germination (conidial NA liquid suspension, conidial YNDH2404 fermentation liquid suspension, conidial pure water suspension at 0h, 3h, 6h, 12h, 24h) of Mycosphaerella tashiroi strain Ml;

[0031] Figure 11 Figure 9 is a comparison chart of the inhibitory effect of strain YNDH2404 on conidial germination (conidial NA liquid suspension, conidial YNDH2404 fermentation liquid suspension, conidial pure water suspension at 0h, 3h, 6h, 12h, 24h) of Colletotrichum coffeicola strain YB-6;

[0032] Figure 12 Figure 10 is a comparison chart of the inhibitory effect of strain YNDH2404 on conidial germination (conidial NA liquid suspension, conidial YNDH2404 fermentation liquid suspension, conidial pure water suspension at 0h, 3h, 6h, 12h, 24h) of Colletotrichum gloeosporioides strain YD-4;

[0033] Figure 13Figure for comparison of inhibition effect of YNDH2404 on conidia germination of Fusarium solani f. sp. coffeae strain YM-4 (conidia NA liquid suspension, conidia YNDH2404 fermentation liquid suspension, conidia pure water suspension 0h, 3h, 6h, 12h, 24h)

[0034] Figure 14 Figure for comparison of potting prevention and treatment effect of YNDH2404 on coffee Figure 14 A is the result figure of inoculating strain MB-5, Figure 14 B is the result figure of inoculation (MB-5+YNDH2404), Figure 14 C is the result figure of inoculation (YNDH2404+MB-5), Figure 14 D is the spread figure of part of leaf infected by inoculating strain MB-5, Figure 14 E is the spread figure of infected leaf of inoculation (MB-5+YNDH2404), Figure 14 F is the spread figure of infected leaf of inoculation (YNDH2404+MB-5);

[0035] Figure 15 Figure for comparison of potting prevention and treatment effect of YNDH2404 on konjac Figure 15 A is the result figure of inoculating strain M1 on konjac, Figure 15 B is the result figure of inoculation (M1+YNDH2404), Figure 15 C is the result figure of inoculation (YNDH2404+M1). DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments, but the present application is not limited to the following technical solutions.

[0037] Coffee Oligoporus luteolus strain MB-5, konjac Oligoporus luteolus strain M1, coffee Colletotrichum cacao strain YB-6, coffee Colletotrichum gloeosporioides strain YD-4, coffee Fusarium solani f. sp. coffeae strain YM-4 are preserved in Yunnan Dehong Institute of Tropical Agricultural Sciences, address: No. 29, Ruijing Road, Ruili City, Dehong Prefecture, Yunnan Province, contact number: 15096646750, contact person: Wang Chunmei.

[0038] Example 1: Obtaining and identifying strains

[0039] 1. Isolation of candidate strains from healthy soil

[0040] (1) Isolation and purification of strains

[0041] Strain YNDH2404 was derived from a soil sample collected from the rhizosphere of a healthy coffee plant in a rubber-coffee intercropping test base of the Yunnan Dehong Tropical Agricultural Science Institute. The specific sampling method was as follows: using a five-point sampling method, 0-20 cm soil was collected using a soil collector, and each treatment was repeated three times. The soil attached to the coffee roots was collected, impurities (roots, stones, etc.) were removed, the collected soil samples were mixed and sealed in sterile self-sealing bags, and then placed in a portable refrigerator and taken back to the laboratory, and stored at -20°C for standby.

[0042] Strain isolation: 10 g of soil sample was weighed and added to a flask containing 90 mL of sterile water, and the soil suspension was prepared by shaking and mixing at 30°C. Using the dilution method, the soil suspension was diluted by 10 times in gradient, and 100 μL of the soil suspension with dilution times of 10 -7 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 was taken with a pipette gun and plated on NA plates, respectively, and incubated at 30°C for 24 h. Different morphological single colonies were picked and purified, and then stored in 50% sterile glycerol (glycerol and NA liquid 1:1 mixture) at -80°C for standby.

[0043] The strain YNDH2404 obtained by the above method was cultured on NA solid medium to form round, milky white, opaque colonies, with a raised center, wavy edges, rough surface, and dry, as shown in the colony morphology of the strain YNDH2404. Figure 1 Gram staining showed that the strain YNDH2404 was positive, and the bacteria were observed under a light microscope under a scanning electron microscope (Cai Si Sigma300, Germany) to be rod-shaped with blunt ends, as shown in the scanning electron micrograph of the strain YNDH2404. Figure 2

[0044] (2) Identification of the strain

[0045] Strain YNDH2404 was inoculated into NA liquid medium and incubated at 30°C, 180 r / min for 16-20 h. 1 mL of bacterial suspension was centrifuged at 12000 r / min for 1 min, and the bacterial cells were collected. The DNA of the strain YNDH2404 was extracted using a bacterial genomic DNA extraction kit (BioTeke), and PCR amplification was performed. The PCR amplification product was outsourced to Beijing Qikang Biotechnology Co., Ltd. Kunming Branch for first-generation sequencing, and the DNA sequence is shown as SEQ ID NO: 1.

[0046] ​After the gene sequences were processed and spliced by ContingExpress software, the homology alignment of the sequences was performed on the NCBI (http: / / www.ncbi.nlm.nih.gov) website by using Blastn; the Neighbor-Joining (NJ) phylogenetic tree was constructed by using MEGA11.0 software, and 1000 bootstrap cycles were sampled to determine the classification status of the isolated strain YNDH2404.

[0047] The results show that the sequence similarity of the strain YNDH2404 with the strain Bacillus velezensis is 100%. The related strain sequences are downloaded, and the phylogenetic tree based on the 16s rRNA gene sequence is constructed (see Figure 3 for details), and the results show that the strain is closest to Bacillus velezensis B. velezensis , and clusters in the same branch.

[0048] According to the culture characteristics, morphological characteristics and 16S rDNA sequence clustering analysis results of the strain YNDH2404, it is determined that the strain is Bacillus velezensis B.velezensis . The strain was preserved in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on December 16, 2024, and the preservation number is CCTCC NO: M 20242825.

[0049] Example Two Inhibition of Bacillus velezensis YNDH2404 on Myceliophthora vermiculata, Colletotrichum coffeatum and Fusarium solani

[0050] 1. Preparation of coffee Myceliophthora vermiculata strain MB-5 P. breviseta , konjac Myceliophthora vermiculata strain M1 P. breviseta , coffee Colletotrichum coffeatum strains YB-6 Colletotrichum theobromicola and YD-4 C. fructicola , and coffee Fusarium solani strain YM-4 Fusarium solani) : The filter paper pieces containing the strains MB-5, M1, YB-6, YD-4 and YM-4 were picked and placed on PDA (200g peeled potatoes, 20g glucose, 15g agar powder, 1000mL water) medium, and cultured in a 25℃ incubator until the mycelium covered the culture dish, and then used.

[0051] 2. Preparation of strain YNDH2404: Bacillus velezensis B.velezensisStrain YNDH2404 was streaked on NA (beef extract 3g, peptone 10g, agar 15g, sodium chloride 5g, water 1000ml, pH 7.0±0.1) solid medium and incubated in a 30℃ incubator. When the colonies grew over the streak, they were ready for use.

[0052] 3. The antibacterial effect was determined by the flat plate confrontation method. Specifically, the colony edge of strains MB-5, M1, YB-6, YD-4, and YM-4 cultured on PDA plates was punched with a sterilized puncher with a diameter of 5mm, and the fungus cake was inoculated in the middle of the PDA medium plate. Strain YNDH2404 cultured on NA plates was inoculated at a distance of 2cm from the edge of the plate (9cm) to the center, with 4 points per dish. The plate inoculated with strain YNDH2404 was placed in an incubator (25℃) for 7d to observe the colony morphology of strains YB-6, YD-4, and YM-4, and 14d to observe the colony morphology of strains MB-5 and M1. The colony diameter was measured by the cross method. The control group was the PDA plate inoculated with only the fungus cake of strains MB-5, M1, YB-6, YD-4, and YM-4. The culture was set up in 5 replicates. The calculation formula of the inhibition rate was: inhibition rate (%) = (control pathogenic fungus colony diameter - treatment pathogenic fungus colony diameter) / control pathogenic fungus colony diameter x 100%.

[0053] The experimental results are shown in Table 1: the average colony diameter of strain MB-5 in the treatment group was 1.5cm, and the average colony diameter of strain MB-5 in the control group was 7.7cm. The average inhibition rate was 80.52% after calculation; the average colony diameter of strain M1 in the treatment group was 1.7cm, and the average colony diameter of strain M1 in the control group was 7.5cm. The average inhibition rate was 77.33% after calculation; the average colony diameter of strain YB-6 in the treatment group was 2.3cm, and the average colony diameter of strain YB-6 in the control group was 8.5cm. The average inhibition rate was 72.94% after calculation; the average colony diameter of strain YD-4 in the treatment group was 2.3cm, and the average colony diameter of strain YD-4 in the control group was 8.3cm. The average inhibition rate was 72.29% after calculation; the average colony diameter of strain YM-4 in the treatment group was 3.0cm, and the average colony diameter of strain YM-4 in the control group was 6.1cm. The average inhibition rate was 50.82% after calculation. From the above results, it can be seen that strains MB-5, M1, YB-6, YD-4, and YM-4 all have a certain inhibitory effect on strain YNDH2404. Figures 4-8It can be seen that the growth of strains MB-5, M1, YB-6, YD-4 and YM-4 in the treatment group is obviously inhibited, and the colony size is significantly smaller than that of the control group, and the conidia produced by strains MB-5 and M1 are significantly less than those of the control group, which indicates that the strain YNDH2404 has a strong inhibitory effect on the short hair-like Kuehneria, Colletotrichum, and Fusarium solani, and has a good prevention and control effect on coffee short hair-like Kuehneria and konjac Kuehneria, coffee cocoa anthracnose, and coffee fruit anthracnose. It is found through comparison that the inhibition rates of the Bacillus velezensis YNDH2404 on the coffee anthracnose YB-6 and YD-4 are 72.94% and 72.53% respectively, which are higher than the inhibition rates of the Bacillus velezensis MC2-1 (55.8%) and BS2C (65.08%) on the coffee anthracnose.

[0054] Table 1 Inhibitory effect of the strain YNDH2404 on coffee pathogenic bacteria (MB-5, YB-6, YD-4, YM-4) and konjac pathogenic bacteria M1

[0055]

[0056] Example Three Influence of the Bacillus velezensis YNDH2404 on the conidium germination of Kuehneria breviapicis, Colletotrichum and Fusarium solani

[0057] 1. Preparation of the biocontrol bacterial liquid, the strain YNDH2404 prepared in Example Two is inoculated into a liquid NA culture solution, and is cultured at 30 DEG C and 180 r / min on a shaking table for 3 days; the culture solution is filtered through 4 layers of sterile gauze to obtain a strain fermentation liquid, at this time, the value of OD 600 is 13.

[0058] 2. Two groups of treatments are set, one of which is a control. The first treatment is a NA (beef extract 3g, peptone 10g, agar 15g, sodium chloride 5g, water 1000 mL, pH value 7.0+ / -0.1) liquid, and the second treatment is a strain YNDH2404 fermentation liquid, and the control is sterile pure water. The strains MB-5, M1, YB-6, YD-4 and YM-4 cultured on PDA culture medium are respectively added with the NA liquid, the YNDH2404 fermentation liquid and the sterile pure water to wash down the conidia, and the conidia are filtered through 4 layers of sterile gauze to obtain a pathogenic bacterial conidium NA liquid suspension, a pathogenic bacterial conidium YNDH2404 fermentation liquid suspension and a pathogenic bacterial conidium pure water suspension, and the concentrations are all adjusted to 1x10 5The conidial suspension was dropped on hydrophobic glass slides, and the conidial germination rate was observed at 3, 6, 12, and 24 h. Each conidial suspension was biologically repeated three times, and each time was technically repeated three times. The experimental results were statistically analyzed by SPSS 26.0 (statistical Product and Service Solutions) software.

[0059] The experimental results: The conidial suspension of YNDH2404 of strains MB-5, M1, YB-6, and YD-4 did not germinate until 24 h. The conidial germination rates of the strain MB-5 in the pure water suspension were 0%, 5%, 10%, and 14% at 3, 6, 12, and 24 h, respectively, and the conidial germination rates in the NA liquid suspension were 0%, 4%, 89%, and 100%, respectively. The conidial germination rates of the strain M1 in the pure water suspension were 0%, 5%, 8%, and 16% at 3, 6, 12, and 24 h, respectively, and the conidial germination rates in the NA liquid suspension were 0%, 5%, 88%, and 100%, respectively, as shown in Table 2, Figures 9-10 ); the conidial germination rates of the strain YB-6 in the pure water suspension were 0%, 1%, 14%, and 46% at 3, 6, 12, and 24 h, respectively, and the conidial germination rates in the NA liquid suspension were 0%, 98%, 100%, and 100%, respectively. The conidial germination rates of the strain YD-4 in the pure water suspension were 0%, 11%, 81%, and 95% at 3, 6, 12, and 24 h, respectively, and the conidial germination rates in the NA liquid suspension were 0%, 88%, 100%, and 100%, respectively, as shown in Table 3, Figures 11-12 ); the conidial germination rates of the strain YM-4 in the pure water suspension were 14%, 59%, 86%, and 100% at 3, 6, 12, and 24 h, respectively, and the conidial germination rates in the NA liquid suspension were 38%, 84%, 100%, and 100%, respectively. The conidial germination rates of the strain YNDH2404 in the fermentation liquid suspension were 0%, 0%, 4%, and 39% at 3, 6, 12, and 24 h, respectively, as shown in Table 4, Figure 13 ). It is shown that the fermentation liquid of the strain YNDH2404 has a strong inhibitory effect on the strains MB-5 and M1 of the short-haired Gonytrichia, the strains YB-6 and YD-4 of the anthracnose, and an inhibitory effect on the strain YM-4 of the Fusarium solani. It is also shown that the NA liquid promotes the conidial germination of the pathogenic bacteria.

[0060] Table 2 Effects of different suspension liquids on the conidial germination of the strains MB-5 and M1 of the short-haired Gonytrichia

[0061]

[0062] Note: The same column in the table indicates a significant difference (p < 0.05), and the same below.

[0063] The inventors also set the OD 600The values are 9, 7.5, 4.5, and the effect of YNDH2404 fermentation broth on the germination of conidia of Mycosphaerella coffeicola strain MB-5 is observed, and the results show that there is no germination.

[0064] Table 3 Effect of different suspension liquids on the germination of conidia of C. arachidicola strains YB-6 and YD-4

[0065]

[0066] Table 4 Effect of different suspension liquids on the germination of conidia of F. solani strain YM-4

[0067]

[0068] Example 4: Prevention and treatment effect of Bacillus velezensis YNDH2404 on coffee leaf spot and konjac leaf spot caused by Mycosphaerella coffeicola (potting)

[0069] 1. Preparation of biocontrol bacterial liquid, same as Example 3.1.

[0070] 2. Preparation of conidial suspension of Mycosphaerella coffeicola strain MB-5 and Mycosphaerella konjac strain M1: The strains MB-5 and M1 cultured on PDA medium were washed with NA liquid to obtain conidia (the biocontrol bacterial liquid was cultured with NA liquid), and the conidial suspension was obtained by filtering with 4 layers of sterile gauze, and the concentration was adjusted to 1x10 6 / mL for standby.

[0071] 3. Potting prevention and treatment effect determination method: 3 treatments were set, ① conidial suspension of strains MB-5 and M1 was sprayed and inoculated on coffee and konjac leaves, respectively, as a control; ② coffee and konjac leaves were sprayed and inoculated with conidial suspension of strains MB-5 and M1, respectively, and 24 h later, biocontrol bacterial strain YNDH2404 liquid was sprayed and inoculated (marked as MB-5+YNDH2404 / M1+YNDH2404); ③ biocontrol bacterial strain YNDH2404 fermentation broth was sprayed and inoculated on coffee and konjac leaves, and 24 h later, conidial suspension of strains MB-5 and M1 was sprayed and inoculated (marked as YNDH2404+MB-5 / YNDH2404+M1); (② treatment test: post-inoculation treatment; ③ prevention test: pre-inoculation treatment). 5 seedlings for each group (coffee variety: Dehe 716, seedling age: 3 months; konjac variety: Zhuyajin konjac, seedling age: 1 month, and the seedlings had basically the same growth), and the leaves were sprayed to cover the front and back surfaces with uniform droplets. Each treatment was repeated 3 times. 10 days after inoculation, the disease conditions were investigated, the coffee and konjac leaf spot disease grading standards are shown in Table 5, the disease index was calculated according to formula (1), and the prevention and treatment effect was calculated according to formula (2).

[0072] Table 5 Coffee, konjac leaf disease grading standards

[0073]

[0074] Formula (1)

[0075] Formula (2)

[0076] The experimental results are as follows: after inoculation for 10 days, the disease incidence is investigated, the disease index of coffee leaves inoculated with strain MB-5 conidial suspension is 85.31, the disease index of konjac leaves inoculated with strain M1 conidial suspension is 100, the disease index of coffee leaves inoculated with (MB-5+YNDH2404) is 4.16, the control effect is 95.12%, the disease index of konjac leaves inoculated with (M1+YNDH2404) is 17.78, the control effect is 82.22%, the disease index of coffee leaves inoculated with (YNDH2404+MB-5) is 2.59, the prevention effect is 96.96%, and the disease index of konjac leaves inoculated with (YNDH2404+M1) is 12.15, the prevention effect is 87.85%. It is shown that the bacillus velezensis YNDH2404 has a good control effect on coffee leaf spot disease and konjac leaf spot disease caused by Leptographium brevipes.

[0077] Table 6 Control effect of bacillus velezensis YNDH2404 on coffee and konjac leaf spot diseases caused by Leptographium brevipes

[0078]

[0079] In conclusion, the bacillus velezensis YNDH2404 provided in the application has a good control effect on coffee leaf spot disease and konjac leaf spot disease caused by Leptographium brevipes.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis YNDH2404, characterized in that: The strain YNDH2404 was deposited in China Center for Type Culture Collection on December 23, 2024, and the deposit number is CCTCC NO.M20242825.

2. The use of the Bacillus velezensis according to claim 1 in the preparation of any one of the following products: 1) inhibiting the germination of Lecythophora hoffmannii and its conidia; 2) preventing the disease caused by Lecythophora hoffmannii.

3. Use according to claim 2, wherein: The disease caused by Lecythophora hoffmannii is coffee leaf spot or konjak leaf spot.

4. A biocontrol bacterium, characterized in that: The active ingredient of the biocontrol agent comprises the Bacillus velezensis YNDH2404 according to claim 1.

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