Antifungal staphylococcus sciuri and application thereof

Abstract

The present invention relates to antifungal bacteria and uses thereof. The invention also relates to a composition comprising said bacteria and to the use of said bacteria in inhibiting aspergillus or insect breeding.

Description

Technical Field

[0001] The invention relates to bacteria and uses thereof, in particular to antifungal Staphylococcus sciuri and uses thereof. Background Art

[0002] Fungi are an important class of insect pathogens. As fungal insecticides, they can be used in biological pest control. However, they also pose a major threat to some large-scale economic insects, such as silkworms and bees, and bring risks and losses to the industrial breeding of silkworms.

[0003] Compared with economic animals such as poultry and livestock, economic insects such as silkworms have a short life cycle, small size, and high stocking density. Once infected, they will die quickly and are difficult to treat. The disease is easy to spread quickly, causing large-scale infection and heavy losses. Therefore, prevention is more important than treatment. Strengthening the resistance of economic insects such as silkworms to diseases will achieve better results. However, taking the prevention of silkworm blast disease as an example, the current means are relatively limited, mainly limited to keeping the silkworm room dry, ventilated, and hygienic, and regularly applying environmental treatment methods such as anti-blast powder and lime powder with chlorine-containing disinfectants as the main ingredients. Once silkworms accidentally eat anti-blast powder, they may be poisoned.

[0004] In addition, insect feeds are also used in the breeding of economic insects such as silkworms. These feeds are generally not sterilized at high temperatures, and low-temperature storage affects energy consumption. When stored at room temperature for a long time, they are easily contaminated by fungi such as Aspergillus and become moldy and rotten, and insects such as silkworms will no longer eat them. On the other hand, once these mold contaminants are accidentally eaten by economic insects such as silkworms, the latter will also cause infection and disease. Therefore, common fungi such as Aspergillus flavus and Aspergillus oryzae are also very common hazards to insects such as silkworms. Summary of the invention

[0005] In view of the defects of the prior art, the inventors found that bacteria on the surface of insects can help insects resist infection by pathogenic fungi. For example, silkworms with relatively abundant bacteria on their surface a few days after molting are more resistant to Metarhizium Robertsii and Beauveria bassiana than silkworms with fewer bacteria on their surface just molting. Therefore, the inventors chose to screen bacteria with antifungal function from the surface of silkworms and add them as probiotics in the process of silkworm breeding to improve the immunity of silkworms to Bombyx batryticatus and the anti-corruption ability of silkworm feed.

[0006] The first aspect of the present invention provides a bacterium, which was deposited in the China General Microbiology Center (CGMCC) under CGMCC No. 28001 on July 24, 2023.

[0007] In one or more embodiments, the bacterial species is in the form of a live bacterial population, a freeze-dried bacterial population, an inanimate bacterial population, or a cell fraction thereof. Preferably, the bacterial species is in the form of a live bacterial population, or a cell fraction thereof.

[0008] The present invention also provides a method for culturing bacteria, comprising incubating the bacteria described herein with a culture medium suitable for culturing bacteria.

[0009] In one or more embodiments, the culture medium comprises peptone, beef extract, NaCl, agar, and sterile defibrinated sheep blood.

[0010] In one or more embodiments, the culture medium comprises peptone, yeast extract, and NaCl.

[0011] In one or more embodiments, the pH of the incubation is 6-8, preferably 7.2-7.4.

[0012] In one or more embodiments, the incubation temperature is 35-40°C, preferably 37°C.

[0013] The present invention also provides a composition comprising the bacterium described in any embodiment herein or a cell-free extract, filtrate, supernatant or lysate thereof.

[0014] In one or more embodiments, the composition can be in the form of a powder, a solid, a liquid, or a formulation.

[0015] In one or more embodiments, the composition is a liquid composition comprising the bacteria. In one or more embodiments, the composition is a buffer comprising the bacteria. In one or more embodiments, the buffer is a phosphate buffer. In one or more embodiments, the OD of the bacteria in the liquid composition is 600 0.01 to 5, preferably, OD 600 is 5.

[0016] The present invention provides a feed composition comprising the bacteria according to any embodiment of the present invention and an insect feed.

[0017] In one or more embodiments, the insect is an insect of the order Lepidoptera.

[0018] In one or more embodiments, the lepidopteran insect is an insect of the family Bombycidae, preferably an insect of the genus Bombyx, and more preferably the silkworm (Bombyx mori Linnaeus).

[0019] In one or more embodiments, the insect is an insect larva.

[0020] In one or more embodiments, the insect feed is mulberry leaves or products thereof.

[0021] In one or more embodiments, the feed composition is at OD 600 The mulberry leaves are soaked and dried in a bacterial solution of 0.01-5 (e.g., 0.1 or 1) of the bacteria. In one or more embodiments, the soaking lasts for at least 1 second, preferably at least 5 seconds, such as 5-10 seconds. In one or more embodiments, the drying is natural air drying.

[0022] In one or more embodiments, the feed composition comprises a commercially available silkworm feed product, such as SilkMate PS from Nippon Nosan Industry Co., Ltd. (NOSAN).

[0023] In one or more embodiments, the ratio of bacteria to insect feed in the feed composition is: 20 ml OD 600 Bacteria in bacterial buffer at 5: (1-1000 g) Insect feed, preferably: 20 ml OD 600 Bacteria in a bacterial buffer at 5: (10-200 g) insect feed, more preferably: 20 ml OD 600 Bacteria in bacterial buffer of 5:50 g insect feed.

[0024] The present invention also provides a method for raising silkworm larvae, comprising feeding the silkworm larvae with the feed composition described herein. In one or more embodiments, the silkworm larvae are 5th instar silkworm larvae. In one or more embodiments, the silkworm larvae are fed 40 grams of the feed composition every 24 hours.

[0025] The present invention also provides use of the bacterium described in any embodiment herein in inhibiting Aspergillus or in preparing an insect feed composition.

[0026] In one or more embodiments, the inhibiting Aspergillus is inhibiting Aspergillus in insect feed.

[0027] In one or more embodiments, the inhibiting Aspergillus is inhibiting the growth and / or spore germination of Aspergillus.

[0028] In one or more embodiments, the Aspergillus is Aspergillus flavus and / or Aspergillus oryzae.

[0029] In one or more embodiments, the insect is an insect of the order Lepidoptera.

[0030] In one or more embodiments, the lepidopteran insect is an insect of the family Bombycidae, preferably an insect of the genus Bombyx, and more preferably the silkworm (Bombyx mori Linnaeus).

[0031] In one or more embodiments, the insect is an insect larva.

[0032] In one or more embodiments, the insect feed is mulberry leaves or products thereof.

[0033] In one or more embodiments, the feed composition is at OD 600 The mulberry leaves are soaked and dried in a bacterial solution of 0.01-5 (e.g., 0.1 or 1) of the bacteria. In one or more embodiments, the soaking lasts for at least 1 second, preferably at least 5 seconds, such as 5-10 seconds. In one or more embodiments, the drying is natural air drying.

[0034] In one or more embodiments, the feed composition comprises a commercially available silkworm feed product, such as SilkMate PS from Nippon Nosan Industry Co., Ltd. (NOSAN).

[0035] In one or more embodiments, the ratio of bacteria to insect feed in the feed composition is: 20 ml OD 600 Bacteria in bacterial buffer at 5: (1-1000 g) Insect feed, preferably: 20 ml OD 600 Bacteria in a bacterial buffer at 5: (10-200 g) insect feed, more preferably: 20 ml OD 600 Bacteria in bacterial buffer of 5:50 g insect feed.

[0036] The present invention also provides a method for inhibiting Aspergillus in insect feed, comprising the step of adding the bacteria or composition described in any embodiment of the present invention to the insect feed.

[0037] In one or more embodiments, the inhibiting Aspergillus is inhibiting Aspergillus in insect feed.

[0038] In one or more embodiments, the inhibiting Aspergillus is inhibiting the growth and / or spore germination of Aspergillus.

[0039] In one or more embodiments, the Aspergillus is Aspergillus flavus and / or Aspergillus oryzae.

[0040] In one or more embodiments, the insect is an insect of the order Lepidoptera.

[0041] In one or more embodiments, the lepidopteran insect is an insect of the family Bombycidae, preferably an insect of the genus Bombyx, and more preferably the silkworm (Bombyx mori Linnaeus).

[0042] In one or more embodiments, the insect is an insect larva.

[0043] In one or more embodiments, the insect feed is mulberry leaves or products thereof.

[0044] In one or more embodiments, the feed composition is at OD 600 The mulberry leaves are soaked and dried in a bacterial solution of 0.01-5 (e.g., 0.1 or 1) of the bacteria. In one or more embodiments, the soaking lasts for at least 1 second, preferably at least 5 seconds, such as 5-10 seconds. In one or more embodiments, the drying is natural air drying.

[0045] In one or more embodiments, the feed composition comprises a commercially available silkworm feed product, such as SilkMate PS from Nippon Nosan Industry Co., Ltd. (NOSAN).

[0046] In one or more embodiments, the ratio of bacteria to insect feed in the feed composition is: 20 ml OD 600 Bacteria in bacterial buffer at 5: (1-1000 g) Insect feed, preferably: 20 ml OD 600 Bacteria in a bacterial buffer at 5: (10-200 g) insect feed, more preferably: 20 ml OD 600 Bacteria in bacterial buffer of 5:50 g insect feed.

[0047] The present invention also provides a kit for identifying the bacterium or its genome as described in any embodiment of the present invention, comprising a reagent for specifically detecting the bacterium.

[0048] In one or more embodiments, the reagent is an oligonucleotide primer, a probe or an antibody that specifically detects the bacteria. The kit also includes other reagents required for identifying bacteria using the primer, probe or antibody, such as a buffer, a nucleic acid polymerase, dNTP, a fluorescent dye, etc.

[0049] In one or more embodiments, the primers are shown as SEQ ID NOs: 1 and 2.

[0050] The present invention also provides a method for improving the resistance of insects to Beauveria or Metarhizium bacteria, comprising contacting the insects with the bacteria or composition described in any embodiment of the present invention. Preferably, the method comprises soaking the insects in a bacterial solution described in any embodiment of the present invention, wherein the concentration of the bacterial solution is in the range of OD600 =0.01~5, preferably, the concentration range of bacterial solution is OD 600 =5.

[0051] In one or more embodiments, the insect is an insect of the order Lepidoptera.

[0052] In one or more embodiments, the lepidopteran insect is an insect of the family Bombycidae, preferably an insect of the genus Bombyx, more preferably a silkworm (Bombyx mori Linnaeus). In one or more embodiments, the insect is an insect larva.

[0053] In one or more embodiments, the Beauveria bacterium is Beauveria bassiana.

[0054] In one or more embodiments, the Metarhizium bacterium is Metarhizium robertsii.

[0055] The present invention also provides a method for identifying the resistance of insects to bacteria of the genus Beauveria or Metarhizium, comprising detecting microorganisms on the surface of the insects. If the microorganisms include a bacterial strain with registration number CGMCC No. 28001, the insect has high resistance to bacteria of the genus Beauveria or Metarhizium.

[0056] In one or more embodiments, the method comprises:

[0057] (1) Isolation of microorganisms from insect surfaces, and

[0058] (2) Identify whether the microorganisms include the bacterial strain with registration number CGMCC No.28001.

[0059] In one or more embodiments, the insect is an insect of the order Lepidoptera.

[0060] In one or more embodiments, the lepidopteran insect is an insect of the family Bombycidae, preferably an insect of the genus Bombyx, more preferably a silkworm (Bombyx mori Linnaeus). In one or more embodiments, the insect is an insect larva.

[0061] In one or more embodiments, step (1) comprises isolating the microorganism from a liquid (including a buffer, such as PBS buffer) in contact with the molting skin of the insect larvae.

[0062] In one or more embodiments, step (2) includes amplifying the genome of the microorganism using SEQ ID NOs: 1 and 2 as primers, and identifying the amplified product.

[0063] Advantages of the present invention:

[0064] The present invention screens a strain of Staphylococcus sciuri (currently classified as Mammaliicoccus sciuri) capable of antagonizing fungi from the body surface of silkworms, and the strain can significantly inhibit the spore germination and colony growth of fungi in mixed culture with Roberts' green muscardine, Beauveria bassiana, Aspergillus flavus and Aspergillus oryzae. After treating silkworms with Staphylococcus sciuri bacterial liquid, the resistance of silkworms to two silkworm pathogens, namely, Roberts' green muscardine and Beauveria bassiana, can be significantly improved. At the same time, adding the Staphylococcus sciuri bacterial liquid into silkworm feed can significantly inhibit the germination of Aspergillus spores in the feed and prevent the feed from becoming moldy. Moreover, artificially adding Staphylococcus sciuri does not affect the growth and development of silkworm larvae, and feeding silkworms with mulberry leaves added with Staphylococcus sciuri has no obvious effect on the weight of their pupae and cocoon silk. The above results indicate that Staphylococcus aureus is an insect probiotic with application prospects. Adding it to insect feed can not only help prevent the feed from mildew, but also increase the colonization of Staphylococcus aureus on the insect surface through food, thereby helping economic insects resist insect pathogenic fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 : Bioassay of normal silkworm larvae at different ages (survival curves of two groups of normal silkworms (L5D0, L5D3) (A) infected with Metarhizium anisopliae, (B) infected with Beauveria bassiana. The spore concentrations of Metarhizium anisopliae and Beauveria bassiana were 5×10 7 conidia / mL. Log-rank test: *, P ≤ 0.05).

[0066] Figure 2 : Changes in the number of microorganisms on the body surface of silkworm larvae at different ages (the eluates of microorganisms on the body surface of silkworms at different ages were cultured on LB plates with no resistance, aztreonam resistance, and nafcillin resistance, and counted. The data among the groups were analyzed by one-way ANOVA: ns (not significant), P>0.05; ****, P≤0.0001).

[0067] Figure 3 :Inhibitory effect of silkworm surface microorganisms on spore germination of insect pathogenic fungi ((A) Spore germination rate of silkworm surface microorganisms after co-culture with Metarhizium Robertsii for 12 hours; (B) Spore germination rate of silkworm surface microorganisms after co-culture with Beauveria bassiana for 16 hours. The spore concentrations of Metarhizium and Beauveria bassiana were 5×10 6 conidia / mL. Each treatment was repeated 10 times. One-way ANOVA: ****, P≤0.0001)

[0068] Figure 4:The inhibitory effect of Staphylococcus sciuri on the spore germination of Aspergillus flavus (left) and Aspergillus oryzae (right) (the spore germination rate of Staphylococcus sciuri after mixed culture with Aspergillus flavus and Aspergillus oryzae for 12 hours, the spore concentration of Aspergillus flavus and Aspergillus oryzae was 5×10 6 conidia / mL, 10 replicates for each treatment, one-way ANOVA: ****, P ≤ 0.0001).

[0069] Figure 5 :Pretreatment with Staphylococcus sciuri can help silkworm larvae resist fungal infection ((A) Survival curves of silkworm larvae infected with Metarhizium anisopliae (B) Beauveria bassiana. Staphylococcus sciuri OD 600 The value is 5, and the spore concentrations of Metarhizium and Beauveria bassiana are both 5×10 7 conidia / mL. Log-rank test: ***, P ≤ 0.001; ****, P ≤ 0.0001).

[0070] Figure 6 : Pretreatment with Staphylococcus sciuri can help sterile silkworms resist fungal infection (the feasibility of obtaining sterile silkworms was verified by (A) 16S DNA fragment amplification and (B) plate coating. Survival curves of sterile silkworms infected with (C) Metarhizium and (D) Beauveria bassiana. Staphylococcus sciuri OD 600 The value is 5, and the spore concentrations of Metarhizium and Beauveria bassiana are both 5×10 7 conidia / mL. Log-rank test: *, P ≤ 0.05, ***, P ≤ 0.001).

[0071] Figure 7 : Staphylococcus australis inhibits the growth of Aspergillus and prevents silkworm feed from mildew (adding Aspergillus flavus and Aspergillus oryzae spores to silkworm feed will cause mildew, while adding Staphylococcus australis at the same time can inhibit the occurrence of mildew. All photos were taken 10 days after inoculation).

[0072] Figure 8 :Adding different concentrations of Staphylococcus sciuri to mulberry leaves had no effect on the growth and development of silkworms ((A) pupa weight and (B) cocoon weight on the fifth day of the pupal stage. Each treatment was repeated ten times, and each repeat was the average of the pupa weight or cocoon weight of 10 silkworms. One-way ANOVA, ns (not significant), P>0.05). DETAILED DESCRIPTION

[0073] The inventors have found that on the one hand, bacteria on the surface of insects can help insects resist infection by pathogenic fungi. For example, silkworms with relatively abundant bacteria on their surface a few days after molting are more resistant to Metarhizium Robertsii and Beauveria bassiana than silkworms with relatively few bacteria on their surface just molting. On the other hand, the bacteria can make insect feed have an antiseptic effect. Therefore,

[0074] The inventors screened out a bacterium by counting, separating and identifying microorganisms on the surface of insects, which is a bacterial strain with a deposit registration number of CGMCC No. 28001, which can inhibit the infection of insect pathogenic fungi. In one or more embodiments, the bacterial species is in the form of a live bacterial population, a freeze-dried bacterial population, an inanimate bacterial population, or a cell component thereof. Preferably, the bacterial species is in the form of a live bacterial population, or a cell component thereof.

[0075] Bacterial culture

[0076] The present invention provides a method for culturing bacteria, comprising incubating the bacteria described herein with a culture medium suitable for culturing bacteria. Exemplarily, the culture medium comprises peptone, beef extract, NaCl, agar and sterile defibrinated sheep blood, or comprises peptone, yeast extract and NaCl. Incubation refers to culturing cells under certain conditions, and the pH of the incubation described herein is 6-8, preferably 7.2-7.4; the temperature of the incubation is 35-40°C, preferably 37°C. Specifically, the sterilization conditions of the culture medium and the buffer are 121°C, 0.1MPa, and 15 minutes.

[0077] Composition

[0078] The present invention provides a composition comprising the bacteria described in any embodiment of the present invention or its cell-free extract, filtrate, supernatant or lysate, wherein the composition can be in powder, solid, liquid or formulation. In one or more embodiments, the composition is a liquid composition comprising the bacteria. In one or more embodiments, the composition is a buffer comprising the bacteria. In one or more embodiments, the buffer is a phosphate buffer. In one or more embodiments, the OD of the bacteria in the liquid composition is 600 0.01 to 5, preferably, OD 600 is 5.

[0079] The composition of the present invention may also be a feed composition, comprising the bacteria described in any embodiment of the present invention and insect feed. Adding the bacteria described in the present invention to the feed can play a role in preventing mold and spoilage. The ratio of bacteria to insect feed is: 20ml OD 600 The bacteria in the bacterial buffer of 5:50g insect feed. Exemplarily, the anti-mold effect described herein includes that the bacteria can inhibit the growth and / or spore germination of Aspergillus. Preferably, the Aspergillus is Aspergillus flavus or Aspergillus oryzae.

[0080] The term "feed" is a product form suitable for insects as known in the art, including hay, straw, silage, compressed and pelleted feed, oil and mixed rations, as well as germinated grains and beans (such as bean sprouts, fresh malt or malt liquor). In a specific embodiment, the feed of the present application is mulberry leaves or their products. Mulberry leaf products generally contain: mulberry leaf powder, protein (such as soy protein), starch, sugar, cellulose, plasticizer, citric acid, vitamins, minerals, preservatives, antibiotics. Generally, the protein content in mulberry leaf products is more than 1%, fat is more than 0.5%, fiber is less than 5%, and ash is less than 5. An example of a mulberry leaf product is SilkMate PS from Nippon Agro-Industry Co., Ltd. (NOSAN), which is in powder form and contains a plasticizer. The powder is heated after adding 2-5 times of water.

[0081] The term "insect" herein refers to Lepidoptera. In one or more embodiments, the Lepidoptera insect is an insect of the family Bombydae, preferably an insect of the genus Bombyx, more preferably a silkworm (Bombyx mori Linnaeus). In one or more embodiments, the insect is an insect larva.

[0082] Methods and uses

[0083] The use of the bacteria described in any embodiment of the present invention in inhibiting Aspergillus or in preparing insect feed. The Aspergillus inhibition is to inhibit Aspergillus in insect feed. In one or more embodiments, the Aspergillus is Aspergillus flavus and / or Aspergillus oryzae. Specifically, the Aspergillus inhibition is to inhibit the growth and / or spore germination of Aspergillus.

[0084] A method for inhibiting Aspergillus in insect feed, comprising the step of adding the bacterium or composition described in any embodiment of the present invention to the insect feed.

[0085] The present invention also provides a method for improving the resistance of insects to Beauveria or Metarhizium bacteria, comprising contacting the insects with the bacteria or composition described in any embodiment of the present invention. Specifically, the "contact" comprises soaking the insects with the bacterial solution described in any embodiment of the present invention, and the concentration of the bacterial solution ranges from OD 600 =0.01~5, preferably, the concentration range of bacterial solution is OD 600 = 5. In one or more embodiments, the bacterium of the genus Beauveria is Beauveria bassiana. The bacterium of the genus Metarhizium is Metarhizium robertsii.

[0086] The present invention also provides a method for identifying the resistance of insects to bacteria of the genus Beauveria or Metarhizium, comprising detecting microorganisms on the surface of the insects. If the microorganisms include a bacterial strain with registration number CGMCC No. 28001, the insect has high resistance to bacteria of the genus Beauveria or Metarhizium.

[0087] In one or more embodiments, the method comprises: (1) soaking the silkworm exuvia formed by insect larvae in 50 ml of sterile buffer to separate microorganisms on the insect body surface, and (2) identifying whether the microorganisms include a bacterial strain with registration number CGMCC No. 28001. Specifically, step (2) further comprises amplifying the genome of the microorganism using SEQ ID NO: 1 and 2 as primers, and identifying the amplified product.

[0088] Reagents

[0089] The present invention also provides a kit for identifying the bacterium or its genome as described in any embodiment of the present invention, comprising a reagent for specifically detecting the bacterium.

[0090] In one or more embodiments, the reagent is an oligonucleotide primer, a probe or an antibody that specifically detects the bacteria. The kit also includes other reagents required for identifying bacteria using the primer, probe or antibody, such as a buffer, a nucleic acid polymerase, dNTP, a fluorescent dye, etc.

[0091] In one or more embodiments, the primers are shown as SEQ ID NOs: 1 and 2.

[0092] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0093] Example

[0094] Example 1: Counting of bacterial colonies on the body surface of silkworm

[0095] In our study, we found that the fifth-instar silkworm (Bombyx mori) larvae (L5D0) just after molting were more susceptible to Metarhizium anisopliae ( Figure 1 , A; Log-rank analysis: χ 2 =5.78, P=0.016) and Beauveria bassiana ( Figure 1 , B; Log-rank analysis: χ 2=4.433, P=0.035) when infected. Since more than half of the fifth instar third day silkworm larvae (L5D3) had pupated but not died during the test period, the half-lethal time LT50 could not be calculated. To maintain consistency, this study no longer calculated the half-lethal time LT50, and directly compared the significance of the difference in survival curves by Log-rank analysis. From the results, it can be seen that the difference between the two is significant, P<0.05, that is, the resistance of the fifth instar third day silkworm larvae (L5D3) to insect pathogenic fungi is significantly stronger than that of the fifth instar silkworm larvae (L5D0) just after molting. We speculate that this may be due to the difference in the surface microorganisms of the two. For this reason, the surface microorganisms of silkworms at different stages were counted:

[0096] 1. The last day of the 4th instar silkworm larvae (L4E), the silkworm exuviae formed from the 4th to the 5th instar (L4S), the 1st day of the 5th instar silkworm larvae (L5D0), the 3rd day of the 5th instar silkworm larvae (L5D3), and the 6th day of the 5th instar silkworm larvae (L5D6) were selected and divided into five groups, each with thirty silkworm larvae. The average value of the microbial counts on the surface of every three silkworm larvae was one replicate, and each group had a total of ten replicates.

[0097] 2. After freezing anesthetization, each group of silkworms was placed in a 50 mL sterile centrifuge tube, sterile PBS solution was added, and vortexed for 30 seconds. The silkworms or silkworm exuviae were taken out, and diluted at a ratio of 1:500 and coated on non-antibiotic LB plates, Aztreonam (Aztreonam, inhibiting Gram-negative bacteria G-) resistant LB plates, and Nafcillin (Nafcillin, inhibiting Gram-positive bacteria G+) resistant LB plates. The antibiotic concentration was 50 μg / mL.

[0098] 3. Count the number of single clones on the plate after 48 hours.

[0099] 4. One-way analysis of variance.

[0100] By counting the colony forming unit (CFU) of the eluted bacterial colonies, we found that the total number of microorganisms on the body surface of silkworm larvae at the end of the fourth instar (L4E) and the total number of microorganisms on the fourth instar molt (L4S) were not much different; but just after the molt entered the fifth instar (L5D0), the number of microorganisms on the body surface of silkworms decreased rapidly. With the increase of time, from just after molting (L5D0) to the third day after molting (L5D3), the number of colonies rebounded rapidly, and reached the same level as the end of the fourth instar (L4S, L4E) on the sixth day of the fifth instar (L5D6) ( Figure 2 The CFU statistics on LB plates with nefcillin and aztreonam showed that the number of monoclonals on the plates with nefcillin was significantly reduced, and the number of CFU on the plates with aztreonam was not significantly different from that on the LB plates without antibiotics, further proving that the microorganisms on the surface of silkworms are mainly Gram-positive bacteria ( Figure 2 ).

[0101] The counting results were consistent with our expectations. Molting caused a significant reduction in the number of microorganisms on the body surface of silkworms, which may have caused the silkworms' resistance to fungal infections to decrease. The body surface of silkworms at the end of the fourth and fifth instars had a relatively rich number of Gram-positive bacteria. We tried to separate, purify, culture and identify the species of these surface bacteria, and screen for strains that have an inhibitory effect on fungal spore germination.

[0102] Example 2: Isolation and identification of microorganisms on the surface of silkworms

[0103] 1. Take the silkworm exuviae formed from the 4th to 5th instar of Bombyx mori, soak them in 50 mL of sterile PBS buffer, vortex and smear them on LB plates.

[0104] 2. After obtaining a single colony through cultivation, a single colony was picked and amplified by colony PCR using primers 518F (SEQ ID NO: 1: 5'-CGGTTACCTTGTTACGACTT-3') and 1452R (SEQ ID NO: 2: 5'-AGAGTTTGATCMTGGCTCAG-3') and high-fidelity enzymes from Nanjing Novozyme 2×Phanta Flash Master Mix (Dye Plus). The reaction system (Table 1) and reaction procedure (Table 2) are shown in the following table. The sequencing results of the amplified products were sequenced using NCBI to identify the species of the symbiotic bacteria, and the species composition was shown in Table 3.

[0105] Table 1. PCR amplification reaction system

[0106]

[0107] Table 2. PCR reaction program

[0108]

[0109] Table 3. Bacterial composition isolated from the fourth instar exuviae of silkworm larvae

[0110]

[0111] The 13 isolated and identified symbiotic bacteria on the surface of silkworms were screened for antifungal activity. The fungi selected were Metarhizium robertsii ARSEF23 and Beauveria bassiana ARSEF2860, the main pathogens of Bombyx batryticatus disease. The method is as follows:

[0112] 1. The isolated symbiotic bacteria were pre-cultured in LB liquid medium at 37°C, 220rpm, for 12 hours until the logarithmic growth phase (OD value was about 1-5 at this time). One single clone was one replicate, and each symbiotic bacteria had a total of ten replicates.

[0113] 2. Will be equipped with 5×10 6 coindia / mL of Metarhizium anisopliae or Beauveria bassiana was transferred to a 60 mm mixing culture dish.

[0114] 3. Add the pre-cultured symbiotic bacteria to the cell culture dish with fungal spores so that the initial OD of the symbiotic bacteria in the dish is 600 The value is 0.01.

[0115] 4. At 30°C, the Metarhizium was mixed cultured for 12 hours and the Beauveria was mixed cultured for 16 hours. The spore germination rate was then counted under a 400x microscope.

[0116] 5. One-way analysis of variance.

[0117] The results showed that Staphylococcus sciuri completely inhibited the spore germination of Metarhizium and Beauveria bassiana ( Figure 3 ). Therefore, Staphylococcus sciuri is the most promising insect probiotic.

[0118] On this basis, we further examined the inhibitory effect of S. sciuri on the spore germination of common saprophytic fungi Aspergillus flavus and Aspergillus oryzae to determine whether it has the potential to prevent insect feed from mold and spoilage.

[0119] The specific method is as follows:

[0120] 1. Pre-culture Staphylococcus aureus in LB medium at 37°C and 220 rpm. One single clone is considered as one replicate.

[0121] 2. Will be equipped with 5×10 6 coindia / mL of Aspergillus flavus or Aspergillus oryzae was transferred to a 60 mm mixing culture dish.

[0122] 3. Add the pre-cultured Staphylococcus sciuridae to the cell culture dish with fungal spores so that the initial OD of Staphylococcus sciuridae in the dish is 600 The value is 0.01.

[0123] 4. At 30℃, Aspergillus flavus and Aspergillus oryzae were mixed and cultured for 12 hours, and the spore germination rate was counted under a microscope at 400 times magnification. Every 100 spores was considered as one replicate.

[0124] The results showed that Staphylococcus sciuri had a significant inhibitory effect on the spore germination of Aspergillus flavus and Aspergillus oryzae, especially on the spore germination of Aspergillus flavus. Figure 4 ), suggesting that it may also have a good effect in preventing insect feed from mildew.

[0125] The squirrel Staphylococcus of the present invention was submitted to the China General Microbiological Culture Collection Center (CGMCC) for preservation on July 24, 2023, with a preservation number of CGMCC No. 28001. The preservation unit is the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The unit issued an acceptance notice and a viability report on August 23, 2023. The sterilization conditions of all the following culture media and buffers are: 121°C, 0.1MPa, 15 minutes.

[0126] 0.1M PBS buffer

[0127]

[0128] Dissolve in 800 mL of distilled water, adjust the pH of the solution to 7.4, and finally add distilled water to make up to 1 L.

[0129] LB solid medium

[0130]

[0131] Add water to make up to 200 mL.

[0132] Example 3: Colonization and proliferation of Staphylococcus sciuri on the body surface of silkworms helps them resist infection by Metarhizium and Beauveria bassiana

[0133] The immersion method was used to bioassay to verify the protective effect of Staphylococcus sciuri on silkworms against infection with Metarhizium and Beauveria bassiana. The silkworms in the experimental group were first soaked in Staphylococcus sciuridae, and then soaked in spore suspension of Metarhizium or Beauveria bassiana 24 hours later; the Ss OD5CK group, Mr CK group, and Bb CK group were soaked in Staphylococcus sciuridae, Metarhizium or Beauveria bassiana spore suspensions, respectively; the Mock group was soaked in 0.05% Tween-20. The specific method is as follows:

[0134] 1. Each group had 50 newly molted fifth-instar silkworm larvae. The experimental group and Ss OD5CK group were placed in OD 600 The cells were immersed in PBS solution of Staphylococcus aureus with a value of 5 for 30 seconds, while the Mr CK group and the Bb CK group were not immersed.

[0135] 2.24 hours later, the experimental group, Mr CK group, and Bb CK group were placed in 5×10 7 The mice were immersed in a spore suspension containing Metarhizium or Beauveria bassiana with spores / mL for 30 seconds, and the Mock group was immersed in a 0.05% Tween-20 solution.

[0136] 3. Record the number of silkworm deaths every 12 hours.

[0137] 4. Log-rank test was used for survival analysis.

[0138] The results showed that pretreatment with Staphylococcus aureus did not affect the survival of silkworm larvae and could significantly prolong the survival of silkworm larvae in the presence of Metarhizium anisopliae ( Figure 5 , A) (Log-rank analysis: 2 = 15.299, P = 0.000), and Beauveria bassiana ( Figure 5 , B) (Log-rank analysis: 2=10.334, P=0.001) survival time during infection, which helps to enhance the resistance of silkworms to Bombyx batryticatus. It can be seen that, whether it is to Metarhizium or Beauveria, the silkworms' resistance is significantly enhanced after pretreatment with Staphylococcus australis, P≤0.001, especially to Metarhizium. After pretreatment with Staphylococcus australis, more than half of the silkworms did not die during the test period, while more than 90% of the control group died. This test used newly molted fifth-instar silkworms, which are relatively sensitive to Metarhizium and Beauveria, and Staphylococcus australis showed a better protective effect.

[0139] Example 4: Staphylococcus sciuri helps sterile silkworms resist infection by Metarhizium and Beauveria bassiana

[0140] To further prove that Staphylococcus sciuridae helps silkworms resist Bombyx batryticatus, we also prepared sterile silkworms and measured the effect of Staphylococcus sciuridae pretreatment on sterile silkworms.

[0141] The culture and verification methods of sterile silkworms are as follows:

[0142] (1) Cultivation of sterile silkworms

[0143] 1. After the silkworms lay eggs, collect them in a sterile 120mm culture dish. Put wet cotton in the dish to keep it moist. Start aseptic treatment after all the silkworm eggs turn green.

[0144] 2. Add an appropriate amount of 75% alcohol to the culture dish in the clean bench, soak for 1 minute, and then discard the 75% alcohol.

[0145] 3. Add 1% sodium hypochlorite solution to the culture dish and soak for 30 seconds, then discard the 1% sodium hypochlorite.

[0146] 4. Then add 75% alcohol to the culture dish again to soak the silkworm eggs.

[0147] 5. Turn on the fan in the clean bench to maximum and let it air dry for 30 minutes. After the alcohol has completely evaporated, transfer it to a new sterile culture dish and put cotton in the sterile culture dish to keep it moist.

[0148] 6. Within 24 hours, almost all silkworm eggs were hatched. Add an appropriate amount of sterile feed Silkmate 2S (Nosan Corporation) sterilized by high-pressure steam for 15 minutes to the culture dish. Add once every 24 hours and replace the sterile culture dish every 48 hours.

[0149] (2) Validation of sterile silkworms

[0150] 1. Take three 5th-instar silkworms and put them in a 2 mL tube and crush them with sterile steel balls.

[0151] 2. Add 1 mL of sterilized PBS solution, vortex and smear the homogenate on the plate. Observe the results after 48 hours. If there is no single colony on the plate, it proves that the sterile silkworm culture is successful.

[0152] 3. The above homogenate was amplified by colony PCR using primers 518F (SEQ ID NO: 1: 5'-CGGTTACCTTGTTACGACTT-3') and 1452R (SEQID NO: 2: 5'-AGAGTTTGATCMTGGCTCAG-3') and Nanjing Novozyme 2×Phanta Flash MasterMix (Dye Plus) high-fidelity enzyme. The reaction system (Table 1) and reaction procedure (Table 2) were the same as before. At the same time, an appropriate amount of the homogenate was applied to the LB plate.

[0153] 4. If there are no colonies on the plate and the amplified product shows no bands, it proves that the sterile silkworm culture is successful.

[0154] Compared with the control group of common silkworms, the sterile silkworm larvae could not amplify the 16sDNA fragment; in addition, when the homogenate was applied to the LB plate, no bacterial clones grew compared with the control group of common silkworms ( Figure 6 , A, B), proving the reliability of the obtained sterile silkworm larvae. On this basis, we used sterile silkworm larvae for further bioassays, the method was the same as in Example 3. The results showed that pretreatment with Staphylococcus aureus can also promote the resistance of sterile silkworm larvae to Metarhizium anisopliae ( Figure 6 , C; Log-rank analysis: λ 2 =3.955, P = 0.047) and Beauveria bassiana infection ( Figure 6 , D; Log-rank analysis: λ 2=16.119, P=0.001). In this test, the protective effect of Staphylococcus sciuri on sterile silkworms was also significant, P<0.05. After pretreatment with Staphylococcus sciuri, about half of the sterile silkworms did not die during the test period.

[0155] Example 5: Inhibitory effect of Staphylococcus sciuri on Aspergillus in silkworm feed

[0156] In order to test the effect of S. squirrelii on preventing insect feed from mildew, we added S. squirrelii bacterial suspension and spore suspension of Aspergillus flavus and Aspergillus oryzae into silkworm feed to observe whether S. squirrelii can inhibit the growth of Aspergillus flavus and Aspergillus oryzae in silkworm feed. The specific method is as follows:

[0157] 1. Experimental group: 10 ml LB culture medium of Staphylococcus aureus, OD 600 For 5, with 10 ml of 5 × 10 6 conidia / mL of Aspergillus spore suspension. Control group: 10 ml of PBS solution and 10 ml of 5×10 6 coindia / mL Aspergillus spore suspension. Mock group: 20 ml PBS solution.

[0158] 2. Add 30 ml of sterile water to 50 g of silkworm feed (SilkMate PS product, Nippon Agricultural Industry Co., Ltd. (NOSAN)) powder, sterilize at 100°C for 30 min, add 20 ml of the corresponding bacteria and spore mixture, Aspergillus spore suspension, and PBS solution to the sterilized feed, stir well, and pour onto a plate.

[0159] 3.Take photos and observe after 10 days.

[0160] The results showed that the silkworm feed plates with only Aspergillus spore suspension added had a large amount of Aspergillus growing, while the plates with Staphylococcus aureus suspension added showed no trace of Aspergillus growing ( Figure 7 ), indicating that the growth of Aspergillus was significantly inhibited by Staphylococcus australis, demonstrating the excellent effect of Staphylococcus australis in preventing silkworm feed from mildew.

[0161] Example 6: Adding Staphylococcus aureus does not affect the growth and development of silkworm larvae

[0162] The above examples show that Staphylococcus sciuri has the function of being a probiotic for silkworms and has the potential for production and application. Although isolated from the surface of silkworm larvae, it is still necessary to answer whether artificial addition of this bacterium affects the growth and development of silkworm larvae. To this end, we used different OD 600Soak mulberry leaves with Staphylococcus aureus liquid with an OD value (OD 0.01, OD 0.1, OD 1, OD 5), and then use the dried mulberry leaves to feed the larvae that have just entered the fifth instar. After feeding until pupation, weigh the pupae and cocoon silk after five days of pupal stage. The specific method is as follows:

[0163] 1. Centrifuge the LB fermentation broth of Staphylococcus aureus at 5000 rpm for 10 minutes, discard the supernatant, and resuspend it in different amounts of sterile PBS buffer to make the OD 600 The values ​​were 0.01, 0.1, 1 and 5. Then, the mulberry leaves were immersed in PBS solutions of four different OD values ​​of Staphylococcus aureus, and the mulberry leaves of the Mock group were immersed in sterile PBS solution, and each group was immersed for 5 seconds.

[0164] 2. Dry the soaked mulberry leaves and feed them to the newly molted 5th instar silkworm larvae at a rate of 40 g every 24 hours. Measure the cocoon weight and pupa weight after 5 days of pupal stage, and obtain the cocoon silk weight by subtracting the pupa weight from the cocoon weight.

[0165] 3. Mock group, OD 0.01, OD 0.1, OD 1, OD 5, five groups in total, 10 replicates in each group, the average value of the weight of 10 silkworm pupae (or cocoon silk weight) represents one replicate.

[0166] 4. One-way analysis of variance.

[0167] One-way ANOVA revealed that there was no significant difference in the weight of pupae or cocoon silk formed under different feeding conditions compared with the Mock group ( Figure 8 ). It can be seen that the addition of Staphylococcus aureus does not affect the growth and development of silkworm larvae, does not affect the silk production, and is safe to use.

Claims

1. A bacterium, which is a bacterial strain with a deposit registration number of CGMCC No.28001.

2. A composition comprising the bacterium according to claim 1 or a cell-free extract, filtrate, supernatant or lysate thereof, Preferably, the composition can be in the form of powder, solid, liquid or formulation.

3. The composition according to claim 2, It is characterized in that The composition is a buffer containing the bacteria, and the OD of the bacteria in the liquid composition is 600 It is 0.01~5.

4. A feed composition comprising the bacterium according to claim 1 and an insect feed, Preferably, the insect is a Lepidoptera insect; Preferably, the insects are insect larvae.

5. Use of the bacterium according to claim 1 in inhibiting Aspergillus, in insect breeding, or in preparing insect feed, Preferably, the Aspergillus is Aspergillus flavus and / or Aspergillus oryzae; Preferably, the insect is a Lepidoptera insect; Preferably, the insects are insect larvae.

6. A kit for identifying the bacterium or its genome according to claim 1, comprising a reagent for specifically detecting the bacterium, Preferably, the reagent is an oligonucleotide primer, a probe or an antibody that specifically detects the bacteria.

7. A method for improving the resistance of insects to Beauveria and / or Metarhizium bacteria, comprising contacting the insects with the bacteria or composition described in any embodiment of the present invention, Preferably, the insect is a Lepidoptera insect; Preferably, the insects are insect larvae.

8. The method according to claim 7, It is characterized in that The bacterium of the genus Beauveria is Beauveria bassiana, and the bacterium of the genus Metarhizium is Metarhizium robertsii.

9. The method according to claim 7 or 8, It is characterized in that The method comprises soaking the insect in the bacterial solution of claim 1, wherein the concentration of the bacterial solution is OD 600 =0.01~5.

10. A method for identifying the resistance of an insect to bacteria of the genus Beauveria or Metarhizium, comprising detecting microorganisms on the surface of the insect. If the microorganisms include a bacterial strain with registration number CGMCC No. 28001, the insect has a high resistance to bacteria of the genus Beauveria or Metarhizium. Preferably, the method include: (1) Isolation of microorganisms from insect surfaces, and (2) identifying whether the microorganisms include a bacterial strain with registration number CGMCC No. 28001, Preferably, the insect is a Lepidoptera insect, Preferably, the insects are insect larvae.

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