Stenotrophomonas maltophilia strain YN4-2 with bacteriostatic function, bacteriostatic agent and application of stenotrophomonas maltophilia strain YN4-2

Abstract

The invention provides a stenotrophomonas maltophilia strain YN4-2 with a bacteriostatic function, a bacteriostatic agent and application of the stenotrophomonas maltophilia strain YN4-2, and belongs to the technical field of microorganisms. The preservation number of the stenotrophomonas maltophilia strain YN4-2 is CGMCC (China General Microbiological Culture Collection Center) No.30474, and the preservation number of the stenotrophomonas maltophilia strain YN4-2 is CGMCC No.30474. A plate confrontation experiment shows that the stenotrophomonas maltophilia strain YN4-2 has broad-spectrum antibacterial activity, can effectively inhibit the activity of pathogenic fungi and pathogenic bacteria of moraceae plants, has the strongest antibacterial activity on rhodosporium virens and botryosphaeria mori, and has the inhibition rate of 88% or above. In addition, the stenotrophomonas maltophilia strain YN4-2 can inhibit silkworm pathogenic bacteria beauveria bassiana, has no toxic or side effect on silkworms, and can be used as an effective strain for biological prevention and control of plant diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Stenotrophomonas maltophilia strain YN4-2 with antibacterial function, an antibacterial agent and application thereof. Background Art

[0002] Mulberry is a perennial economic crop with a long history of cultivation in my country. It can not only be used for silkworm breeding, but also has edible and medicinal value and ecological functions. However, during the growth process, mulberry trees are often threatened by a variety of pathogenic microorganisms, resulting in a decrease in the yield and quality of mulberry leaves, which in turn affects the stable development of the sericulture industry. It is reported that there are about 200 types of pathogens that cause mulberry diseases, mainly including fungal diseases, bacterial diseases and viral diseases, among which fungal diseases are the most common. For the prevention and control of mulberry diseases, chemical prevention is mainly used. Although chemical pesticides can effectively prevent and control the occurrence of diseases in the short term, the long-term use of chemical pesticides will not only make pathogens resistant and reduce the prevention effect, but also destroy the ecology, pollute the environment and increase the risk of silkworm poisoning, death or inability to cocoon normally, thus affecting the sustainable development of the sericulture industry.

[0003] In view of this, finding a more environmentally friendly, safe and sustainable pest control technology has become a key issue that needs to be urgently addressed in the current silk industry. In recent years, biological control, as a green control concept, has received more and more attention due to its unique advantages. Biological control uses biological resources in nature, such as natural enemies, competitive microorganisms, and biogenic pesticides, to control the occurrence and spread of diseases. Compared with chemical control, biological control has the significant advantages of being highly targeted, environmentally friendly, and not easy to induce drug resistance. Therefore, the development and optimization of biological control technology is of great significance to promoting the green development of the silk industry, ensuring ecological and environmental safety, and improving the yield and quality of silk cocoons. Summary of the invention

[0004] The object of the present invention is to provide a strain of Stenotrophomonas maltophilia YN4-2 with antibacterial function, which has a broad-spectrum antibacterial activity and can effectively inhibit the activity of pathogenic fungi of Moraceae plants (Mulberry Brown Spot Shell Sporangia, Mori Stem Spot Mildew Leaf Spot Fungus, Mori Ring Spot Fungus, Mori Paint Spot Fungus and Pink Trichothecene) and pathogenic bacteria, among which the antibacterial activity against Mulberry Brown Spot Shell Sporangia and Mori Ring Spot Fungus is the strongest, with an inhibition rate of more than 88%. In addition, the Stenotrophomonas maltophilia strain YN4-2 can also inhibit the silkworm pathogen Beauveria bassiana and has no toxic side effects on silkworms.

[0005] The invention provides a Stenotrophomonas maltophilia strain YN4-2, whose deposit number is CGMCC No.30474.

[0006] The invention provides an antibacterial agent, comprising the Stenotrophomonas maltophilia strain YN4-2 and auxiliary materials.

[0007] Preferably, the concentration of the Stenotrophomonas maltophilia strain YN4-2 in the antibacterial agent is 1×10 7 CFU / g and above.

[0008] The present invention provides the use of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in inhibiting the activity of pathogenic bacteria of Moraceae plants.

[0009] The present invention provides the use of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in preventing and controlling diseases caused by pathogenic bacteria of Moraceae plants.

[0010] Preferably, the Moraceae plant pathogens include Moraceae plant pathogenic fungi and / or Moraceae plant pathogenic bacteria.

[0011] Preferably, the plant pathogenic fungi of the Moraceae family include at least one of the following species: Phloeospora maculans, Boeremia exigua, Gonatophragmium mori, Myrothecium rorideum and Trichothecium roseum;

[0012] The plant pathogenic bacteria of the Moraceae family include at least one of the following species: Kosakonia cowanii, Enterobacter cloacae, and Stenotrophomonas sp.

[0013] Preferably, the diseases caused by the pathogenic fungi of the Moraceae family include at least one of the following: mulberry brown spot disease, mulberry ring disease, mulberry leaf spot disease and mulberry stem spot mildew leaf spot disease.

[0014] The present invention provides application of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in preparing a medicine for inhibiting Beauveria bassiana.

[0015] The present invention provides application of the Oligotrophomonas maltophilia strain YN4-2 or the antibacterial agent in silkworm breeding.

[0016] Beneficial Effects

[0017] The invention provides a strain of Stenotrophomonas maltophilia YN4-2 with antibacterial function, and the deposit number of the strain of Stenotrophomonas maltophilia YN4-2 is CGMCC No. 30474. Plate confrontation experiments show that the strain of Stenotrophomonas maltophilia YN4-2 has a broad-spectrum antibacterial activity, and can effectively inhibit the activity of pathogenic fungi of Moraceae plants (Mulberry Brown Spot Shell Sporangiophora, Mulberry Stem Spot Mildew Leaf Spot Fungus, Mulberry Ring Pattern Fungus, Mulberry Paint Spot Fungus and Pink Trichothecene) and pathogenic bacteria (Cossackia corylifolia, Enterobacter cloacae and Stenotrophomonas oligotrophica), among which the antibacterial activity against Mulberry Brown Spot Shell Sporangiophora and Mulberry Ring Pattern Fungus is the strongest, and the inhibition rate is above 88%. In addition, the strain of Stenotrophomonas maltophilia YN4-2 can also inhibit the pathogenic bacteria of silkworm Beauveria bassiana and has no toxic side effects on silkworms. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony images of Stenotrophomonas maltophilia strain YN4-2 on PDA and LB medium;

[0019] Figure 2 The phylogenetic tree of the Stenotrophomonas maltophilia strain YN4-2 constructed based on the 16S rDNA sequence;

[0020] Figure 3 The diagram shows the inhibitory effects of different strains of YN4-2 on pathogenic fungi;

[0021] Figure 4 This is a graph showing the inhibitory effects of different strains of YN4-2 on pathogenic bacteria.

[0022] Biomaterial Deposit Information

[0023] Stenotrophomonas maltophilia strain YN4-2 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on April 29, 2024. The address of the deposit center is: Institute of Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 30474. DETAILED DESCRIPTION

[0024] The invention provides a Stenotrophomonas maltophiliam strain YN4-2, whose deposit number is CGMCC No.30474.

[0025] In the present invention, the maltophilia strain YN4-2 is isolated from the current year branches of Nongsang No. 8. The colonies of the maltophilia strain YN4-2 are milky white, smooth, with irregular colony edges and opaque on PDA medium; and yellow, smooth, sticky, opaque, with irregular colony edges ( Figure 1

[0026] In the present invention, the method for activating and expanding the culture of the maltophilia strain YN4-2 is preferably: the maltophilia strain YN4-2 is inoculated into a potato dextrose agar (PDA) medium, and the colony of the activated maltophilia strain YN4-2 is obtained, and a single colony is inoculated into potato dextrose water (PDB) with an inoculation needle for expansion culture, so as to obtain the seed liquid of the maltophilia strain YN4-2. The culture temperature of the activation and expansion culture is independently preferably 24 to 29°C, and most preferably 25°C; the culture time of the activation and expansion culture is independently preferably 1 to 4 days, and more preferably 2 days.

[0027] In the present invention, the maltophilia strain YN4-2 has a broad-spectrum antibacterial activity, and can effectively inhibit the activity of pathogenic fungi of the Moraceae family (Mulberry Brown Spot Shell Sporangia, Mori Stem Spot Mildew Leaf Spot Fungus, Mori Ring Pattern Fungus, Mori Paint Spot Fungus and Pink Trichothecene) and pathogenic bacteria (Cossackia corylifolia, Enterobacter cloacae and Xanthomonas oligotrophica), among which the antibacterial activity against Mulberry Brown Spot Shell Sporangia and Mori Ring Pattern Fungus is the strongest, with an inhibition rate of more than 88%. In addition, the maltophilia strain YN4-2 can also inhibit the silkworm pathogen Beauveria bassiana and has no toxic side effects on silkworms.

[0028] Based on the fact that the strain YN4-2 has a broad-spectrum antibacterial function, the present invention provides an antibacterial agent, comprising the Stenotrophomonas maltophilia strain YN4-2 and auxiliary materials.

[0029] In the present invention, the concentration of the strain YN4-2 of Stenotrophomonas maltophilia in the antibacterial agent is 1×10 7 CFU / g or more. The antibacterial agent preferably includes bacterial powder and / or aqueous solution. There is no special limitation on the preparation method of the antibacterial agent, and the preparation scheme of the antibacterial agent well known in the art can be adopted. For example, the activated and expanded cultured Stenotrophomonas maltophilia strain YN4-2 is mixed with auxiliary materials to obtain the obtained product. The present invention does not specifically limit the composition of the auxiliary materials, and the auxiliary materials of the antibacterial agent well known in the art can be adopted. When the antibacterial agent is bacterial powder, the auxiliary materials preferably include at least one of the following: oligoxylose, soluble starch and sodium salt. When the antibacterial agent is an aqueous solution, the auxiliary materials preferably include at least one of the following: water, glycerol, sorbic acid, zein and glycerol.

[0030] The present invention provides the use of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in inhibiting the activity of pathogenic bacteria of Moraceae plants.

[0031] In the present invention, the Moraceae plant pathogenic fungi preferably include at least one of the following species: Phloeospora maculans, Boeremia exigua, Gonatophragmium mori, Myrothecium rorideum and Trichothecium roseum; more preferably include at least one of the following species: Phloeospora maculans, Boeremia exigua and Trichothecium roseum; most preferably include Phloeospora maculans and / or Boeremia exigua.

[0032] The plant pathogenic bacteria of the Moraceae family preferably include at least one of the following species: Kosakonia cowanii, Enterobacter sp. and Stenotrophomonas sp.

[0033] In one embodiment of the present invention, the plate confrontation experiment verified that the strain YN4-2 of Stenotrophomonas maltophilia had an inhibitory effect on a variety of mulberry disease bacteria, wherein the inhibition rate of mulberry brown spot shell spore fungus and mulberry ring spot fungus was above 88%, and the inhibition rates of pink trichothecene, mulberry stem spot mildew leaf spot fungus and mulberry paint spot fungus were 71.06%, 62.25% and 49.36% respectively. It can be seen that the strain YN4-2 of Stenotrophomonas maltophilia has the effect of inhibiting plant pathogens of the mulberry family.

[0034] The present invention provides the use of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in preventing and controlling diseases caused by pathogenic bacteria of Moraceae plants.

[0035] The present invention provides the use of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in preventing and controlling diseases caused by pathogenic bacteria of Moraceae plants.

[0036] In the present invention, the type of the plant pathogen of the Moraceae family is preferably the same as the above-mentioned application. The method of using the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent is preferably to apply the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent to the aerial part of the plant.

[0037] In the present invention, the diseases caused by the pathogenic fungi of the Moraceae family include at least one of the following: mulberry brown spot disease, mulberry ring disease, mulberry leaf spot disease and mulberry stem spot disease, and more preferably include mulberry brown spot disease and / or mulberry ring disease.

[0038] The present invention provides application of the Stenotrophomonas maltophilia strain YN4-2 or the antibacterial agent in preparing a medicine for inhibiting Beauveria bassiana.

[0039] In one embodiment of the present invention, a plate confrontation experiment showed that the Stenotrophomonas maltophilia strain YN4-2 can effectively inhibit the pathogenic bacteria Beauveria bassiana of silkworms, with an inhibition rate of 78.32%.

[0040] The present invention provides application of the Oligotrophomonas maltophilia strain YN4-2 or the antibacterial agent in silkworm breeding.

[0041] In one embodiment of the present invention, the fermentation liquid of the Stenotrophomonas maltophilia strain YN4-2 was fed to silkworms. The results showed that the Stenotrophomonas maltophilia strain YN4-2 had no toxic side effects on silkworms and could be used in silkworm breeding to inhibit the infection of Beauveria bassiana to silkworms.

[0042] To further illustrate the present invention, the following is a detailed description of a Stenotrophomonas maltophilia strain YN4-2 with antibacterial function, an antibacterial agent and its application provided by the present invention 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] Test culture medium (pH 6-6.5):

[0044] Potato agar medium (PDA): 200 g potato, 15 g agar, 20 g glucose, 1000 ml distilled water;

[0045] LB solid medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, distilled water 1000ml;

[0046] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 ml distilled water;

[0047] Nutrient agar (NA): peptone 10g, beef extract 5g, sodium chloride 3g, agar 15g, distilled water 1000ml.

[0048] The pathogenic fungi tested were: Phloeospora maculans, Boeremia exigua, Gonatophragmium mori, Myrothecium rorideum, Trichothecium roseum and Beauveria bassiana.

[0049] Pathogenic bacteria of mulberry trees tested: Kosakonia cowanii, Enterobacter cloacae, and Stenotrophomonas sp.

[0050] The test strains were isolated (hosts were from mulberry trees and silkworms), identified and preserved in our laboratory, and were activated for later use.

[0051] Example 1

[0052] 1. Strain Screening

[0053] (1) Mulberry varieties: In a mulberry orchard where mulberry brown spot disease is serious, branches of the mulberry brown spot disease-resistant variety Nongsang No. 8 (2-4 cm thick) were selected for strain screening

[0054] Isolation and purification of endophytic biocontrol Bacillus of mulberry: Cut the branches of Nongsang No. 8 into 3-4cm long sections, rinse with tap water, then immerse in 75% alcohol for 1min for surface disinfection, wash with sterile water 3 times, then disinfect with 0.1% mercuric chloride hydrochloric acid solution for 1min, finally rinse with sterile water 4 times and dry with sterile filter paper. Cut the surface disinfected branches into small sections with a surgical blade, inoculate on different PDA, NA and Gao's No. 1 culture media, and place the plates at 25℃ for constant temperature culture. Finally, the isolated and purified strains were obtained.

[0055] Screening of antagonistic strains: First, using Morizocystis as an indicator bacteria, the plate confrontation culture method was used to determine the antibacterial activity of the isolated strains, that is, the pathogen and the isolated and purified endophytic biocontrol bacteria were inoculated 2 cm in the middle of the PDA culture dish, repeated three times, and placed in a 25°C incubator for 15 days. The diameter of the colony was measured and the antibacterial rate was calculated according to formula I.

[0056] Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100% Formula I

[0057] The strains with significant antibacterial effects on Psoralea corylifolia were further evaluated for their inhibitory effects on other pathogens using the same method. Finally, strains YN4-2 and G3-4 were screened out, which had good inhibitory effects on a variety of pathogenic fungi. The results of the inhibition rates of strains YN4-2 and G3-4 on different pathogens are shown in Tables 1, 2, Figure 3 and Figure 4 .

[0058] Table 1 Inhibition rate of strains YN4-2 and G3-4 against different pathogens

[0059]

[0060]

[0061] Table 2 Inhibitory effect of strain 2FG3-4 on bacterial diseases of mulberry

[0062] Test strains YN4-2 antibacterial zone diameter (mm) G3-4 Inhibition zone diameter (mm) Enterobacter cloacae 20.50±0.30 16.51±0.49 Xanthomonas oligotrophicus 25.67±0.68 1.53±0.87 Coxsackia coli 17.35±0.97 4.52±0.83

[0063] According to the results in Tables 1 and 2, strain YN4-2 has good antibacterial effects on a variety of different pathogens. Strain YN4-2 has the strongest antibacterial activity against Mori brown spot Aspergillus and Mori ring pattern pathogens, with an inhibition rate of more than 88%. In addition, it also has a high inhibition rate against other pathogens such as Mori stem spot, Beauveria bassiana, Pink Synechocystis, Cossackia coryna, Enterobacter cloacae and Xanthomonas oligotrophicus.

[0064] Safety evaluation of bacterial solution for silkworms: The strain was inoculated into PDB medium and cultured at 29°C and 200 r / min for 48 h. The fermentation solution was centrifuged and the bacterial cells were retained. The cells were resuspended in sterile water and the concentration was adjusted to OD 600 =0.8 for standby use. The mulberry leaves were treated with the bacterial suspension by the dipping method. After dipping 10g of mulberry leaves in 10ml of bacterial suspension for 8 seconds, the mulberry leaves were placed in a ventilated place to dry naturally. The mulberry leaves soaked in clean water were used as a blank control. Then, healthy silkworms from the third instar were fed with dried mulberry leaves once a day until they formed cocoons on the cocoon nest. During this period, the growth of the silkworms was continuously monitored. The experimental results showed that feeding with bacterial suspension had no effect on the survival rate and cocooning rate of silkworms.

[0065] 2. Classification and identification of strain YN4-2:

[0066] (1) Colony morphology observation

[0067] The strain YN4-2 was streaked onto LB and PDA solid media and cultured in a 28°C incubator for 48 h. The colony morphology was observed. Figure 1 shown.

[0068] Depend on Figure 1 It can be seen that the colonies of strain YN4-2 are milky white, smooth and opaque on PDA medium; they are yellow, smooth, sticky, opaque, and have irregular colony edges on LB medium; and they are yellow, smooth, sticky, opaque, and have irregular colony edges on LB medium.

[0069] (2) Gram staining

[0070] Strain YN4-2 was subjected to Gram staining and observed under a microscope, and strain YN4-2 was found to be a Gram-negative bacterium.

[0071] (3) Physiological and biochemical characteristics

[0072] The physiological and biochemical characteristics of strain YN4-2 were determined. The test results are shown in Table 3.

[0073] Table 3 Physiological and biochemical characteristics of strain YN4-2

[0074] project result project result Oxidase + Starch hydrolysis + Catalase + Indole test - Citrate Utilization + Hydrogen sulfide test - Gelatin liquefaction + Propionate - Methyl red test - D-Xylitol - VP test + L-arabinose - Nitrate reduction + D-Mannitol - 7% NaCl growth - Glucose fermentation tube + Growth at pH 5.7 + Phenylalanine deaminase -

[0075] Note: + indicates a positive reaction, - indicates a negative reaction.

[0076] As shown in Table 3, strain YN4-2 is oxidase positive, can produce catalase (catalase positive), can hydrolyze starch, can utilize citrate, is negative in the methyl red test, can produce gelatinase, and is positive in the VP test.

[0077] (4) Molecular Biological Identification

[0078] Extraction of genomic DNA of strain YN4-2: Use a sterilized toothpick to pick up a single colony and place it in LB liquid culture medium, shake and culture it at 30°C, 200 rpm overnight, then use a bacterial genomic DNA extraction kit (Shanghai Bioengineering Co., Ltd.) to extract bacterial genomic DNA, and use an ultra-micro nucleic acid protein analyzer to determine the concentration and purity of total DNA.

[0079] 16SrDNA gene amplification and sequence determination: Amplify the 16SrDNA gene using genomic DNA as a template.

[0080] The primers used for amplification were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3);

[0081] The PCR amplification reaction system was 25 μL: 10× buffer 2.5 μL, 2.5 mmol / L dNTP 0.5 μL, 2.5 U / L Taq polymerase 0.5 μL, 1 μL each of upstream and downstream primers, 18.5 μL ddH2O, and 1 μL template.

[0082] Amplification reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, 30 cycles, and extension at 72°C for 2 min.

[0083] The amplified product was detected by 1% agarose gel electrophoresis and then sent to BGI for sequencing. The obtained sequence was compared for homology with the sequence registered in the GenBank database. The sequencing results were spliced ​​to obtain the nearly full-length sequence of the 16S rRNA gene of the YN4-2 strain. The 16S rDNA sequence of the strain YN4-2 is shown in SEQ ID NO: 1, and the length is 1450bp. The obtained sequence was compared for homology and phylogenetic analysis with the sequence registered in the GenBank database. The strain YN4-2 had a high similarity with Stenotrophomonas maltophilia, with a similarity of 99%. The evolutionary tree showed that it was clustered with Stenotrophomonas maltophilia. Therefore, combined with the morphological characteristics and molecular identification results, the strain YN4-2 was identified as Stenotrophomonas maltophilia.

[0084] 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. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Stenotrophomonas maltophilia strain YN4-2, characterized in that: The deposit number is CGMCC No.30474.

2. An antibacterial agent, characterized in that The method comprises the Stenotrophomonas maltophilia strain YN4-2 according to claim 1 and auxiliary materials.

3. The antibacterial agent according to claim 2, characterized in that The concentration of the strain YN4-2 of Stenotrophomonas maltophilia in the antibacterial agent is 1×10 7 CFU / g and above.

4. Use of the Stenotrophomonas maltophilia strain YN4-2 according to claim 1 or the antibacterial agent according to claim 2 or 3 in inhibiting the activity of plant pathogens of the Moraceae family.

5. Use of the Stenotrophomonas maltophilia strain YN4-2 according to claim 1 or the antibacterial agent according to claim 2 or 3 in preventing and controlling diseases caused by pathogenic bacteria of Moraceae plants.

6. The use according to claim 4 or 5, characterized in that: The Moraceae plant pathogens include Moraceae plant pathogenic fungi and / or Moraceae plant pathogenic bacteria.

7. The use according to claim 6, characterized in that: The plant pathogenic fungi of the Moraceae family include at least one of the following species: Phloeospora maculans, Boeremia exigua, Gonatophragmium mori, Myrothecium rorideum and Trichothecium roseum; The plant pathogenic bacteria of the Moraceae family include at least one of the following species: Kosakonia cowanii, Enterobacter cloacae, and Stenotrophomonas sp.

8. The use according to claim 6, characterized in that: The diseases caused by the pathogenic fungi of the Moraceae family include at least one of the following: mulberry brown spot disease, mulberry ring disease, mulberry leaf spot disease and mulberry stem spot mildew leaf spot disease.

9. Use of the Stenotrophomonas maltophilia strain YN4-2 according to claim 1 or the antibacterial agent according to claim 2 or 3 in the preparation of a medicament for inhibiting Beauveria bassiana.

10. Use of the Stenotrophomonas maltophilia strain YN4-2 according to claim 1 or the antibacterial agent according to claim 2 or 3 in silkworm breeding.

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