A strain of Streptomyces fusiforme LS159 and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to *Streptomyces fusiforme* and its applications in the biological control of plant diseases and food preservation. The LS159 strain obtained in this invention has the preservation number CGMCC No. 19503. It possesses broad-spectrum antibacterial properties and can be used both as a biocontrol agent and to prevent food spoilage, thus having wide-ranging application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to microbial strains and their applications in the fields of biological control of plant diseases and food preservation, and more specifically to a strain of Streptomyces fusiforme and its applications. Background Technology

[0002] Microorganisms are ubiquitous and widespread in nature. Most of them are harmless or beneficial to humans, utilizing live bacterial cells for food fermentation and vaccine development, and their physiological metabolism for the production of organic acids, enzymes, and antibiotic metabolites. They also play a vital role in the natural cycle of matter and resource utilization. However, a small number of microorganisms can cause plant diseases and food spoilage, resulting in harm and losses for humans. For harmful microorganisms causing plant diseases and food spoilage, the use of chemical pesticides and chemical preservatives has traditionally been the primary method. With the improvement of people's living standards and the gradual increase in awareness of food safety and environmental protection, the demand for green and low-toxicity prevention and control measures is constantly increasing.

[0003] Actinomycetes, a class of Gram-positive prokaryotic microorganisms, are widely distributed in soil, water, and on and inside plants. Many species possess antimicrobial properties, making them commonly used in plant disease control and food preservation. Studies have shown that some actinomycetes inhibit the growth of harmful microorganisms by producing antibiotics or chitinases, or by limiting and preventing pathogen growth through niche and nutrient competition, thereby reducing disease infection and damage. Among actinomycetes, Streptomyces (…) are the most studied and applied. Streptomyces spp.), such as Streptomyces flavus ( S. microflavus ), kanamycin streptomycin ( S. kanamyceticus ), Streptomyces coccidioides ( S. rimosus ), Streptomyces coccidioides ( S. globisporus ), etc., but regarding Streptomyces spindleii ( S. netropsis There are few reports on the control and related properties of harmful microorganisms. Chinese patent (publication number: CN107058131A) discloses a strain of *Streptomyces fusiforme* that controls wheat scab and reduces gibberellin content in wheat; this bacterium was isolated and screened from soil collected from rice paddies. Numerous studies have shown that different strains of the same microorganism exhibit significant differences in biological characteristics and antagonistic effects against different diseases. Therefore, it is necessary to continue screening for more strains capable of controlling other diseases to provide a richer source of microbial resources for the application of this bacterium.

[0004] The fermentation of microbial agents requires the addition of suitable nutrients such as carbon and nitrogen sources. To reduce costs, waste products from industrial and agricultural production are often considered when selecting culture media. These waste products contain certain amounts of carbon and nitrogen sources, and with appropriate adjustments, they can serve as excellent culture media sources for microbial agent fermentation. Beer production processes, including saccharification, fermentation, and bottling, generate large amounts of wastewater. This beer wastewater contains abundant organic matter such as sugars and proteins. my country is a major beer producer and consumer, generating 3-10 tons of wastewater per ton of beer produced. The large volume of beer wastewater discharge means breweries spend significant funds and consume substantial amounts of energy annually to treat it. However, from another perspective, the organic matter in beer wastewater (including sugars and proteins) provides excellent nutrient substrates for microbial growth and can be used to create microbial culture media for fermentation and production of microbial products. Summary of the Invention

[0005] Based on this, this invention, while screening for *Streptomyces fusiforme* strains that inhibit plant pathogens and food spoilage microorganisms, also conducts physiological and molecular identification of these strains and utilizes brewery wastewater for fermentation culture of *Streptomyces fusiforme*. This will be of great significance for reducing the use of chemical agents, lowering environmental pollution, improving food safety, and achieving sustainable agricultural development. The inventors ultimately screened and identified one strain of *Streptomyces fusiforme* (…). Streptomyces netropsis The invention was completed by studying LS159 and its functions in depth.

[0006] This invention first provides a strain of Streptomyces spindleii ( Streptomyces netropsis The LS159 strain has the accession number CGMCC No.19503.

[0007] The results showed that strain LS159 was effective against the pathogen of chestnut blight (… Cryphonectria parasitica Poplar tree sclerotium ( Rhizoctonia solani ), Carrot soft rot pathogen ( Pectobacterium carotovorum ) and the pathogen of bacterial wilt of tomato ( Ralstonia solanacearum It has a strong inhibitory effect; in addition, it has a strong inhibitory effect on the pathogen of root rot of Chinese cabbage (… Fusarium oxysporum ) and bacterial poplar canker pathogen ( Botryosphaeria dothidea It also has a certain inhibitory effect. Moreover, it has a better antibacterial spectrum and antibacterial effect against these eight pathogens than the same *Streptomyces fusiforme* strain (strain number LS286). Compared with other *Streptomyces* strains isolated from the same region, its antibacterial spectrum and antibacterial effect also differ. Therefore, this invention further provides the application of the LS159 strain as a biocontrol bacterium. More specifically, it is used to control the pathogen of chestnut blight (… Cryphonectria parasitica Poplar tree sclerotium ( Rhizoctonia solani ), Carrot soft rot pathogen ( Pectobacterium carotovorum ) or tomato bacterial wilt pathogen ( Ralstonia solanacearum Fungal diseases caused by ); or used for the pathogen of root rot of Chinese cabbage ( Fusarium oxysporum ), bacterial poplar canker pathogen ( Botryosphaeria dothidea It is used to control bacterial diseases caused by bacteria. More specifically, it is used for the prevention and control of diseases in chestnuts, poplars, carrots, tomatoes, or Chinese cabbage.

[0008] Further research showed that strain LS159 had antagonistic effects against eight common fungi and bacteria that cause food spoilage, including Penicillium expansum (…). Penicillium expansum Staphylococcus aureus ( Staphylococcus aureus ) and Escherichia coli ( Escherichia coli It has the best antagonistic effect against Aspergillus flavus ( ). Aspergillus flavus Alternaria ( Alternaria alternata ) and Bacillus cereus ( Bacillus cereus It also has a good inhibitory effect. Therefore, this invention further provides the application of the LS159 strain in the prevention and control of food spoilage. More specifically, it is used to inhibit Aspergillus niger (…). Aspergillus niger Aspergillus flavus Aspergillus flavus ), extended Penicillium ( Penicillium expanse Alternaria ()), Alternaria ( Alternaria alternata ), or Staphylococcus aureus ( Staphylococcus golden ), Escherichia coli ( Escherichia coli ), Bacillus cereus ( Bacillus cereus Bacillus coagulans ( Bacillus coagulans ).

[0009] Further research in this invention shows that the LS159 strain can effectively utilize a culture medium with beer wastewater as the main component. Compared to ISP2 medium, in the initial 1-3 days of cultivation, the growth rate of *Streptomyces fusiforme* fermented with beer wastewater is slower than that treated with ISP2 medium fermentation, but the growth rates of both gradually approach each other in the later stages of fermentation. Therefore, this invention also provides a method for fermenting the LS159 strain using beer wastewater. Specifically, the LS159 strain is fermented using a fermentation culture broth with beer wastewater as the basic component. More specifically, the fermentation culture broth is prepared by mixing beer wastewater and tap water at a volume ratio of 1:3-8, then adding glucose and yeast extract, adjusting the pH to 7.0-7.4, and sterilizing and cooling. The fermentation conditions are 30-34 ℃ and 100-200 rpm for 3-7 days. Preferably, the fermentation broth is prepared by mixing beer wastewater and tap water at a volume ratio of 1:5, then adding 0.5% glucose and 0.5% yeast extract, adjusting the pH to 7.2, and sterilizing and cooling; the culture conditions are 32 ℃ and 150 rpm for 5 days.

[0010] Furthermore, studies have shown that the antibacterial function of *Streptomyces fusiforme* LS159 bacterial suspension fermented from beer wastewater is consistent with that of bacterial suspension cultured on ISP2 medium, indicating that *Streptomyces fusiforme*, an antagonistic bacterium, can be fermented from beer wastewater. Therefore, this invention also provides a bacterial suspension obtained by the above fermentation method. Furthermore, this invention provides the application of the bacterial suspension as a biocontrol bacterium or in the prevention of food spoilage.

[0011] The LS159 strain obtained by screening in this invention has broad-spectrum antibacterial activity and can be used as a biocontrol agent as well as to prevent food spoilage, thus having wide application value. Attached Figure Description

[0012] Figure 1 Antagonistic effects of different strains of actinomycetes on the antibacterial activity of Fusarium fusarium FD1.

[0013] Figure 2 Phylogenetic tree of 16S rDNA of strain LS159.

[0014] Figure 3 The effect of fermenting Streptomyces spindleii LS159 in beer wastewater.

[0015] Biological material preservation information: The LS159 strain of this invention is named *Streptomyces fusiforme* (…). Streptomyces netropsis The specimen was deposited on March 24, 2020, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101), abbreviated as CGMCC, with accession number CGMCC No. 19503. Detailed Implementation

[0016] The present invention will be described below through specific embodiments in order to provide a good understanding of the present invention, but this does not constitute a limitation thereof.

[0017] Example 1: Isolation and screening of Streptomyces with antagonistic functions

[0018] (1) Source of soil samples

[0019] Soil samples were taken from the oil peony planting base in Xiangyuan County, Changzhi City, Shanxi Province. Healthy 5-year-old oil peony plants were selected, the plants were pulled up by the roots, and the soil adhering to the roots (i.e., rhizosphere soil) was gently shaken off and brushed with a sterile brush. Samples from 5 plants were mixed into one soil sample. The soil was placed in a sterile plastic bag and brought back to the laboratory for storage in a 4°C refrigerator.

[0020] (2) Isolation of Streptomyces strains

[0021] Take 10 g of rhizosphere soil sample and place it in 90 mL of sterile distilled water. Shake evenly for 30 min, then dilute the soil suspension to 10 mL. -3 10 -4 10 -5 Take 100 μL of the diluted solution and spread it onto Gao's No. 1 solid medium (Gao's No. 1 synthetic medium 37.5 g, 3% potassium dichromate (aq) 3 mL, distilled water 1000 mL, pH 7.4-7.7). Incubate upside down in a 30℃ incubator. After 3-7 days, observe the plates and select colonies with different morphology, color, and size to streak on Gao's No. 1 medium plates. Store the purified strain at 4℃ for the next step of antagonistic bacteria screening.

[0022] (3) Screening for antagonistic function of strains

[0023] The antagonistic effects of the above-mentioned isolated strains were screened using the plate confrontation culture method. The pathogen used was *Fusarium solani*, a strain highly pathogenic to oil peony, preserved in the laboratory. Fusarium solani For Fusarium oxysporum FD1, in the center of a modified PDA medium (i.e., a 1:1 mixture of PDA and ISP2 medium: 2 g yeast extract, 5 g malt extract, 2 g glucose, 5 g NaCl, 2.5 g yeast extract, 5 g peptone, and 1000 mL distilled water), a 0.5 cm diameter circular pathogen block was taken using a sterile punch and placed in the center of the plate. The actinomycetes to be tested were then inoculated 2.5 cm away from the pathogen block. Three types of actinomycetes were inoculated on each plate. The plates were incubated at 28 ℃ for 5-7 days. The inhibition of the pathogen by the actinomycetes was observed and recorded daily. The diameter of the pathogen and the antagonistic radius were measured using calipers, and the inhibition rate was calculated. Inhibition rate = (diameter of control pathogen - diameter of treated pathogen) / diameter of control pathogen * 100%.

[0024] The results showed that 650 pure microbial strains were isolated from the rhizosphere soil of *Peony oleracea* collected in the field in Changzhi, Shanxi Province. Based on colony morphology, 42 strains were identified as actinomycetes. Among them, 20 strains showed antagonistic activity against *Fusarium solani* FD1, and 5 strains (LS2, LS62, LS159, LS164, and LS286) exhibited inhibition rates greater than 50%. The strains with the highest inhibition rates were LS159 and LS164. (See details below.) Figure 1 .

[0025] Example 2: Physicochemical characteristics and 16S rRNA molecular identification of Streptomyces LS159

[0026] The strain LS159, which showed the best antagonistic effect against Fusarium solani, was subjected to morphological observation and physiological and biochemical identification in accordance with the "Handbook of Systematic Identification of Common Bacteria". At the same time, the strain was subjected to 16S rRNA gene amplification and sequencing analysis.

[0027] The physicochemical characteristics of strain LS59 are as follows: On ISP2 solid medium (4 g yeast extract, 10 g malt extract, 4 g glucose, 20 g agar, 1 L distilled water, pH 7.4-7.6), the mycelia of this strain are light yellow, and the aerial mycelia are light brown and opaque. The spores are long and columnar with a smooth surface. Wrinkles appear after 48 h of culture, and spores are produced after 3-7 days of culture. It is Gram-positive, can utilize starch, produces catalase, and is positive for VP assay (see Table 1).

[0028] Table 1. Morphological characteristics and physiological and biochemical assay results of strain LS159

[0029]

[0030] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0031] After 16S rRNA gene sequencing, BLAST alignment analysis was performed on NCBI, and the results showed that this bacterium was similar to Streptomyces fusiforme. Streptomyces netropsis (NR 112494.1) showed the highest homology (99.86%), which is quite different from other Streptomyces species. Based on these characteristics, strain LS159 was named *Streptomyces fusiforme*. Streptomyces netropsis The results of constructing the phylogenetic tree are shown below. Figure 2 The bacterium was deposited on March 24, 2020, at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101), with accession number CGMCC No. 19503.

[0032] Example 3: Antagonistic effect of Streptomyces fusiforme LS159 on various plant pathogenic microorganisms

[0033] Four pathogenic microorganisms each of common plant fungal and bacterial diseases were selected as experimental materials. The antagonistic functional spectrum of *Streptomyces fusiforme* LS159 was determined using the plate confrontation method. Simultaneously, four other *Streptomyces* strains isolated with high inhibition rates against *Fusarium solani* FD1 were used for comparison. The four pathogenic fungi were the pathogens of *Phytophthora capsulatum* (a type of plant disease). Phytophthora capsicum ), Chestnut blight pathogen ( Cryphonectria parasitica Poplar tree sclerotium ( Rhizoctonia solani ), the pathogen of Chinese cabbage root rot ( Fusarium oxysporumThe four pathogenic bacteria were *Bacterium tumefaciens*, the pathogen causing bacterial spot disease in tomatoes. Pseudomonas syringae ), Carrot soft rot pathogen ( Pectococcus of carrots ), Tomato bacterial wilt pathogen ( Ralstonia solanacearum Bacterial poplar canker ( Botryosphaeria dothidea ).

[0034] Take a single colony of Streptomyces and inoculate it in ISP2 liquid medium. Incubate at 30 ℃ and 150 rpm for 3-5 days. Use the cultured bacterial solution for plate confrontation experiments. To investigate the antagonistic effect against pathogenic fungi, a plate confrontation experiment was conducted. A modified PDA medium (a 1:1 mixture of PDA and ISP2 media: PDA medium consisted of 200 g peeled potatoes cut into small pieces, boiled in distilled water, filtered through 8 layers of gauze, and supplemented with distilled water to 1 L, along with 20 g glucose and 16 g agar powder; ISP2 medium consisted of 4 g yeast extract, 10 g malt extract, 4 g glucose, and 1 L water) was inoculated into the center of a plate. Symmetrical wells were punched on both sides at a distance of 2.5 cm from the pathogen, and 100 μL of Streptomyces bacterial suspension was added to each well. Each treatment was repeated three times. The plates were incubated at 28 ℃ for 5-7 days, and the antagonistic radius against the pathogen was measured using calipers. To investigate the antagonistic effect of pathogenic bacteria, a plate confrontation experiment was conducted. 200 μL of each of the four cultured pathogenic bacteria were spread onto LB modified medium (a 1:1 mixture of LB medium and ISP2 medium: LB medium consisted of 10 g NaCl, 5 g yeast extract, 10 g peptone, 1 L distilled water, pH 7.0-7.5; ISP2 medium consisted of 4 g yeast extract, 10 g malt extract, 4 g glucose, 1 L water). Three 0.7 cm wells were made in the center of each plate, and 100 μL of Streptomyces bacterial suspension was added to each well. The plates were incubated at 30 ℃ for 24-36 h, and the inhibition diameter was measured using calipers.

[0035] The results showed that LS159 was effective against the pathogen of chestnut blight (… Cryphonectria parasitica Poplar tree sclerotium ( Rhizoctonia solani ), Carrot soft rot pathogen ( Pectobacterium carotovorum ) and the pathogen of bacterial wilt of tomato ( Ralstonia solanacearum It has a strong inhibitory effect; in addition, it has a strong inhibitory effect on the pathogen of root rot of Chinese cabbage (… Fusarium oxysporum ) and bacterial poplar canker pathogen ( Botryosphaeria dothidea It also exhibits some inhibitory effect. Furthermore, its antibacterial spectrum and efficacy are better than those of the same *Streptomyces fusiforme* strain (strain number LS286) against these eight pathogens. Compared with other *Streptomyces* strains isolated from the same region, its antibacterial spectrum and efficacy also differ. Specific results are shown in Table 2.

[0036] Table 2. Antimicrobial effects of different Streptomyces on 8 plant pathogenic microorganisms.

[0037]

[0038] Note: Pathogenic fungus 1. Phytophthora capsici ( ) Phytophthora capsicum ); 2. Chestnut blight pathogen ( Cryphonectria parasitica ); 3. Rhizoctonia solani ( ) Rhizoctonia solani ); 4. Pathogen of Chinese cabbage root rot ( Fusarium oxysporum Pathogenic bacteria: 1. Bacterial spot pathogen of tomato ( Pseudomonas syringes ); 2. Carrot soft rot pathogen ( Pectobacterium carotovorum ); 3. Bacterial wilt pathogen of tomato ( Ralstonia solanacearum ); 4. Bacterial poplar canker ( Botryosphaeria dothidea "-" indicates no antagonism; "+" indicates 0 < antagonistic radius < 5mm; "++" indicates 5 < antagonistic radius < 10mm; "+++" indicates antagonistic radius > 10mm.

[0039] Example 4: Antagonistic effect of Streptomyces fusiforme LS159 on various food spoilage microorganisms

[0040] Four common fungi and four common bacteria that cause food spoilage were selected as experimental materials. The antimicrobial spectrum of *Streptomyces fusiforme* LS159 was determined using the plate confrontation method, with another *Streptomyces fusiforme* strain, LS286, used as a control. The four fungi were *Aspergillus niger* (…). Aspergillus niger Aspergillus flavus Aspergillus flavus ), extended Penicillium ( Penicillium expanse Alternaria ()), Alternaria ( Alternaria alternata The four bacteria were Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Bacillus cereus ( Bacillus candle Bacillus coagulans ( Bacillus coagulans Fungi were cultured in PDA medium for 3-5 days, and bacteria were cultured in LB liquid medium for 12-18 hours.

[0041] Single colonies of *Streptomyces* were inoculated into ISP2 liquid medium and cultured at 30 ℃ and 150 rpm for 3-5 days. The cultured bacterial solutions were then used for plate confrontation experiments. For the antagonistic effect against food spoilage fungi, plate confrontation experiments were conducted on modified PDA medium (a 1:1 mixture of PDA and ISP2 medium: PDA medium consisted of 200 g peeled potatoes cut into small pieces, boiled in distilled water, filtered through 8 layers of gauze, and diluted to 1 L with distilled water, 20 g glucose, and 16 g agar powder; ISP2 medium consisted of 4 g yeast extract, 10 g malt extract, 4 g glucose, and 1 L water). Activated 0.5 cm × 0.5 cm fungal blocks were inoculated into the center of plates. Symmetrical wells were punched on both sides, 2.5 cm away from the spoilage fungus. 100 μL of *Streptomyces* bacterial solution was added to each well. Each treatment was repeated three times. The plates were incubated at 28 ℃ for 5-7 days. The diameter of the spoilage fungus and the antagonistic radius were measured using calipers. To investigate the antagonistic effect of food spoilage bacteria, a plate confrontation experiment was conducted. 200 μL of each of the four cultured food spoilage bacteria were spread onto LB modified medium (a 1:1 mixture of LB medium and ISP2 medium: LB medium consisted of 10 g NaCl, 5 g yeast extract, 10 g peptone, 1 L distilled water, pH 7.0-7.5; ISP2 medium consisted of 4 g yeast extract, 10 g malt extract, 4 g glucose, 1 L water). Three 0.7 cm diameter wells were made in the center of each plate, and 100 μL of Streptomyces bacterial solution was added to each well. The plates were incubated at 30 ℃ for 24-36 h, and the inhibition diameter was measured.

[0042] The results showed that *Streptomyces fusiforme* LS159 had antagonistic effects against eight fungi and bacteria that cause food spoilage, including *Penicillium expansum* (…). Penicillium expansum Staphylococcus aureus ( Staphylococcus aureus ) and Escherichia coli ( Escherichia coli It has the best antagonistic effect against Aspergillus flavus ( ). Aspergillus flavus Alternaria ( Alternaria alternata ) and Bacillus cereus ( Bacillus cereus It also showed good inhibitory effects. Moreover, LS59 had better inhibitory effects on all eight microorganisms that cause food spoilage than LS286 of Streptomyces fusiforme, as shown in Table 3.

[0043] Table 3. Antimicrobial effects of Streptomyces fusiforme LS159 on eight food spoilage microorganisms.

[0044]

[0045] Note: Food spoilage fungi 1. Aspergillus niger ( Aspergillus niger ), 2. Aspergillus flavus ( Aspergillus flavus ), 3. Extended Penicillium ( Penicillium expansum ), 4. Alternaria ( Alternaria alternate Food spoilage bacteria: 1. Staphylococcus aureus ( Staphylococcus aureus ), 2. Escherichia coli ( Escherichia coli ), 3. Bacillus cereus ( Bacillus cereus ), 4. Bacillus coagulans ( Bacillus coagulating "-" indicates no antagonism; "+" indicates 0 < antagonistic radius < 5mm; "++" indicates 5 < antagonistic radius < 10mm; "+++" indicates antagonistic radius > 10mm.

[0046] Example 5: Fermentation of Streptomyces spindleii LS159 using beer wastewater

[0047] Single colonies of *Streptomyces fusiforme* LS159 were inoculated into ISP2 liquid medium and cultured at 30 ℃ and 150 rpm for 3-5 days. The cultured bacterial suspension was then set aside for use. A certain amount of tap water was added to beer wastewater (beer wastewater to tap water volume ratio 1:5), followed by the addition of 0.5% glucose and 0.5% yeast extract. The pH was adjusted to 7.0-7.4, and the solution was dispensed into 250ml Erlenmeyer flasks (50ml per flask). After sterilization and cooling, 2% of the solution was inoculated and cultured at 32 ℃ and 150 rpm for 1, 2, 3, 5, and 7 days. The OD600 value was measured using a microplate reader. A control group was prepared by inoculating LS159 onto ISP2 medium under the same conditions.

[0048] The results showed that *Streptomyces fusiforme* LS159 could utilize the culture medium with beer wastewater as the main component well. Compared with ISP2 medium, in the initial 1-3 days of culture, the growth rate of *Streptomyces fusiforme* fermented with beer wastewater was slower than that treated with ISP2 medium, but in the middle and later stages of fermentation, the growth rates of the two treatments gradually approached each other. See below for details. Figure 3 .

[0049] Example 6: Antibacterial effect of Streptomyces spindleii LS159 fermented in beer wastewater

[0050] To confirm whether the antibacterial function of *Streptomyces fusiforme* LS159 fermentation broth fermented in beer wastewater was affected, *Streptomyces fusiforme* LS159 fermentation broth cultured in beer wastewater for 5 days was obtained according to the method in Example 5, and its antibacterial effect was studied. The harmful microorganisms used in the antibacterial experiment included one plant pathogenic fungus, *Phytophthora indicum*, the pathogen of *Phytophthora indicum* blight (…). Cryphonectria parasitic ), 1 plant pathogenic bacterium, carrot soft rot pathogen ( Pectobacterium carotovorum ), Penicillium expansum, a fungus that causes food spoilage ( Penicillium expansum ) and one type of food spoilage bacterium, Staphylococcus aureus ( Staphylococcus aureus ).

[0051] The results (see Table 4) showed that the antibacterial function of the Streptomyces fusiforme LS159 bacterial culture fermented with beer wastewater was consistent with that of the bacterial culture cultured on ISP2 medium, indicating that the antagonistic bacteria Streptomyces fusiforme can be fermented using beer wastewater.

[0052] Table 4. Antibacterial effect of *Streptomyces fusiforme* LS159 bacterial suspension fermented from beer wastewater.

[0053]

[0054] Note: 1. Chestnut blight pathogen ( Cryphonectria parasitica ), 2. Carrot soft rot pathogen ( Pectobacterium carotovorum ), 3. Extended Penicillium ( Penicillium expansum ), 4. Staphylococcus aureus ( Staphylococcus aureus ), 3. Escherichia coli ( Escherichia coli ), 3. Bacillus cereus ( Bacillus cereus ), 4. Condensed spore rod ( Bacillus coagulans "++" indicates an antagonistic radius >10mm.

Claims

1. A Streptomyces netropsis strain LS159, with accession number CGMCCNo.19503.

2. The application of the LS159 strain as described in claim 1 as a biocontrol bacterium, characterized in that, It is used to control fungal diseases caused by *Cryphonectria parasitica*, *Rhizoctonia solani*, *Pectobacterium carotovorum*, or *Ralstonia solanacearum*; or to control bacterial diseases caused by *Fusarium oxysporum*, the pathogen causing root rot in Chinese cabbage, or *Botryosphaeria dothidea*, the pathogen causing bacterial canker in poplar.

3. The application as described in claim 2, characterized in that, It is used for the prevention and control of diseases in chestnuts, poplars, carrots, tomatoes, or Chinese cabbage.

4. The application of the LS159 strain as described in claim 1 in the prevention and control of food spoilage, characterized in that, It is used to inhibit Aspergillus niger, Aspergillus flavus, Penicillium expansum, Alternaria alternata, or Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Bacillus coagulans.

5. A method for fermenting the LS159 strain as described in claim 1 using beer wastewater, wherein the LS159 strain is fermented using a fermentation culture broth with beer wastewater as the basic component.

6. The method as described in claim 5, characterized in that, The fermentation broth is made by mixing beer wastewater and tap water at a volume ratio of 1:3-8, then adding glucose and yeast extract, adjusting the pH to 7.0-7.4, and then sterilizing and cooling.

7. The method as described in claim 6, characterized in that, The fermentation broth is prepared by mixing beer wastewater and tap water at a volume ratio of 1:5, then adding 0.5% glucose and 0.5% yeast extract, adjusting the pH to 7.2, and sterilizing and cooling. The fermentation conditions are 30-34℃ and 100-200rpm for 3-7 days.

8. The method as described in claim 7, characterized in that, The culture conditions were 32℃ and 150rpm for 5 days.

9. The bacterial solution obtained by the method according to any one of claims 5 to 8.

10. The bacterial solution as described in claim 9, used as a biocontrol agent or in the prevention and control of food spoilage, is used to inhibit Aspergillus niger, Aspergillus flavus, Penicillium expansum, Alternaria alternata, or Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Bacillus coagulans.

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