Bacillus velezensis zhu-12 and application thereof in inhibiting penaeus vannamei black spot caused by aspergillus fumigatus
By screening and identifying Bacillus Velezii zhu-12, the problem that there is no Bacillus in the existing technology to inhibit the pathogenic fungi of black spot disease of white shrimp was solved, and effective inhibition of Aspergillus fumigatus was achieved, significantly reducing the incidence of black spot disease of white shrimp.
Patent Information
- Application Number
- CN202510891207.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-09-30
AI Technical Summary
There is no biological control method in the prior art for using Bacillus to inhibit the black spot disease of white shrimp caused by pathogenic fungi such as Aspergillus.
Bacillus velezensis zhu-12 was screened out. The extracted Bacillus velezensis zhu-12 can effectively inhibit the growth of Aspergillus fumigatus. It was classified and named Bacillus velezensis and preserved in the China Center for Type Culture Collection.
The inhibition rate of Bacillus Velez zhu-12 against Aspergillus fumigatus exceeds 50%, significantly inhibiting the growth of pathogenic bacteria causing black spot disease of white shrimp, providing a theoretical basis for biological control.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus velez zhu-12 and its application in inhibiting black spot disease of whiteleg shrimp caused by Aspergillus fumigatus. Background Art
[0002] Penaeus vannamei is the most widely farmed shrimp species worldwide due to its large scale, high yield and economic value. However, its aquaculture is plagued by frequent disease outbreaks that cause rapid and large-scale mortality. Aspergillus ) Fungal infection causes whiteleg shrimp ( Litopenaeus vannamei, Black spot disease (Penaeus vannamei, a species of shrimp in the family Penaeidae, order Decapoda) causes the appearance of black mycelium on the body surface (especially the gills and body). Ulceration and necrosis of the infected areas can occur, and in severe cases, the carapace can be damaged, leading to decreased vitality, reduced feeding, and slowed swimming. Dissections may reveal black lesions in the hepatopancreas and muscle tissue. This disease is more common during the nursery and juvenile stages of Penaeus vannamei, resulting in high mortality and negatively impacting aquaculture profitability.
[0003] At present, the Aspergillus species known to cause black spot disease in white shrimp mainly include Aspergillus flavus ( Aspergillus flavus ), Aspergillus awamori ( Aspergillus awamori )、Aspergillus polymorpha( Aspergillus sydowii ) etc., there is no literature report on Aspergillus fumigatus causing black spot disease in whiteleg shrimp. Aspergillus fumigatus ) as a pathogenic bacterium causes great harm in the farming process of whiteleg shrimp. First, it can directly cause black spot disease in whiteleg shrimp; secondly, it can cause growth stunting, nutritional metabolism disorders, immune system collapse, etc., leading to decreased immunity and susceptibility to secondary bacterial infections or viral diseases.
[0004] Bacillus velez ( Bacillus velezensis), Firmicutes, Bacilales, Bacillaceae, Bacillus genus. It is a type of spore-forming Gram-positive bacteria. Most Bacillus spores have the advantages of broad antimicrobial spectrum, rapid growth, easy isolation and cultivation, strong stress resistance and high biosafety. Therefore, they are widely studied as probiotics in agriculture, food, industry, medicine, metallurgy, forestry, environmental protection and military. The application of Bacillus velez in aquaculture has attracted increasing attention. Its excellent antibacterial properties and high temperature resistance have brought many benefits to aquaculture. Bacillus velez can effectively inhibit the growth of various aquatic pathogens, such as Streptococcus and Vibrio, which are the main factors causing diseases in aquatic animals. By reducing the number of pathogens, the incidence and mortality of diseases can be reduced, and the breeding efficiency can be improved. During the growth process, Bacillus velez secretes a variety of bioactive substances, including enzymes, antimicrobial proteins, etc. These substances help to decompose organic matter and harmful substances in water bodies, thereby improving the water quality environment and providing a healthier living environment for aquatic animals.
[0005] Kondee Chauyod,Santi Rattanavarin,Pongsak Sarapukdee,SupanitPorntheeraphat,Kallaya Sritunyalucksana,Numfon Khemthongcharoen. Bacillus velezensis suppression on the growth of Vibrio parahaemolyticus The results showed that Bacillus velezensis could inhibit the growth of Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis disease in marine shrimp. Journal of AppliedAquaculture, 2022, 35(4): 1202-1216. ,KunsookC., Kanjanavas P.,Kompatiparn.,Leeruksakiat P. .Decreased Severity of AcuteHepatopancreatic Necrosis Disease in White Shrimp ( Litopenaeus vannamei ) byMixed Culture of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium.Tropical Life Sciences Research, 2023, 34(1): 85-98. In the study, the severity of acute hepatopancreatic necrosis of whiteleg shrimp (Penaeus vannamei) was reduced by mixed culture of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium. Xu Zhenquan, Han Guowei, Ren Jing, Zhang Zixuan, Sun Jingfeng, Han Zhuoran. Isolation and identification of Bacillus licheniformis from the intestine of Litopenaeus vannamei and its biological characteristics. Journal of Dalian Ocean University, 2024, (1): 74-82. In the study, Bacillus licheniformis RHVJ in the intestine of Litopenaeus vannamei was found to have an inhibitory effect on the growth of Vibrio parahaemolyticus and Aeromonas vermiformis.
[0006] Chinese patent CN111057659A discloses the preparation and use of a methylotrophic Bacillus LW-6 bacterial agent for the biological control of shrimp diseases. Chinese patent CN119662452A discloses a thermotolerant Bacillus pumilus P2-7 and its use in the prevention and control of shrimp aquaculture diseases. The thermotolerant Bacillus pumilus P2-7 described in this patent is effective in preventing and controlling Shewanella disease in shrimp and is also effective in preventing septicemia, acute hepatopancreatic necrosis, white spot syndrome, and infectious subcutaneous and hematopoietic necrosis in shrimp aquaculture.
[0007] However, the Bacillus mentioned in existing literature and patents mainly inhibits the growth of various aquatic pathogenic bacteria, and there is no report on the use of Bacillus for the biological control of black spot disease of white shrimp caused by pathogenic fungi such as Aspergillus. Summary of the Invention
[0008] In response to the deficiencies in the prior art, the present invention provides a Bacillus velez zhu-12 and its use in inhibiting black spot disease of whiteleg shrimp caused by Aspergillus fumigatus. The Bacillus velez zhu-12 of the present invention can effectively inhibit the growth of Aspergillus fumigatus, the pathogenic fungus that causes black spot disease of whiteleg shrimp.
[0009] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: A strain of Bacillus velez zhu-12, taxonomically designated as Bacillus velez Bacillus velezensis , deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is May 9, 2025, and the deposit number is CCTCC NO: M 20251007.
[0010] The invention relates to an application of the Bacillus Velezii zhu-12 in the preparation of a product for inhibiting the black spot disease of whiteleg shrimp caused by Aspergillus fumigatus.
[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention isolates Aspergillus fumigatus, namely strain XQM-1, from a sample of whiteleg shrimp with typical symptoms of black spot disease, verifies that Aspergillus fumigatus is the pathogen of black spot disease of whiteleg shrimp, and screens out a Bacillus velezinis, namely strain zhu-12, which can effectively inhibit the growth of strain XQM-1, thereby providing a theoretical basis for the biological control of black spot disease of whiteleg shrimp using Bacillus velezinis. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 This is a symptom diagram of black spot disease of whiteleg shrimp in Example 2; Figure 2 This is a microscopic observation image of strain XQM-1 in Example 2; In the figure, Figure 2 a and Figure 2 b correspond to before and after spore shedding, respectively, and the magnification is 400 times; Figure 3 This is a genetic evolution analysis diagram of strain XQM-1 in Example 2; Figure 4 The growth graph of strain XQM-1 on the PDA plate corresponding to strain zhu-12 in Example 2 and the PDA plate serving as a blank control (CK); Figure 5 This is the genetic evolution analysis diagram of strain zhu-12 in Example 3. DETAILED DESCRIPTION
[0013] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.
[0014] Example 1 Obtaining strain zhu-12 The Bacillus velezensis zhu-12 of the present invention was isolated from pig manure collected from a pig farm in Shanglin Town, Linqu County. The specific isolation method is as follows: 1. Pig manure collection: Three fresh pig manure samples were randomly collected from a pig farm in Shanglin Town, Linqu County; 2. Culture medium preparation: Prepare solid NA culture medium (dissolve 10 g peptone, 5 g beef extract powder, 5 g sodium chloride, and 15 g agar in 1000 mL distilled water, adjust the pH to 7, and sterilize at 120°C for 25 min). Pour the medium into sterilized culture dishes to make plates.
[0015] 3. Separation and purification: (1) Take 1 g of each of the three pig manure samples, add 10 mL of sterile water, and shake on a shaker to obtain a suspension (28°C, 120 rpm, 30 min); (2) Pipette 100 μL of the suspension and apply it to the NA medium plate using a spreader, and culture at 37°C for 24 h; (3) Select typical colonies (uniform turbid growth, forming round protrusions on the agar plate, with neat edges, smooth surface, moist, opaque gray-white colonies), pick colonies with a colony loop and streak on solid NA culture medium, and culture at 37°C for 24 h. Continue to select colonies and streak them one by one to obtain 6 pure culture strains, which are numbered as zhu-12, zhu-15, zhu-16, zhu-21, zhu-22, and zhu-31 respectively.
[0016] Example 2 Growth inhibition test on the pathogen of black spot disease of white shrimp 1. Isolation and purification of the pathogen of black spot disease of white shrimp: Select white shrimp samples with typical symptoms of black spot disease (black appearance on the shrimp surface, especially the gills and body) (see Figure 1 ), pick the hyphae, inoculate them into PDA medium, and culture them in a constant temperature box at 28℃ until the hyphae germinate. Select typical colonies that meet the morphological characteristics of Aspergillus and transfer them three times. After 7 days, use a puncher with an inner diameter of 0.5 cm to take a bacterial cake from the edge of the colony, transfer it to a PDA plate for purification and culture, and mark the purified strain as XQM-1. The strain with dense hyphae growth, regular colonies and no contamination on the PDA plate was stored in a 1.8mL centrifuge tube and stored in a refrigerator at 4℃.
[0017] The colonies of strain XQM-1 on PDA medium are white and fluffy at the beginning, gradually turning into dark smoke green, and the color deepens after maturity; the back is pale or light yellow; the conidiophores are upright, with the top swelled to form an apicocapsule, on which phialides grow clusters of conidia, which are mostly spherical or nearly spherical. The colony morphology is shown in Figure 2 .
[0018] 2. ITS sequence analysis and identification: Using the extracted genomic DNA of the strain as a template, amplification was performed using the fungal ITS sequence primers ITS1 (sequence shown in SEQ ID No. 1) and ITS4 (sequence shown in SEQ ID No. 2). The PCR product was run on agar gel. The relative position of the markers was used to preliminarily determine the size of the target fragment. The DNA fragment obtained by PCR was between 500 and 750 bp in length, similar to the Aspergillus ITS target fragment (see SEQ ID No. 3). The amplified PCR product was sent to BGI for DNA sequencing, and the determined base sequence was compared with GenBank by BLAST.
[0019] By Aspergillus fumigatus) gene sequences (MW604211.1, KX664376.1), the homology was 100%, and the strain XQM-1 could be identified as Aspergillus fumigatus ( Aspergillus fumigatus )(See Figure 3 ).
[0020] 3. Inoculation: Use a toothpick to pick up the 6 pure culture strains from Example 1 and place them in NA liquid culture medium. Shake thoroughly on a shaker to obtain a suspension (28°C, 120 rpm, 24 hours). Use an ultraviolet spectrophotometer to measure the absorbance at 600 nm (OD600), and then dilute until the bacterial count in the suspension is 10. 7 pieces / mL.
[0021] Prepare 7 PDA plates, labeled as PDA plates 1-7, and punch four holes on PDA plates 1-7 using the cross method. The distance between the center point and the hole is 2.5 cm. Inoculate the center of PDA plates 1-7 with a bacterial cake (0.5 cm) of the pathogenic bacteria strain XQM-1 of white spot disease of white shrimp. Inoculate the four holes of PDA plates 1-6 with the corresponding suspensions of 6 pure culture strains (the number of bacteria is 10 7 100 μL / mL) was inoculated into each well, and PDA plate #7 was used as a blank control (CK). After 4 days, the diameters of the XQM-1 bacterial cakes on PDA plates #1-7 were measured using the cross-hatch method. Three replicates were performed, and the average value was calculated.
[0022] 4. Result statistics: After 4 days, the growth graphs of strain XQM-1 on the PDA plate corresponding to strain zhu-12 and the PDA plate as blank control (CK) are shown in Figure 4 As shown in Table 1, the antibacterial rate calculated by the diameter of the bacterial cake of strain XQM-1 in PDA plates No. 1-7 is shown in Table 1. Figure 4 As can be seen from Table 1, the inhibition rate of strain zhu-12 against strain XQM-1 is >50%, and it can effectively inhibit the mycelial growth of XQM-1.
[0023] Table 1
[0024] Example 3 Morphology and 16S rDNA Identification of Strain zhu-12 Morphological identification of strain zhu-12 was performed using Gram staining: 1. Use an inoculating loop to dip sterile water onto a clean glass slide. Take a small amount of fresh culture of strain zhu-12 and make a uniform smear. Dry it by flame three times to fix it.
[0025] 2. Staining (1) Initial staining: Add crystal violet staining solution, stain for 1 minute, and wash with water.
[0026] (2) Mordanting: Add Gram's iodine solution, mordanting for 1 minute, and washing with water.
[0027] (3) Decolorization: Add decolorizing alcohol dropwise, decolorize for 30 seconds until the effluent is colorless, and then wash with water.
[0028] (4) Restaining: Add safranin restaining solution and restain for 1 minute. Rinse with water, let dry, and examine under an oil immersion lens.
[0029] Results: Gram-positive bacteria appear purple, while Gram-negative bacteria appear red. Microscopic observation revealed that strain zhu-13 was Gram-positive and spherical.
[0030] 16S rDNA sequence analysis and identification of strain zhu-12: The extracted strain genomic DNA was used as a template and bacterial 16S rDNA was used as a primer for amplification. The upstream primer was 27F (sequence shown in SEQ ID No. 4) and the downstream primer was 1492R (sequence shown in SEQ ID No. 5). The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles; and final extension at 72°C for 10 min.
[0031] PCR products were examined using 1.0% agarose gel electrophoresis. The relative position of the markers was used to preliminarily determine the size of the target fragment. The DNA fragment obtained by PCR was between 750 and 1500 bp in length. The 16S rDNA sequence is shown in SEQ ID NO. 6, which is similar in length to the target bacterial 16S rDNA fragment. The amplified PCR product was sent to BGI for DNA sequencing, and the determined base sequence was compared with GenBank by BLAST.
[0032] Through with Bacillus velezensis The gene sequence (MT634404.1) was compared and the homology was 100%, which could identify strain zhu-12 as Bacillus velezensis ( Bacillus velezensis )(See Figure 5 ).
[0033] The strain zhu-12 screened by the present invention is classified and named Bacillus velezensis Bacillus velezensis , deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is May 9, 2025, and the deposit number is CCTCC NO: M 20251007.
Claims
1. A Bacillus velezensis zhu-12, characterized in that The Velez Bacillus zhu-12 is classified as Velez Bacillus Bacillus velezensis , deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is May 9, 2025, and the deposit number is CCTCC NO: M 20251007.
2. Use of the Bacillus Velezii zhu-12 according to claim 1 in the preparation of a product for inhibiting black spot disease of whiteleg shrimp caused by Aspergillus fumigatus.