Bacillus Warrensis strain and application thereof

Abstract

The invention discloses a bacillus wurtzeri strain and application thereof, and belongs to the technical field of microorganisms. The bacillus wurtzeri strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20242289, the preservation date is October 22, 2024, and the preservation address is Wuhan University, Wuhan, China. The bacillus wurtzeri strain disclosed by the invention can effectively inhibit vegetative growth and vegetative propagation of pathogenic bacteria such as colletotrichum gloeosporioides and paecilomyces radinieri, has a remarkable inhibition effect on citrus liberobacter asiaticum and has a good prevention and treatment effect on the citrus liberobacter asiaticum.

Description

Technical Field

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

[0002] Due to its straight and round trunk, fast growth rate, insect resistance, decay resistance, and high economic value, Chinese fir (Cunninghamia lanceolata) is an important fast-growing timber tree species. The afforestation area and wood volume of Chinese fir both rank first among the main afforestation tree species. Chinese fir anthracnose caused by Colletotrichum gloeosporioides has caused serious harm to the growth of Chinese fir in cultivation areas. When the disease is mild, the needles or tender shoots of Chinese fir seedlings turn brown and wither. When it is severe, the young Chinese fir forests turn yellow and die in patches, causing devastating damage. The nutritional mode of Colletotrichum gloeosporioides is a hemibiotrophic mode mediated by appressoria and the like. The process of its infecting host plants includes: (1) Spores germinate to form germ tube hyphae; (2) Appressoria are differentiated at the top of the germ tube hyphae; (3) The appressoria differentiate to produce infection pegs that penetrate the cell wall of the plant surface cells and differentiate to form infection hyphae to infect living cells; (4) The infection hyphae differentiate to produce secondary hyphae that secrete degrading enzymes to kill host cells.

[0003] At present, the prevention and control of Chinese fir anthracnose mainly rely on chemical control. Chemical control methods are prone to problems such as environmental pollution, accidental injury to natural enemies, health risks to humans, livestock and poultry, and drug resistance of pathogens. Biological control has the advantages of being green and environmentally friendly, having a long-lasting control effect, and being harmless to humans and livestock, making it an ideal way for plant pest control. One application method of biological control is to directly release microorganisms in the natural environment and play a biological control role through the contact between biocontrol microorganisms and target pests and diseases; another way is to purify and apply the antibacterial substances produced by the metabolism of biocontrol microorganisms. This method has the advantages of high control efficiency and little influence by environmental factors, and has broad development and application prospects.

[0004] Huanglongbing (HLB), also known as yellow shoot disease and yellow blight, is a worldwide bacterial disease of citrus caused by infection with phloem bacteria. It seriously affects yield and quality and can even cause citrus trees to wither and die. Since the pathogen of citrus huanglongbing cannot be cultured in vitro and Koch's postulates cannot be completed, based on the source, pathogenic ability, and genomic information of the pathogen, the huanglongbing pathogen is currently tentatively classified into 4 subspecies under the genus Candidatus Liberibacter, namely Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, Candidatus Liberibacter americanus, and Candidatus Liberibacter caribbeanus reported in 2015. Among the 4 subspecies, Candidatus Liberibacter asiaticus has the widest distribution and is the subspecies that poses the greatest threat to the citrus industry.

[0005] Curvularia muehlenbeckiae is a fungus that mainly causes leaf spot disease in plants and has a significant competitive impact on crops during the rainy season and autumn, thus affecting crop yields.

[0006] Therefore, developing new and more effective biocontrol strains is a technical problem to be solved. Summary of the Invention

[0007] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a strain of Bacillus velezensis SMXJ16 and its application in controlling Chinese fir anthracnose, citrus huanglongbing, and Curvularia muehlenbeckiae to meet the usage requirements of biological control.

[0008] To achieve the above invention purpose, the technical scheme adopted by the present invention is as follows:

[0009] The present invention provides a strain of Bacillus sp., characterized in that: the strain of Bacillus is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242289, the deposit date: October 22, 2024, and the deposit address: Wuhan University, Wuhan, China.

[0010] The present invention also provides the application of the above-mentioned strain of Bacillus velezensis in controlling Chinese fir anthracnose.

[0011] Wherein, the Bacillus Varezzia strain is capable of secreting protease, glucanase and chitinase.

[0012] The fermentation filtrate of the Bacillus Varezii strain can inhibit the spore germination and the development of appressorium of Colletotrichum spruce.

[0013] The present invention also provides use of the Bacillus velezensis strain in inhibiting Curvularia muehlenbeckiae.

[0014] The present invention also provides the use of the Bacillus velezensis strain in preventing and treating citrus Huanglongbing disease.

[0015] Wherein, the pathogenic bacteria of citrus Huanglongbing include Candidatus Liberibacter asiaticus.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) The antifungal components produced by the B. velezensis SMXJ16 strain can effectively inhibit the vegetative growth and asexual reproduction of pathogens such as Colletotrichum spruce and Curvularia muehlenbeckiae, and have a good and broad-spectrum control effect on a variety of pathogenic fungi. The antifungal components contained in the fermentation filtrate of this strain can significantly inhibit the germination and appressorium formation of Colletotrichum spruce.

[0018] (2) After high temperature treatment, the aseptic fermentation filtrate of B. velezensis SMXJ16 can still significantly inhibit the germination of spores and the development of appressorium of C. velezensis and reduce the pathogenicity of the fungus, achieving a good disease prevention and control effect.

[0019] (3) The bacterial suspension of B. velezensis SMXJ16 inhibited the infection of C. truncatula SMCG1#C on detached leaves of different host plants and the development of lesions, and had a good control effect on C. truncatula anthracnose.

[0020] (4) B. velezensis SMXJ16 has a significant inhibitory effect on four indicator pathogens in vitro. In living citrus seedlings, SMXJ16 has a significant inhibitory effect on citrus Huanglongbing (Candidatus Liberibacter asiaticus), and has a good preventive and control effect on citrus Huanglongbing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0022] Figure 1 The results show the secretion of protease, glucanase and chitinase by SMXJ16 strain.

[0023] Figure 2 The SMXJ16 strain was plate-confronted with pathogenic fungi such as Colletotrichum truncatula and Paecilomyces japonicus.

[0024] Figure 3 The SMXJ16 strain has a fermentation filtrate that can significantly inhibit the spore germination and appressorial development of Colletotrichum sprue both in its original form and after dilution.

[0025] Figure 4 The SMXJ16 strain has a fermentation filtrate that can still significantly inhibit the spore germination and appressorial development of Colletotrichum spruce after high temperature treatment.

[0026] Figure 5 After treatment with the SMXJ16 strain, the expression levels of seven chitin synthase (CHS) family genes of Colletotrichum SMCG1#C changed significantly.

[0027] Figure 6 This shows the effect of SMXJ16 strain in controlling Colletotrichum spruce SMCG1#C in plants.

[0028] Figure 7 The maximum likelihood phylogenetic tree.

[0029] Figure 8 The SMXJ 16 strain was plated against four indicator pathogens of citrus Huanglongbing.

[0030] Figure 9 This is the control effect of SMXJ 16 strain on citrus Huanglongbing fungus in living citrus seedlings. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0032] The Bacillus velezensis strain SMXJ16 of the present invention is deposited at the China Center for Type Culture Collection (CCTCC). The deposit address is Wuhan University, Wuhan, China. Its taxonomic name is Bacillus SMXJ16, and its deposit number is CCTCC NO: M 20242289. The deposit date is October 22, 2024.

[0033] Example 1:

[0034] The strain of the present invention was isolated from healthy Chinese fir collected at E118°45', N32°04′. After identification, the strain was determined to be Bacillus velezensis. Strain identification: The extraction and purification of genomic DNA was carried out according to the method of cetyltrimethylammonium bromide (CTAB) for extracting genomic DNA. The bacterial universal primer 16S rRNA was used for PCR amplification of the strain DNA, and then BLAST homology alignment analysis was performed on NCBI, and the sequences of similar strains and sequences of other related species were downloaded as references; the universal primer sequences were F: 5′-AGAGTTTGATCCTGGCTCAG-3′, R: 5′-GGTTACCTTGTTACGACTT-3′; multiple sequence alignment was carried out using BioEdit, and then the best tree-building model was established using the Model Finder program of PhyloSuite 1.2.1 software, and the maximum likelihood phylogenetic tree was constructed using IQ-TREE.

[0035] From Figure 7 The results showed that the 16S rRNA universal primer was used to amplify its sequence, and the sequences of its related strains were downloaded as reference strains according to the NCBI alignment results. Using Streptomyces_herbaricolor_NBRC_12876 as an outgroup, the maximum likelihood method (mL) was used to construct a phylogenetic tree. The results showed that the strain B.velezensis SMXJ16 and the standard strain Bacillus velezensis_CR_502 clustered into the same branch and had the closest genetic relationship. Combining morphological characteristics (analysis of hyphae, spores, and colony morphology), the strain Bacillus velezensis SMXJ16 was identified as Bacillus_velezensis under the phylum Bacillota, class Bacilli, order Bacillales, family Bacillaceae, and genus Bacillus.

[0036] Example 2:

[0037] The Bacillus strain is capable of secreting proteases, glucanases and chitinases.

[0038] (1) The fir biocontrol bacteria B. velezensis SMXJ16 was streaked onto LB solid medium in a clean bench and cultured in a 30°C constant temperature incubator in the dark. A single colony was picked and placed in LB liquid medium and cultured overnight at 30°C and 200 rpm. 2 μL of the overnight culture was taken for primary screening and inoculated into PDA-3% skim milk powder SMP medium. Figure 1 As shown, the strain produced a clearing zone on the PDA-3% skim milk powder SMP medium, indicating that the strain B. velezensis SMXJ16 had the property of producing protease.

[0039] (2) The bacterial solution obtained in step (1) was inoculated into PDA-0.2% β-glucan and PDA-0.5% colloidal chitin culture medium in a clean bench. Figure 1 As shown, the strain produced clear zones on both PDA-0.2% β-glucan and PDA-0.5% colloidal chitin media, indicating that the strain B. velezensis SMXJ16 has the characteristics of producing glucanase and chitinase.

[0040] Example 3

[0041] The effect of the Bacillus strain on the nutritional growth of fir anthracnose and other multiple pathogenic fungi.

[0042] According to Example 2, a Bacillus suspension was obtained. A plate standoff method was used to culture pathogenic fungi such as Colletotrichum gloeosporioides SMCG 1#C, Curvularia muehlenbeckiae, and Fusarium. A bacterial disc (d = 6 mm) was punched at the edge of the colony and inoculated in the center of a PDA culture medium. A cross-cross method was used to inoculate a B. velezensis SMXJ16 bacterial solution at four points 3 cm from the center on the diagonal of the culture medium. A control group was set up at the same time, and each treatment was repeated 3 times. The culture was cultured at a constant temperature of 30°C, the colony diameter of the pathogenic fungus was recorded, and the growth of the pathogenic fungus was observed. When the pathogenic fungus in the control group completely grew the PDA culture medium, the inhibition rate of the B. velezensis SMXJ16 strain on the pathogenic fungus was calculated.

[0043]

[0044] Depend on Figure 2It can be seen that the strain B. velezensis SMXJ16 has a significant inhibitory effect on the vegetative growth of C. truncatula, with an inhibition rate of 55.49%. In addition, it also has an inhibitory effect on the vegetative growth of various pathogenic fungi such as Curvularia muehlenbeckiae and Fusarium. The inhibition rate for Curvularia muehlenbeckiae JS1-1 is 59.26%, and the inhibition rate for Fusarium hunanens HN33-8-2 is 33.01%.

[0045] Example 4

[0046] The effects of the fermentation filtrate of the Bacillus strain, the original solution and the diluted solution on the spore germination and appressorium development of C.

[0047] The Chinese fir biocontrol bacterium B. velezensis SMXJ16 was streaked on LB solid culture medium in a clean bench and cultured in a constant temperature incubator at 30°C in the dark. A single colony was picked and inoculated into a 50 mL Erlenmeyer flask containing 10 mL of PDA liquid culture medium. The culture was shaken at 30°C and 200 rpm / min for 48 h, and the supernatant was collected by centrifugation at 10,000 × g for 5 min. The supernatant was filtered through a 0.22 μm bacterial filter to obtain a sterile fermentation filtrate.

[0048] Mycelial blocks of Colletotrichum sempervirens SMCG1#C were cut and placed in CM liquid culture medium. The culture was shaken at 200 rpm for 24 h at 25 °C, filtered through two filter membranes, and centrifuged at 8000 rpm for 8 min to collect spores. The spores were washed three times with sterile water and then adjusted to a spore concentration of 2 × 10 5 / mL, for future use;

[0049] The three treatment groups were as follows: ① mixing equal volumes of sterile fermentation broth of the biocontrol bacteria B. velezensis SMXJ16 with spore liquid; ② diluting the sterile fermentation broth of the biocontrol bacteria B. velezensis SMXJ16 5-fold with PDA liquid and then mixing it with equal volumes of spore liquid; ③ diluting the sterile fermentation broth of the biocontrol bacteria B. velezensis SMXJ16 10-fold with PDA liquid and then mixing it with equal volumes of spore liquid. PDB liquid medium mixed with spore liquid in equal volumes was used as a control. The final concentration of spore liquid in both the control and treatment groups was 1×10 5 20 μL of the mixture was dripped onto a hydrophobic glass slide for each of the control and treatment groups. The cells were incubated at 25°C and observed at 2, 4, 8, and 12 hours. Each experiment was replicated three times. Approximately 150 data sets were measured for each time period and compared using SPSS.

[0050] Depend on Figure 3 It can be seen that in the control group where PDA liquid culture medium and spore liquid were mixed in equal volumes, the spores of C. truncatum began to germinate at 2 hours, with a germination rate of 83.48%. At 4 hours, the spore germination rate had reached 93.92% and infection structures - appressoriums had formed at the tops of some spore germ tubes. At 8 hours, appressoriums had formed at the tops of most spore germ tubes. At 12 hours, the appressoriums formed by spore germination turned black. In the treatment group, the aseptic fermentation liquid of the biocontrol bacteria B. velezensis SMXJ16 made the spores of C. truncatum completely unable to germinate. When the aseptic fermentation filtrate of B. velezensis SMXJ16 was diluted 5 and 10 times with PDA liquid respectively and then mixed with equal volumes of spore liquid, the spores of C. truncatum could germinate, but the spore germination rate was significantly lower than that of the control group. The spore germination rate of the treatment group with SMXJ16 sterile fermentation filtrate diluted 10 times was only 77.8% at 2 hours and 87.56% at 4 hours. The spore germination rate of the treatment group with SMXJ16 sterile fermentation filtrate diluted 5 times was only 63.3% at 2 hours and 74.18% at 4 hours. In addition, the cell walls of the germ tube hyphae formed by spore germination formed a swollen body, and the spores in the treatment group did not form the infection structure - appressorium until 12 hours.

[0051] Example 5

[0052] The invention relates to the influence of the fermentation filtrate of the Bacillus strain on the spore germination and the development of the appressorium of the fir anthracnose fungus after being treated with high temperature.

[0053] The sterile fermentation filtrate of the Chinese fir biocontrol bacterium B. velezensis SMXJ16 was obtained according to Example 3. The sterile fermentation filtrate was subjected to high temperature treatment (121°C, 20 min). The treatment groups were as follows: ① the sterile fermentation filtrate was mixed with the spore solution in equal volumes; ② the sterile fermentation filtrate after high temperature treatment was mixed with the spore solution in equal volumes. The final concentration of the spore solution in all treatment groups was 1×10 5 The control group and the treatment group were each incubated at 25°C for 2, 4, 8, and 12 hours. Three replicates were used for each experiment. Approximately 150 data sets were measured for each time period and compared using SPSS.

[0054] Depend on Figure 4It can be seen that in the control group where the PDA liquid medium was mixed with the spore liquid in equal volume, the spores of Colletotrichum higginsianum began to germinate after 2 h, the germ tubes elongated, infection structures - appressoria were formed at the tips of the germ tubes of some spores at 4 h, appressoria were formed at the tips of the germ tubes of most spores at 8 h, and the appressoria formed by the germination of spores turned black at 12 h. However, the sterile fermentation broth stock solution of the biocontrol bacterium B. velezensis SMXJ16 completely inhibited the germination of the spores of Colletotrichum higginsianum, and the spores of Colletotrichum higginsianum were still completely unable to germinate in the fermentation broth after heat treatment.

[0055] Example 6

[0056] Effect of the fermentation filtrate of the Bacillus strain on the expression levels of 7 chitin synthase (CHS) family genes of Colletotrichum higginsianum SMCG1#C.

[0057] Chitin is a homopolymer composed of N-acetylglucosamine (GlcNAc) and linked by β-1,4-microfibrils. It is one of the main components of the fungal cell wall. The primary polysaccharide can fold up to form antiparallel chains, and intra-chain hydrogen bonds are formed to further harden the carbohydrate into super-strong microfibrils. The 3D network of chitin microfibrils is covalently bound to β(1,3)-glucan to form the fungal skeleton. Chitin synthase is the key enzyme that catalyzes the polymerization of GlcNAc. They are usually located on the cytoplasmic membrane and are ideal targets for the development of fungicides. Chitin synthase (CHS) genes from various fungi have been classified into 7 classes. Current research shows that among the 7 chitin synthases of Colletotrichum higginsianum SMCG1#C, CgChs1, CgChs5, and CgChs6 are involved in regulating the vegetative growth, cell wall integrity, asexual reproduction, and pathogenicity of Colletotrichum higginsianum.

[0058] Streak the biocontrol bacterium B. velezensis SMXJ16 of Chinese fir on the LB solid medium in a laminar flow cabinet, place it in a constant temperature incubator at 30 °C for dark cultivation, pick a single colony, inoculate it into a triangular flask (250 mL) containing 100 mL of PDA liquid medium, shake and cultivate at 30 °C and 200 rpm / min for 24 h, centrifuge at 10,000×g for 5 min to collect the supernatant, and filter the supernatant through a 0.22 μm bacterial filter to obtain a sterile fermentation filtrate.

[0059] Cut the mycelial blocks of Colletotrichum higginsianum SMCG1#C that have been cultured in the dark at 25 °C for 3 d, inoculate them into 100 mL of PDA liquid medium, culture at 25 °C and 100 rpm for 16 h, collect the mycelial pellets with a single-layer filter membrane, transfer the mycelial pellets to 100 mL of sterile water, shake and culture at 25 °C and 100 rpm for 5 min, and filter to collect the mycelial pellets. The above operations are repeated 2 times.

[0060] The treatment group was to place the mycelial pellets in 100 mL of sterile fermentation filtrate of B. velezensis SMXJ16, and the control group was to place the mycelial pellets in 100 mL of PDA liquid medium. Both groups were treated for 6 h, and the treated mycelial pellets were obtained by filtration through a filter membrane, ground to a fine powder in liquid nitrogen, and RNA was extracted using the Trizol method and reverse transcribed into cDNA. For qRT-PCR, 18S was used as the internal reference gene, and 7 chitin synthase (CHS1-7) primers were designed according to the qPCR primer design principle. The relative quantification of each transcript was calculated using the 2 -ΔΔCt method. Each experiment was set with 3 replicates, and the experiment was repeated 3 times.

[0061] As Figure 5 shown, compared with the control group, CgChs1, CgChs5, and CgChs6 in the chitin synthase (CHS) family after treatment with the sterile fermentation filtrate of B. velezensis SMXJ16 showed extremely significant fold upregulation.

[0062] Example 7

[0063] The control efficacy of the said Bacillus bacterial suspension against Colletotrichum gloeosporioides on different host plants.

[0064] 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 dish and moistened with an appropriate amount of sterile water. 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 experimental settings were as follows:

[0065] The experimental settings for the preventive group inoculation were: the control group was inoculated with 10 μL of PDA liquid medium, and the treatment group was inoculated with 10 μL of B. velezensis SMXJ16 bacterial suspension. After 3 d, the SMCG1#C spore suspension (concentration of 1x10 5 cells / mL) was inoculated simultaneously. In addition, a group was set up that was only inoculated with 10 μL of B. velezensis SMXJ16 bacterial suspension. Filter paper fragments were added to the bases of the leaves of Cunninghamia lanceolata, Liriodendron chinense, and Populus in all groups for moisture preservation treatment. The culture dishes were sealed and incubated at a constant temperature of 25°C, and the disease conditions of the leaves were observed and the lesion sizes were recorded. Each treatment had 3 replicates.

[0066] The experimental settings for the simultaneous inoculation were: 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 SMXJ16 bacterial suspension and SMCG1#C spore suspension. The final concentration of the SMCG1#C spore suspension inoculated in both groups was 1x10 510 μL of B. velezensis SMXJ16 suspension alone was used. For all groups, filter paper fragments were added to the bases of leaves from Chinese fir, Ligusticum chuanxiong, and Poplar trees. After moisturizing, the petri dishes were sealed and incubated at 25°C. The leaves were observed for disease progression and the size of the lesions was recorded. Three replicates were used for each treatment.

[0067] The treatment group inoculation experiment was set up as follows: first inoculate SMCG1#C spore solution (concentration of 1x10 5 3 days later, the control group was inoculated with 10 μL of PDA liquid culture medium, the treatment group was inoculated with 10 μL of B. velezensis SMXJ16 suspension, and another group was inoculated with only 10 μL of B. velezensis SMXJ16 suspension. For all groups, filter paper fragments were added to the bases of leaves of Chinese fir, Ligusticum chuanxiong, and Poplar trees to maintain moisture. The culture dishes were sealed and incubated at 25°C. The leaves were observed for lesion development and the size of the lesions was recorded. Each treatment was repeated three times.

[0068] Figure 6 The prevention group was pre-inoculated with the strain suspension for 3 days and then inoculated with C. fir SMCG1#C; the mixed group was inoculated with the strain suspension and C. fir SMCG1#C spore solution. Both treatments completely inhibited the infection of C. fir SMCG1#C on detached leaves of different host plants and the development of lesions. However, the treatment that first inoculated with C. fir SMCG1#C spore solution and then inoculated with the strain suspension could not prevent the continued expansion of lesions. Figure 6 As shown in Figure 1, the control group in the preventive experiment (inoculated with 10 μL of PDA liquid medium first, followed by SMCG1#C spores 3 days later) developed obvious lesions on the leaves of Chinese fir, Ligusticum ovata, and Poplar trees. However, the treatment group (inoculated with 10 μL of B. velezensis SMXJ16 suspension first, followed by SMCG1#C spores 3 days later) was completely incapable of infecting C. truncatum, with no lesions forming on the leaves of any of the host plants (Chinese fir, Ligusticum ovata, and Poplar trees). Inoculation with only 10 μL of the B. velezensis SMXJ16 suspension also failed to form lesions on the leaves of the host plants (Chinese fir, Ligusticum ovata, and Poplar trees). This demonstrates that pre-inoculation with the B. velezensis SMXJ16 suspension completely inhibits C. truncatum infection and lesion development, and that the B. velezensis SMXJ16 suspension itself is harmless to the plants.

[0069] Depend on Figure 6As can be seen from B, in the control group of the co-inoculation experiment (the mixture of PDA liquid medium and SMCG1#C spore liquid), obvious lesions were formed on the leaves of Chinese fir, Liriodendron chinense, and Populus. In the treatment group (the mixture of B. velezensis SMXJ16 bacterial suspension and SMCG1#C spore liquid), the Chinese fir anthracnose fungus could not infect at all, and no lesions were formed on the leaves of the host plants (Chinese fir, Liriodendron chinense, Populus). Only inoculating 10 μL of B. velezensis SMXJ16 bacterial suspension also did not form lesions on the leaves of the host plants (Chinese fir, Liriodendron chinense, Populus). Thus, it can be seen that the co-inoculation of B. velezensis SMXJ16 bacterial suspension and SMCG1#C spore liquid completely inhibited the infection of Chinese fir anthracnose fungus and the development of lesions, and the B. velezensis SMXJ16 bacterial suspension itself was harmless to plants.

[0070] It can be seen from Figure 6 As can be seen from C, in the control group of the treatment experiment (first inoculating SMCG1#C spore liquid and then inoculating 10 μL of PDA liquid medium after 3 days), obvious lesions were formed on the leaves of the host plants (Chinese fir, Liriodendron chinense, Populus). In the treatment group (first inoculating SMCG1#C spore liquid and then inoculating 10 μL of B. velezensis SMXJ16 bacterial suspension after 3 days), obvious lesions were also formed on the leaves of the host plants (Chinese fir, Liriodendron chinense, Populus) by the Chinese fir anthracnose fungus, and there was no obvious difference in the statistical data of the lesions between the control group and the treatment group. However, only inoculating 10 μL of B. velezensis SMXJ16 bacterial suspension still did not form lesions on the leaves of the host plants (Chinese fir, Liriodendron chinense, Populus). Thus, it can be seen that the B. velezensis SMXJ16 bacterial suspension could not inhibit the expansion of lesions on the host plants after first inoculating SMCG1#C spore liquid, and had no obvious therapeutic effect on anthracnose, but the B. velezensis SMXJ16 bacterial suspension itself was harmless to plants.

[0071] Example 8

[0072] The antagonistic effect of the said Bacillus bacterial suspension against four indicator pathogenic bacteria.

[0073] The biocontrol bacterium B. velezensis SMXJ16 was co-cultured with the aforementioned four indicator pathogenic bacteria to determine whether it had biocontrol potential. It included the following steps:

[0074] Indicator pathogens: Agrobacterium tumefaciens, Xanthomonas Campestris pv. citri, Erwinia carotovora subsp. carotovora Dye, Ralstonia solanacearum; Biocontrol strain: Biocontrol bacterium B. velezensis SMXJ16.

[0075] Obtain the Bacillus suspension according to Example 2. Then, use a pipette to add the indicator pathogenic bacterial solution to the PDA solid medium at a ratio of 1:100 to prepare a bacteria-containing plate. Then, take 20 μl of the biocontrol bacterial suspension and inoculate it in the center of the bacteria-containing plate. The bacteria-containing plate inoculated with LB liquid medium is used as a control, and each treatment is repeated 3 times. Observe whether a clear antagonistic zone is formed around the colonies of the biocontrol potential bacteria on the surface of the bacteria-containing plate (containing the pathogen). If not, it indicates that the biocontrol potential bacteria have no antagonistic effect on the indicator pathogen. If a clear antagonistic zone is formed, it indicates that the endophytic bacteria have an antagonistic effect on the pathogen.

[0076] From Figure 8 it can be seen that B. velezensis SMXJ16 produced clear antagonistic zones in the in vitro plate confrontation experiments with the four indicator pathogens of citrus huanglongbing bacteria, indicating that it has an obvious inhibitory effect on the four indicator pathogens.

[0077] Example 9

[0078] Effect of the inoculation of the described Bacillus strain on the content of citrus huanglongbing bacteria in living citrus seedlings.

[0079] The Bacillus bacterial suspension was obtained according to Example 2. In the treatment group, the biocontrol bacterium B. velezensis SMXJ 16 bacterial suspension was inoculated into healthy citrus, while in the control group, only the LB blank medium was inoculated. Then, Candidatus Liberibacter asiaticus was inoculated into the citrus seedlings in the treatment group and the control group. qPCR was used to continuously track the accumulation of the number of Candidatus Liberibacter asiaticus pathogens in the citrus seedlings in the treatment group and the control group. Sampling was performed once every 1-2 months after inoculating Candidatus Liberibacter asiaticus. Leaves of the treated plants in each group were collected (mixed sampling on the inoculated seedlings within the same treatment), and DNA was 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 primers used were the upstream primer CQULas F03 and the downstream primer CQULas R03. The base sequence of the upstream primer CQULas F03 was 5'-CAAGGAAAGAGCGTAGAA-3', and the base sequence of the downstream primer CQULas R03 was 5'-CCTCAAGATCGGGTAAAG-3'. The amplified specific gene sequence fragment of Candidatus Liberibacter asiaticus Asian species rpLJ / rpLL was 382 bp. The real-time fluorescence quantitative PCR reaction system was as follows: SYBR 10 μL, 0.4 μL of the upstream primer CQULas F03 was added, 0.4 μL of the downstream primer CQULas R03 was added, 7.2 μL of ddH2O, and 2.0 μL of template DNA. Each sample was set with 3 replicates, and cross-contamination was avoided during sample addition. The negative control was added last. The real-time fluorescence quantitative PCR reaction conditions were: 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 was automatically collected at the extension stage of each cycle; the final extension was at 72°C for 7 min. If, compared with the control group, the number of Candidatus Liberibacter asiaticus pathogens in the citrus seedlings did not show a significant increase after treatment with the biocontrol bacterium, it was judged that the biocontrol bacterium had a preventive effect on Candidatus Liberibacter asiaticus disease.

[0080] As Figure 9 shown, compared with the control group, in the treatment group inoculated with the biocontrol bacterium SMXJ16, the CT value showed a significant increase, indicating that the content of Candidatus Liberibacter asiaticus in the citrus seedlings in the treatment group was significantly lower than that in the control group. Thus, it can be seen that SMXJ16 has a significant inhibitory effect on the proliferation of Candidatus Liberibacter asiaticus in citrus, and it has a control effect on Candidatus Liberibacter asiaticus disease.

[0081] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. 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 they should all be covered by the scope of the claims of the present invention.

Claims

1. A Bacillus velezensis strain, characterized in that: The Varese Bacillus strain is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242289, the deposit date: October 22, 2024, and the deposit address: Wuhan University, Wuhan, China.

2. Use of the Bacillus velezensis strain according to claim 1 in preventing and treating fir anthracnose.

3. The application according to claim 2, characterized in that: The Bacillus Varezzia strain is capable of secreting protease, glucanase and chitinase.

4. The application according to claim 2, wherein: The fermentation filtrate of the Varese Bacillus subtilis strain can inhibit the spore germination and the development of appressorium of C. fir.

5. Use of the Bacillus velezensis strain according to claim 1 in inhibiting Curvularia muehlenbeckiae.

6. Use of the Bacillus velezensis strain according to claim 1 in preventing and treating citrus Huanglongbing.

7. The application according to claim 6, characterized in that: The pathogenic bacteria of citrus Huanglongbing include Candidatus Liberibacter asiaticus.

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