Pseudomonas orientalis strain LJ-6 and application thereof in preventing and controlling diseases of mulberry trees

Abstract

The application discloses a strain of Pseudomonas extremorientalis LJ-6 and application of the strain in preventing and controlling mulberry diseases. The Pseudomonas extremorientalis in the application is preserved in the Guangdong Microbial Culture Collection Center on June 18, 2024, and the preservation number is GDMCC NO: 64766. The Pseudomonas extremorientalis LJ-6 has good antagonistic effects on bacterial pathogenic bacteria such as Caulobacter segnis, Pantoea ananatis, Klebsiella michiganensis and Enterobacter cloacae, and fungal pathogenic bacteria such as Fusarium oxysporum, Curvularia lunata and Guignardia bidwellii. The Pseudomonas extremorientalis LJ-6 has good prevention and control effects on plant diseases caused by the above pathogenic bacteria invading plants, such as mulberry bacterial wilt, mulberry bacterial blight, mulberry leaf spot and mulberry anthracnose. Therefore, the Pseudomonas extremorientalis LJ-6 has good application prospect and value in preventing and controlling plant diseases such as mulberry and preparing disease prevention and control preparation products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant disease prevention and control. More specifically, it relates to a strain of Pseudomonas extremorientalis LJ-6 and its application in preventing and controlling mulberry diseases. BACKGROUND

[0002] Plant pathogenic bacteria, plant pathogenic fungi, after being transmitted and invading plants, will interfere with the normal physiological and biochemical functions of plants, and cause various pathological changes in the physiology or tissue structure of plants, leading to plant diseases and causing serious economic losses. For example, Ralstonia pseudosolanacearum, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae and other bacterial pathogenic bacteria, Fusarium oxysporum, Curvularia sp., Colletotrichum truncatum and other fungal pathogenic bacteria are common plant pathogenic bacteria.

[0003] Mulberry trees, as perennial woody plants used for both food and medicine, have high economic value. Traditionally, their main use is as feed for silkworms, making them an indispensable feed ingredient for the sericulture industry. At the same time, mulberry branches, roots, and other parts can be used in medicine, possessing 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. Bacterial diseases of mulberry trees mainly include bacterial wilt, bacterial blight, and mulberry downy mildew. Bacterial wilt is caused by a variety of pathogens, such as *Enterobacter cloacae*, *Klebsiella michiganensis*, and *Pantoea ananatis*. Bacterial blight is caused by *Ralstonia pseudosolanacearum* (of the Solanaceae family), while downy mildew is caused by *Pseudomonas syringae*. Bacterial diseases (syringae) are prone to outbreaks in many mulberry-producing areas due to their diverse pathogens, short disease cycles, and high infectivity, often leading to the withering and death of the entire plant. In addition, fungal diseases of mulberry trees are equally serious, affecting a wide variety of parts of the plant. Leaf diseases include brown spot, ring spot, anthracnose, red rust, powdery mildew, leaf blight, and leaf curl; mulberry diseases include sclerotium rot (both enlarged and shrunken); mulberry branch and trunk diseases include wilt, pseudo-wilt, and bud blight; and root diseases include purple feather disease. Both of these major categories of mulberry diseases are difficult to control effectively due to their complex and diverse pathogens and high infectivity.

[0004] Currently, the main control methods for these two types of mulberry diseases are traditional chemical pesticides. 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. Therefore, a green and effective control method is urgently needed. Ye Jingjing et al. (2014) screened a Bacillus subtilis X-16 strain that has a strong inhibitory effect on mulberry spores, providing a technical reference for the large-scale production of microbial agents; however, its antibacterial spectrum is relatively narrow, and its effectiveness in dealing with complex pathogens in practice may not be optimistic. Mulberry endophytic flora is rich in species, and with the gradual promotion and application of microbial agents, the concept of "treating bacteria with bacteria" has attracted more attention. Therefore, isolating more mulberry endophytic bacteria with a broader antibacterial spectrum that have antagonistic and control effects on mulberry pathogens is an important means to address the future green and ecological control of mulberry diseases. Summary of the Invention

[0005] The present application aims to develop new biocontrol bacteria, especially those capable of inhibiting multiple pathogenic bacteria, thereby coping with the complex and diverse mulberry diseases.

[0006] The first object of the present application is to provide a Pseudomonas extremorientalis LJ-6 strain.

[0007] The second object of the present application is to provide a biological agent containing the above-mentioned Pseudomonas extremorientalis LJ-6 or its fermentation product.

[0008] The third object of the present application is to provide the application of the above-mentioned Pseudomonas extremorientalis LJ-6 and the above-mentioned biological agent.

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

[0010] The above-mentioned objects of the present application are achieved by the following technical solutions:

[0011] The present application provides a Pseudomonas extremorientalis LJ-6, which was deposited with the Guangdong Microbial Culture Collection Center on June 18, 2024, and the deposit number is GDMCC NO: 64766.

[0012] Based on the bacteria, the present application also provides a biological agent containing the above-mentioned Pseudomonas extremorientalis LJ-6, or containing the fermentation broth of the Pseudomonas extremorientalis, or containing the fermentation product of the Pseudomonas extremorientalis LJ-6. The fermentation broth refers to the fermentation broth obtained by fermentation culture of Pseudomonas extremorientalis LJ-6 in the culture medium. The fermentation product refers to the product obtained after removing the bacterial cells from the fermentation broth.

[0013] The Pseudomonas extremorientalis LJ-6 of the present application has good antagonistic effect on Solanaceae Ralstonia, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia, and Colletotrichum gloeosporioides. Therefore, the following applications are also within the protection scope of the present application:

[0014] The application of the above-mentioned Pseudomonas extremorientalis LJ-6 or the above-mentioned biological agent in inhibiting the growth of plant pathogenic bacteria or preparing bacteriostatic agents.

[0015] The application of the above-mentioned Pseudomonas extremorientalis LJ-6 or the above-mentioned biological agent in preventing and controlling plant diseases caused by plant pathogenic bacteria.

[0016] The application of the above-mentioned Pseudomonas extremorientalis LJ-6 or the above-mentioned biological agent in preparing products for preventing and controlling plant diseases caused by plant pathogenic bacteria.

[0017] The plant pathogenic bacteria include one or more of Pseudomonas solanacearum, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata and Colletotrichum gloeosporioides.

[0018] As a specific implementation case, when the above pathogen infects mulberry, the plant disease includes mulberry bacterial wilt, mulberry bacterial blight, mulberry leaf spot, mulberry anthracnose and the like.

[0019] Therefore, the application also provides a method for preventing and treating mulberry diseases, which utilizes the above Pseudomonas extremorientalis LJ-6 or the above biological preparation to prevent and treat mulberry diseases.

[0020] Specifically, the mulberry diseases are caused by one or more of Pseudomonas solanacearum, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata and Colletotrichum gloeosporioides. Specifically, the mulberry diseases are mulberry bacterial wilt, mulberry bacterial blight, mulberry leaf spot, mulberry anthracnose and the like.

[0021] The application has the following beneficial effects:

[0022] The application separates a biocontrol bacterium which has good antagonistic effect on Pseudomonas solanacearum, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata and Colletotrichum gloeosporioides, and is identified and named as Pseudomonas extremorientalis LJ-6.

[0023] Taking mulberry as an example, the fermentation liquor of Pseudomonas extremorientalis LJ-6 is used to prevent and control plant diseases caused by the above pathogenic bacteria infecting plants, and the results show that the prevention and control effect is very good, safe and effective, and various problems such as soil environment deterioration and pathogen resistance enhancement caused by chemical pesticides can be avoided.

[0024] Therefore, the Pseudomonas extremorientalis LJ-6 or the fermentation liquor or the fermentation product after removing the bacterium body of the application has good application prospect and value in preventing and controlling plant diseases or preparing preparations for preventing and controlling plant diseases. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is an effect diagram of the inhibition effect of the strain LJ-6 on different bacterial pathogenic bacteria.

[0026] Figure 2 It is an effect diagram of the inhibition effect of the strain LJ-6 on different fungal pathogenic bacteria.

[0027] Figure 3 It is the growth condition of the strain LJ-6 on LB medium.

[0028] Figure 4 It is the gram staining result of the strain LJ-6.

[0029] Figure 5 The scanning electron microscope (12000x) results of strain LJ-6.

[0030] Figure 6 The potting effect diagram of strain LJ-6 for controlling mulberry bacterial wilt (A: extreme east Pseudomonas solatane LJ-6 bacterial liquid treatment; B: extreme east Pseudomonas solatane LJ-6 bacterial liquid + Solanaceae Ralstonia bacterial liquid treatment; C: sterile water treatment; D: Solanaceae Ralstonia bacterial liquid treatment). DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the 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. (Item No.: XKL0211).

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

[0035] Ralstonia pseudosolanacearum was isolated and preserved by the laboratory. It has been disclosed in the article "Yuan T, Li JH, Liu JP. The latest research progress of virulence genes and regulatory networks of Ralstonia pseudosolanacearum complex [J]. Microbiology Bulletin, 2023, 50(05).".

[0036] Fusarium oxysporum was isolated and preserved by the laboratory. It has been disclosed in the patent "Liu JP. 202310254957.8 A strain of Bacillus subtilis and a biocontrol agent and its application. [2023.08.25].".

[0037] Enterobacter cloacae, Klebsiella michiganensis, Pantoea ananatis, Curvularia sp., Colletotrichum truncatum and other pathogenic bacteria were isolated and preserved by the laboratory.

[0038] The culture medium information used in the following examples is as follows:

[0039] Beef extract proteose peptone agar medium (NA): beef extract 3 g, bacteriological peptone 10 g, NaCl powder 5 g, technical agar powder 18 g, distilled water 1000 mL, pH 7.4-7.6;

[0040] Beef extract proteose peptone liquid medium: beef extract 3 g, bacteriological peptone 5 g, glucose 10 g, yeast powder 0.5 g, distilled water 1000 mL, pH 7.0;

[0041] 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;

[0042] LB liquid medium: tryptone 10 g, yeast powder 5 g, NaCl powder 10 g, distilled water 1000 mL, pH 7.0;

[0043] TTC medium: bacteriological peptone 10 g, beef extract powder 3 g, NaCl powder 5 g, technical agar powder 18 g, pH 7.1-7.5;

[0044] MS medium: MS powder 4.74 g, sucrose 20 g, agar 16 g, distilled water 1000 mL, pH 5.8-6.0;

[0045] The raw materials of the above culture media are purchased from Guangdong Huanke Microbial Science and Technology Co., Ltd. and need to be sterilized at 121°C for 15 min before use.

[0046] Example 1 Isolation and purification of antagonistic bacterial strains

[0047] I. Experimental method

[0048] (1) Sample collection: 40 samples of mulberry stem of variety Lenzhou 40 were collected from Sangyuan (North latitude 23°9'43", East longitude 113°20'46") in Zengcheng District, Guangzhou, Guangdong Province.

[0049] (2) Isolation and purification of antagonistic bacteria

[0050] The 40 collected mulberry stem samples were respectively washed with tap water to clean the surface, dried and cut into small pieces with a length of 3 cm. The cut samples were sterilized with 75% ethanol for 3 min and then washed with sterile water for 4 times. Then the samples were sterilized with 0.1% mercuric chloride solution for 5 min and then washed with sterile water for 5 times. 0.2 mL of the sterile water from the last washing of the samples was taken and spread on LB medium, and incubated at 28°C for 1 day to test the surface sterilization effect of the samples.

[0051] The sample mulberry stem surface moisture was dried with sterile filter paper, and the remaining segment was cut in half to obtain the xylem. The xylem was cut into 0.1 cm 3 pieces under sterile conditions using scissors, placed in 10 mL sterile water, and shaken at 28°C and 140 r / min for 15 min on a shaker to obtain an endophyte-containing bacterial solution. According to the 10-fold gradient dilution method, the bacterial solution was diluted by 8 times to obtain 10 -1 ~ 10 -8 dilutions. 100 μL of the 10 -1 ~ 10 -8 dilutions were taken and uniformly coated on NA medium, MS medium, and LB medium using a coater, and each dilution was coated 3 times. The plates were placed in a 28°C incubator and incubated for 2 days. Single colonies were picked and streaked on LB agar medium plates, and pure strains were obtained and stored in glycerol at -80°C.

[0052] (3) Screening of antagonistic bacteria strains

[0053] The single colonies obtained above were cultured in LB liquid medium at 28°C and 180 r / min for 48 h to obtain the antagonistic bacteria solution. Then, the agar diffusion method was used to test the antibacterial activity using the pathogenic bacteria C. michiganensis, E. asburiae, K. michiganensis, P. panonensis, and the pathogenic fungi F. oxysporum, C. curvula, and C. gloeosporioides as indicator bacteria. 20 μL of the antagonistic bacteria solution was added to a small hole punched with a 5 mm punch, and 20 μL of sterile water was added as a negative control. The plates were incubated at 25°C for 4 days, and the presence of inhibition zones and their diameters were measured.

[0054] II. Experimental results

[0055] Through the screening of antagonistic bacteria, a strain with the best antagonistic effect on pathogenic bacteria (C. michiganensis, P. panonensis, K. michiganensis, and E. asburiae) and pathogenic fungi (F. oxysporum, C. curvula, and C. gloeosporioides) was obtained, and was designated as strain LJ-6.

[0056] The specific data of the antibacterial effect of strain LJ-6 on different pathogenic bacteria are shown in Tables 1 and 2, and the inhibition effect of strain LJ-6 on each pathogenic bacteria is shown in Figure 1 and Figure 2As shown in the figure, it can be seen that LJ-6 has good antibacterial effect on bacterial pathogens of Caulobacter segnis, Pantoea ananatis, Klebsiella michiganensis and Enterobacter cloacae, among which the antagonistic effect on Caulobacter segnis is the most obvious, and the diameter of the inhibition zone reaches 22.65±1.24mm, so the strain LJ-6 can be applied to the prevention and control of bacterial wilt of mulberry. The results of the plate agar diffusion method for antagonizing fungi show that the strain LJ-6 has good antibacterial effect on fungal pathogens of Fusarium oxysporum, Curvularia lunata and Colletotrichum gloeosporioides, so the strain LJ-6 can be applied to the prevention and control of leaf spot disease and anthracnose disease of mulberry.

[0057] Table 1: Antibacterial effect of strain LJ-6 on different pathogenic bacteria

[0058]

[0059] Table 2: Antibacterial effect of strain LJ-6 on different pathogenic fungi

[0060]

[0061] Example 2: Identification of strain LJ-6

[0062] According to the colony morphological characteristics of strain LJ-6 on the culture medium, gram staining, scanning electron microscope observation, and physiological and biochemical index test, the classification of the bacterial strain was preliminarily judged, and finally the evolutionary analysis was carried out through 16S rRNA, gyrB, rpoB and 23S rRNA gene sequences.

[0063] I. Experimental method

[0064] The strain LJ-6 selected in Example 1 was inoculated on LB agar medium and cultured at 28°C for 48h, and the colony morphology, growth condition and the like were observed and recorded. The determination of related physicochemical parameters such as gram staining and morphological observation (scanning electron microscope) was carried out according to the "Common Bacteria System Identification Manual".

[0065] The 16S rRNA, gyrB, rpoB and 23S rRNA genes of strain LJ-6 were sequenced, and the obtained sequences were compared with the sequences with high homology downloaded from NCBI.

[0066] 16S rRNA primer sequence:

[0067] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0068] 1492R: 5'-GGTTACCTTGTTACGACTT-3'

[0069] GyrB primer sequence:

[0070] gyrB-F: 5'-TCCGGCGGTCTGCACGGCGT-3'

[0071] gyrB-R: 5'-TTGTCCGGGTTGTACTCGTC-3'

[0072] rpoB primer sequences:

[0073] rpoB-F: 5'-GCAGTGAAAGARTTCTTTGGTTC-3'

[0074] rpoB-R: 5'-GTTGCATGTTNGNACCCAT-3'

[0075] 23S rRNA primer sequences:

[0076] 23S-F: 5'-TGGAGGTATCAGAAGTGCGAAT-3'

[0077] 23S-R: 5'-GGAACTTACCCGACAAGGAAT-3'

[0078] PCR amplification system: 1 μL of primer, 2 μL of DNA template, 12.5 μL of 2x Taq PCR Master Mix II, and ultra-pure water to 25 μL.

[0079] PCR amplification conditions are shown in Table 3:

[0080] Table 3

[0081]

[0082] The PCR amplification products were sent to Beijing Genki Biological Company for sequencing.

[0083] II. Experimental results

[0084] (1) The growth condition of strain LJ-6 on LB medium is shown in Figure 3 It can be seen that the colony diameter of strain LJ-6 on LB medium is about 1-2 mm, round, white and slightly transparent, and the surface is smooth.

[0085] (2) The gram staining result of strain LJ-6 is shown in Figure 4 It can be seen that the colony is rod-shaped, light red, and is determined as gram-negative bacteria.

[0086] (3) The scanning electron microscope (12000x) result of strain LJ-6 is shown in Figure 5 It can be seen that the colony length is about 1-2 microns, long rod-shaped, oval at both ends, and the surface is smooth.

[0087] (4) The physiological and biochemical characteristics of strain LJ-6 are shown in Table 4. It can be seen that strain LJ-6 can grow on Simons citrate agar medium, utilize citrate, utilize gluconate, diffuse in semi-solid agar medium, have motility, decompose starch, and utilize glucose and other carbon sources.

[0088] Table 4. Physiological and biochemical characteristics of strain LJ-6

[0089]

[0090] Note: "+" represents a positive reaction, and "-" represents a negative reaction

[0091] (5) Based on the sequence alignment results of 16S rRNA, gyrB, rpoB and 23S rRNA of strain LJ-6, as shown in Table 5, the sequencing alignment results have the highest similarity to Pseudomonas extremorientalis.

[0092] Table 5. Sequence alignment results of strain LJ-6

[0093]

[0094] In summary, combined with morphology, physiological and biochemical characteristics and molecular identification, strain LJ-6 is identified as Pseudomonas extremorientalis, and was preserved in the Guangdong Microbial Culture Collection Center on June 18, 2024, with the preservation number GDMCC NO: 64766.

[0095] Example 3 Pot experiment of antagonistic bacteria LJ-6 liquid on the prevention and control of bacterial wilt of mulberry

[0096] I. Experimental method

[0097] According to the results of the plate inhibition experiment of Example 1, the prevention and control effect of Pseudomonas extremorientalis LJ-6 on mulberry bacterial wilt was detected. 40 mulberry seedlings (120d) with the same growth vigor were divided into A, B, C and D groups, and after root injury treatment, the following treatments were carried out:

[0098] Group A: 20 mL of Pseudomonas extremorientalis LJ-6 liquid cultured in LB medium for 2 days was added;

[0099] Group B: 20 mL of Pseudomonas extremorientalis LJ-6 liquid cultured in LB medium for 2 days was added, and 7 days later, 20 mL of L. solanacearum liquid with a concentration of 10 9 CFU / mL was added;

[0100] Group C: 20 mL of sterile water was added;

[0101] Group D: 20 mL of Pseudomonas extremorientalis strain LJ-6 liquid with a concentration of 10 9 CFU / mL of Solanaceae Ralstonia was applied.

[0102] After four groups of potted co-cultivation for 14 days, the growth and disease of mulberry seedlings were investigated.

[0103] II. Experimental results

[0104] The results of the potted prevention and control experiment are shown in Figure 6 and Tables 6-7.

[0105] Table 6 Potted growth performance data of strain LJ-6 for preventing and treating mulberry bacterial wilt

[0106]

[0107] Table 7 Potted growth performance data of strain LJ-6 for preventing and treating mulberry bacterial wilt

[0108]

[0109]

[0110] The results show that the mulberry trees in group A and group C grow normally, and the growth of the mulberry trees in group A is better than that in group C, which indicates that the Pseudomonas extremorientalis LJ-6 of the application does not cause mulberry wilt disease and can promote the growth of mulberry trees.

[0111] However, 10 mulberry trees in group D all show different degrees of wilting, of which 7 are severely wilted. In comparison, only one mulberry tree in group B is wilted, which indicates that the Pseudomonas extremorientalis LJ-6 isolated by the application has a good prevention and control effect on mulberry bacterial wilt caused by Ralstonia of Solanaceae.

[0112] The above examples are the preferred embodiments of the application, but the embodiments of the application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the application shall be equivalent replacement methods and shall be included in the protection scope of the application.

Claims

1. An extremely orientalized Pseudomonas ( Pseudomonas extremorientalis LJ-6, characterized in that, The bacterium has been deposited with the Guangdong Microbial Culture Collection Center on June 18, 2024, and the deposit number is GDMCC NO: 64766.

2. A biological agent, characterized in that, The Pseudomonas extremorientalis LJ-6 of claim 1.

3. The use of the Pseudomonas extremorientalis LJ-6 of claim 1 or the biological preparation of claim 2 in inhibiting plant pathogenic bacteria or in preparing bacteriostatic agents, wherein the plant pathogenic bacteria are one or more of Xanthomonas campestris, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata, and Colletotrichum truncatum.

4. The use of the Pseudomonas extremorientalis LJ-6 of claim 1 or the biological preparation of claim 2 in preventing and controlling plant diseases caused by plant pathogenic bacteria, wherein the plant pathogenic bacteria are one or more of Xanthomonas campestris, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata, and Colletotrichum truncatum.

5. The use of the Pseudomonas extremorientalis LJ-6 of claim 1 or the biological preparation of claim 2 in preparing products for preventing and controlling plant diseases caused by plant pathogenic bacteria, wherein the plant pathogenic bacteria are one or more of Xanthomonas campestris, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata, and Colletotrichum truncatum.

6. A method for controlling a disease of mulberry characterized by, The use of the Pseudomonas extremorientalis LJ-6 of claim 1 or the biological preparation of claim 2 in preventing and controlling mulberry diseases caused by one or more of Xanthomonas campestris, Pantoea ananatis, Klebsiella michiganensis, Enterobacter cloacae, Fusarium oxysporum, Curvularia lunata, and Colletotrichum truncatum.

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