Pantoea KG-43 and application thereof in prevention and control of mulberry diseases

Abstract

The invention discloses pantoea KG-43 and application of the pantoea KG-43 in prevention and control of mulberry diseases. The Pantoea coffeiphila KG-43 is obtained and is preserved in the Guangdong Microbial Culture Collection Center on June 18, 2024, and the preservation number of the Pantoea coffeiphila KG-43 is GDMCC (China General Microbiological Culture Collection Center) NO: 64765. The pantoea KG-43 has a good antagonistic effect on lauraceae lauraceae and pantoea ananatis, has a good prevention and control effect on plant diseases, including mulberry ralstonia solanacearum and mulberry bacterial wilt, caused by plants infected by the pathogenic bacteria, and is safe and effective. Therefore, the pantoea KG-43 or the fermentation product thereof has good application prospect and value in prevention and control of plant diseases such as mulberries and the like or preparation of preparations for preventing and controlling plant diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease prevention and control, and more specifically, relates to a strain of Pantoeacoffeiphila KG-43 and its application in preventing and controlling mulberry diseases. Background Art

[0002] Mulberry tree is a deciduous tree or shrub of the genus Morus in the family Moraceae. As a perennial woody plant with both medicinal and edible properties, it has high economic value. Its traditional main use is to feed silkworms, and it is an indispensable feed raw material to support the sericulture industry. At the same time, mulberry branches, roots and other parts can be used as medicine, and have high medicinal economic value. With the continuous deepening of mulberry industry research, mulberry trees are widely used in various industries such as food, animal husbandry, and ecology, playing an increasingly important role. However, mulberry tree diseases, especially bacterial and fungal diseases, have devastating effects on the production and development of mulberry trees, seriously restricting the healthy and stable development of the mulberry industry.

[0003] The main bacterial diseases of mulberry trees include mulberry bacterial wilt, mulberry bacterial wilt, etc. Among them, the pathogens of mulberry bacterial wilt are of various types, such as Pantoea ananatis and other pathogens, and the pathogen of mulberry bacterial wilt is Ralstonia pseudosolanacearum, and there is often cross infection, which makes the disease situation more complicated. Bacterial diseases have complex pathogens, strong infectivity, and short disease cycle. They frequently break out in many sericulture production areas, usually causing the whole plant to wither and die, and are difficult to effectively prevent and control. At present, the prevention and control methods for this type of mulberry disease are mainly based on traditional chemical agents, but the long-term use of chemical pesticides is prone to deterioration of the soil environment, a decrease in the richness of soil microbial populations, and an increase in the drug resistance of pathogens, which cannot fundamentally solve the problem.

[0004] Therefore, a green and effective prevention and control method is urgently needed. Liu Ji et al. (2023) screened out a strain of Bacillus tequilensis K3-11, which has a good antagonistic effect on Ralstonia solanacearum and Pantoea pineapple, and can be used to prevent and control mulberry bacterial wilt and mulberry wilt. The endophytic flora of mulberry is rich in species, especially with the gradual promotion and application of microbial agents, "using bacteria to treat bacteria" has attracted more attention. Therefore, the isolation of endophytic bacteria with antagonistic and control effects on mulberry pathogens is an important means to deal with the green ecological prevention and control of mulberry diseases in the future. Summary of the invention

[0005] The present invention aims to overcome the defects and shortcomings of existing methods for preventing and controlling mulberry diseases, especially mulberry bacterial diseases, and provide a biocontrol bacterium that can simultaneously target mulberry bacterial wilt pathogens such as Pantoea ananatis and mulberry bacterial wilt pathogen Ralstonia pseudosolanacearum, which can cope with more complex mulberry bacterial diseases in practice.

[0006] The first object of the present invention is to provide a Pantoea KG-43 strain.

[0007] The second object of the present invention is to provide a biological preparation containing the above-mentioned Pantoea KG-43 or its fermentation product.

[0008] The third object of the present invention is to provide the application of the above-mentioned Pantoea KG-43 and the above-mentioned biological preparation.

[0009] The fourth object of the present invention is to provide a method for preventing and controlling mulberry tree diseases.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention provides a strain of Pantoea coffeiphila KG-43, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 18, 2024, with a deposit number of GDMCC NO: 64765.

[0012] The present invention provides a biological agent for preventing and controlling plant diseases, wherein the biological agent contains the above-mentioned Pantoea KG-43, or contains the fermentation liquid of the Pantoea KG-43, or contains the fermentation product of the Pantoea KG-43.

[0013] The fermentation broth refers to the fermentation broth obtained by fermenting Pantoea KG-43 in a culture medium.

[0014] The fermentation product refers to the product obtained after removing the bacteria from the fermentation liquid.

[0015] The Pantoea KG-43 of the present invention has a good antagonistic effect on Ralstonia sanguineus and Pantoea ananas, so the following applications should also be within the scope of protection of the present invention:

[0016] The use of the above-mentioned Pantoea KG-43 or the above-mentioned biological preparation in inhibiting the growth of plant pathogens.

[0017] The use of the above-mentioned Pantoea KG-43 or the above-mentioned biological agent in preventing and controlling plant diseases caused by plant pathogens.

[0018] The use of the above-mentioned Pantoea KG-43 or the above-mentioned biological agent in the preparation of products for preventing and controlling plant diseases caused by plant pathogens.

[0019] The plant pathogenic bacteria include Ralstonia sanguineus and / or Pantoea ananas.

[0020] The plant diseases include mulberry bacterial wilt and mulberry bacterial wilt.

[0021] The present invention also provides a method for preventing and controlling mulberry tree diseases, which comprises using the above-mentioned Pantoea KG-43 or the above-mentioned biological agent to prevent and control mulberry tree diseases.

[0022] Specifically, the mulberry tree disease is caused by one or both of Ralstonia thunbergii and Pantoea ananas.

[0023] The present invention has the following beneficial effects:

[0024] The present invention has isolated a biocontrol bacterium that has a good antagonistic effect on Ralstonia mulberryi and Pantoea pineapple, and it has been identified and named Pantoea KG-43. Pantoea KG-43 or its fermentation liquid or the fermentation product after removing the bacterial body has a good control effect on plant diseases (mulberry wilt and mulberry bacterial wilt) caused by the above-mentioned pathogens infecting plants. It is safe and effective and can avoid various problems such as soil environmental deterioration caused by chemical pesticides and enhanced drug resistance of pathogens. Therefore, Pantoea KG-43 or its fermentation liquid or the fermentation product after removing the bacterial body has a good application prospect and value in preventing and controlling plant diseases or preparing preparations for preventing and controlling plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the inhibitory effect of strain KG-43 on different pathogens.

[0026] Figure 2 The growth status of strain KG-43 on LB agar medium.

[0027] Figure 3 The Gram staining results of strain KG-43.

[0028] Figure 4 The scanning electron microscopy (50000×) results of strain KG-43.

[0029] Figure 5 These are potted plant effects of strain KG-43 in preventing and controlling mulberry bacterial wilt (Figure A shows the treatment with Pantoea KG-43 bacterial solution; Figure B shows the treatment with Pantoea KG-43 bacterial solution + Ralstonia solanacearum bacterial solution; Figure C shows the treatment with sterile water; and Figure D shows the treatment with Ralstonia solanacearum bacterial solution). DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.

[0031] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field.

[0032] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] The PCR reagents used in the following examples were purchased from Guangzhou Xinkailai Biotechnology Co., Ltd. (Cat. No.: XKL0211).

[0034] The pathogen information used in the following examples:

[0035] Ralstonia pseudosolanacearum was isolated and preserved by our laboratory and has been disclosed in the article "Yuan Ting, Li Jinhao, Liu Jiping. Latest research progress on virulence genes and regulatory networks of Ralstonia pseudosolanacearum complex [J]. Bulletin of Microbiology, 2023, 50(05).".

[0036] Pantoea ananatis was isolated and preserved by our laboratory and has been disclosed in the article [1] Wang Jicheng, Sun Xunxun, Luo Longhui, Liu Jiping. Isolation and identification of four primary antibacterial agents for soil-borne diseases of mulberry trees [J]. Sericultural Science, 2020, 46(1): 31-36.

[0037] The culture medium information used in the following examples:

[0038] Beef extract peptone agar medium (NA): 3g beef extract, 10g bacteriological peptone, 5g NaCl powder, 18g technical agar powder, 1000mL distilled water, pH 7.4-7.6;

[0039] Beef extract peptone liquid medium: beef extract 3g, bacteriological peptone 5g, glucose 10g, yeast powder 0.5g, distilled water 1000mL, pH 7.0;

[0040] LB medium: tryptone 10 g, yeast powder 5 g, NaCl powder 10 g, technical agar powder 18 g, distilled water 1000 mL, pH 7.0;

[0041] LB liquid medium: tryptone 10 g, yeast powder 5 g, NaCl powder 10 g, distilled water 1000 mL, pH 7.0;

[0042] TTC medium: bacteriological peptone 10g, beef extract powder 3g, NaCl powder 5g, technical agar powder 18g, pH 7.1-7.5;

[0043] MS medium: MS powder 4.74 g, sucrose 20 g, agar 16 g, distilled water 1000 mL, pH 5.8-6.0;

[0044] The above culture medium raw materials were purchased from Guangdong Huankai Microbiological Technology Co., Ltd. and were sterilized at 121°C for 15 minutes before use.

[0045] Example 1 Isolation and purification of antagonistic bacterial strains

[0046] 1. Experimental Methods

[0047] (1) Sample collection: In February 2022, the author Li Jinhao collected 40 roots of mulberry trees of the Kangqing 10 variety in the mulberry garden of Xiahua Village, Zengcheng District, Guangzhou City, Guangdong Province (23°9'43”N, 113°20'46”E).

[0048] (2) Isolation and purification of antagonistic bacteria

[0049] The collected mulberry root samples were cleaned by brushing the surface with tap water, and the branches were cut into small sections of 3 cm in length after drying. Under sterile conditions, the cut sample roots were disinfected with 75% ethanol for 3 minutes and then rinsed with sterile water for 4 times. They were then disinfected with 0.1% mercuric chloride solution for 5 minutes and then rinsed with sterile water for 5 times. 0.2 mL of sterile water from the last rinse of the sample was taken and spread on LB culture medium, and cultured at 28°C for 1 day to test the disinfection effect of the sample surface.

[0050] According to the method of Liang Jiajun (2018), the surface moisture of the sample mulberry root was absorbed with sterile filter paper, and the two ends were cut off respectively. The remaining section was cut in the middle to remove the xylem, and the xylem was cut into 0.1 cm pieces with scissors under sterile conditions. 3 The fragments were placed in 10 mL of sterile water and shaken in a shaker at 28°C and 140 rpm for 15 min to obtain a bacterial solution containing endophytes. According to the 10-fold gradient dilution method, the bacterial solution was gradiently diluted 8 times to obtain 10 -1 ~10 -8 Take 100 μL of 10 -1 ~10 -8 The bacterial dilution was evenly spread on NA medium, MS medium and LB medium with a spreader, and each dilution was repeated 3 times. Incubate inverted in a 28℃ constant temperature incubator for 2 days, pick a single colony and streak it on an LB agar medium plate, purify the pure strain, and store it in glycerol, and place a portion in a -80℃ refrigerator.

[0051] (3) Screening of antagonistic bacterial strains

[0052] The single colonies isolated above were cultured in LB liquid medium at 28°C and 180r / min for 48h to obtain the selected antagonistic bacteria solution. Then, the pathogenic bacteria Ralstonia solanacearum and Pantoea ananas were used as indicator bacteria, and the antibacterial activity was tested by agar diffusion method. 20μL of the selected antagonistic bacteria solution was taken with a micropipette and added to the small hole punched with a 5mm puncher. 20μL of sterile water was added to the negative control, and the solution was cultured in a 25°C incubator for 4d to detect whether there was an antibacterial zone and measure its diameter.

[0053] 2. Experimental Results

[0054] Through the screening experiment of antagonistic bacteria, a strain with antagonistic effect on pathogenic Ralstonia solanacearum and Pantoea ananas and obvious antibacterial effect was obtained, which was recorded as strain KG-43.

[0055] The specific data of the antibacterial effect of strain KG-43 on the two pathogens are shown in Table 1, and the inhibitory effect of strain KG-43 on the two pathogens is shown in Figure 1 As shown in the figure, it can be seen that KG-43 has good antibacterial effects on bacterial pathogens Ralstonia solanacearum and Pantoea anacardiae, among which the antagonistic effect on Ralstonia solanacearum is the most obvious, with the diameter of the inhibition zone reaching 23.75±1.69mm. Therefore, strain KG-43 can be used for the prevention and control of mulberry bacterial wilt and mulberry bacterial wilt.

[0056] Table 1 Antibacterial effect of strain KG-43 on different pathogens

[0057]

[0058] Example 2 Identification of strain KG-43

[0059] The classification of the bacterial strain was preliminarily determined based on the colony morphology of strain KG-43 on the culture medium, Gram staining, scanning electron microscopy observation, and various physiological and biochemical index tests, and finally an evolutionary analysis was performed using the 16S rDNA gene sequence.

[0060] 1. Experimental Methods

[0061] The strain KG-43 obtained in Example 1 was inoculated on LB agar medium and cultured at 28°C for 48 hours, and the colony morphology, growth status, etc. were observed and recorded. The determination of relevant physical and chemical parameters such as Gram staining and morphological observation (scanning electron microscopy) was referred to the "Handbook of Identification of Common Bacterial Systems".

[0062] The 16S rDNA gene of strain KG-43 was sequenced, and the obtained sequences were compared with the sequences with high homology downloaded from NCBI.

[0063] 16S rDNA Primer Sequences:

[0064] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0065] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0066] PCR amplification system: 1 μL of each primer, 2 μL of DNA template, 12.5 μL of 2×Taq PCR Master MixⅡ, and ultrapure water to 25 μL.

[0067] PCR amplification conditions are shown in Table 2:

[0068] Table 2

[0069]

[0070] The PCR amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0071] 2. Experimental Results

[0072] (1) The growth status of strain KG-43 on LB medium is as follows Figure 2 As shown, it can be seen that the colony diameter of strain KG-43 on LB medium is about 3 to 4 mm, round, milky white, and watery.

[0073] (2) Gram staining results of strain KG-43 are as follows Figure 3 As shown, it can be seen that the colonies are short rod-shaped and light-colored, and are determined to be Gram-negative bacteria.

[0074] (3) The results of scanning electron microscopy (50000×) of strain KG-43 are as follows Figure 4 As shown, it can be seen that the long diameter of the colony is about 0.5 to 1.5 microns, short columnar, with a rough and wrinkled surface, and both ends are concave towards the center.

[0075] (4) The results of physiological and biochemical characteristics of strain KG-43 are shown in Table 3. It can be seen that strain KG-43 can grow on Simon's citrate agar medium, utilize citrate, can diffuse in semisolid agar medium, has motility, can decompose starch and gelatin, utilize most carbon such as glucose, and can fix nitrogen, dissolve potassium, and dissolve inorganic and organic phosphorus.

[0076] Table 3 Physiological and biochemical characteristics of strain KG-43

[0077]

[0078] Note: "+" represents a positive reaction, "-" represents a negative reaction

[0079] (5) The 16S rDNA sequencing results of strain KG-43 showed the highest similarity with Pantoea coffeiphila (as shown in Table 4).

[0080] Table 4 Gene sequence comparison results of strain KG-43

[0081]

[0082] (6) The results of 16S rDNA gene sequencing of strain KG-43 are as follows:

[0083] CCCGTATCAAAAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTTATGAGATCCGCTTGCCCTCGCGGGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCCCTGAGTTCCCGACCGAATCGCTGGCAACAGAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTGTGGATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAA CGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCAAGCCTGCCAGTTTCAAATGCAGTTCCCGGGTTGAGCCCGGGGATTTCACATCTGACTTAACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCCGGG

[0084] In summary, combined with morphological, physiological and biochemical characteristics and molecular identification, strain KG-43 was identified as Pantoeacoffeiphila and was deposited in Guangdong Provincial Microbiological Culture Collection on June 18, 2024, with the deposit number GDMCCNO: 64765.

[0085] Example 3 Potted plant control experiment of Pantoea KG-43 bacterial solution on mulberry bacterial wilt

[0086] 1. Experimental Methods

[0087] According to the results of the plate antibacterial experiment in Example 1, the control effect of Pantoea KG-43 on mulberry bacterial wilt was detected. 40 mulberry seedlings (120 days) with the same growth were divided into four groups, A, B, C, and D. After the root injury treatment, they were treated as follows:

[0088] Group A: 20 mL of Pantoea KG-43 cultured in LB medium for 2 days was applied;

[0089] Group B: 20 ​​mL of Pantoea KG-43 cultured in LB medium for 2 days was applied, and 20 mL of 10 9 CFU / mL of Ralstonia solanacearum solution;

[0090] Group C: 20 mL of sterile water was applied;

[0091] Group D: 20 mL of 10 9 CFU / mL of Ralstonia solanacearum solution.

[0092] After the four groups of potted plants were cultivated for 14 days, the growth and disease of the mulberry seedlings were investigated.

[0093] 2. Experimental Results

[0094] The results of potted control experiment are as follows Figure 5 As shown in Tables 5 and 6.

[0095] Table 5 Potted growth performance data of strain LJ-6 for controlling mulberry bacterial wilt

[0096]

[0097]

[0098] Table 6 Potted growth performance data of strain LJ-6 for controlling mulberry bacterial wilt

[0099]

[0100] The results showed that the mulberry trees in groups A and C grew normally, and in comparison, the growth of group A was better than that of group C, indicating that the Pantoea KG-43 of the present invention would not cause mulberry wilt disease and could promote the growth of mulberry trees.

[0101] All 10 mulberry trees in group D wilted to varying degrees, of which 6 wilted severely. In contrast, only 2 mulberry trees in group B wilted, indicating that the Pantoea KG-43 isolated by the present invention has a good control effect on mulberry wilt caused by Ralstonia solanacearum.

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Pantoea coffeiphila KG-43, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 18, 2024, with the deposit number GDMCC NO: 64765.

2. A biological agent, characterized in that: Contains the Pantoea KG-43 described in claim 1.

3. A biological agent, characterized in that: A fermentation broth containing the Pantoea KG-43 according to claim 1.

4. A biological agent, characterized in that: A fermentation product containing the Pantoea KG-43 described in claim 1.

5. Use of Pantoea KG-43 as claimed in claim 1 or any biological preparation as claimed in claims 2 to 4 in inhibiting the growth of plant pathogens or preparing antibacterial agents.

6. Use of Pantoea KG-43 described in claim 1 or any biological agent described in claims 2 to 4 in preventing and controlling plant diseases caused by plant pathogens.

7. Use of Pantoea KG-43 described in claim 1 or any biological agent described in claims 2 to 4 in the preparation of products for preventing and controlling plant diseases caused by plant pathogens.

8. The use according to any one of claims 5 to 7, characterized in that: The plant pathogenic bacteria are one or two of Ralstonia sanguineus and Pantoea ananas.

9. A method for preventing and controlling mulberry tree diseases, characterized in that: The Pantoea KG-43 described in claim 1 or any biological agent described in claims 2 to 4 is used to prevent and control mulberry tree diseases.

10. The method according to claim 9, characterized in that: The mulberry tree disease is caused by one or both of Ralstonia thunbergii and Pantoea ananas.

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