A strain of pan-mycobacterium KG-43 and its application in the control of mulberry diseases

Abstract

This invention discloses a strain of Pantoea coffeiphila KG-43 and its application in the control of mulberry diseases. A strain of Pantoea coffeiphila KG-43 was obtained and deposited at the Guangdong Provincial Microbial Culture Collection Center on June 18, 2024, with accession number GDMCC NO: 64765. This Pantoea coffeiphila KG-43 exhibits excellent antagonistic activity against *Ralstonia solanaceae* and *Pantoea coffeiphila*, demonstrating good control effects against plant diseases caused by these pathogens, including *Ralstonia solanaceae* and bacterial wilt of mulberry, and is safe and effective. Therefore, Pantoea coffeiphila KG-43 or its fermentation products have promising applications and value in the control of plant diseases such as mulberry and in the preparation of formulations for controlling plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology. More specifically, it relates to a strain of Pantotheca officinale KG-43 and its application in controlling mulberry diseases. Background Technology

[0002] Mulberry trees are deciduous trees or shrubs belonging to the genus *Morus* in the family Moraceae. As a perennial woody plant used for both food and medicine, it has high economic value. Traditionally, its main use was as feed for silkworms, making it an indispensable feed ingredient for the sericulture industry. At the same time, mulberry branches, roots, and other parts can be used in medicine, giving it high medicinal and economic value. With the continuous deepening of research on the mulberry industry, mulberry trees are widely used in various industries such as food, animal husbandry, and ecology, playing an increasingly important role. However, mulberry diseases, especially bacterial and fungal diseases, have a devastating impact on mulberry production and development, seriously restricting the healthy and stable development of the mulberry industry.

[0003] Bacterial diseases of mulberry trees mainly include bacterial wilt and bacterial wilt. Bacterial wilt has numerous pathogens, such as *Pantoea ananatis*, while bacterial wilt is caused by *Ralstonia pseudosolanacearum* (of the Solanaceae family). Cross-infection is common, further complicating the disease situation. These bacterial diseases are characterized by complex pathogens, high infectivity, and short disease cycles, frequently breaking out in many mulberry-producing areas. They often lead to the death of the entire plant, making effective control difficult. Currently, the main control method for these mulberry diseases is traditional chemical pesticide application. However, long-term use of chemical pesticides can deteriorate the soil environment, reduce the richness of soil microbial communities, and increase the drug resistance of pathogens, failing to fundamentally solve the problem.

[0004] Therefore, a green and effective prevention and control method is urgently needed. Liu et al. (2023) screened a strain of Bacillus tequilensis K3-11, which has good antagonistic effects against Solanaceae's Raulella and Pantotheca pineapple, and can be used to control bacterial wilt and Fusarium wilt of mulberry. Mulberry has a rich variety of endophytic flora, and with the gradual promotion and application of microbial agents, the "using bacteria to control bacteria" approach has attracted more attention. Therefore, isolating endophytic bacteria with antagonistic and control effects against mulberry pathogens is an important means to address future green and ecological prevention and control of mulberry diseases. Summary of the Invention

[0005] This invention aims to overcome the defects and shortcomings of existing methods for controlling mulberry diseases, especially bacterial diseases, and to provide a biocontrol agent that can simultaneously target both Pantoea ananatis and Ralstonia pseudosolanacearum, the pathogen of bacterial wilt in mulberry, thus addressing the more complex bacterial diseases of mulberry in practice.

[0006] The first objective of this invention is to provide a pan-bacterial KG-43 strain.

[0007] A second objective of this invention is to provide a biological agent containing the aforementioned pantothecin KG-43 or its fermentation products.

[0008] A third objective of this invention is to provide applications of the aforementioned pantothecin KG-43 and the aforementioned biological agents.

[0009] The fourth objective of this invention is to provide a method for preventing and controlling diseases of mulberry trees.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a strain of Pantoea coffeiphila KG-43, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 18, 2024, with accession number GDMCC NO: 64765.

[0012] This invention provides a biological agent for controlling plant diseases, wherein the biological agent contains the above-mentioned pantothecin KG-43, or a fermentation broth containing the pantothecin KG-43, or a fermentation product containing the pantothecin KG-43.

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

[0014] The fermentation product refers to the product obtained after removing the bacterial cells from the fermentation broth.

[0015] The pantothecin KG-43 of this invention has a good antagonistic effect on *Raylorhizium anisopliae* and *Pantothecinium pineapple*, therefore the following applications should also be within the scope of protection of this invention:

[0016] The application of the above-mentioned pan-yeast KG-43 or the above-mentioned biological agents in inhibiting the growth of plant pathogens.

[0017] Application of the above-mentioned Pan-Mycobacterium KG-43 or the above-mentioned biological agents in the prevention and control of plant diseases caused by plant pathogens.

[0018] The application of the above-mentioned Pan-Mycobacterium KG-43 or the above-mentioned biological agents in the preparation of products for the prevention and control of plant diseases caused by plant pathogens.

[0019] The plant pathogens include *Laurella multocida* and / or *Panthera pineapple*.

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

[0021] The present invention also provides a method for preventing and controlling mulberry diseases, wherein the method involves using the above-mentioned Pantothecin KG-43 or the above-mentioned biological agents to prevent and control mulberry diseases.

[0022] Specifically, the mulberry diseases mentioned are caused by one or two of the following: *Laurella multocida* and *Panthera pineapple*.

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

[0024] This invention isolated a biocontrol bacterium exhibiting strong antagonistic activity against *Laurella multocida* and *Pantotheca acuminata*, which was identified and named *Pantotheca acuminata* KG-43. *Pantotheca acuminata* KG-43, its fermentation broth, or the fermentation product after removing the bacterial cells, demonstrates excellent control effects against plant diseases (mulberry bacterial wilt and mulberry bacterial wilt) caused by the aforementioned pathogens. It is safe and effective, avoiding the problems associated with chemical pesticides, such as soil degradation and increased pathogen resistance. Therefore, *Pantotheca acuminata* KG-43, its fermentation broth, or the fermentation product after removing the bacterial cells have promising applications and value in controlling plant diseases and preparing formulations for plant disease control. Attached Figure Description

[0025] Figure 1 The image shows 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 results of Gram staining for strain KG-43.

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

[0029] Figure 5 The potted plant effects of strain KG-43 in controlling bacterial wilt of mulberry are shown in Figure A (KG-43 bacterial solution treatment); Figure B (KG-43 bacterial solution + Ralstonia solanaceae bacterial solution treatment); Figure C (sterile water treatment); and Figure D (Ralstonia solanaceae bacterial solution treatment). Detailed Implementation

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

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

[0032] Unless otherwise specified, all 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. (Catalog No.: XKL0211).

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

[0035] Ralstonia pseudosolanacearum, a member of the Solanaceae family, was isolated and preserved in 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 in our laboratory and has been disclosed in the article "[1] Wang Jicheng, Sun Xunxun, Luo Longhui, Liu Jiping. Isolation and identification of four soil-borne disease-preventing bacteria of mulberry [J]. Sericultural Science, 2020, 46(1):31-36".

[0037] Information on the culture media 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 culture medium: 3g beef extract, 5g bacteriological peptone, 10g glucose, 0.5g yeast powder, 1000mL distilled water, pH 7.0;

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

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

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

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

[0044] All the above culture medium raw materials were purchased from Guangdong Huankai Microbial Technology Co., Ltd., and all were sterilized at 121℃ for 15 minutes before use.

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

[0046] I. Experimental Methods

[0047] (1) Sample collection: In February 2022, the author, Li Jinhao, collected 40 samples of the roots of the Kangqing No. 10 mulberry tree in the mulberry garden of Xiahua Village, Zengcheng District, Guangzhou City, Guangdong Province (23°9'43"N"E).

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

[0049] The collected mulberry root samples were washed clean with tap water, dried, and the branches were cut into 3cm sections. Under aseptic conditions, the cut root samples were disinfected with 75% ethanol for 3 minutes, rinsed 4 times with sterile water, then disinfected with 0.1% mercuric chloride solution for 5 minutes, and rinsed 5 times with sterile water. 0.2mL of the sterile water from the last rinse was taken and spread on LB medium and incubated at 28℃ for 1 day to test the surface disinfection effect of the samples.

[0050] Following the method of Liang Jiajun (2018), after blotting the surface moisture of the mulberry root sample with sterile filter paper, the two ends were cut off. The remaining section was split open from the middle to remove the xylem. Under sterile conditions, the xylem was cut into 0.1 cm pieces with scissors. 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 suspension containing endophytic bacteria. The bacterial suspension was then serially diluted 8 times according to the 10-fold serial dilution method to obtain 10... -1 ~10 -8 The bacterial culture dilution. Take 100 μL of each of the 10... -1 ~10 -8 The bacterial suspension was diluted and evenly spread on NA, MS, and LB agar plates using a spreader, with each dilution repeated three times. The plates were incubated upside down at 28°C for 2 days. Single colonies were then picked and streaked onto LB agar plates for purification to obtain pure strains. These pure strains were then stored in glycerol at -80°C.

[0051] (3) Screening of antagonistic bacterial strains

[0052] The isolated single colonies were cultured in LB liquid medium at 28°C and 180 rpm for 48 h to obtain candidate antagonistic bacterial suspensions. Then, using the pathogenic bacteria *Raulella solanaceae* and *Panthera pineapple* as indicator bacteria, the antibacterial activity was tested using the agar diffusion method. 20 μL of the candidate antagonistic bacterial suspension was added to wells punched with a 5 mm punch using a micropipette. For the negative control, 20 μL of sterile water was added. The mixtures were incubated at 25°C for 4 days, and the presence and diameter of inhibition zones were measured.

[0053] II. Experimental Results

[0054] Through screening experiments of antagonistic bacteria, a strain with significant antibacterial effect against pathogens such as *Lauroceras sorbifolium* and *Panthera pineapple* was obtained, and it was designated as strain KG-43.

[0055] The specific data on the antibacterial effect of strain KG-43 against the two pathogens are shown in Table 1, and the graphs showing the inhibitory effect of strain KG-43 against the two pathogens are shown in Figure 2. Figure 1 As shown in the figure, KG-43 has a good inhibitory effect on the bacterial pathogens *Raulella solanaceae* and *Panthera pineapple*. The antagonistic effect on *Raulella solanaceae* is the most obvious, with the inhibition zone diameter reaching 23.75±1.69 mm. Therefore, strain KG-43 can be used for the prevention and control of mulberry bacterial wilt and mulberry bacterial wilt.

[0056] Table 1. Antibacterial effects of strain KG-43 against different pathogens.

[0057]

[0058] Example 2: Identification of strain KG-43

[0059] Based on the colony morphology of strain KG-43 on the culture medium, Gram staining, scanning electron microscopy, and various physiological and biochemical indicators, the bacterial strain classification was initially determined. Finally, evolutionary analysis was performed using the 16S rDNA gene sequence.

[0060] I. Experimental Methods

[0061] The strain KG-43 obtained in Example 1 was inoculated onto LB agar medium and cultured at 28°C for 48 hours. Colony morphology and growth status were observed and recorded. Gram staining, morphological observation (scanning electron microscopy), and other relevant physicochemical parameters were determined according to the "Handbook of Systematic Identification of Common Bacteria".

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

[0063] 16S rDNA primer sequence:

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

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

[0066] PCR amplification system: 1 μL each of primers, 2 μL of DNA template, 12.5 μL of 2×Taq PCR Master Mix II, and ultrapure water to a final volume of 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] II. Experimental Results

[0072] (1) The growth status of strain KG-43 on LB medium is as follows: Figure 2 As shown, the colonies of strain KG-43 on LB medium are approximately 3–4 mm in diameter, round, milky white, and milky in appearance.

[0073] (2) Gram staining results of strain KG-43 are as follows Figure 3 As shown, the colonies are short rod-shaped and light-colored, indicating they are Gram-negative bacteria.

[0074] (3) The scanning electron microscope (50000×) results of strain KG-43 are as follows: Figure 4 As shown, the colony length is about 0.5 to 1.5 micrometers, short columnar, with a rough and wrinkled surface, and both ends are concave towards the center.

[0075] (4) The 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, diffuse in semi-solid agar medium, has motility, can decompose starch and gelatin, utilize glucose and most carbons, can fix nitrogen, solubilize potassium, dissolve inorganic phosphorus and organic phosphorus.

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

[0077]

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

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

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

[0081]

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

[0083] CCCGTATCAAAAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTTATGAGATCCGCTTGCCCTCGCGGGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCCCTGAGTTCCCGACCGAATCGCTGGCAACAGAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTGTGGATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAA CGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCAAGCCTGCCAGTTTCAAATGCAGTTCCCGGGTTGAGCCCGGGGATTTCACATCTGACTTAACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCCGGG

[0084] In summary, based on morphological, physiological and biochemical characteristics and molecular identification, strain KG-43 was identified as Pantotheacoffeiphila and deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 18, 2024, with accession number GDMCCNO: 64765.

[0085] Example 3: Pot experiment on the control of bacterial wilt of mulberry by pan-mycelium KG-43 bacterial solution.

[0086] I. Experimental Methods

[0087] Based on the results of the plate inhibition experiment in Example 1, the control effect of pan-mycelium KG-43 on mulberry bacterial wilt was tested. Forty mulberry seedlings (120 days old) with similar growth were divided into four groups: A, B, C, and D. After root damage treatment, they were treated as follows:

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

[0089] Group B: 20 ​​mL of pantothenic acid KG-43 cultured on LB medium for 2 days was applied. 7 days later, 20 mL of a 10% concentration was applied. 9 CFU / mL of *Raulella solanaceae* suspension;

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

[0091] Group D: Apply 20 mL of a 10% concentration. 9 CFU / mL of *Laurella multocida* (society of solanaceous bacteria).

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

[0093] II. Experimental Results

[0094] Results of potted plant control experiment as follows Figure 5 As shown in Tables 5 and 6.

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

[0096]

[0097]

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

[0099]

[0100] The results showed that the mulberry trees in both groups A and C grew normally, and compared with group C, the growth of group A was better; indicating that the pantothecin KG-43 of the present invention does not cause mulberry wilt disease and can promote mulberry growth.

[0101] All 10 mulberry trees in group D showed varying degrees of wilting, with 6 of them severely wilted. In contrast, only 2 mulberry trees in group B showed wilting, indicating that the pantothecin KG-43 isolated in this invention has a good control effect on bacterial wilt of mulberry caused by *Raphettich. solanaceae*.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of pantothecin KG-43, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 18, 2024, with accession number GDMCC NO: 64765.

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

3. The application of the pan-mycelium KG-43 of claim 1 or the biological agent of claim 2 in inhibiting the growth of plant pathogens or in preparing antimicrobial agents that inhibit the growth of plant pathogens, wherein the plant pathogen is one or both of Solanaceae Raulella and Pan-mycelium pineapple.

4. The application of the pan-mycelium KG-43 of claim 1 or the biological agent of claim 2 in the prevention and control of plant diseases caused by plant pathogens, wherein the plant pathogen is one or both of Solanaceae Raulella and Pan-mycelium pineapple.

5. The use of the Pan-Mycobacterium KG-43 of claim 1 or the biological agent of claim 2 in the preparation of products for controlling plant diseases caused by plant pathogens, wherein the plant pathogen is one or both of Solanaceae Raulella and Pan-Mycobacterium pineapple.

6. A method for preventing and controlling mulberry tree diseases, characterized in that, The pantothecin KG-43 of claim 1 or the biological agent of claim 2 is used to control mulberry diseases, wherein the mulberry diseases are caused by one or two of the following: Solanaceae Raulella and Pantothecin pineapple.

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