Application of Paenibacillus polymyxa ZJU11 and its intracellular extracts in controlling mango anthracnose

Abstract

The present invention relates to the technical field of biocontrol bacteria, and in particular to the application of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose. The present invention provides the application of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose. The taxonomic nomenclature of the Paenibacillus polymyxa ZJU11 is Paenibacillus polymyxa ZJU11, with a deposit number of CCTCC NO: M 20241847. The polymyxa bacillus ZJU11 of the present invention is a biocontrol strain isolated from the root system of watermelon and has good antagonistic effect on mango anthracnose. The intracellular extract of the strain has good antagonistic effect on the dominant pathogens of mango postharvest anthracnose and inhibits mango anthracnose, and can be used to prevent and control mango postharvest anthracnose and alleviate the symptoms of mango postharvest anthracnose.

Description

Technical Field

[0001] The invention relates to the technical field of biocontrol bacteria, and in particular to application of Paenibacillus polymyxa ZJU11 and intracellular extracts thereof in preventing and controlling mango anthracnose. Background Art

[0002] Mango fruit anthracnose (MFA) is a fungal disease caused by the anthracnose pathogen. The anthracnose pathogens identified in mangoes include the Colletotrichum gloeosporioides complex and the Colletotrichum oxysporum complex. 暹罗炭疽菌 )、Fruit Anthrax( 炭疽菌属 果生炭疽菌 ) and Asian anthrax ( 亚洲炭疽菌 ) are the dominant pathogens. The mango anthracnose caused by these three dominant pathogens develops quickly, has strong pathogenicity, is difficult to prevent and control, and has a high incidence rate.

[0003] The control of mango anthracnose is mainly based on chemical prevention and control. Commonly used chemical agents include prochloraz, azoxystrobin, thiophanate-methyl, bromothionil, etc. Long-term use of chemical agents not only causes environmental pollution and damages human health, but also causes a series of problems such as drug resistance of pathogens and outbreaks of diseases. Therefore, the research and development of efficient, safe, and environmentally friendly green preservation and fresh-keeping technologies has important theoretical and practical significance for reducing the occurrence of mango anthracnose and increasing farmers' income.

[0004] Biological control is safe, broad-spectrum, sustainable and green, and plays an important role in the prevention and control of mango anthracnose. Among the currently developed biological pesticides, Paenibacillus polymyxa ( 多粘类芽孢杆菌 ) is a type of PGPRR strain with great application potential in the field of agricultural production. It is reported that Bacillus polymyxa can significantly inhibit plant diseases such as tomato bacterial wilt, pepper wilt, cotton wilt, rapeseed rot, and black pine root rot. However, different Bacillus polymyxa have different and specific effects on the prevention and control of plant diseases, and there are large differences in both the antibacterial spectrum and the antagonistic effect of bacteria. Moreover, the previous research on Bacillus polymyxa was limited to the antibacterial properties of its fermentation supernatant and the nutritional and spatial competition of the bacteria, and no research on its intracellular extracts has been reported. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose. The Paenibacillus polymyxa ZJU11 provided by the present invention is a biocontrol strain with good antagonistic effect on mango anthracnose, and the intracellular extract of the strain has good antagonistic effect on the dominant pathogens of postharvest anthracnose of mango and inhibits mango anthracnose.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an application of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose. The taxonomic nomenclature of the Paenibacillus polymyxa ZJU11 is 多粘类芽孢杆菌 ZJU11, the deposit number is CCTCC NO: M20241847.

[0008] Preferably, the mango anthracnose is postharvest mango anthracnose.

[0009] Preferably, the pathogens of mango anthracnose include: one or more of Siamese anthracnose, fruit anthracnose and Asian anthracnose.

[0010] The present invention provides a method for preventing and controlling postharvest anthracnose of mangoes, comprising the following steps:

[0011] The harvested mangoes are immersed in a biocontrol agent, or the biocontrol agent is sprayed on the surface of the harvested mangoes; the biocontrol agent comprises an intracellular extract of Paenibacillus polymyxa ZJU11; the preparation method of the intracellular extract comprises the following steps: using methanol to extract Paenibacillus polymyxa ZJU11 to obtain an alcohol extract; drying the alcohol extract to obtain the intracellular extract; the taxonomic nomenclature of Paenibacillus polymyxa ZJU11 is 类芽孢杆菌属 多粘(的) ZJU11, the deposit number is CCTCC NO: M 20241847.

[0012] Preferably, the concentration of the intracellular extract in the biocontrol agent is ≥1 mg / mL.

[0013] Preferably, the soaking time is 5 to 10 min.

[0014] Beneficial effects:

[0015] The present invention provides an application of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose. The taxonomic nomenclature of the Paenibacillus polymyxa ZJU11 is 多粘类芽孢杆菌 ZJU11, the deposit number is CCTCC NO: M20241847. The polymyxa bacillus ZJU11 of the present invention is a biocontrol strain isolated from the root system of watermelon and has good antagonistic effect on mango anthracnose. The intracellular extract of the strain has good antagonistic effect on the dominant pathogens of mango postharvest anthracnose and inhibits mango anthracnose, and can be used to prevent and control mango postharvest anthracnose and alleviate the symptoms of mango postharvest anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 The colony characteristics and scanning electron micrograph of Paenibacillus polymyxa ZJU11;

[0018] Figure 2 This is the phylogenetic tree of Paenibacillus polymyxa ZJU11 constructed based on 16S rRNA;

[0019] Figure 3 The results of the in vitro antibacterial activity test of intracellular extracts of Paenibacillus polymyxa ZJU11;

[0020] Figure 4 These are the results of the experiment on the control of anthracnose of “Jinhuang” silvergrass by Paenibacillus polymyxa ZJU11.

[0021] Biological Deposit Description

[0022] Paenibacillus polymyxa ZJU11, classified as 多粘类芽孢杆菌 ZJU11 was deposited in the China Center for Type Culture Collection (CCTCC) on August 26, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20241847.

[0023] Siamese anthrax HNI2, taxonomic name 暹罗炭疽菌 HNI2 was deposited in the China Center for Type Culture Collection (CCTCC) on August 28, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20241875.

[0024] Colletotrichum niger HNI5, classified as 果生炭疽菌 HNI5 was deposited in the China Center for Type Culture Collection (CCTCC) on August 28, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20241874.

[0025] Asian anthrax HNI6, named 亚洲炭疽菌 HNI6 was deposited in the China Center for Type Culture Collection (CCTCC) on August 28, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20241873. DETAILED DESCRIPTION

[0026] The present invention provides an application of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose. The taxonomic nomenclature of the Paenibacillus polymyxa ZJU11 is 多粘类芽孢杆菌 ZJU11, with a deposit number of CCTCC NO: M20241847. The polymyxa bacillus ZJU11 provided by the present invention is a biocontrol strain isolated from the root system of watermelon and has good antagonistic effect on mango anthracnose. The colonies are white, colloidally translucent, raised, with irregular edges, short rod-shaped cells, and the surface is full of wrinkles. The intracellular extract of the strain has good antagonistic effects on the dominant pathogens of mango postharvest anthracnose and inhibiting mango anthracnose, and can be used to prevent and control mango postharvest anthracnose and alleviate the symptoms of mango postharvest anthracnose.

[0027] As an embodiment, the mango anthracnose is postharvest mango anthracnose.

[0028] As an embodiment, the pathogens of mango anthracnose include: one or more of Siamese anthracnose, fruit anthracnose and Asian anthracnose.

[0029] As an embodiment, the mango varieties include but are not limited to 'Golden Glory' mango.

[0030] The present invention provides a method for preventing and controlling postharvest anthracnose of mangoes, comprising the following steps:

[0031] The harvested mangoes are immersed in a biocontrol agent, or the biocontrol agent is sprayed on the surface of the harvested mangoes; the biocontrol agent comprises an intracellular extract of Paenibacillus polymyxa ZJU11; the preparation method of the intracellular extract comprises the following steps: using methanol to extract Paenibacillus polymyxa ZJU11 to obtain an alcohol extract; drying the alcohol extract to obtain the intracellular extract; the taxonomic nomenclature of Paenibacillus polymyxa ZJU11 is 类芽孢杆菌属 多粘(的) ZJU11, the deposit number is CCTCC NO: M 20241847.

[0032] As an embodiment, the alcohol extraction temperature is 28°C, the rotation speed is 200 r / min, and the extraction time is 24 h.

[0033] As an embodiment, the concentration of the intracellular extract in the biocontrol agent is ≥1 mg / mL. As another embodiment, the concentration of the intracellular extract in the biocontrol agent is 1-50 mg / mL.

[0034] As an embodiment, the biocontrol agent can be prepared by dissolving or dispersing the intracellular extract. As an embodiment, the solvent of the biocontrol agent can be methanol. As an embodiment, the biocontrol agent dispersant can be water.

[0035] As an implementation mode, the soaking time is 5 to 10 minutes.

[0036] To further illustrate the present invention, the application of the Bacillus polymyxa ZJU11 and its intracellular extracts provided by the present invention in preventing and controlling mango anthracnose is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 Identification of Paenibacillus polymyxa strain ZJU11

[0038] A biocontrol strain with good antagonistic effect against mango anthracnose was isolated from the watermelon root system and numbered ZJU11. The strain ZJU11 was identified as follows:

[0039] 1. Morphological identification

[0040] The inoculation loop was dipped into the bacterial solution cultured in PDB medium for 24 hours and streaked on the PDA solid medium. After the plate was sealed, it was inverted and cultured at 28°C for 48 hours to observe the colony characteristics of strain ZJU11. Another 1 mL of culture solution was taken and centrifuged at 5000 r / min for 5 minutes. The precipitate was collected and washed three times with sterile water to remove the culture medium attached to the surface of the bacteria. Subsequently, 1 mL of 2.5% glutaraldehyde solution was added to the precipitated bacteria, fixed overnight, and the cell morphology of the strain was observed under a scanning electron microscope after preparation. The results are shown in Figure 1 , where A is the colony of strain ZJU11, and B is the scanning electron microscopy result of strain ZJU11.

[0041] The results showed that the colonies of strain ZJU11 were white, gelatinous and translucent, with raised colonies and irregular edges. Electron microscopy showed that the cells of strain ZJU11 were short rods with wrinkles on the surface, which may be related to its good colonization and attachment ability.

[0042] 2. Molecular identification

[0043] The genomic DNA of strain ZJU11 was extracted by CTAB method, and the extracted genomic DNA was used as a template for PCR amplification of conserved genes. The selected conserved gene was 16S rRNA, and the universal primers used for PCR amplification were synthesized by Shanghai Bioengineering Technology Co., Ltd., as follows:

[0044] Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1;

[0045] Reverse primer: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2.

[0046] The PCR reaction system was 50 μL, and the reaction system consisted of 25 μL KOD One Mix, 1.5 μL forward primer, 1.5 μL reverse primer, 1 μL template DNA, and 21 μL deionized water.

[0047] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 60°C for 55 s, and extension at 68°C for 10 s, for 35 cycles; and final extension at 68°C for 7 min.

[0048] After 1% agarose gel electrophoresis, the PCR product was selected for target band excision and recovery, and the product was purified using EZNAGel Extraction Kit (Omega BIO-TEK, D2500-02).

[0049] The gel-recovered product was ligated to the cloning vector pEASY-Blunt Zero Cloning Vector and transformed into Trans 1-T1 competent cells. Pick the positive clone strain in sterile water and use primers M13F and M13R to identify the positive clone. The primer sequences are as follows:

[0050] M13F: 5'-GTAAAACGACGGCCAGT-3', SEQ ID NO.3;

[0051] M13R: 5'-CAGGAAACAGCTATGAC-3', SEQ ID NO. 4.

[0052] The successfully identified strains were commissioned to Shanghai Bioengineering Technology Co., Ltd. to sequence the target gene, and the sequencing results were compared using the software SnapGene. The 16S rRNA gene sequencing results of strain ZJU11 are shown in SEQ ID NO.5, and the sequence has been uploaded to the NCBI database with the accession number PQ146574, as follows:

[0053]

[0054] The sequence was aligned by Blast in the NCBI database, and its similarity with Bacillus polymyxa was over 99%. The sequence was aligned by Mafft and pruned by Gblock using PhyloSuite software. A phylogenetic tree based on 16SrRNA sequence was constructed using Mega-X software. The phylogenetic tree file was beautified using ChiPlot online platform and Adobe Illustrator software. The beautified phylogenetic tree result is shown in the figure. Figure 2 shown.

[0055] From the constructed phylogenetic tree, we can see that ZJU11 and 多粘类芽孢杆菌 They belong to the same genetic branch. Combined with the morphological characteristics of the strain, strain ZJU11 was finally identified as Bacillus polymyxa ( 类芽孢杆菌属 多粘(的) ).

[0056] Example 2 Antagonistic efficacy of intracellular extracts of Paenibacillus polymyxa ZJU11 against mango anthracnose pathogens

[0057] The mango pathogenic bacteria strain used in the present invention is:

[0058] Siamese Anthrax ( 暹罗炭疽菌 HNI2), isolated from the fruit of 'Jinhuang' mango, was deposited in China Center for Type Culture Collection with the deposit number CCTCC M 20241875;

[0059] Fruit Anthracnose 果生炭疽菌 HNI5), isolated from the fruit of 'Jinhuang' mango, was deposited in China Center for Type Culture Collection with the deposit number CCTCC M 20241874;

[0060] Asian anthrax ( 亚洲炭疽菌 HNI6), isolated from the fruit of 'Jinhuang' mango, was deposited in China Center for Type Culture Collection with the deposit number CCTCC M 2024187.

[0061] 1. Preparation of intracellular extract of Paenibacillus polymyxa ZJU11: Use a sterile inoculation loop to pick a loop of Paenibacillus polymyxa ZJU11 from the PDA plate and inoculate it into 50 mL of PDB medium. Incubate it at 28°C and 200 r / min for 24 h as seed liquid. Pipette 6 mL of bacterial suspension at a 2% (v / v) inoculation volume and inoculate it into a 1L conical flask containing 300 mL of PDB medium. Incubate it at 28°C and 200 r / min for 96 h. Centrifuge the bacterial suspension at 12000 r / min and 4°C for 30 min, discard the supernatant, and collect the precipitate. Wash the bacterial precipitate with sterile water 3 times to completely remove the culture medium on the surface of the bacteria. Combine all the bacteria, add 300 mL of anhydrous methanol, and extract at 28°C and 200 r / min for 24 h. Subsequently, the extract was centrifuged at 12000 r / min and 4°C for 15 min, and the methanol phase was collected and concentrated and evaporated to dryness using a rotary evaporator with the parameters of vacuum degree 60 hPa, temperature 50°C, and speed 78 r / min. The extract was collected and stored in a -20°C refrigerator for later use.

[0062] 2. Preparation of anthrax spore suspension: Pick the hyphae of the above three mango pathogens stored at 4°C and inoculate them into 50 mL of PDB medium, and culture them at 28°C and 200 r / min shaking for 72 h. Filter the hyphae with a nylon membrane to collect the spore suspension. Centrifuge at 4°C and 5000 r / min for 10 min to collect the bacterial precipitate, and wash it three times with sterile water. Finally, use a hemocytometer to count the spores of the three pathogens under an optical microscope, and adjust the spore concentration to 1×10 with sterile water. 6 Pieces / mL.

[0063] 3. Preparation of intracellular extract solution of Paenibacillus polymyxa: Take the extract preserved in step 1 and dry it thoroughly in a solvent evaporator. The dried extract is dissolved in anhydrous methanol to prepare a 1 mg / mL methanol solution and filtered with a 0.22 μm filter membrane to obtain a sterile intracellular extract solution.

[0064] 4. Plate antibacterial test: Take 100 μL of the above concentration of 1×10 6 Spread a spore suspension of 100 / mL on a PDA plate. After the surface of the plate is dry, use a blue 1 mL pipette with a diameter of about 8 mm to make a hole on the surface of the culture medium, remove the agar block, add 60 μL of the above-mentioned intracellular extract solution of Bacillus polymyxa to the hole, and add an equal volume of methanol solution to the control group. After the solution is absorbed, seal the plate with a sealing film and culture it in a biochemical incubator at 28°C in the dark for 5 days. Count the diameter of the inhibition zone and take pictures. The results are shown in Figure 3 ,in, 暹罗炭疽菌 : Siamese Anthrax; 果生炭疽菌 : Fruit Anthracnose; 亚洲炭疽菌 : Asian anthrax.

[0065] Depend on Figure 3 It can be seen that the methanol solution in the control group had no inhibitory effect on the three anthracnose pathogens, and no inhibition zone appeared on the plate, while the intracellular extract treatment group showed an obvious inhibition zone, and the inhibition zones for Siamese anthracnose, fruit anthracnose and Asian anthracnose were 16.97 ± 1.14 mm, 14.65 ± 0.59 mm and 20.22 ± 0.88 mm, respectively, indicating that the intracellular extract of Bacillus polymyxa can significantly inhibit the growth of mango anthracnose pathogens.

[0066] Example 3 Antagonistic efficacy of intracellular extracts of Paenibacillus polymyxa ZJU11 against golden mango anthracnose

[0067] The method of Example 2 was used to prepare a 50 mg / mL intracellular extract solution.

[0068] The "Golden Brilliant" mango fruits of uniform maturity and free of pests and diseases were picked from the mango plantation and transported to the laboratory for surface disinfection within 2 hours. First, the dust on the surface of the fruit was washed off with tap water, and then soaked in a 2% sodium hypochlorite solution for 5 minutes. Then, the residual sodium hypochlorite was washed with tap water and dried at room temperature. Three wounds were made on the surface of the fruit with an inoculation needle, and a circular filter paper soaked in a 50 mg / mL intracellular extract solution was attached to the wound of the fruit. A filter paper soaked in anhydrous methanol was used as a control. Subsequently, a sterilized 1 mL pipette tip was used to beat a bacterial cake from a 7-day-old plate of Colletotrichum anthracis HNI5 (CCTCCM 20241874) and attached to the surface of the filter paper. The fruit was wrapped with plastic wrap and placed in a climate chamber at 25°C and 95% relative humidity. After 3 days, the expansion of the fruit lesions was observed, and the diameter of the lesions was counted and photographed. Results are shown in Figure 4 .

[0069] Depend on Figure 4 It can be seen that the diameter of anthracnose lesions on mango fruits inoculated with Colletotrichum siamese expanded rapidly, reaching 8.29 ± 0.74 mm on the third day, while the diameter of lesions on fruits treated with the intracellular extract of Paenibacillus polymyxa ZJU11 was significantly reduced to only 4.37 ± 0.31 mm, indicating that the intracellular extract of Paenibacillus polymyxa ZJU11 has significant antagonistic potential for preventing and controlling postharvest anthracnose of mangoes.

[0070] In summary, the Bacillus polymyxa ZJU11 provided by the present invention has good antagonistic effects on the dominant pathogens of postharvest anthracnose of mangoes and inhibits mango anthracnose, and can be used to prepare a bacterial agent for preventing and treating postharvest anthracnose of mangoes and alleviate the symptoms of postharvest anthracnose of mangoes.

[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Paenibacillus polymyxa ZJU11 in preventing and controlling mango anthracnose, and taxonomic nomenclature of Paenibacillus polymyxa ZJU11 Paenibacillus polymyxa ZJU11, the deposit number is CCTCC NO: M 20241847.

2. The use according to claim 1, characterized in that: The mango anthracnose is postharvest mango anthracnose.

3. The use according to claim 1 or 2, characterized in that: The pathogens of mango anthracnose include: Siamese anthracnose Colletotrichum siamense , Colletotrichum Colletotrichum fructicola and Asian anthrax Colletotrichum asianum One or more of .

4. A method for preventing and treating postharvest anthracnose of mangoes, characterized in that: The following steps are involved: Soaking the harvested mangoes in a biocontrol agent, or spraying the biocontrol agent on the surface of the harvested mangoes; the biocontrol agent includes an intracellular extract of Paenibacillus polymyxa ZJU11; The preparation method of the intracellular extract comprises the following steps: using methanol to extract the polymyxa bacillus ZJU11 to obtain an alcohol extract; drying the alcohol extract to obtain the intracellular extract; the alcohol extract further comprises culturing the polymyxa bacillus ZJU11; the culturing time is 96 hours; the taxonomic nomenclature of the polymyxa bacillus ZJU11 Paenibacillus polymyxa ZJU11, the deposit number is CCTCC NO: M 20241847.

5. The method according to claim 4, characterized in that The concentration of the intracellular extract in the biocontrol agent is ≥1 mg / mL.

6. The method according to claim 4, characterized in that The soaking time is 5 to 10 minutes.

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