Novel bacillus and application thereof in prevention and treatment of bacterial diseases of crops

Abstract

The invention discloses a novel bacillus and application thereof in prevention and treatment of bacterial diseases of crops. The bacterial strain is a potential new species of bacillus, is named as Bacillus sp.B088, has been preserved in Guangdong Microbial Culture Collection Center on January 16, 2025, and has a preservation number of GDMCC No: 65817. The novel bacillus B088 disclosed by the invention can be used for efficiently inhibiting the growth of pathogenic bacteria of bacterial diseases of various crops in a broad-spectrum manner. Therefore, the novel bacillus B088 disclosed by the invention has good application potential in the aspect of preventing and treating bacterial diseases of crops.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a novel bacillus and application thereof in the prevention and treatment of bacterial diseases of crops. Background Art

[0002] Bacterial diseases of crops refer to a series of physiological lesions of cells and tissues caused by infection of pathogenic bacteria in crops. In recent years, due to frequent extreme climates and changes in crop planting structure, the outbreak trend of bacterial diseases of crops has become more and more serious, and the economic losses caused have increased year by year. According to incomplete statistics, the direct economic losses caused by bacterial diseases of crops in my country to agricultural production exceed tens of billions of yuan each year, posing a serious threat to my country's agricultural production and food security. To solve this problem, chemical control methods based on chemical preparations are widely used due to their low cost, wide control spectrum, and good effect. Among them, copper preparations and antibiotics are the main agents. However, due to the rapid release of copper ions in the environment, it is very easy to cause soil and water pollution, which in turn leads to excessive heavy metals in fruits and vegetables. At the same time, the large-scale use of antibiotics can cause bacterial resistance problems, resulting in a significant reduction in their efficiency. Therefore, the development of new economic and environmentally friendly bacterial disease control methods is of great significance for stabilizing agricultural production and ensuring food security.

[0003] There are many relationships between organisms in nature, including competitive relationships. Antagonism between bacteria is essentially a competitive relationship, which usually refers to a bacterium obtaining more resources and living space by producing biologically active substances (such as toxins, metabolites, enzymes, etc.) or other methods to inhibit or kill competitors. Compared with chemical control methods, the method of controlling bacterial diseases of crops by using competitive relationships between bacteria has the advantages of no residue, no pollution, and environmental friendliness, and has attracted the attention of more and more researchers. Among the many biocontrol bacteria that have been reported to antagonize bacterial diseases of crops, Bacillus is one of the most concerned groups. Bacillus can multiply in large numbers under suitable conditions, and it has strong adaptability and can survive and reproduce under a variety of extreme conditions. Therefore, it can function in a variety of complex environments. The Bacillus groups with biocontrol effects that are widely concerned at present mainly include Bacillus subtilis, Bacillus velez and Bacillus brevis. For example, Bacillus subtilis can prevent and control fungal diseases such as wilt, anthrax and blight, while Bacillus velez can prevent and control bacterial diseases such as bacterial wilt and leaf spot. However, there is no report on Bacillus with broad-spectrum control of bacterial diseases of crops such as canker, bacterial wilt, leaf gall and leaf spot. Summary of the invention

[0004] The first object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a potential new species of Bacillus sp. B088. The strain has been deposited in Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, deposit number is GDMCC No: 65817, deposit date is January 16, 2025.

[0005] The morphological characteristics of the Bacillus B088 are as follows: Gram staining is positive, and the single colony is nearly round, white, smooth, opaque, and has irregular edges.

[0006] The second object of the present invention is to provide the use of the novel Bacillus B088 in the preparation of drugs for preventing and treating ulcer disease, bacterial wilt, leaf gall disease and leaf spot disease.

[0007] The third object of the present invention is to provide a microbial preparation for preventing and treating ulcer disease, comprising the above-mentioned Bacillus B088 or its fermentation liquid as an active ingredient.

[0008] The fourth object of the present invention is to provide a microbial preparation for preventing and controlling bacterial wilt, which contains the above-mentioned Bacillus B088 or its fermentation liquid as an active ingredient.

[0009] A fifth object of the present invention is to provide a microbial preparation for preventing and treating leaf gall disease, comprising the above-mentioned Bacillus B088 or its fermentation liquid as an active ingredient.

[0010] The sixth object of the present invention is to provide a microbial preparation for preventing and controlling leaf spot disease, comprising the above-mentioned Bacillus B088 or its fermentation liquid as an active ingredient.

[0011] The seventh object of the present invention is to provide an active substance that inhibits the pathogen of ulcer disease, Xanthomonas citri subspecies citrus, the pathogen of bacterial wilt, Ralstonia solanacearum, the pathogen of leaf gall disease, and the pathogen of leaf spot disease, Pseudomonas syringae, which is derived from the fermentation broth of the above-mentioned Bacillus B088 and is used to prepare inhibitors, agents, etc. for preventing and treating ulcer disease, bacterial wilt disease, leaf gall disease and leaf spot disease.

[0012] The present invention also provides a method for preventing and controlling ulcer disease, bacterial wilt disease, leaf gall disease and leaf spot disease, which comprises spraying Bacillus B088 or its fermentation liquid to the area to be prevented and controlled.

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

[0014] The present invention reports for the first time a potential new species in the genus Bacillus, which has good control effects on canker, bacterial wilt, leaf gall and leaf spot. The first discovery of this strain enriches the microbial resources with biological control effects in my country. Its stable antibacterial effect, high efficiency and environmental friendliness have broad application prospects in the control of bacterial diseases of crops.

[0015] Bacillus sp.B088 has been deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit number is GDMCC No: 65817, and the deposit date is January 16, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the colony morphology of the new Bacillus B088 in TSA medium.

[0017] Figure 2 This is the phylogenetic tree of the new Bacillus sp. B088.

[0018] Figure 3 It is the inhibitory effect of the new Bacillus B088 on the ulcer pathogen Xanthomonas citri subsp. citri.

[0019] Figure 4 This is the inhibitory effect of the new Bacillus B088 on the bacterial wilt pathogen Ralstonia solanacearum.

[0020] Figure 5 It is the inhibitory effect of the new Bacillus B088 on the leaf gall pathogen Rhodococcus fascicularis.

[0021] Figure 6 This is the inhibitory effect of the new Bacillus B088 on the leaf spot pathogen Pseudomonas syringae. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] Example 1: Isolation and identification of novel Bacillus sp. B088

[0024] Select healthy fruit trees in the citrus orchard in Danxia Street, Renhua County, Shaoguan City, Guangdong Province (N 25°3′29″, E 113°45′18″), use sterile scissors to cut fresh citrus leaves and put them in a sterile sampling bag. The leaf samples were then stored in a 4°C sampling box. Bring the samples back to the laboratory, weigh 10g of leaves on the clean bench, add them to a 500mL Erlenmeyer flask containing 190mL of sterile PBS buffer (0.2mol / L, pH=7.0), shake well and place them in a 28°C constant temperature shaker, shaking at 200rpm for 30min. Place the flask in an ultrasonic oscillator, 40KHz, work for 8s, rest for 2s, and sonicate for 10min. Take 1mL of the eluate and add it to a test tube containing 9mL of PBS buffer, and dilute to 10 in sequence. -1 , 10 -2 , 10 -3 and 10 -4 , draw the original solution of eluent, 10 -1 , 10 -2 , 10 -3 and 10 -4 100 μL of each diluted sample was spread on TSA medium (Qingdao Haibo, catalog number HB0177) and placed in a 28°C biochemical incubator for 7 days. The colony size, color, dryness, smoothness, and whether a halo is produced were observed, and different single colonies were picked from the culture medium to a new TSA medium, and streaked multiple times until a pure culture was obtained. The obtained strains were numbered, mixed with 25% glycerol (v / v), and stored in an ultra-low temperature refrigerator, thereby obtaining strain B088.

[0025] The strain B088 was Gram-positive. The colony morphology of the strain after culturing in TSA medium for 3 days was as shown in the attached figure. Figure 1 As shown, a single colony is nearly round, 2 to 6 mm in diameter, white, with a smooth, opaque surface and irregular edges.

[0026] Example 2: 16S rRNA gene sequence analysis of novel Bacillus sp. B088

[0027] The HiPure bacterial DNA extraction kit (Cat. No. D3146-03, Guangzhou Meiji Biotechnology Co., Ltd.) was used to extract and purify the genomic DNA of strain B088. Using this DNA as a template, the 16S rRNA gene sequence was amplified using the bacterial 16S rRNA gene amplification universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). Take an appropriate amount of PCR product for gel electrophoresis detection. When the presence of the target band is confirmed, the remaining PCR product is sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1, with a length of 1420bp. The 16S rRNA gene sequence of strain B088 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results show that strain B088 is similar to the model strain Bacillus rugosus SPB7 T The 16S rRNA gene sequence of Bacillus tequilensis KCTC13622 (accession number, JABUXO010000041) has the highest similarity of 99.93%; T (Accession No. AYTO01000043), Bacillus halotolerans ATCC 25096 T (Accession number, LPVF01000003), Bacillus cabrialesii TE3 T (Accession No. MK462260), Bacillus inaquosorum KCTC 13429 T (Accession No., AMXN01000021) and Bacillus stercoris JCM 30051 T The 16S rRNA gene sequence of strain B088 was the second highest, both at 99.86%; the similarity with the 16S rRNA gene sequence of other model strains in the genus Bacillus was less than 99.70%. These analysis results showed that strain B088 had a similarity with the model strain Bacillus rugosus SPB7 T However, since the similarity between strain B088 and multiple model strains was significantly higher than the species classification threshold of 98.65%, the accurate taxonomic status of strain B088 could not be determined by 16S rRNA gene analysis.

[0028] Example 3: Analysis of the complete gene sequence of the novel Bacillus sp. B088

[0029] The novel Bacillus B088 genome extracted in Example 2 was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for genome sequencing. By constructing an Illumina PE (~400bp) library, the Illumina Novasek platform was used for sequencing. After the original Reads were quality-checked, the high-quality data obtained was spliced ​​with SPAdes v3.15.3 for genome sequence splicing. According to the parameters of splicing contig coverage ≥100 and length ≥500bp, low-quality contig sequences were filtered and removed. After removing the low-quality sequences, the strain B088 genome was quality-checked using the software CheckM v1.1.3. The results showed that the Bacillus B088 genome integrity was 99.75% and the contamination was 0.25%. According to the criteria of integrity ≥95% and contamination ≤5% for high-quality genomes, the quality of the strain B088 genome is high, which can ensure the accuracy of subsequent analysis. The strain B088 genome was analyzed using the software QUAST v5.0.2. The results showed that the genome sequence of strain B088 contained 25 contigs, the total genome length was 3980254bp, the N50 length was 294377bp, and the G+C content of genomic DNA was 43.82%.

[0030] The 70% digital DNA-DNA hybridization value (dDDH) is the gold standard for the classification and identification of prokaryotic species. The online tool Genome-to-Genome Distance Calculator 3.0 (https: / / ggdc.dsmz.de / ggdc.php#) was used to calculate the distance between the genome sequence of strain B088 and the closest model strain Bacillus rugosus SPB7. T The dDDH value between genome sequences was 60.7%, which was lower than the 70% species classification standard, indicating that strain B088 did not belong to the species Bacillus rugosus. The phylogenetic tree of strain B088 and closely related reference strains was constructed using the software UBCG v3.0. The results are shown in the attached figure. Figure 2As shown in the figure, strain B088 formed an independent phylogenetic branch, and the support bootstrap value was 100, indicating that strain B088 has an independent taxonomic status. Based on the results of dDDH and phylogenetic analysis, strain B088 is a potential new species of the genus Bacillus, so it was named Bacillus sp. B088. The strain has been deposited in Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Postal Code: 510070, the deposit number is GDMCC No: 65817, and the deposit date is January 16, 2025.

[0031] Example 4: Identification of the inhibitory effect of the novel Bacillus sp. B088 on ulcer pathogens

[0032] The double-layer agar diffusion method was used to determine the inhibitory effect of strain B088 on the pathogen of citrus canker, Xanthomonas citri subsp. citri (Xcc). Xcc and strain B088 were inoculated into NB medium (Qingdao Haibo, catalog number HB0108) for activation, and the fermentation broth was collected after 24 hours of culture. Prepare a bottom NA medium (Qingdao Haibo, catalog number HB0109) of moderate thickness. Place Oxford cups on the bottom NA medium, 3 per plate. Take 1 mL of Xcc fermentation broth and add it to 300 mL NA medium. The final concentration of Xcc is 10 6 ~10 7 cfu / mL, and after solidification, take out the Oxford cup, which is the upper culture medium. Take 30 μL of the fermentation liquid of strain B088 and add it to the well, culture it at 28℃ for 36h, and use a ruler to measure the diameter of the inhibition zone.

[0033] As attached Figure 3 As shown, after adding the novel Bacillus sp. B088, Xcc formed a clear transparent circle around the hole, with diameters of 15.3, 14.6 and 14.2 mm, respectively, and an average of 14.7 mm. The results showed that the novel Bacillus sp. B088 had a significant inhibitory effect on the growth of Xcc, the pathogen of ulcer disease.

[0034] Example 5: Identification of the inhibitory effect of the novel Bacillus sp. B088 on bacterial wilt pathogens

[0035] The double-layer agar diffusion method was used to determine the inhibitory effect of strain B088 on bacterial wilt pathogens. The bacterial wilt pathogen Ralstonia solanacearum GMI 1000 and strain B088 were inoculated into NB medium for activation, and the fermentation broth was collected after 24 hours of culture. Prepare a bottom NA medium with a moderate thickness. Place Oxford cups on the bottom NA medium, 3 per plate. Take 1mL of GMI1000 fermentation broth and add it to 300mL NA medium. The final concentration of GMI 1000 is 10 6 ~107 cfu / mL, and after solidification, take out the Oxford cup, which is the upper culture medium. Take 30 μL of the fermentation liquid of strain B088 and add it to the well, culture it at 28℃ for 36h, and use a ruler to measure the diameter of the inhibition zone.

[0036] As attached Figure 4 As shown, after adding strain B088, the bacterial wilt pathogen Ralstonia solanacearum GMI 1000 formed a clear transparent circle around the hole, with diameters of 15.5, 15.7 and 14.6 mm, respectively, and an average of 15.2 mm. The results show that strain B088 has a significant inhibitory effect on the growth of bacterial wilt pathogen Ralstonia solanacearum GMI 1000.

[0037] Example 6: Identification of the inhibitory effect of the novel Bacillus sp. B088 on leaf gall pathogens

[0038] The double-layer agar diffusion method was used to determine the inhibitory effect of strain B088 on leaf gall pathogens. The leaf gall pathogen Rhodococcus fascicularis GDMCC 1.839 and strain B088 were inoculated into NB medium for activation, and the fermentation broth was collected after 24 hours of culture. Prepare a bottom NA medium with moderate thickness. Place Oxford cups on the bottom NA medium, 3 per plate. Take 1 mL of GDMCC 1.839 fermentation broth and add it to 300 mL of NA medium. The final concentration of GDMCC 1.839 is 10 6 ~10 7 cfu / mL, and after solidification, take out the Oxford cup, which is the upper culture medium. Take 30 μL of the fermentation liquid of strain B088 and add it to the well, culture it at 28℃ for 36h, and use a ruler to measure the diameter of the inhibition zone.

[0039] As attached Figure 5 As shown in the figure, after adding strain B088, the leaf gall pathogen Rhodococcus fascicularis GDMCC 1.839 formed a clear transparent circle around the hole, with diameters of 14.7, 14.6 and 14.9 mm, respectively, and an average of 14.7 mm. The results showed that strain B088 had a significant inhibitory effect on the growth of leaf gall pathogen Rhodococcus fascicularis GDMCC 1.839.

[0040] Example 7: Identification of the inhibitory effect of the novel Bacillus sp. B088 on leaf spot pathogens

[0041] The double-layer agar diffusion method was used to determine the inhibitory effect of strain B088 on leaf spot pathogens. The bacterial leaf spot pathogen Pseudomonas syringae GDMCC 1.330 and strain B088 were inoculated into NB medium for activation, and the fermentation broth was collected after 24 hours of culture. Prepare a bottom NA medium with moderate thickness. Place Oxford cups on the bottom NA medium, 3 per plate. Take 1 mL of GDMCC 1.330 fermentation broth and add it to 300 mL of NA medium. The final concentration of GDMCC 1.330 is 10 6 ~10 7 cfu / mL, and after solidification, take out the Oxford cup, which is the upper culture medium. Take 30 μL of the fermentation liquid of strain B088 and add it to the well, culture it at 28℃ for 36h, and use a ruler to measure the diameter of the inhibition zone.

[0042] As attached Figure 6 As shown, after adding strain B088, the pathogen of fruit tree canker Pseudomonas syringae GDMCC 1.330 formed a clear transparent circle around the hole, with diameters of 12.2, 11.9 and 12.6 mm, respectively, and an average of 12.3 mm. The results showed that strain B088 had a significant inhibitory effect on the growth of leaf spot pathogen Pseudomonas syringae GDMCC 1.330.

[0043] The above results show that the new Bacillus sp. B088 is a potential new species of the genus Bacillus, which has the ability to significantly inhibit the growth of bacterial pathogens of crops such as the ulcer pathogen Xcc, the bacterial wilt pathogen Ralstonia solanacearum GMI 1000, the leaf gall pathogen Rhodococcus fascicularis GDMCC 1.839 and the leaf spot pathogen Pseudomonas syringae GDMCC 1.330. It is a broad-spectrum and highly efficient biocontrol functional strain, which can be used for the prevention and control of crop ulcers, bacterial wilt, leaf gall and leaf spot through direct spraying, or used in the production of microbial fertilizers, pesticides, microbial agents, etc., and then applied to the prevention and control of the above-mentioned bacterial diseases of crops.

[0044] SEQ ID NO.1

[0045]

[0046]

[0047] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Bacillus sp. B088, deposit number: GDMCC No: 65817.

2. Use of the Bacillus B088 or its fermentation liquid according to claim 1 in the preparation of a medicament for preventing and treating ulcer disease, bacterial wilt, leaf gall disease and leaf spot disease.

3. A microbial preparation for preventing and treating ulcer disease, characterized in that: Contains the Bacillus B088 or its fermentation liquid as claimed in claim 1 as an active ingredient.

4. A microbial preparation for preventing and treating bacterial wilt, characterized in that: Contains the Bacillus B088 or its fermentation liquid as claimed in claim 1 as an active ingredient.

5. A microbial preparation for preventing and treating leaf gall disease, characterized in that: Contains the Bacillus B088 or its fermentation liquid as claimed in claim 1 as an active ingredient.

6. A microbial preparation for preventing and controlling leaf spot disease, characterized in that: Contains the Bacillus B088 or its fermentation liquid as claimed in claim 1 as an active ingredient.

7. An active substance for inhibiting canker, bacterial wilt, leaf gall and leaf spot, characterized in that: The active substance is the fermentation broth of Bacillus B088 according to claim 1.

8. A method for preventing and treating canker, bacterial wilt, leaf gall and leaf spot, characterized in that: The method comprises spraying the Bacillus B088 or its fermentation liquid described in claim 1 to the area to be controlled.

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