A strain of Gluconobacter freundii AY1 and its application in controlling mango anthracnose

Abstract

The present invention relates to the technical field of biocontrol bacteria, and in particular to a strain of Gluconobacter freundii AY1 and its application in preventing and controlling mango anthracnose. The present invention provides a strain of Gluconobacter freundii ( Gluconobacter frateurii )AY1, with a deposit number of CCTCC NO: M 20241846. The Gluconobacter freundii AY1 of the present invention is isolated and purified by the inventor from the surface of the "Golden Glory" mango fruit picked from the mango plantation area in Jiyang District, Sanya City, Hainan Province, and is a biocontrol strain with good antagonistic effect on mango postharvest anthracnose (inhibition index is 0.513-0.555). The strain has good antagonistic effect on the dominant pathogens of mango postharvest anthracnose and inhibits mango anthracnose, and can be used to prepare a bacterial agent for preventing and controlling 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, in particular to a strain of Gluconobacter freundii AY1 and application thereof in preventing and controlling mango anthracnose. Background Art

[0002] mango( Mangifera indica Lnn.) belongs to the Anacardiaceae ( Anacardiaceae ) Mangifera indica Mangifera L.), is one of the world's top five fruits and is known as the "King of Tropics". However, the mango picking season is usually hot and rainy, and the mango's physiological metabolic activity is vigorous after harvest, which makes it very easy to be infected by microorganisms during transportation, storage and sales, causing rot and deterioration, resulting in huge economic losses. Among them, Anthracnose ( Colletotrichum ) Anthracnose caused by pathogenic fungi is the main cause of post-harvest rot in mangoes. Mango anthracnose control is mainly based on chemical control, which mainly interferes with, kills or inhibits the growth of pathogens by soaking or spraying chemical fungicides to achieve the purpose of disease prevention and control. Some synthetic chemical fungicides such as methyl thiophanate, carbendazim, and fenpropimorph have been widely used in the prevention and control of mango anthracnose. However, long-term use of chemical fungicides can easily make anthracnose bacteria resistant, and chemical fungicides can easily pollute the environment and pose a threat to human health.

[0003] The use of biological control to control postharvest fruit diseases has become a hot topic of research at home and abroad, and biological control has become an inevitable trend to replace chemical fungicides. The use of biological measures to control postharvest diseases can make up for the shortcomings of chemical treatments, without pollution to the environment, without pesticide residues, and not easy to make pathogens resistant. Therefore, it is of great practical significance to explore efficient biocontrol strains for the prevention and control of postharvest anthracnose disease in mangoes. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a strain of Gluconobacter freundii AY1 and its application in preventing and controlling mango anthracnose. The Gluconobacter freundii AY1 provided by the present invention is a biocontrol strain with good antagonistic effect on mango anthracnose, and the strain has good antagonistic effect on the dominant pathogens of postharvest anthracnose of mango and inhibits mango anthracnose.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Gluconobacter freundii ( Gluconobacter frateurii )AY1, the deposit number is CCTCC NO: M 20241846.

[0007] The present invention provides a biocontrol agent, comprising the Gluconobacter freundii AY1 described in the above technical solution.

[0008] Preferably, the number of viable bacteria of Gluconobacter freundii AY1 in the biocontrol agent is ≥ 1.0×10 8 CFU / mL.

[0009] The present invention provides a method for preparing the biocontrol agent described in the above technical solution, comprising: inoculating the Gluconobacter freundii AY1 described in the above technical solution into a culture medium for cultivation to obtain the biocontrol agent.

[0010] Preferably, the culture medium is PDB culture medium; and the culture temperature is 25-30°C.

[0011] The present invention provides use of the Gluconobacter freundii AY1 described in the above technical solution or the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution in preventing and controlling mango anthracnose.

[0012] Preferably, the mango anthracnose is postharvest mango anthracnose.

[0013] Preferably, the pathogens of mango anthracnose include: Siamese anthracnose ( Colletotrichum siamense )、Fruit Anthrax( Colletotrichum fructicola ) and Asian anthrax ( Colletotrichum asianum )

[0014] The present invention provides a method for preventing and controlling postharvest anthracnose of mangoes, comprising the following steps:

[0015] The harvested mangoes are soaked in a biocontrol agent, or the biocontrol agent is sprayed on the surface of the harvested mangoes; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.

[0016] Preferably, the soaking time is 5 to 10 minutes.

[0017] Beneficial effects:

[0018] The present invention provides a strain of Gluconobacter freundii AY1, with a deposit number of CCTCC NO: M 20241846. The Gluconobacter freundii AY1 provided by the present invention is separated and purified from the surface of "Jinhuang" mango fruits picked from a mango plantation in Jiyang District, Sanya City, Hainan Province, and the obtained strain has a good antagonistic effect on mango postharvest anthracnose (inhibition index is 0.513-0.555). The strain has good antagonistic effects on the dominant pathogens of mango postharvest anthracnose and inhibits mango anthracnose, and can be used to prepare a bacterial agent for preventing and controlling mango postharvest anthracnose, and alleviate the symptoms of mango postharvest anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 The colony characteristics and scanning electron micrograph of Gluconobacter freundii AY1 are shown;

[0021] Figure 2 This is the phylogenetic tree of Gluconobacter freundii AY1 constructed based on the concatenation of 16S rRNA and ISR;

[0022] Figure 3 The results of the in vitro antagonistic activity test of Gluconobacter freundii AY1 against the dominant pathogen of mango anthracnose;

[0023] Figure 4 The results are shown in the experimental results of the control of Gluconobacter freundii AY1 against anthracnose of golden mango.

[0024] Biological Deposit Description

[0025] Gluconobacter freundii AY1, taxonomically named Gluconobacter frateurii AY1 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 20241846.

[0026] Siamese anthrax HNI2, taxonomic name Colletotrichum siamense 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.

[0027] Colletotrichum niger HNI5, classified as Colletotrichum fructicola 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.

[0028] Asian anthrax HNI6, named Colletotrichum asianum 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

[0029] The present invention provides a strain of Gluconobacter freundii ( Gluconobacter frateurii)AY1, with a deposit number of CCTCC NO: M 20241846. The Gluconobacter freundii AY1 provided by the present invention is isolated and purified from the surface of the "Golden Brilliant' mango fruit picked from the mango plantation in Jiyang District, Sanya City, Hainan Province. The obtained biocontrol strain has good antagonistic effect on mango post-harvest anthracnose, grows rapidly on PDA culture medium, and the colonies are white and milky, with a smooth surface and a short cylindrical shape. It is monospore, and the inhibition index of siam anthracnose, fruit anthracnose and Asian anthracnose is 0.513~0.555. The results of the embodiment show that compared with the control group, the diameter of the fruit anthracnose lesions after the treatment of Gluconobacter freundii AY1 is significantly reduced, and the diameter of the fruit lesions is reduced from 14.13±2.45 mm in the control group to 5.67±1.30 mm, indicating that Gluconobacter freundii AY1 has the potential to prevent and control mango anthracnose as a biocontrol agent.

[0030] Based on the above advantages, the present invention provides a biocontrol agent, including the Gluconobacter freundii AY1 described in the above technical solution.

[0031] As an embodiment, the number of viable bacteria of Gluconobacter freundii AY1 in the biocontrol agent is ≥ 1.0×10 8 CFU / mL.

[0032] The present invention provides a method for preparing the biocontrol agent described in the above technical solution, comprising: inoculating the Gluconobacter freundii AY1 described in the above technical solution into a culture medium for cultivation to obtain the biocontrol agent.

[0033] As an embodiment, the culture medium is PDB medium.

[0034] In one embodiment, the culture temperature is 25-30° C. In another embodiment, the culture temperature is 28° C.

[0035] Based on the above advantages, the present invention provides the use of Gluconobacter freundii AY1 described in the above technical scheme or the biocontrol agent described in the above technical scheme or the biocontrol agent prepared by the preparation method described in the above technical scheme in preventing and controlling mango anthracnose.

[0036] As an embodiment, the mango anthracnose is postharvest mango anthracnose.

[0037] As an embodiment, the pathogens of mango anthracnose include: one or more of Siamese anthracnose, fruit anthracnose and Asian anthracnose.

[0038] As an embodiment, the mango varieties include but are not limited to 'Golden Glory' mango.

[0039] The present invention provides a method for preventing and controlling postharvest anthracnose of mangoes, comprising the following steps:

[0040] The harvested mangoes are soaked in a biocontrol agent, or the biocontrol agent is sprayed on the surface of the harvested mangoes; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.

[0041] As an implementation mode, the soaking time is 5 to 10 minutes.

[0042] To further illustrate the present invention, a strain of Gluconobacter freundii AY1 provided by the present invention and its application in preventing and controlling mango anthracnose are 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.

[0043] Example 1 Identification of Gluconobacter freundii strain AY1

[0044] The inventors isolated and purified the surface of "Jinhuang" mango fruits picked from the mango plantation in Jiyang District, Sanya City, Hainan Province, and obtained a biocontrol strain with good antagonistic effect on post-harvest anthracnose of mangoes, numbered AY1, and identified the strain AY1 as follows:

[0045] 1. Morphological identification

[0046] The isolated strain AY1 was streaked on PDA medium and inverted at 28°C to observe morphological characteristics. A ring of AY1 strain was picked up with an inoculation loop and inoculated into PDB medium. The culture was shaken at 28°C and 200 rpm for 24 h. 1 mL of bacterial solution was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the bacteria were washed three times with sterile water, fixed with 2.5% glutaraldehyde overnight, and used for scanning electron microscopy observation. The results are shown in Figure 1 , where A is the colony of strain AY1, and B is the scanning electron microscopy result of strain AY1.

[0047] The results showed that strain AY1 grew rapidly on PDA medium, and the colonies were white, milky, and smooth. Electron microscopy showed that strain AY1 was short cylindrical and monospore.

[0048] 2. Molecular identification

[0049] The genomic DNA of strain AY1 was extracted by CTAB method, and the extracted genomic DNA was used as a template for PCR amplification of conserved genes. The selected conserved genes were 16S rRNA and ISR, and the universal primers used for PCR amplification were synthesized by Shanghai Bioengineering Technology Co., Ltd., as follows:

[0050] Forward primer for 16S rRNA: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1;

[0051] Reverse primer for 16S rRNA: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2;

[0052] ISR forward primer: 5'-AGTCGTAACAAGGTAGCCGT-3', SEQ ID NO.3;

[0053] Reverse primer of ISR: 5'-GTGCCAAGGCATCCACC-3', SEQ ID NO.4.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] Ligation of gel-recovered products into cloning vectors pEASY ® -Blunt Zero Cloning Vector, converted to Trans 1-T1 competent cells. Pick the positive clone strain in sterile water and use primers M13 Forward Primer and M13 Reverse Primer to identify the positive clone. The primer sequences are as follows:

[0058] M13F: 5'-GTAAAACGACGGCCAGT-3', SEQ ID NO.5;

[0059] M13R: 5'-CAGGAAACAGCTATGAC-3', SEQ ID NO. 6.

[0060] The successfully identified strains were commissioned to Shanghai Bioengineering Technology Co., Ltd. to sequence the target genes. Eight positive clones were tested for each gene, and the sequencing results were compared using the software SnapGene. The 16S rRNA gene sequencing results of strain AY1 are shown in SEQ ID NO.7. The sequence has been uploaded to the NCBI database with the accession number PQ106868, as follows:

[0061]

[0062] The ISR gene sequencing result of strain AY1 is shown in SEQ ID NO.8. The sequence has been uploaded to the NCBI database with accession number PQ110013. The details are as follows:

[0063] 5'--3';

[0064] Phylosuite software was used to perform MAFFT alignment, GBlocks trimming and concatenation of the 16S rRNA and ISR sequences, and the phylogenetic tree was constructed using Mega7.0 software. The results are shown in Figure 2 .

[0065] The 16S rRNA and ISR gene sequences of AY1 were compared with those in Genebank by BLAST. The strain with the highest similarity to AY1 was Gluconobacter frateurii , with a homology of 99%. The phylogenetic tree was constructed by connecting the two sequences in series. The phylogenetic tree results show that strain AY1 and Gluconobacter freundii ( Gluconobacter frateurii ) were clustered into one branch, with a bootstrap value of 0.744, which was greater than 0.500 and reached the critical value for microbial species classification. Combined with the morphological characteristics and molecular identification of AY1, strain AY1 was finally identified as Gluconobacter freundii ( Gluconobacter frateurii ).

[0066] Example 2 In vitro antagonism of Gluconobacter freundii AY1 against dominant pathogenic fungi of mango anthracnose in 'Jinhuang'

[0067] The mango pathogenic bacteria strain used in the present invention is:

[0068] Siamese Anthrax ( Colletotrichum siamense HNI2), isolated by the inventor from the fruit of 'Jinhuang' mango, was deposited in China Center for Type Culture Collection with the deposit number of CCTCC M 20241875;

[0069] Fruit Anthracnose Colletotrichum fructicola HNI5), isolated by the inventor from the fruit of 'Jinhuang' mango, and deposited in China Center for Type Culture Collection with the deposit number of CCTCC M 20241874;

[0070] Asian anthrax ( Colletotrichum asianum HNI6), isolated by the inventor from the fruit of Jinhuang mango, and deposited in China Center for Type Culture Collection with the deposit number of CCTCC M 2024187;

[0071] Activation of strains: Gluconobacter frateurii AY1, Colletotrichum siamense HNI2, Colletotrichum fructicola HNI5 and Colletotrichum asianum HNI6 was taken out from the -80℃ freezer. Gluconobacter frateuriiAY1 was streaked and inoculated on the PDA medium, and the other strains were spread on the surface of the PDA medium and inverted in a 28°C biochemical incubator. After culturing for 48 h, strain AY1 was picked up and inoculated on the PDB medium with an inoculation loop and cultured on a shaker at 28°C and 200 rpm for 24 h. When the pathogen strains grew all over the plate, a sterile punch was used to punch out 8 mm bacterial blocks and inoculated in the center of another new PDA plate, and static culture was performed for 7 days for plate antagonism test. The formula of the PDA medium is as follows: 200 g of peeled potatoes were boiled for 20 min, and the filtrate was taken, 20 g of anhydrous glucose, and the volume was adjusted to 1000 mL with distilled water, pH 7.0.

[0072] Use a puncher with a diameter of 8 mm to punch out bacterial blocks from the activated 3 pathogen culture media, inoculate them in the center of the PDA plate, and use an inoculation loop to draw straight lines twice on the pathogen with Gluconobacter freundii AY1, which are recorded as the treatment group. Each group has 3 replicates, and the group without Gluconobacter freundii AY1 is the control group. After the Para film plate is sealed, it is placed in a 28℃ incubator for static culture. Observe the growth status of the pathogen, count the colony diameter of the pathogen, and calculate the inhibition index according to formula I and formula II. The results are shown in Figure 3 , where different lowercase letters represent significant differences between the treatment group and the control group.

[0073] The inhibition index of the control group = D1 / D3 formula Ⅰ;

[0074] The inhibition index of the treatment group = (D3-D2) / D3 formula II;

[0075] Among them, D1 is the radius of the pathogen; D2 is the radius of the pathogen of the Gluconobacter freundii AY1 group; and D3 is the distance between the center of the pathogen of the Gluconobacter freundii AY1 group and Gluconobacter freundii AY1.

[0076] The results showed that the pathogens in the control group expanded evenly in the PDA medium, and the colonies were round, while in the treatment group inoculated with Gluconobacter freundii AY1, the colony diameters of the three anthrax pathogens were significantly inhibited, and the pathogen colonies were elliptical, indicating that Gluconobacter freundii AY1 can significantly antagonize the growth of anthrax pathogens.

[0077] Example 3 The control effect of Gluconobacter freundii AY1 on anthracnose of Golden Miscanthus

[0078] Gluconobacter freundii AY1 was inoculated into PDB medium and cultured in a shaking incubator at 200 rpm and 28 °C for 36 h to obtain a viable cell count of 1.0 × 10 8 CFU / mL, and a bacterial suspension of Gluconobacter freundii AY1 was obtained.

[0079] Select the "Golden Brilliant" fruits of uniform size, maturity, and no mechanical damage and pests and diseases. First, wash the surface twice with 0.1% sodium hypochlorite solution, and then disinfect the surface of the fruit with 75% ethanol by volume. After the fruit is dried, use an inoculation needle to make mechanical wounds on the surface of the fruit (3 holes, 1 mm wide × 3 mm deep), and inoculate each hole with 10 μL of 1.0× 10 8 CFU / mL of Gluconobacter freundii AY1 suspension, and set the "Jinhuang" fruit with 10 μL PDB culture medium in each well as the control group. After 8 hours, the mycelium block was taken from the edge of the colony of Siamese anthracnose bacteria HNI2 (deposit number CCTCC M 20241875) cultured for 7 days with an 8 mm puncher, inoculated into the wound of the fruit, and fixed with plastic wrap. The mango fruit was placed in a biochemical incubator with a temperature of 28°C and a relative humidity of 95% for dark culture, and the disease of the mango fruit was observed and the size of the lesions was counted. The results are shown in Figure 4 .

[0080] The efficacy of Gluconobacter freundii AY1 against anthracnose of "Jinhuang" mangoes showed that the diameter of the fruit anthracnose lesions after treatment with Gluconobacter freundii AY1 was significantly reduced compared with the control group. The diameter of the fruit lesions decreased from 14.13 ± 2.45 mm in the control group to 5.67 ± 1.30 mm, indicating that Gluconobacter freundii AY1 has the potential to control anthracnose of "Jinhuang" mangoes after harvest as a biocontrol agent.

[0081] In summary, the Gluconobacter freundii AY1 provided by the present invention has good effects of antagonizing the dominant pathogens of postharvest anthracnose of mango and inhibiting mango anthracnose, and can be used to prepare a bacterial agent for preventing and treating postharvest anthracnose of mango and alleviate the symptoms of postharvest anthracnose of mango.

[0082] 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. A strain of Gluconobacter freundii ( Gluconobacter frateurii )AY1, the deposit number is CCTCC NO: M20241846.

2. A biocontrol agent, characterized in that: It includes the Gluconobacter freundii AY1 described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that The number of viable bacteria of Gluconobacter freundii AY1 in the biocontrol agent is ≥1.0×10 8 CFU / mL.

4. The method for preparing the biocontrol agent according to claim 2 or 3, characterized in that: include: The Gluconobacter freundii AY1 described in claim 1 is inoculated into a culture medium and cultured to obtain the biocontrol agent.

5. The preparation method according to claim 4, characterized in that: The culture medium is PDB culture medium; the culture temperature is 25-30°C.

6. Use of the Gluconobacter freundii AY1 according to claim 1, the biocontrol agent according to claim 2 or 3, or the biocontrol agent prepared by the preparation method according to claim 4 or 5 in preventing and controlling mango anthracnose.

7. The use according to claim 6, characterized in that: The mango anthracnose is postharvest mango anthracnose.

8. The use according to claim 6 or 7, characterized in that: The pathogens of mango anthracnose include: Siamese anthracnose ( Colletotrichum siamense )、Fruit Anthrax( Colletotrichum fructicola ) and Asian anthrax ( Colletotrichum asianum ) 9. A method for preventing and controlling postharvest anthracnose of mangoes, characterized in that: The following steps are involved: The harvested mangoes are soaked in a biocontrol agent, or the biocontrol agent is sprayed on the surface of the harvested mangoes; the biocontrol agent is the biocontrol agent according to claim 2 or 3, or is prepared by the preparation method according to claim 4 or 5.

10. The method according to claim 9, characterized in that The soaking time is 5 to 10 minutes.

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