A kind of Paenibacillus polymyxa J-04-06 and its use

Abstract

The present invention discloses a polymyxa Paenibacillus J‑04‑06 and its use, and belongs to the technical field of agricultural biological control. The polymyxa Paenibacillus (Paenibacillus polymyxa) J‑04‑06 strain is deposited in the China Microbiological Culture Collection Center, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation time of September 25, 2023, and a preservation number of CGMCC No. 28540. The polymyxa Paenibacillus (P. polymyxa) J‑04‑06 obtained by screening the present invention is found to have a good inhibitory effect on a variety of pathogens, especially on watermelon wilt and post-harvest citrus anthracnose.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and specifically relates to Paenibacillus polymyxa J-04-06 and a use thereof. Background Art

[0002] With the deepening of disease research in my country, biological control has received great attention, mainly through the screening of antagonistic microorganisms or genetic technology to construct microbial strains with antagonistic effects. The most studied microorganisms include bacteria, fungi, actinomycetes, etc. Among them, the bacteria are mainly Bacillus and Pseudomonas, and Paenibacillus, which is closely related to Bacillus, has also received great attention. Bacteria compete with pathogens for nutrients and ecological niches, or produce antibacterial substances to inhibit the growth of pathogens.

[0003] Bacillus has the advantages of fast reproduction, spore production, high effective viable count, and strong survival ability. The strong survival of Bacillus is mainly due to its multi-layer cell wall structure and stress-resistant spores. Except for Bacillus anthracis and Bacillus cereus, they are generally considered to be beneficial and safe to plants and the ecological environment. Therefore, a lot is known about the genus Bacillus, and the most common ones are Bacillus subtilis (B. subbilis) and Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus subtilis and Bacillus amyloliquefaciens can inhibit plant pathogens and promote plant root growth.

[0004] Paenibacillus polymyxa belongs to the rhizosphere bacteria and is used as a biological control agent for plant diseases. As a biocontrol bacterium, Paenibacillus polymyxa can not only directly secrete active metabolites to antagonize pathogens, but also play a disease-resistant effect through interactions with plants. Paenibacillus polymyxa can induce systemic resistance in plants, which can increase the activity of defense-related enzymes in plants. After the systemic resistance of plants is induced, they have broad-spectrum resistance. Tong Yunhui et al. found that the suspension of Paenibacillus polymyxa W3 strain and its filtrate can induce systemic resistance of tomato leaves to gray mold fungi, and Paenibacillus polymyxa W3 colonized in the rhizosphere or leaves can induce and activate plant systemic resistance and produce resistance to subsequent pathogen infection. In addition, the biocontrol Paenibacillus polymyxa can also provide nutrients to host plants through nitrogen fixation and phosphorus solubilization. Since Bacillus polymyxa can effectively prevent plant diseases caused by bacteria, fungi and nematodes, it is usually screened out and used to prepare biocontrol agents. However, most of the Bacillus polymyxa disclosed in the prior art have unsatisfactory control effects on diseases and pests. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a polymyxa bacillus J-04-06 and its use, which can effectively prevent and treat diseases caused by pathogens such as watermelon wilt and citrus anthracnose.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] A Paenibacillus polymyxa (Paenibacillus polymyxa) J-04-06, which is deposited in the China Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is September 25, 2023, and the preservation number is CGMCC No.28540.

[0008] Furthermore, the nucleotide sequence of 16S rDNA of the strain is shown in SEQ ID NO.1.

[0009] Furthermore, the suitable growth temperature of the bacteria is 33°C to 37°C, the optimal growth temperature is 35°C, the pH tolerance range is 1 to 10, and the optimal pH is 7.

[0010] The bacterial agent is prepared using the above-mentioned Bacillus polymyxa J-04-06.

[0011] Furthermore, the active ingredient in the bacterial agent is Bacillus polymyxa J-04-06, or its bacterial suspension, fermentation broth, fermentation product or secondary metabolite.

[0012] The application of the above-mentioned Paenibacillus polymyxa J-04-06 or bacterial agent in the preparation of medicine for preventing and treating watermelon wilt disease.

[0013] The use of the above-mentioned Bacillus polymyxa J-04-06 or bacterial agent in the preparation of drugs for preventing and treating citrus anthracnose.

[0014] The use of the above-mentioned Bacillus polymyxa J-04-06 or bacterial agent in the preparation of drugs for preventing and treating tobacco bacterial wilt.

[0015] . A fungicide, comprising the above-mentioned Bacillus polymyxa J-04-06, or a bacterial agent.

[0016] Furthermore, the fungicide can inhibit pathogens including Atractylodes macrocephala root rot, apple leaf spot pathogen, wheat fusarium rust, cucumber cotton rot, rice sheath blight, cabbage soft rot, kiwifruit canker and tobacco bacterial wilt pathogen.

[0017] Beneficial effects of the present invention:

[0018] The polymyxa (P. polymyxa) J-04-06 obtained by the present invention is screened and found to have a good inhibitory effect on a variety of pathogens, especially on watermelon wilt and post-harvest citrus anthracnose. Therefore, developing the P. polymyxa J-04-06 strain into a new microbial pesticide is expected to reduce the use of chemical pesticides and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the colony picture of P. polymyxa J-04-06;

[0020] Figure 2 The inhibitory effect of P. polymyxa J-04-06 on the growth of different pathogens;

[0021] Figure 3 This is the electrophoresis diagram of P.polymyxa J-04-06 in 16S rDNA PCR product; among them, band 1 is DNA Marker, and band 2 is 16S rDNA PCR product;

[0022] Figure 4 This is the 16SrDNA phylogenetic tree of strain P. polymyxa J-04-06. DETAILED DESCRIPTION

[0023] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0024] Example 1 Obtaining Paenibacillus polymyxa J-04-06

[0025] 1. Isolation of strains

[0026] 1. Collection of soil samples

[0027] Before sampling, you need to use a sampling shovel to remove the loose soil and weeds on the surface of the soil. If the soil sample is deep, you can use a soil drill to collect the soil sample. Sieve the collected soil sample through a 2mm soil sieve to remove fine roots, stones, insects and other debris in the soil. If the soil sample is very sticky, use tweezers to remove the debris in the sample. Put the collected soil sample into the prepared sterile self-sealing bag and kraft paper.

[0028] 2. Isolation and purification of strains

[0029] Using the dilution method, 2 g of soil was weighed and placed in a sterilized mortar. 10 mL of sterile water was added for thorough grinding. Then, the ground juice of 1 mL was taken and added to a test tube containing 9 mL of sterile water. After mixing evenly, the mixture was diluted to 10 -6 Concentration, 10 -4 , 10 -5 , 10 -6 The concentrated bacterial solution was kept in a water bath at 80°C for 30 minutes to kill most non-bacillus bacteria, and then coated on beef extract peptone solid culture medium plates, and the growth of strains on the plates after overnight culture was observed, and the colony morphology and colony color were observed. Colonies with different colony morphologies were picked and purified multiple times until a single colony appeared.

[0030] 3. Screening of antagonistic strains

[0031] (1) Initial screening of antagonistic Bacillus

[0032] Three plant pathogens in the laboratory, namely, wheat fusarium wilt, watermelon wilt, and tobacco brown spot pathogen, were used as target bacteria. After activation, they were inoculated on PDA plates and cultured at 28°C until they were full. A 5mm diameter pathogen block was made from the plant pathogen plate using a hole puncher and placed in the center of the pathogen screening plate. The purified Bacillus was picked up with an inoculation loop and inoculated on the four corners 3cm from the center of the plate. Three replicates were set for each treatment, and the antibacterial effect was observed after culturing at 28°C for 7 days. The strains that had a significant inhibitory effect on the growth of pathogens, and the edges of the inhibited pathogenic hyphae were flat and regular and the antagonistic effect was long-lasting were selected, and the colony diameter was recorded and the inhibition rate was calculated.

[0033]

[0034] Table 1 Screening of antagonistic strains

[0035]

[0036] Through preliminary screening, four strains with antagonistic effects were obtained, and the antibacterial effects of strains Q-01, Q-05, P-14, and J-04-06 on three pathogens were determined by the confrontation plate method. As shown in Table 1, the four antagonistic strains had obvious inhibitory effects on tobacco brown spot pathogen, watermelon wilt pathogen, and wheat fusarium, and the inhibitory effects were all above 42%.

[0037] (2) For strains that enter the secondary screening, follow the primary screening method:

[0038] Fungal pathogens: Use a 0.5 cm punch to punch the fungus cakes of apple leaf spot pathogen, cucumber cotton rot pathogen, citrus anthracnose pathogen, rice sheath blight pathogen, and Atractylodes macrocephala root rot pathogen. Set up 3 replicates for each treatment and culture at 25℃ for 3-5 days until the plant pathogens cover the entire plate. Record the colony diameter and calculate the inhibition rate.

[0039]

[0040] Bacterial pathogens: Take 5mL of liquid cultured for 18 hours of strains, kiwifruit canker pathogens and tobacco bacterial wilt pathogens (37℃, 180r / min), shake well in LB medium at 40℃, pour on a plate and blow dry. Then, take 5μL of liquid cultured for 18 hours of Paenibacillus polymyxa J-04-06 (37℃, 180r / min), drop it on a plate pre-placed with a sterile filter paper disc (7mm in diameter), and culture at 37℃ for 20 hours. Set up 3 replicates for each treatment, and culture at 37℃ for 20 hours. Record the colony diameter and transparent circle diameter, and calculate the size of the inhibition circle.

[0041] Table 2 Inhibitory effect of strain J-04-06 on different pathogens

[0042]

[0043]

[0044] From Table 2 and Figure 2 It can be seen that the antibacterial activity test results show that strain J-04-06 has different degrees of inhibitory effect on tobacco brown spot pathogen, watermelon wilt pathogen, Atractylodes root rot pathogen, apple leaf spot pathogen, wheat ergot pathogen, cucumber cotton rot pathogen, citrus anthracnose pathogen, rice sheath blight pathogen, cabbage soft rot pathogen, kiwi fruit canker pathogen and tobacco bacterial wilt pathogen.

[0045] II. Identification of J-04-06

[0046] 1. Morphological identification

[0047] After morphological observation of J-04-06, the morphology of the strain is as follows: the colony is white in color, with ridges around it that are higher than the middle part, shiny, and Gram-positive; the bacteria produce spores, are oval in shape, and the body is in the center.

[0048] 2. Physiological and biochemical identification

[0049] The strain J-04-06 was physiologically and biochemically identified with reference to the 9th edition of Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems (edited by Dong Xiuzhu, Science Press). The results are shown in Table 3.

[0050] Table 3J-04-06 Physiological and biochemical identification results

[0051]

[0052] Note: + indicates a positive reaction; - indicates a negative reaction.

[0053] 3. 16S rDNA sequence determination and analysis

[0054] (1) DNA extraction

[0055] Inoculate the purified single colonies of different morphologies into the bacterial suspension liquid culture medium, culture at 35°C overnight, draw 2 mL of culture medium into a sterile centrifuge tube, centrifuge at 13,000 r / min for 10 min, discard the supernatant, and collect the bacterial precipitate (2 to 3 times in total). Extract genomic DNA from the bacterial precipitate using a bacterial genomic DNA rapid extraction kit and follow the operating procedures.

[0056] (2) PCR amplification and phylogenetic tree construction

[0057] The DNA solution obtained in step (1) is used as an amplification template and PCR amplification is performed using bacterial universal primer 27F-1492R;

[0058] PCR amplification system (using Taq enzyme amplification): 25 μl system: 1 μl cDNA, 1 μl upstream primer F, 1 μl downstream primer R, 12.5 μl 2× Taq Master Mix, 9.5 μl ddH2O,

[0059] PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min (number of cycles: 35 cycles), termination extension at 72°C for 10 min, and 12°C ∞.

[0060] After 1% agarose gel electrophoresis, the PCR amplification products were sent to Bioengineering for sequencing. The 16S rDNA gene sequences were analyzed using the NCBI database, and the phylogenetic tree was constructed using the software MEGA 7.0. Figure 4 shown.

[0061] PCR amplification results are shown in Figure 3 The PCR product size was 1458 bp. After sequencing, the 16SrDNA sequence of strain J-04-06 was shown in SEQ ID NO. 1. After morphological characteristics, physiological and biochemical characteristics and 16SrDNA sequencing of strain J-04-06, it was identified that J-04-06 was Paenibacillus polymyxa.

[0062] The bacteria was isolated from the soil and cultured in beef extract peptone solid medium at 35°C for 3 days and cultured for several generations. There was no significant change in its culture characteristics, morphological characteristics, physiological and biochemical characteristics, and other biological characteristics, showing good biological trait stability. The strain was identified by 16S rDNA and named Paenibacillus polymyxa J-04-06. It was deposited in the China Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​September 25, 2023, with a deposit number of CGMCC No. 28540).

[0063] Example 2 Bactericidal activity of Paenibacillus polymyxa J-04-06

[0064] 1. Control of watermelon wilt by Paenibacillus polymyxa J-04-06

[0065] Test material: Watermelon seeds (Jingyu No. 2)

[0066] The isolated biocontrol bacteria were propagated in LB medium and diluted into a bacterial suspension of 109 CFU / mL for subsequent potted plant control efficacy tests.

[0067] The potted plant protection test was conducted in the greenhouse of the Institute of Plant Protection, Sichuan Agricultural University, with the temperature controlled at 28-35℃ and natural light. The watermelon seeds were surface sterilized with 75% alcohol for 5 minutes and washed with sterile water three times. The seeds were planted in disposable plastic cups containing mixed soil (110g of bottom soil / cup, 5×10 suspension of spores of watermelon wilt pathogen) and the mixture was stirred for 2-3 hours. 5 spores / g of the middle layer of fungus soil 45g / cup, surface soil 35g / cup), 5 spores / cup, biocontrol bacteria were used for root irrigation, 30mL of 10-fold diluted biocontrol bacteria solution was poured into each cup, 5 replicates were used for each treatment, and clean water treatment was used as the control. After the control was infected, the watermelon emergence rate, plant height and diseased plant rate were investigated, and the control effect of the biocontrol bacteria was calculated.

[0068]

[0069] Table 5 The protective effect of Paenibacillus polymyxa and its growth-promoting effect on watermelon

[0070]

[0071] Note: L: Bacillus polymyxa J-04-06 bacterial suspension; L×5: J-04-06 bacterial suspension diluted 5 times; L×10: J-04-06 bacterial suspension diluted 10 times

[0072] Statistical analysis was performed using IBM SPSS Statistics 25 data processing software, and Duncan's new multiple range method was used for difference significance analysis. Data in the same column with different lowercase letters indicate significant differences (P<0.05).

[0073] The test results (Table 4 and Table 5) show that after being treated with the bacterial suspension and sterile filtrate of Paenibacillus polymyxa J-04-06, the strain has a certain control effect on watermelon wilt. After treatment, the watermelon plants can be uniformly emerged and the plants grow robustly, while the occurrence of watermelon wilt can be effectively inhibited. The experimental results are shown in Table 5, among which the J-04-06 bacterial suspension has the best comprehensive performance, with a seedling rate of 95%, an increase of 18.75% over the control; the control effect on watermelon wilt is 59.86%.

[0074] 2. Control of postharvest citrus anthracnose with Paenibacillus polymyxa J-04-06

[0075] Stab inoculation: Take fresh citrus fruit, stab a wound (3 mm × 3 mm) at the waist of the fruit, and inoculate 20 μL of biocontrol bacteria suspension (1 × 10 9 CFU / mL), sterile water was used as blank control, and 20 μL of prochloraz (concentration 200 mg / L) was used as positive control. After inoculation, the fruit was placed in a plastic box at 25°C. After 24 hours, 20 μL of 10 4 Conidia suspension of anthracnose fungi with spores / mL was placed in a plastic box in a plastic bag (relative humidity about 95%) at 28°C for 24 hours. Observation began on the second day and was conducted once a day to investigate the fruit rot rate and disease index of citrus anthracnose and calculate the prevention effect. Each treatment was repeated 3 times for 15 fruits. Citrus fruit disease severity classification standard:

[0076] Table 6 Disease classification standards

[0077]

[0078]

[0079]

[0080]

[0081] Table 7 Statistics on the disease index and control effect of J-04-06 on post-harvest citrus anthracnose

[0082]

[0083] Note: CK: clean water; L: J-04-06 bacterial suspension stock solution

[0084] Statistical analysis was performed using IBM SPSS Statistics 25 data processing software, and Duncan's new multiple range method was used for difference significance analysis. Data in the same column with different lowercase letters indicate significant differences (P<0.05).

[0085] As shown in Table 7, after the citrus fruits were inoculated with puncture wounds and stored at room temperature for 20 days, the incidence of citrus anthracnose in the control fruits reached 93.3%, while the rotten fruit rate in the fruits treated with the suspension of Paenibacillus polymyxa J-04-06 was 43.3%. Although there was a significant difference between the protective effect of strain J-04-06 (52.1%) and prochloraz (85.7%), the rotten fruit rate and disease index were significantly lower than those of the control.

[0086] In summary, the polymyxa bacillus J-04-06 screened in the present application has excellent bactericidal efficacy, especially against citrus anthracnose and watermelon wilt.

[0087] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A polymyxa bacillus ( Paenibacillus polymyxa ) J-04-06, characterized in that, The strain is deposited in the China Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is September 25, 2023, and the preservation number is CGMCC No.28540.

2. A bacterial agent prepared by using the polymyxa bacillus J-04-06 described in claim 1, characterized in that: The active ingredient in the bacterial agent is Bacillus polymyxa J-04-06.

3. Use of the Paenibacillus polymyxa J-04-06 described in claim 1 or the bacterial agent described in claim 2 in the preparation of a medicament for preventing and treating watermelon wilt.

4. Use of the Paenibacillus polymyxa J-04-06 described in claim 1 or the bacterial agent described in claim 2 in the preparation of a medicament for preventing and treating citrus anthracnose.

5. Use of the Paenibacillus polymyxa J-04-06 described in claim 1 or the bacterial agent described in claim 2 in the preparation of a medicament for preventing and treating tobacco bacterial wilt.

6. A bactericide, characterized in that: It includes the Bacillus polymyxa J-04-06 described in claim 1, or the bacterial agent described in claim 2.

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