Bacillus and application thereof

Abstract

The invention discloses bacillus and application thereof, and belongs to the technical field of microorganisms, the bacillus is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20242290, and the preservation date is October 22, 2024. The composition can effectively inhibit vegetative growth and vegetative propagation of pathogenic bacteria such as colletotrichum gloeosporioides and paecilomyces japonicus, and has a good and broad-spectrum control effect on various pathogenic fungi. Bacteriostatic components contained in fermentation filtrate of the bacillus can remarkably inhibit germination of colletotrichum spruce spores and formation of appressorium. And the bacillus has a good prevention and treatment effect on the citrus huanglongbing.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus and its application. Background Art

[0002] Fusarium wilt of Chinese fir caused by Colletotrichum gloeosporioides is a common disease in Chinese fir planting areas. Almost all places where Chinese fir grows may be invaded by this disease, especially in artificial young forests in low mountain and hilly areas. Its pathogen is Glomerella cingulata, belonging to Ascomycetes, Sphaeriales fungi. This disease mainly harms the leaves and branches of Chinese fir, usually occurring in spring and early summer, which is just the germination period of new shoots of Chinese fir. The symptoms are that the tips of new shoots turn brown and wither, and in severe cases, it extends downward until the needles completely wither. Many small black dots can be seen on the withered needles, especially obvious on the stomatal bands on the back of the needles. When the humidity is too high, light pink spore masses can also be seen on the small black dots. Under natural conditions, this disease has a latent infection phenomenon, that is, it infects in autumn and only develops in the following spring. Generally, it starts to develop in early April, reaches the peak from late April to early May, stops after June, and there is also a small amount of development on the yellowed new shoots in autumn.

[0003] Huanglong disease of citrus mainly harms citrus fruit trees and is not a disease of Chinese fir, but it is representative in the field of plant disease research. Its pathogen is Candidatus Liberibacter, belonging to Gram-negative bacteria. This bacterium parasitizes in the phloem of citrus fruit trees and cannot be artificially cultured at present, but can be detected by scientific methods. Huanglong disease can cause symptoms such as yellowing of citrus fruit tree leaves, weakening of tree vigor, and fruit deformity, seriously affecting the yield and quality of citrus. Its transmission routes are mainly through vector insects such as Diaphorina citri, and can also be transmitted over long distances through diseased seedlings, scions, etc. At present, biological control methods show certain potential in the control of Huanglong disease. For example, endophytes are used to antagonize Candidatus Liberibacter, and the growth and reproduction of pathogens are inhibited by substances such as antibiotics and siderophores secreted by endophytes, so as to achieve the purpose of disease control. At present, Candidatus Liberibacter is divided into 4 subspecies, namely Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, Candidatus Liberibacter americanus, and Candidatus Liberibacter caribbeanus. Among them, the Asian subspecies has the widest distribution.

[0004] Curvularia muehlenbeckiae mainly damages leaves. The typical lesions are round or oval, 1-2 mm in size, with a central pale white or yellowish-brown color, a dark brown margin, and a light yellow halo around. When the humidity is high, grayish-black mold layers can be produced on both the front and back of the diseased parts. Developing new biological control strains is a technical problem to be solved. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the object of the present invention is to provide the application of a biocontrol bacterium B.velezensis SMXJ22 in controlling Colletotrichum gloeosporioides SMCG1#C and citrus huanglongbing. As a plant endophytic bacterium, compared with other environmental microorganisms, the antibacterial components produced by this bacterium can exist and be transported in plants for a long time, and have a more lasting control effect. This bacterium has a good control effect on Colletotrichum gloeosporioides and citrus huanglongbing.

[0006] To achieve the above invention object, the technical solution adopted by the present invention is as follows: A Bacillus sp., which is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, China, and its deposit number is CCTCC NO: M 20242290, deposit date: October 22, 2024. The Bacillus sp. is Bacillus velezensis.

[0007] The present invention also provides the application of the Bacillus sp. in inhibiting Curvularia muehlenbeckiae.

[0008] The present invention also provides the application of the Bacillus sp. in controlling citrus huanglongbing.

[0009] Among them, the pathogenic bacteria of citrus huanglongbing include Candidatus Liberibacter asiaticus.

[0010] The present invention also provides the application of the Bacillus sp. in controlling Colletotrichum gloeosporioides.

[0011] Among them, the Bacillus sp. can secrete protease, glucanase and chitinase.

[0012] Among them, the fermentation filtrate of the Bacillus sp. can inhibit the spore germination and appressorium development of Colletotrichum gloeosporioides.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] (1) The antibacterial components produced by B. velezensis SMXJ22 can effectively inhibit the vegetative growth and asexual reproduction of pathogenic fungi such as Colletotrichum gloeosporioides and Curvularia muehlenbeckiae, and have good and broad-spectrum control effects on a variety of pathogenic fungi. The antibacterial components contained in the fermentation filtrate of this bacterium can significantly inhibit the spore germination and appressorium formation of Colletotrichum gloeosporioides.

[0015] (2) The sterile fermentation filtrate of B. velezensis SMXJ22 can still significantly inhibit the spore germination of Colletotrichum gloeosporioides, the development of appressoria and reduce the pathogenicity of this bacterium after being treated at a high temperature of 121 °C, showing excellent disease control effects.

[0016] (3) The bacterial suspension of B. velezensis SMXJ22 completely inhibits the infection of Colletotrichum gloeosporioides SMCG1#C on the detached leaves of different host plants and the development of lesions, showing excellent control effects on Colletotrichum gloeosporioides.

[0017] (4) B. velezensis SMXJ22 has good control effects on citrus huanglongbing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0019] Figure 1 It shows the secretion of protease, glucanase and chitinase by SMXJ22.

[0020] Figure 2 It shows the confrontation on the plate between SMXJ22 and Colletotrichum gloeosporioides and other various pathogenic fungi.

[0021] Figure 3 It shows that both the original fermentation filtrate of SMXJ22 and the diluted one can completely inhibit the spore germination and the development of appressoria of Colletotrichum gloeosporioides.

[0022] Figure 4 It shows that the fermentation filtrate of SMXJ22 can still significantly inhibit the spore germination and the development of appressoria of Colletotrichum gloeosporioides after being treated at a high temperature.

[0023] Figure 5 It shows that after being treated with SMXJ22, the expression levels of key genes in 7 chitin synthase (CHS) family genes of Colletotrichum gloeosporioides SMCG1#C change significantly.

[0024] Figure 6 For the inhibitory effect of SMXJ22 on Sphaeropsis sapinea SMCG1#C in plants.

[0025] Figure 7 For the maximum likelihood phylogenetic tree.

[0026] Figure 8 For the colony of biocontrol bacterium SMXJ22 and its antibacterial zone (the middle colony), as well as the indicator pathogens (the rest are Agrobacterium tumefaciens, Xanthomonas citri subsp. citri, Ralstonia solanacearum, and soft rot bacteria).

[0027] Figure 9 For the detection process of the colonization test of the biocontrol bacterium in citrus.

[0028] Figure 10 For the detection results of the colonization of the biocontrol bacterium in citrus.

[0029] Figure 11 For the fold change of the relative content of Candidatus Liberibacter asiaticus in citrus seedlings of different treatment groups detected by qPCR. Detailed implementation manners

[0030] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following specific embodiments are used to describe the detailed implementation manners of the present invention in detail.

[0031] The Bacillus SMXJ22 of the present invention is deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, China, and its deposit number is CCTCC NO: M 20242290, and the deposit date is October 22, 2024.

[0032] Example 1:

[0033] The strain of the present invention was isolated from collected Chinese fir, and it was identified as Bacillus velezensis through identification. The strain identification process is as follows: First, the cetyltrimethylammonium bromide (CTAB) method was used to complete the extraction and purification of the strain's DNA genome. Then, the universal bacterial primer 16S rRNA was used to carry out PCR amplification reaction on the extracted strain DNA. Subsequently, the amplified product was uploaded to the NCBI database for BLAST homology comparison analysis, and at the same time, the sequences of similar strains and related closely related species sequences were downloaded as the reference basis for subsequent analysis. After that, these sequences were subjected to multiple sequence alignment using the BioEdit software. Then, with the help of the PhyloSuite1.2.1 software, the best tree-building model was determined through the ModelFinder program, and finally, the IQ-TREE software was used to construct a phylogenetic tree based on the maximum likelihood method. The universal primer sequences are: F: 5’-AGAGTTTGATCCTGGCTCAG-3’, R: 5’-GGTTACCTTGTTACGACTT-3’.

[0034] As Figure 7 shown, in this study, the universal primer of 16S rRNA was used to perform PCR amplification on the target sequence, and according to the comparison results of the NCBI database, the strain sequence with a relatively close genetic relationship to the target strain was selected as the reference sequence. When constructing the phylogenetic tree, the strain Streptomyces_herbaricolor_NBRC_12876 was used as the outgroup, and the maximum likelihood method (abbreviated as ML) was used to construct the phylogenetic tree. The analysis results showed that the strain B.velezensis SMXJ22 and the standard strain Bacillus velezensis_CR_502 clustered into the same branch on the phylogenetic tree, indicating the closest genetic relationship between the two. Combining morphological characteristics (analysis of hyphae, spores, and colony morphology), the strain B.velezensis SMXJ22 was identified as Bacillus_velezensis of the genus Bacillus, family Bacillaceae, order Bacillales, class Bacilli, and phylum Bacillota.

[0035] Example 2:

[0036] The Bacillus strain SMXJ22 of the present invention has the ability to secrete protease, glucanase, and chitinase, and its screening and identification process is as follows:

[0037] (1) First, in a laminar flow hood environment, the fir biocontrol bacterium - B. velezensis SMXJ22 was placed on the surface of an LB solid medium by streaking inoculation, and then it was put into an incubator with a constant temperature of 30 °C and cultured under completely dark conditions. After the colonies grew to an appropriate size, single colonies were picked and inoculated into an LB liquid medium, and cultured overnight with shaking at a rotation speed of 200 revolutions per minute at a temperature of 30 °C. Then, 2 μL of the overnight cultured bacterial solution was taken for preliminary screening. The specific method was to inoculate this bacterial solution onto a PDA-SMP (special medium for protease screening) medium containing 3% skim milk powder. As Figure 1 shown, obvious transparent zones were formed by the inoculated strains on the PDA-3% skim milk powder SMP medium, and this phenomenon indicated that the strain B. velezensis SMXJ22 had the ability to produce protease.

[0038] (2) Next, the bacterial solution obtained in step (1) was inoculated again in the laminar flow hood. The media inoculated this time were PDA-0.2% β-glucan medium and PDA-0.5% colloidal chitin medium. As Figure 1 shown, transparent zones were formed by the inoculated strains on both of these media, and this result showed that the strain B. velezensis SMXJ22 not only had the ability to produce protease, but also had the characteristics of producing glucanase and chitinase.

[0039] Example 3

[0040] Prepare a Bacillus bacterial suspension according to the procedure of Example 2, and conduct experiments using the plate confrontation method. The specific operations are as follows: Select maturely cultured pathogenic fungi such as Colletotrichum fir SMCG 1#C, Curvularia muehlenbeckiae, and Fusarium, and use a puncher to obtain disks with a diameter of 6 mm at the edge of the colonies. Then, inoculate these disks at the center position of a PDA medium. Using the cross-cross method, inoculate the B. velezensis SMXJ22 bacterial solution at four points on the diagonal of the medium, 3 cm away from the center. At the same time, set up a control group, and set 3 replicate experiments for each treatment group. Place the inoculated medium in an incubator at a constant temperature of 30 °C for culture, and regularly record the colony diameter of the pathogenic fungi and observe their growth conditions during this period. After the pathogenic fungi in the control group completely covered the PDA medium, calculate the inhibition rate of the B. velezensis SMXJ22 strain against the pathogenic fungi.

[0041]

[0042] From Figure 2As can be clearly seen from the presented data, the strain B. velezensis SMXJ22 showed an extremely obvious inhibitory effect on the vegetative growth of Colletotrichum gloeosporioides SMCG1#C, with an inhibition rate reaching 54.87%. Moreover, this strain also has the ability to inhibit the vegetative growth of various pathogenic fungi such as Curvularia muehlenbeckiae and Fusarium. Specifically, the inhibition rate of SMXJ22 against Curvularia muehlenbeckiae JS1-1 was 56.45%, and the inhibition rate against Fusarium HN33-8-2 was 46.38%.

[0043] Example 4

[0044] Effects of the original fermentation filtrate of the Bacillus strain of the present invention and after dilution on the spore germination and appressorium development of Colletotrichum gloeosporioides:

[0045] In a laminar flow hood, B. velezensis SMXJ22 was streaked on an LB solid medium and then placed in an incubator at 30°C for culturing in the dark. After single colonies grew, they were inoculated into a 50 mL Erlenmeyer flask containing 10 mL of PDA liquid medium and cultured on a shaker at 30°C and 200 rpm for 2 days. Then, it was centrifuged at a centrifugal force of 10,000×g for 5 minutes, and the supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain a sterile fermentation filtrate.

[0046] Mycelial blocks of Colletotrichum gloeosporioides SMCG1#C were cut and placed in CM liquid medium, and cultured with shaking at 200 rpm at 25°C for 24 hours. Then, it was filtered through two layers of filter membranes and centrifuged at a centrifugal force of 8000×g for 8 minutes to collect spores. The spores were washed 3 times with sterile water, and the spore concentration was adjusted to 2×10^5 cells / mL with 5% PDA liquid medium for subsequent use.

[0047] Three treatment groups were set up in the experiment as follows: ① Mix the sterile fermentation broth of the biocontrol bacterium B. velezensis SMXJ22 and the spore suspension in equal volumes; ② Dilute the sterile fermentation broth of the biocontrol bacterium B. velezensis SMXJ22 5-fold with PDA liquid and then mix it with the spore suspension in equal volumes; ③ Dilute the sterile fermentation broth of the biocontrol bacterium B. velezensis SMXJ22 10-fold with PDA liquid and mix it with the spore suspension in equal volumes. At the same time, mix PDA liquid medium and spore suspension in equal volumes as the control group to ensure that the final concentration of the spore suspension in both the control group and the treatment groups is 1×10^5 cells / mL. Take 20 μL of the mixed solution from the control group and the treatment groups respectively, drop it on a hydrophobic glass slide, and culture it under the constant temperature condition of 25°C. Observe at different time points such as 2 hours, 4 hours, 8 hours, and 12 hours, and set 3 replicates for each experiment. At the same time, measure and analyze about 150 groups of data for each time period.

[0048] According to Figure 3 As shown in the results, when PDA liquid medium and spore suspension were mixed in equal volumes as the control group, the spores of Colletotrichum cunninghamiicola began to germinate after 2 hours, and the germination rate reached 83.48%; at 4 hours, the spore germination rate was as high as 93.92%, and infection structures - appressoria had formed at the tips of the germ tubes of some spores; at 8 hours, appressoria had formed at the tips of most spore germ tubes; after 12 hours, the appressoria formed by spore germination turned black. In the treatment groups, the sterile fermentation broth of the biocontrol bacterium B. velezensis SMXJ22 completely inhibited the germination of Colletotrichum cunninghamiicola spores; after diluting the sterile fermentation filtrate of B. velezensis SMXJ22 5-fold and 10-fold with PDA liquid respectively and mixing it with the spore suspension in equal volumes, the spores of Colletotrichum cunninghamiicola SMCG 1#C still could not germinate and no infection structures were produced.

[0049] Example 5

[0050] The present invention aims to explore the effects of the fermentation filtrate of the described Bacillus strain after undergoing high-temperature treatment on the spore germination of Colletotrichum cunninghamiicola and the development of appressoria.

[0051] According to the operation process of Example 3, the sterile fermentation filtrate of the biocontrol bacterium B. velezensis SMXJ22 of Chinese fir was successfully obtained. Subsequently, the sterile fermentation filtrate was placed in a high-temperature environment for treatment (specific conditions: 121 °C for 20 minutes). Different treatment groups were set up in the experiment, and the specific arrangements were as follows: ① The sterile fermentation broth without high-temperature treatment was mixed with the spore suspension in equal volume; ② The sterile fermentation broth after high-temperature treatment was mixed with the spore suspension in equal volume. The final concentration of the spore suspension in all treatment groups was uniformly set at 1×10^5 cells / mL. At the same time, the PDA liquid medium was mixed with the spore suspension in equal volume as the control group. 20 μL of the mixed solution was taken from the control group and the treatment group respectively and dropped on the hydrophobic glass slide, and cultured under the constant temperature condition of 25 °C. Observations were made at different time points such as 2 hours, 4 hours, 8 hours, and 12 hours, and 3 replicates were set for each test. Moreover, about 150 groups of data in each time period were measured, and the data were compared and analyzed using SPSS software.

[0052] According to Figure 4 The results presented show that in the experiment with the PDA liquid medium mixed with the spore suspension in equal volume as the control group, the spores of Colletotrichum gloeosporioides Penz. SMCG 1#C began to germinate after 2 hours, and the germ tubes gradually elongated; at 4 hours, infection structures - appressoria had formed at the tips of some spore germ tubes; at 8 hours, appressoria had formed at the tips of most spore germ tubes; after 12 hours, the appressoria formed by spore germination showed a melanization phenomenon. In the treatment group, the original sterile fermentation broth of the biocontrol bacterium B. velezensis SMXJ22 completely inhibited the germination of Colletotrichum gloeosporioides Penz. spores; the fermentation broth after high-temperature treatment also completely inhibited the germination of Colletotrichum gloeosporioides Penz. SMCG 1#C spores.

[0053] Example 6

[0054] This study focused on the effect of the fermentation filtrate of the Bacillus strain on the expression levels of 7 chitin synthase (CHS) family genes in Colletotrichum gloeosporioides Penz. SMCG1#C.

[0055] According to gene characteristics, chitin synthase (CHS) genes from different fungi have been classified into 7 categories. Existing studies have shown that among the 7 chitin synthase genes of Colletotrichum gloeosporioides Penz. SMCG1#C, CgChs5 and CgChs6 play important roles in regulating the vegetative growth of the fungus, maintaining cell wall integrity, promoting asexual reproduction, and enhancing pathogenicity.

[0056] Fermentation filtrate preparation: Inside a laminar flow hood, inoculate the Chinese fir biocontrol bacterium Bacillus velezensis (B.velezensis SMXJ22) onto an LB solid medium. After streaking, place it in a constant temperature incubator at 30°C and culture it in the dark. After single colonies grow, inoculate them into a 250 mL Erlenmeyer flask containing 100 mL of PDA liquid medium, and culture them on a shaker at 30°C and 200 rpm for 24 hours. Then, centrifuge at a centrifugal force of 10,000×g for 5 minutes, collect the supernatant, and filter the supernatant using a 0.22 μm bacterial filter to obtain a sterile fermentation filtrate.

[0057] Cut a mycelial block of Colletotrichum firense SMCG1#C cultured for 3 days at 25°C in the dark, inoculate it into 100 mL of PDA liquid medium, and culture it at 25°C and 100 rpm for 16 hours. Then, collect the mycelial pellets using a single-layer filter membrane, transfer the mycelial pellets to 100 mL of sterile water, shake and culture them on a shaker at 25°C and 100 rpm for 5 minutes, and then collect the mycelial pellets by filtration. Repeat the above operations 2 times to ensure the purity and activity of the mycelial pellets.

[0058] Experimental grouping and treatment: Set up a treatment group and a control group. In the treatment group, place the mycelial pellets in 100 mL of sterile fermentation filtrate of B.velezensis SMXJ22, and in the control group, place the mycelial pellets in 100 mL of PDA liquid medium. Treat both groups for 6 hours. After the treatment, filter with a filter membrane to obtain the treated mycelial pellets, and place them in liquid nitrogen and grind them into a fine powder.

[0059] RNA extraction and qRT-PCR detection: Extract RNA from the ground powder using the Trizol method and reverse transcribe it into cDNA. Using 18S as an internal reference gene, design primers for 7 chitin synthases (CHS1-7) according to the qPCR primer design principle. Using the qRT-PCR technique, calculate the relative quantification of each transcript using the 2-ΔΔCt method. Set 3 replicates for each experiment, and repeat the experiment 3 times to improve the reliability and accuracy of the experimental results.

[0060] Experimental results: According to Figure 5 the results shown, compared with the control group, after treatment with the sterile fermentation filtrate of B.velezensis SMXJ22, the expression levels of the CgChs5 and CgChs6 genes in the chitin synthase (CHS) family of Colletotrichum firense SMCG1#C showed significant fold-upregulation, indicating that this fermentation filtrate has an obvious promoting effect on the expression of these two genes.

[0061] Example 7

[0062] The control effect of the Bacillus bacterial suspension on Colletotrichum gloeosporioides on different host plants.

[0063] The Bacillus bacterial suspension was obtained according to Example 1, and the SMCG1#C spore suspension was obtained according to Example 3. The culture dishes were surface-sterilized with 75% anhydrous ethanol, filter paper was placed at the bottom of the dishes, and an appropriate amount of sterile water was added to moisten it. The leaves of Cunninghamia lanceolata, Liriodendron chinense, and Populus were surface-sterilized with 75% anhydrous ethanol; the leaves of Cunninghamia lanceolata, Liriodendron chinense, and Populus were scalded with a sterile needle. The experiments were set up as follows:

[0064] For the preventive group inoculation experiment: The control group was inoculated with 10 μL of PDA liquid medium, and the treatment group was inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension. After 3 days, both groups were inoculated with the SMCG1#C spore suspension (concentration of 1×10 5 CFU / mL). In addition, one group was only inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension. Filter paper fragments were added to the bases of the leaves of Cunninghamia lanceolata, Liriodendron chinense, and Populus in all groups for moisturizing treatment. The culture dishes were sealed and incubated at a constant temperature of 25°C. The disease incidence of the leaves was observed, and the lesion size was recorded. Each treatment was repeated 3 times.

[0065] For the simultaneous inoculation experiment: The control group was inoculated with a mixture of PDA liquid medium and SMCG1#C spore suspension, and the treatment group was inoculated with a mixture of B. velezensis SMXJ22 bacterial suspension and SMCG1#C spore suspension. The final concentration of the SMCG1#C spore suspension inoculated in both groups was 1×10 5 CFU / mL. In addition, one group was only inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension. Filter paper fragments were added to the bases of the leaves of Cunninghamia lanceolata, Liriodendron chinense, and Populus in all groups for moisturizing treatment. The culture dishes were sealed and incubated at a constant temperature of 25°C. The disease incidence of the leaves was observed, and the lesion size was recorded. Each treatment was repeated 3 times.

[0066] For the treatment group inoculation experiment: First, the SMCG1#C spore suspension (concentration of 1×10 5 CFU / mL) was inoculated. After 3 days, the control group was inoculated with 10 μL of PDA liquid medium, and the treatment group was inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension. In addition, one group was only inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension. Filter paper fragments were added to the bases of the leaves of Cunninghamia lanceolata, Liriodendron chinense, and Populus in all groups for moisturizing treatment. The culture dishes were sealed and incubated at a constant temperature of 25°C. The disease incidence of the leaves was observed, and the lesion size was recorded. Each treatment was repeated 3 times.

[0067] By Figure 6It can be seen that in the control group of the prevention group experiment (first inoculated with 10 μL of PDA liquid medium and then inoculated with SMCG1#C spores after 3 days), obvious lesions were formed on the leaves of Chinese fir, tulip tree, and poplar. In the treatment group (first inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension and then inoculated with SMCG1#C spores after 3 days), the Chinese fir anthracnose fungus could not infect at all, and no lesions were formed on the leaves of the host plants (Chinese fir, tulip tree, poplar). Only inoculating 10 μL of B. velezensis SMXJ22 bacterial suspension also did not form lesions on the leaves of the host plants (Chinese fir, tulip tree, poplar). Thus, it can be seen that pre-inoculating with the B. velezensis SMXJ22 bacterial suspension completely inhibited the infection of Chinese fir anthracnose fungus and the development of lesions, and the B. velezensis SMXJ22 bacterial suspension itself was harmless to plants.

[0068] It can be seen from Figure 6 that in the control group of the co-inoculation group experiment (a mixed solution of PDA liquid medium and SMCG1#C spore solution), obvious lesions were formed on the leaves of Chinese fir, tulip tree, and poplar. In the treatment group (a mixed solution of B. velezensis SMXJ22 bacterial suspension and SMCG1#C spore solution), the Chinese fir anthracnose fungus could not infect at all, and no lesions were formed on the leaves of the host plants (Chinese fir, tulip tree, poplar). Only inoculating 10 μL of B. velezensis SMXJ22 bacterial suspension also did not form lesions on the leaves of the host plants (Chinese fir, tulip tree, poplar). Thus, it can be seen that co-inoculating with the B. velezensis SMXJ22 bacterial suspension and SMCG1#C spore solution completely inhibited the infection of Chinese fir anthracnose fungus and the development of lesions, and the B. velezensis SMXJ22 bacterial suspension itself was harmless to plants.

[0069] It can be seen from Figure 6 that in the control group of the co-inoculation group experiment (first inoculated with SMCG1#C spore solution and then inoculated with 10 μL of PDA liquid medium after 3 days), obvious lesions were formed on the leaves of the host plants (Chinese fir, tulip tree, poplar). In the treatment group (first inoculated with SMCG1#C spore solution and then inoculated with 10 μL of B. velezensis SMXJ22 bacterial suspension after 3 days), obvious lesions were also formed on the leaves of the host plants (Chinese fir, tulip tree, poplar) by the Chinese fir anthracnose fungus, and there was no significant difference in the statistical data of the lesions between the control group and the treatment group. However, only inoculating 10 μL of B. velezensis SMXJ22 bacterial suspension still did not form lesions on the leaves of the host plants (Chinese fir, tulip tree, poplar). Thus, it can be seen that the B. velezensis SMXJ22 bacterial suspension could not inhibit the expansion of lesions on the host plants after pre-inoculating with SMCG1#C spore solution, and had no obvious therapeutic effect on anthracnose, but the B. velezensis SMXJ22 bacterial suspension itself was harmless to plants.

[0070] Example 8

[0071] The antagonistic culture was carried out between the biocontrol bacterium Bacillus velezensis SMXJ22 and four indicator pathogenic bacteria of Candidatus Liberibacter asiaticus to determine whether it has biocontrol potential. The steps are as follows:

[0072] ① Culture of biocontrol strains: The bacteria were cultured on LB medium by streaking method in an incubator at 28 - 30 °C. The bacterial suspension was obtained by shaking culture at 30 °C and 200 rpm for 24 h.

[0073] ② Culture of indicator pathogenic strains: Pathogenic bacterial strains such as Agrobacterium tumefaciens, Xanthomonas Campestris pv. citri, Erwinia carotovora subsp. carotovora Dye, and Ralstonia solanacearum were cultured on LB medium by streaking method in the dark in an incubator at 28 - 30 °C; the bacterial suspension was obtained by shaking culture at 30 °C and 200 rpm for 24 h.

[0074] ③ Petri dish antagonism: After co - culturing the biocontrol bacterium and the pathogenic bacterium, observe whether there is an obvious antagonistic zone or inhibition zone to screen for biocontrol bacteria with significant biocontrol effects against pathogenic bacteria such as A. tumefaciens, X. Campestris pv. citri, E. carotovora, and R. solanacearum. The specific steps are as follows: Dip a sterile inoculation needle into the alternative pathogenic bacteria and the single colony of the biocontrol bacterium respectively, and streak them on the LB medium plate. Incubate them at 30 °C in an inverted position to obtain the single colonies of the pathogenic bacteria and the biocontrol bacterium. Then dip a sterile white pipette tip into the single colonies of the alternative pathogenic bacteria and the biocontrol bacterium respectively, and put them into 10 mL of LB liquid medium respectively. Shake - culture them at 30 °C and 200 rpm for 24 h to obtain the bacterial suspensions of the alternative pathogenic bacteria and the biocontrol bacterium. Then use a pipette to add the indicator pathogenic bacteria liquid to the PDA solid medium at a ratio of 1:100 to prepare a bacteria - containing plate, and then take 20 μL of the biocontrol bacteria suspension and inoculate it in the center of the bacteria - containing plate. The bacteria - containing plate inoculated with LB liquid medium was used as a control, and each treatment was repeated 3 times.

[0075] ④ Evaluation of the Petri dish antagonistic effect of biocontrol bacteria and indicator pathogenic bacteria:

[0076] On the surface of the bacterial-containing plate (containing pathogenic bacteria), observe whether a clear antagonistic zone is produced around the colonies of the biocontrol potential bacteria. If not, it indicates that the biocontrol potential bacteria have no antagonistic effect on the indicator pathogenic bacteria.

[0077] It can be seen from Figure 8 that B.velezensis SMXJ22 produced clear antagonistic zones against all four indicator pathogenic bacteria of Candidatus Liberibacter asiaticus, indicating that it has antagonistic effects against all four indicator pathogenic bacteria of Candidatus Liberibacter asiaticus.

[0078] Example 9

[0079] The colonization situation of the said Bacillus strain in living citrus potted seedlings.

[0080] Inoculate the selected dominant bacteria into healthy citrus plants to evaluate their colonization ability in the host. Obtain the biocontrol bacterial suspension, inject it into the healthy citrus potted seedlings by trunk inoculation, take samples every month after inoculation for tracking detection, and detect according to the process shown in Figure 9 as follows. As shown in Figure 10 take samples of citrus leaves from 5 healthy citrus potted seedlings after inoculation, extract DNA and sequence it, and compare the sequence with B.velezensis SMXJ22. It is found that the sequence results of the re-isolated strains are completely consistent with B.velezensis SMXJ22, indicating that B.velezensis SMXJ22 has successfully colonized in the citrus seedlings.

[0081] Example 10

[0082] The effect of inoculating the said Bacillus strain on the content of Candidatus Liberibacter asiaticus in living citrus seedlings.

[0083] First, obtain a Bacillus spore suspension. In the treatment group, inoculate the biocontrol bacterium B. velezensis SMXJ22 spore suspension into healthy citrus plants. In the control group, only inoculate the LB blank medium. Then, inoculate Candidatus Liberibacter asiaticus into the citrus seedlings in both the treatment group and the control group. qPCR continuously tracks the accumulation of the number of Candidatus Liberibacter asiaticus pathogens in the citrus seedlings in the treatment group and the control group. Samples are taken once every 1-2 months after inoculating Candidatus Liberibacter asiaticus. Leaves of the treated plants in each group are collected (mixed sampling on the inoculated seedlings within the same treatment), and DNA is extracted and then detected for the pathogen load using qPCR. The method for real-time fluorescence quantitative PCR to detect Candidatus Liberibacter asiaticus pathogens: The citrus internal reference primers used are as follows: The base sequence of the upstream primer Citrus UBQ-F is 5-TGGACGCTTCAGTCTGTTTG-3’, and the base sequence of the downstream primer Citrus UBO-R is 5-TCGTCAATCACCCCTTCTTT-3’. The detection primers for Candidatus Liberibacter asiaticus are the upstream primer CQULas F 03 and the downstream primer CQULas R 03. The base sequence of the upstream primer CQULas F 03 is 5-CAAGGAAAGAGCGTAGAA-3’, and the base sequence of the downstream primer CQULas R 03 is 5-CCTCAAGATCGGGTAAAG-3’. The amplified specific gene sequence fragment of Candidatus Liberibacter asiaticus Asian species rpLJ / rpLL is 382 bp. The real-time fluorescence quantitative PCR reaction system is as follows: 10 μL of SYBR, 0.4 μL of the upstream primer CQULas F 03 is added, 0.4 μL of the downstream primer CQULas R 03 is added, 7.2 μL of ddH2O, and 2.0 μL of template DNA; 3 replicates are set for each sample. Avoid cross-contamination during sample loading, and the negative control is loaded last. The real-time fluorescence quantitative PCR reaction conditions: Pre-denaturation at 94 °C for 5 min; denaturation at 95 °C for 5 s, then annealing at 59 °C for 15 s, extension at 72 °C for 45 s, for a total of 40 cycles; fluorescence is automatically collected at the extension stage of each cycle; the final extension is at 72 °C for 7 min. If compared with the control group, after treatment with the biocontrol bacterium, the number of Candidatus Liberibacter asiaticus pathogens in the citrus seedlings does not show a significant increase (such as Figure 8 ), it is judged that the biocontrol bacterium has a preventive effect on Candidatus Liberibacter asiaticus.

[0084] As Figure 11 shown, the content of Candidatus Liberibacter asiaticus in the control group plants is classified as 1. Compared with the control group, the content of Candidatus Liberibacter asiaticus in the citrus seedlings of the treatment group pre-inoculated with the biocontrol bacterium B. velezensis SMXJ22 is extremely significantly reduced compared to the control group. Thus, it can be seen that B. velezensis SMXJ22 has a significant inhibitory effect on the proliferation of Candidatus Liberibacter asiaticus in citrus plants, and it has a control effect on Candidatus Liberibacter asiaticus.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered by the scope of the claims of the present invention.

Claims

1. A Bacillus sp., characterized in that: The Bacillus is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, China, and its deposit number is CCTCC NO: M 20242290. The deposit date is October 22, 2024. The Bacillus is Bacillus velezensis.

2. Use of the Bacillus sp. according to claim 1 in inhibiting Curvularia muehlenbeckiae.

3. Use of the Bacillus sp. according to claim 1 in controlling citrus huanglongbing.

4. The application according to claim 3, wherein: The pathogen causing citrus huanglongbing includes Candidatus Liberibacter asiaticus.

5. Use of the Bacillus sp. according to claim 1 in controlling Chinese fir anthracnose.

6. The application according to claim 2, characterized in that: The Bacillus can secrete protease, glucanase and chitinase.

7. The application according to claim 6, characterized in that: The fermentation filtrate of the Bacillus can inhibit the spore germination and appressorium development of Colletotrichum higginsianum.

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