Bacillus altitudinis and application thereof in preventing and treating plant diseases

Abstract

The invention discloses bacillus altitudinis and an application of the bacillus altitudinis in prevention and treatment of plant diseases. The bacillus altitudinis CDJ1 is preserved in Guangdong Microbial Culture Collection Center on October 18, 2024, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 65289. The bacillus altitudinis CDJ1 disclosed by the invention can be used for effectively resisting pectobacterium taro, and in the formula, the bacillus altitudinis CDJ1 can be used for effectively resisting Desia Dabesii, Xanthomonas citrus pathogenic variants, Xanthomonas citrus mango pathogenic variants, Xanthomonas campestris, Xanthomonas sp., Xanthomonas sp., Xanthomonas sp. And Xanthomonas arborescens plum pathogenic variants, so that the bacillus altitudinis CDJ1 can be used for effectively resisting pectobacterium taro and the bacillus altitudinis CDJ1 can be used for effectively resisting pectobacterium taro. The composition has a good prevention and treatment effect on various plant diseases. When the bacillus altitudinis CDJ1 is prepared into a microbial agent, the use of chemical pesticides can be reduced, and the bacillus altitudinis CDJ1 has a wide application prospect in the field of agricultural biocontrol.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease control, and specifically, to a Bacillus altitudinis and its application in controlling plant diseases. Background Art

[0002] Diseases and pests can damage the roots, stems, leaves and fruits of crops, causing damage to plant growth, reduction in yield and even death. In global agricultural production, plant diseases are one of the important factors restricting the high and stable yield of crops, which can directly lead to the decline of crop yield and quality. Although traditional chemical control methods can control plant diseases to a certain extent, they also bring problems such as environmental pollution, ecological damage and food residues.

[0003] Using beneficial microorganisms and their metabolites or plant-derived bioactive substances to control plant diseases is a sustainable green method. Compared with chemical control, biological control has many advantages, such as being able to selectively target pests or pathogens, being degradable in the environment, not generating drug resistance, and not having a long-term impact on the ecosystem.

[0004] A large number of studies have shown that many biocontrol microorganisms can form a strong symbiotic relationship with host plants, promoting the growth and development of host plants, enhancing the tolerance of host plants to harsh environments, and improving the disease resistance of host plants. Among them, the biocontrol bacteria used more are bacteria of the genus Bacillus, which can produce two types of antagonistic factors, lipopeptide antibiotics and antagonistic proteins, and have an antibiotic effect on pathogenic bacteria. Bacillus has been proven to be effective against a variety of plant pathogens and can be used as a plant growth promoter and systemic resistance inducer. The main species include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, etc. Bacillus has a wide antibacterial range and has achieved remarkable results in the control of a variety of plant diseases due to its outstanding advantages, and is widely used in the field of biocontrol. For example, Chinese invention patent CN109136157A discloses that Bacillus velezensis can be used to prevent rice blast, Chinese invention patent CN102367429A discloses that Bacillus subtilis can be used to control tobacco anthracnose, and Chinese invention patent CN110669812A discloses that Bacillus amyloliquefaciens can be used to control cucumber anthracnose. However, there are many types of plant diseases, and it is still necessary to continue to search for biocontrol bacteria that can control more disease types at the same time, so as to improve plant disease resistance and promote healthy plant growth. Summary of the Invention

[0005] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a Bacillus altitudinis and its application in preventing and controlling plant diseases.

[0006] The first object of the present invention is to provide a Bacillus altitudinis CDJ1.

[0007] The second object of the present invention is to provide the application of the above-mentioned Bacillus altitudinis CDJ1 in treating and / or preventing plant diseases.

[0008] The third object of the present invention is to provide the application of the above-mentioned Bacillus altitudinis CDJ1 in preparing a biological agent for treating and / or preventing plant diseases.

[0009] The fourth object of the present invention is to provide the application of the above-mentioned Bacillus altitudinis CDJ1 in treating and / or preventing plant diseases caused by Xanthomonas.

[0010] The fifth object of the present invention is to provide the application of the above-mentioned Bacillus altitudinis CDJ1 in preparing a biological agent for treating and / or preventing plant diseases caused by Xanthomonas.

[0011] The sixth object of the present invention is to provide the application of the above-mentioned Bacillus altitudinis CDJ1 in preparing a fungicide.

[0012] The seventh object of the present invention is to provide a biocontrol agent.

[0013] The eighth object of the present invention is to provide a method for treating and / or preventing plant diseases.

[0014] The present invention claims the following:

[0015] A Bacillus altitudinis CDJ1, which was deposited at the Guangdong Provincial Culture Collection of Microorganisms on October 18, 2024, with the deposit number GDMCC NO: 65289.

[0016] The application of the above-mentioned Bacillus altitudinis CDJ1 in treating and / or preventing plant diseases, wherein the plant diseases include one or more of soft rot of taro, citrus canker, mango angular leaf spot, black rot of cruciferous vegetables, tomato scab, gummosis of sugarcane, bacterial wilt of walnut, and bacterial leaf spot of plum.

[0017] Use of the above-mentioned Bacillus altitudinis CDJ1 in the preparation of a biological agent for treating and / or preventing plant diseases, wherein the plant diseases include one or more of soft rot of taro, citrus canker, mango angular leaf spot, black rot of cruciferous vegetables, tomato scab, gummosis of sugarcane, bacterial wilt of walnut, and bacterial leaf spot of plum.

[0018] Preferably, the soft rot of taro is caused by Pectobacterium colocasium and / or Dickeya fangzhongdai;

[0019] The citrus canker is caused by Xanthomonas citri pv. citri;

[0020] The mango angular leaf spot is caused by Xanthomonas citri pv. mangiferaeindicae;

[0021] The black rot of cruciferous vegetables is caused by Xanthomonas campestris pv. campestris;

[0022] The tomato scab is caused by Xanthomonas perforans;

[0023] The gummosis of sugarcane is caused by Xanthomonas axonopodis;

[0024] The bacterial wilt of walnut is caused by Xanthomonas euroxanthea;

[0025] The bacterial leaf spot of plum is caused by Xanthomonas arboricola pv. pruni.

[0026] Use of the above-mentioned Bacillus altitudinis CDJ1 in treating and / or preventing plant diseases caused by Xanthomonas.

[0027] Use of the above-mentioned Bacillus altitudinis CDJ1 in the preparation of a biological agent for treating and / or preventing plant diseases caused by Xanthomonas.

[0028] Preferably, the Xanthomonas includes one or more of Xanthomonas citri pv. citri, Xanthomonas citri pv. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea, and Xanthomonas arboricola pv. pruni.

[0029] The application of the above-mentioned Bacillus altitudinis CDJ1 in the preparation of a fungicide, wherein the fungicide is a fungicide for killing one or more of Pectobacterium colocasium, Dickeya fangzhongdai, Xanthomonas citri pv. citri, Xanthomonas citri pv. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea, and Xanthomonas arboricola pv. pruni.

[0030] A biocontrol agent comprising the above-mentioned Bacillus altitudinis CDJ1.

[0031] A method for treating and / or preventing plant diseases, comprising applying the above-mentioned Bacillus altitudinis CDJ1 and / or the biocontrol agent to plants.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention discloses a Bacillus altitudinis and its application in controlling plant diseases. The Bacillus altitudinis CDJ1 was deposited at the Guangdong Microbial Culture Collection Center on October 18, 2024, with the deposit number GDMCC NO: 65289 and the deposit address being the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. The Bacillus altitudinis CDJ1 of the present invention can effectively resist Pectobacterium carotovorum subsp. carotovorum, Dickeya fangzhongdai, Xanthomonas citri subsp. citri, Xanthomonas citri subsp. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis pv. vasculorum, Xanthomonas euroxanthea, and Xanthomonas arboricola pv. pruni, and has good control effects on soft rot of taro, citrus canker, mango angular leaf spot, black rot of cruciferous vegetables, tomato bacterial speck, sugarcane gummosis, walnut bacterial blight, and bacterial leaf spot of plum. Preparing the Bacillus altitudinis CDJ1 of the present invention into a microbial inoculant can reduce the use of chemical pesticides and has broad application prospects in the field of agricultural biological control. Brief Description of the Drawings

[0034] Figure 1 Figure 6 shows the colony morphology of strain CDJ1 on LB medium.

[0035] Figure 2 Figure 10 shows the Neighbor-Joining phylogenetic tree constructed based on the 16S rRNA gene of strain CDJ1.

[0036] Figure 3 Figure 14 shows the determination results of the antibacterial spectrum of Bacillus altitudinis CDJ1 against plant pathogens.

[0037] Figure 4 Figure 18 shows the control effect diagram of Bacillus altitudinis CDJ1 on citrus canker in detached leaves; a: control group, b: prevention group, c: treatment group. Detailed Embodiments

[0038] The following specific embodiments are used to further illustrate the present invention, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0040] LB liquid medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of water, pH = 7.0; sterilized at 121 °C for 20 min.

[0041] LB solid medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar, 1000 mL of water, pH = 7.0; sterilized at 121 °C for 20 min.

[0042] Screening of biocontrol strains in Example 1

[0043] 1. Leaf source

[0044] Citrus leaves collected from Zengcheng District, Guangzhou City, Guangdong Province on January 9, 2023.

[0045] 2. Strain screening

[0046] (1) Leaf collection

[0047] Collect leaves in the citrus orchard, 5 samples from each tree. Collect 3 trees from each plot, and record the sampling time, location and variety. The collected samples are stored at room temperature for bacterial isolation.

[0048] (2) Bacterial isolation

[0049] Plate dilution method: Weigh 3 g of leaves into a 50 mL centrifuge tube, surface disinfect with 1% sodium hypochlorite solution (v / v) and 75% ethanol solution (v / v), then add 27 mL of sterile water, and then shake in a shaker at 200 rpm and 28 °C. After 20 h, take it out and let it stand at room temperature for 10 min to prepare the original leaf bacterial suspension. Dilute this original solution stepwise to obtain dilution solutions with a total of 6 gradients of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Take 100 μL of each leaf bacterial suspension dilution solution and spread it evenly on LB and R2A plates, with 3 replicates for each gradient. Place the plates in an incubator at 28 °C and incubate for 48 h, then observe.

[0050] 3. Bacterial purification

[0051] Observe and pick out single colonies, streak purify on LB and R2A plates, incubate them upside down in an incubator at 28 °C, pick out single colonies after 12 h, and number them in sequence.

[0052] 4. Bacterial preservation

[0053] Inoculate the single colonies of the strain into LB and R2A liquid media, culture in a shaker at 28 °C and 180 rpm for 12 h, then pipette 1 mL of the bacterial liquid and mix it with 0.5 mL of 60% sterile glycerol solution (v / v), gently shake and mix well, and store at -80 °C for long term.

[0054] 5. Strain screening

[0055] The method of using the flat plate bacteriostatic circle to screen biocontrol strains is as follows:

[0056] The citrus canker pathogen (Xanthomonas citri pv. citri) and the candidate biocontrol strains were activated on LB plates and then inoculated into liquid LB medium and cultured until the OD 600 value reached 1.0. Using LB medium as the screening medium, add 49 mL of LB medium that has been fully dissolved by heating to a 50 mL centrifuge tube. When the temperature of the medium drops to about 50 °C, mix the citrus canker pathogen bacterial solution and the LB medium in a volume ratio of 1:49, pour it into a square dish (13 cm × 13 cm) and spread it flat. After cooling and solidifying, use a 5 mm punch to punch holes in the medium and use a sterile toothpick to pick out the agar blocks. Add 10 μL of the candidate bacterial solution to the holes, wait for the bacterial solution to dry, seal the plate, and culture it upside down in an incubator at 28 °C. After culturing for 24 h, observe whether there is a transparent bacteriostatic circle, take pictures and record the results.

[0057] Through the above steps for primary screening and re-screening, a strain with the best bacteriostatic effect was obtained, named CDJ1. The colony morphology of the CDJ1 strain is as Figure 1 shown. Observing the strain morphology, it was found that the CDJ1 strain grew rapidly on LB medium. Obvious colonies could be seen in the incubator at 28 °C in about 12 h. The colonies were white, opaque, nearly round, with a rough and dry surface and irregular edges. There was an obvious strange smell after the bacterial solution was fermented and cultured for 24 h.

[0058] Example 2 Identification of the 16S rRNA gene of the biocontrol strain

[0059] I. Experimental method

[0060] Use a pipette tip to pick a small amount of fresh colonies as a template, and use the bacterial 16S rRNA gene primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3) for PCR amplification to obtain the target fragment. The PCR reaction system is: 2×Taq PCR Mix 12.5 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, sterile water 9.5 μL, fresh colonies.

[0061] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, for 34 cycles; pre-extension at 72°C for 10 min, and storage at 16°C. After the reaction, the PCR products were all examined by 1% agarose gel electrophoresis. The PCR stock solution was sent to Sangon Biotech for sequencing. The sequencing results were subjected to BLAST alignment on the NCBI website, and a phylogenetic tree was constructed using MEGA 11 software to determine the genus and species of the closely related bacterial strains.

[0062] II. Experimental Results

[0063] The nucleotide sequence of the 16S rRNA gene of strain CDJ1 is shown in SEQ ID NO: 1. A phylogenetic tree was constructed by neighbor-joining analysis. The results showed that the 16S rRNA gene of strain CDJ1 had 99.24% similarity with Bacillus altitudinis and was on the same branch of the phylogenetic tree as Bacillus altitudinis ( Figure 2 ), so strain CDJ1 belongs to Bacillus altitudinis.

[0064] Strain CDJ1 was deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC NO: 65289, the deposit address being the 5th floor of Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, and the deposit date being October 18, 2024. The taxonomic name is Bacillus altitudinis.

[0065] Example 3 Determination of the Antibacterial Spectrum of Bacillus altitudinis CDJ1

[0066] I. Experimental Method

[0067] Single colonies of plant pathogenic bacteria (Table 1) were separately picked into LB medium, and at the same time, a single colony of Bacillus altitudinis CDJ1 (GDMCC NO: 65289) from Example 2 was picked into LB medium. After culturing in a shaker at 28°C and 180 rpm for 12 h, the concentration of the plant pathogenic bacteria suspension was adjusted to OD 600 = 1, and the concentration of the Bacillus altitudinis CDJ1 suspension was adjusted to OD 600 = 1. 1 mL of the plant pathogenic bacteria suspension was separately taken and thoroughly mixed with about 49 mL of LB solid medium, then poured into plates. Then, holes with a diameter of 8 mm were punched in the center of the LB plates, and 5 μL of the Bacillus altitudinis CDJ1 suspension was inoculated into the holes. Three replicates were made and placed in an incubator at 28°C. After culturing for 48 h, the presence or absence of inhibition zones was observed, the size of the inhibition zones was recorded, and the results were organized and photographed.

[0068] Table 1 Plant Pathogenic Bacteria

[0069]

[0070] Note: (1) The citrus canker pathogen was collected by our laboratory and identified as Xanthomonas citri pv. citri.

[0071] (2) Dickeya oryzeae EC1 was disclosed in PMID: 30336787.

[0072] (3) Dickeya fangzhongdai CL3 was disclosed in PMID: 35273588.

[0073] II. Experimental Results

[0074] The results of the antibacterial spectrum determination of Bacillus altitudinis CDJ1 are as Figure 3 shown in Table 1 and Table 2. The results indicate that Bacillus altitudinis CDJ1 has significant antagonistic effects against the pathogens of taro soft rot, citrus canker, mango angular leaf spot, cruciferous vegetable black rot, tomato scab, sugarcane gummosis, walnut bacterial wilt, and plum bacterial leaf spot.

[0075] Table 2 The diameter of the antibacterial circle of Bacillus altitudinis CDJ1 against plant pathogens

[0076]

[0077]

[0078] Example 4 Determination of the control effect of Bacillus altitudinis CDJ1 on citrus canker in detached leaves

[0079] I. Experimental Method

[0080] Pick a single colony of the citrus canker pathogen Xanthomonas citri pv. citri into LB medium, and at the same time pick a single colony of Bacillus altitudinis CDJ1 (GDMCC NO: 65289) into LB medium. After culturing in a shaker at 28 °C and 180 rpm for 24 h, adjust the concentration of the citrus canker pathogen Xanthomonas citri pv. citri bacterial solution to OD 600 = 1, and the concentration of the Bacillus altitudinis CDJ1 bacterial solution to OD 600 = 1.

[0081] Control group: Select the leaves of living plants with consistent growth status, puncture wounds with needles, spray and inoculate the citrus canker pathogen Xanthomonas citri pv. citri, with 3 replicates, bag them, and observe the disease occurrence of the leaves (canker spots, yellow halos) after growing for 14 d, take pictures and record.

[0082] Prevention group: Select the leaves of living plants with consistent growth status, puncture wounds with a needle, spray and inoculate with Bacillus altitudinis CDJ1, with 3 replicates, bag overnight, and then spray and inoculate with the pathogen of citrus canker, Xanthomonas citri pv. citri, 24 hours later. Observe the disease incidence of the leaves (canker spots, yellow halos) after growing for 14 days, take pictures and record.

[0083] Treatment group: Select the leaves of living plants with consistent growth status, puncture wounds with a needle, spray and inoculate with the pathogen of citrus canker, Xanthomonas citri pv. citri, with 3 replicates, bag overnight, and then spray and inoculate with Bacillus altitudinis CDJ1, 24 hours later. Observe the disease incidence of the leaves (canker spots, yellow halos) after growing for 13 days, take pictures and record.

[0084] The formula for calculating the control effect is as follows: (the number of diseased wounds in the control group - the number of diseased wounds in the treatment group) / the number of diseased wounds in the control group × 100%.

[0085] II. Experimental results

[0086] The schematic diagram of the control effect of Bacillus altitudinis CDJ1 on citrus canker is as Figure 4 shown. The results show that Bacillus altitudinis CDJ1 has a good control effect on citrus canker, and the control effect reaches 100%.

[0087] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Bacillus altitudinis CDJ1, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 18, 2024, with the deposit number GDMCC NO: 65289.

2. The use of Bacillus altitudinis CDJ1 according to claim 1 in treating and / or preventing plant diseases, characterized in that: The plant diseases include one or more of taro soft rot, citrus canker, mango angular spot, cruciferous vegetable black rot, tomato scab, sugarcane gummosis, walnut bacterial wilt and plum bacterial spot.

3. Use of Bacillus altitudinis CDJ1 according to claim 1 in the preparation of biological agents for treating and / or preventing plant diseases, characterized in that: The plant diseases include one or more of taro soft rot, citrus canker, mango angular spot, cruciferous vegetable black rot, tomato scab, sugarcane gummosis, walnut bacterial wilt and plum bacterial spot.

4. The use according to claim 2 or 3, characterized in that: The taro soft rot is caused by Pectobacterium colocasium and / or Dickeya fangzhongdai; The citrus canker is caused by Xanthomonas citri pv. citri; The mango angular spot disease is caused by Xanthomonas citripv. mangiferaeindicae; The black rot of cruciferous vegetables is caused by cruciferous black rot fungus (Xanthomonas campestrispv. campestris); The tomato scab disease is caused by Xanthomonas perforans; The sugarcane gummosis is caused by Xanthomonas axonopodis; The walnut bacterial wilt is caused by Xanthomonas euroxanthea; The plum bacterial spot disease is caused by Xanthomonas arborifolia pruni (Xanthomonas arborifolia pruni).

5. Use of Bacillus altitudinis CDJ1 according to claim 1 in treating and / or preventing plant diseases caused by Xanthomonas.

6. Use of Bacillus altitudinis CDJ1 according to claim 1 in the preparation of biological preparations for treating and / or preventing plant diseases caused by Xanthomonas.

7. The use according to claim 5 or 6, characterized in that: The xanthomonas include one or more of Xanthomonas citri pv. citri, Xanthomonas citripv. mangiferaeindicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea and Xanthomonas arboricolapv. pruni.

8. Use of Bacillus altitudinis CDJ1 according to claim 1 in the preparation of a fungicide, characterized in that: The fungicide is a fungicide that kills one or more of Pectobacterium colocasium, Dickeya fangzhongdai, Xanthomonas citripv. citri, Xanthomonas citri pv. mangiferae indicae, Xanthomonas campestris pv. campestris, Xanthomonas perforans, Xanthomonas axonopodis, Xanthomonas euroxanthea and Xanthomonas arboricola pv. pruni.

9. A biocontrol agent, characterized in that: The method comprises the Bacillus saltitudinis CDJ1 according to claim 1.

10. A method for treating and / or preventing plant diseases, characterized in that: Applying the Bacillus altitudinis CDJ1 of claim 1 and / or the biocontrol agent of claim 9 to plants.

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