Serratia marcescens s1 and culture method thereof, agricultural pest control agent and application

Abstract

The present application belongs to the field of agricultural microbial technology, and particularly relates to a strain of Serratia marcescans S1, a culture method thereof, an agricultural pest control agent and application. The Serratia marcescans S1 has been preserved in the Guangdong Provincial Microbial Culture Collection Center on September 2, 2022, and the preservation number is GDMCC No:62756. The strain can be used as an environmentally friendly green pesticide to replace chemical pesticides and other environmentally harmful pesticides to control the Bradysia hydrelli, and provides a new microbial strain and control method strategy for the control of agricultural pests. The strain is environmentally friendly, does not affect human health, is easy to prepare, has low cost, is easy to use, is easy to popularize, and has good biological control effect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Serratia marcescens S1 and its culture method, an agricultural pest control agent and its application. Background Technology

[0002] Existing research on the insect toxicity of *Serratia marcescens* shows that it has strong toxicity to Lepidoptera, Coleoptera, Hemiptera, and Diptera, but research on Lepidoptera is more abundant, while research on Coleoptera, Hemiptera, and Diptera is less. For example, patents or existing technology reports on a broad-spectrum toxic *Serratia marcescens* strain SfSm-1 and its applications show that *Serratia marcescens* has strong toxicity to Lepidoptera species such as beet armyworm, fall armyworm, diamondback moth, cabbage caterpillar, bollworm, Asian corn borer, and beet armyworm; Coleoptera species such as flea beetle; and Hemiptera species such as brown planthopper (isolation, identification, and insecticidal activity of *Serratia marcescens* pathogenic to potato tuber moth larvae). *Serratia marcescens* isolated from potato tuber moth larvae has high insecticidal activity against potato tuber moth. Currently, some researchers have found that *Serratia marcescens* is also effective against Hemiptera species such as citrus psyllids (…). Diaphorina citri ) and brown planthopper ( Nilaparvata lugens The mortality rate is relatively high; the citrus fruit fly ( Bactrocera dorsalis (Hendel) Ingestion of Serratia marcescens can lead to infection of intestinal cells.

[0003] Mushroom mosquito (Heterophthalmos spp.) Bradysia impatiens The fungus gnatula, belonging to the family Sciaridae in the order Diptera, is widely distributed in my country and is one of the most common and important pests of edible fungi, medicinal fungi, wild fungi, and ornamental plants. Controlling the fungus gnatula has long been a difficult problem for mushroom farmers, lacking effective control methods. Because of its small size, concealed nature, highly synchronized adult emergence, and strong reproductive capacity, it is difficult to detect in the early stages of damage. Once adults are discovered, most of the substrate has already been used for egg production, making timely control impossible. Due to the biological characteristics of this pest, its control is extremely difficult, necessitating the extensive use of chemical pesticides. This leads to increased pesticide resistance in the fungus gnatula, deterioration of the edible fungi production environment, and excessive pesticide residues in fresh mushrooms, resulting in unstable product quality. Cases of excessive pesticide residues in exported mushrooms are frequent, putting the industry at a disadvantage in international market competition and posing a serious threat to the sustainable development of the edible fungi industry.

[0004] The edible fungi industry plays a vital role in increasing farmers' income, improving agricultural efficiency, and protecting public health, and is the fifth largest industry in my country's agricultural planting sector. With the continuous expansion of edible fungi cultivation and the increasing economic benefits, mushroom farmers are planting more intensively and continuously, leading to increasingly serious pest and disease problems. Pest damage has resulted in a 20-30% reduction in total edible fungi production. In particular, squalids (fungi larvae) feed on mycelium, fruiting bodies, young mushroom buds, or bore into the stipe. The feeding and movement of both larvae and adults can spread pathogens, contaminating the fruiting bodies and severely impacting the development of the edible fungi industry. Because most edible fungi have short cultivation periods and are directly edible, and because many mushroom farmers lack a thorough understanding of the population dynamics of these pests in relation to temperature, humidity, and seasons, and lack knowledge of pest control, their methods are often inappropriate. They rely heavily on chemical control and neglect agricultural, physical, and biological methods, leading to numerous environmental and safety problems. Especially during the fruiting period, pesticide residues can easily remain in the fruiting bodies, posing a threat to human and animal health. Currently, very few pesticides are registered for use on edible fungi. Therefore, developing and utilizing highly efficient, low-toxicity, low-residue, harmless to humans and animals, and environmentally compatible biological pesticides to replace chemical pesticides is crucial for the development of edible fungi. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Serratia marcescens S1, named as follows: Serratia marcescen S1, deposited on September 2, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No: 62756), exhibits the following colony characteristics: opaque, red, round, raised, smooth, moist, viscous, and glossy. When cultured on LB agar at 28°C, the colony color gradually deepens until it turns red with increasing incubation time. It is Gram-negative, and the bacterial cells are short rods.

[0006] The nucleotide sequence of the 16S rDNA of Serratia marcescens S1 is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1: The *Serratia marcescens* disclosed in this invention is derived from diseased *Myxobolus hygrophorus*. B.peraffinis Biological agents isolated from larvae, prepared using live Serratia marcescens or its metabolites as active ingredients, are applied to control Aegilops spp. (a type of venomous mosquito). The inhibition rate can reach over 75% after 4 days of application, reducing the damage of Aegilops spp. to edible fungi and improving the yield and quality of edible fungi.

[0008] The present invention also provides a method for culturing the aforementioned Serratia marcescens S1, comprising the following steps: Method 1: Solid culture method: Serratia marcescens diluted with sterile water was plated on LB solid medium and incubated in the dark at 28±1℃ for 48 h. Method 2: Liquid culture method: First, inoculate Serratia marcescens into LB liquid medium, and then place it on a shaker at 28±1℃, 80 rpm~180 rpm for 15 h~25 h.

[0009] As a further preferred embodiment, the LB solid culture medium comprises the following components: 15 g to 20 g agar, 10 g sodium chloride, 10 g tryptone, 5 g yeast extract, 1 L water, and the pH of the culture medium is 7.0.

[0010] As a further preferred embodiment, the LB liquid culture medium comprises the following components: 10g sodium chloride, 10g tryptone, 5g yeast extract, 1L water, and the pH of the culture medium is 7.0.

[0011] The *Serratia marcescens* S1 and / or its fermentation broth provided by this invention can be used in the preparation of agricultural pest control agents. Furthermore, this invention also provides an agricultural pest control agent containing the aforementioned *Serratia marcescens* S1 and / or its fermentation broth; the concentration of *Serratia marcescens* S1 is 4.0 × 10⁻⁶. 5 cfu / mL~4.0×10 9 cfu / mL; and the above-mentioned agricultural pest control agent can be used to control agricultural pests; the above-mentioned agricultural pest is *Heterophthalmus fasciatus*.

[0012] The beneficial effects of this invention are as follows: This invention provides a *Serratia marcescens* S1 strain, which is effective against *Myxocytosporum tobira*. Bradysia peraffinis A new strain was isolated from the organism. This strain can serve as an environmentally friendly green agent to replace environmentally harmful pesticides, including chemical pesticides, for the control of *Heterophthalmus oryzae*, providing a new microbial strain and control strategy for agricultural pests. This strain is environmentally friendly, does not affect human health, is easy to prepare, low in cost, convenient to use, and easy to promote, exhibiting excellent biological control effects. Experimental results show that the fermentation broth obtained from this strain at a concentration of 4.0 × 10⁻⁶...9 cfu / mL, 4 × 10 8 cfu / mL, 4 × 10 7 cfu / mL, 4×10 6 cfu / mL, 4×10 5 The CFU / mL treatment resulted in a mortality rate of over 90% for *Agrostis spp.* on day 6. Attached Figure Description

[0013] Figure 1 The image shows a colony diagram of Serratia marcescens S1. Figure 2 The diagram shown is a phylogenetic analysis of the 16S rDNA of Serratia marcescens S1 (Note: Each branch is labeled with: strain name + GenBank sequence number). Figure 3 The figure shown is a bioassay diagram of Serratia marcescens S1 fermentation broth on Myxobolus fasciatus. Figure 4 The diagram shows the bioassay of Serratia marcescens S1 suspension on Myxobolus fasciatus. Figure 5 The diagram shows the bioassay of Serratia marcescens S1 fermentation supernatant on Myxobolus 'Heteromorphus'. Figure 6 The image shown is a comparison between normal larvae and larvae infected with strain S1. Detailed Implementation

[0014] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0015] The culture medium used in this embodiment is: LB liquid medium: 10g sodium chloride, 10g tryptone, 5g yeast extract, 1 L water, pH 7.0.

[0016] LB solid medium: 15-20g agar, 10g sodium chloride, 10g tryptone, 5g yeast extract, 1L water, pH 7.0.

[0017] Example 1 Isolation and culture of Serratia marcescens S1 Larvae of *Serratia marcescens* that died from infection and turned red were inoculated into LB agar plates and incubated at 28±1 ℃. After colonies formed, colonies of the same color as the infected larvae were picked and transferred to fresh LB agar plates. Pure cultures were obtained by streak plating. The morphology of the bacteria was then examined under a microscope to confirm their similarity. The cultures were then transferred to LB agar slants in test tubes to obtain *Serratia marcescens* S1, which was then stored at 4 ℃.

[0018] Example 2 Identification and phylogenetic analysis of Serratia marcescens S1 (1) Traditional biological identification: Serratia marcescens S1 colonies on LB agar are opaque, red, round, with a raised, smooth, moist, viscous, and glossy surface. When this strain is cultured on LB solid agar at 28°C, the colony color gradually deepens until it turns red with prolonged incubation (Fig. 1b). It is Gram-negative, and the bacterial cells are short rod-shaped (Fig. 1c).

[0019] (2) Molecular biological identification: DNA was extracted using the TSINGKE Plant DNA Extraction Kit (Universal Type) as a template, following the instructions for use. Universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3') were used. The amplification system consisted of 1 μL each of primers 27F and 1492R, 1 μL of template DNA, and 47 μL of TSINGKE Gold Mix (green). The PCR program was as follows: 98℃ pre-denaturation for 2 min; 98℃ for 10 s; 55℃ for 10 s; 72℃ for 20 s, for a total of 35 cycles; extension at 72℃ for 5 min; and storage at 4℃. PCR products were detected by 1.0% agarose gel electrophoresis. Qualified PCR products were sent to TSINGKE Biotechnology Co., Ltd. for bidirectional sequencing.

[0020] Based on the 16S sequencing results, the nucleotide sequence of the strain's 16S rDNA is shown in SEQ ID NO.1. The sequence returned by the company was assembled using DNAstar, and the primers at both ends were removed. The sequencing results were compared for homology using the BLAST tool on NCBI (National Center for Biotechnology Information), and combined with homologous sequences from GenBank, a phylogenetic tree was constructed using MEGA 7.0 software with the Neighbor-Joining (NJ) method for molecular identification and genetic distance analysis. Simultaneously, the obtained sequence was uploaded to the NCBI database to obtain the corresponding sequence number (OP168187). The results are as follows: Figure 2 As shown, the strain has the highest similarity to (NR113236.1) Serratia marcescens and (NR114043.1) Serratia marcescens. It is classified as Serratia marcescens and named strain S1. This strain was deposited on September 2, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCCNo: 62756. The address of the depository is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0021] Example 3 Toxicity determination of Serratia marcescens S1 against Myxobolus aegyptiacus Preparation of bacterial suspension and determination of insecticidal activity: *Serratia marcescens*, diluted with sterile water, was plated onto LB agar plates and incubated at 28 °C in the dark for 48 h. Single bacterial strains grown on the plates were then rinsed into sterile Erlenmeyer flasks, shaken thoroughly, and counted using the plate count method to prepare a bacterial suspension with a concentration of 4.0 × 10⁻⁶. 9 CFU / mL. The original bacterial suspension was serially diluted to a concentration of 4.0 × 10⁻⁶. 9 cfu / mL, 4.0×10 8 cfu / mL, 4.0×10 7 cfu / mL, 4.0×10 6 cfu / mL, 4.0×10 5 Different concentrations of bacterial suspensions were obtained using cfu / mL for insecticidal activity testing, with sterile water used as a blank control.

[0022] Fermentation broth preparation and insecticidal activity determination: *Serratia marcescens* was inoculated into LB liquid medium and then cultured on a shaker at 28℃ and 180 rpm for 25 h. The activated strain was then inoculated into an Erlenmeyer flask containing 100 mL of LB liquid medium and cultured on a shaker (28℃, 180 r / min) for 24 h. The concentration of the bacterial suspension was determined by zeroing the culture with sterile LB liquid medium. When the OD625 value of the fermentation stock diluted 10 times was 0.22, the corresponding concentration for the spread plate method was 4 × 10⁻⁶. 8 CFU / mL. The original fermentation broth was serially diluted to a concentration of 4.0 × 10⁻⁶ CFU / mL. 9 cfu / mL, 4.0 × 10 8 cfu / mL, 4.0×10 7 cfu / mL, 4.0 × 10 6 cfu / mL, 4.0 × 10 5 Different concentrations of bacterial suspensions were obtained using cfu / mL for insecticidal activity testing, with sterile LB liquid medium used as a blank control.

[0023] Preparation of fermentation supernatant and determination of insecticidal activity: 4.0 × 10⁻⁶ μL of the above-prepared test strain was used. 9 cfu / mL, 4.0 × 10 8 cfu / mL, 4.0 × 10 7 cfu / mL, 4.0 × 10 6 cfu / mL, 4.0×10 5 The fermentation broth was centrifuged at 10,000 r / min for 10 min, and the supernatant was then filtered through a 0.22 μm sterile filter membrane to obtain the fermentation supernatant. Sterile LB liquid medium was used as a blank control.

[0024] Immerse PDA culture medium covered with *Agrocybe aegerita* mycelium in the above-mentioned mycelial solution for 5 seconds, air dry in a ventilated area, and place in petri dishes (9 cm in diameter) lined with moistened filter paper. Inoculate 10 third-instar larvae of *Agrocybe aegerita* in each dish, repeating 3 times. Separately, PDA culture medium covered with *Agrocybe aegerita* mycelium, soaked in sterile water (or sterile LB liquid medium), serves as a blank control (CK). Incubate at 28℃, and record the number of larval deaths daily. Analyze the data using SPSS statistical software.

[0025] In this embodiment, *Serratia marcescens* strain GDMCC 1.218 was used as a control strain and the experiment was conducted under the same conditions as the *Serratia marcescens* S1 experiment described above. This *Serratia marcescens* strain GDMCC 1.218 was purchased from the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Center for Microbiological Analysis and Testing). The results are shown in Table 1, along with the *Serratia marcescens* S1 experimental data. Table 1. Results of toxicity assays of Serratia marcescens S1 fermentation broth and GDMCC 1.218 strain fermentation broth to Myxobolus heliotropium. Note: Average mortality rate is expressed as mean ± standard error. Different letters in the same column indicate significant differences between treatments (P<0.05). Figure 3 The image shows a bioassay of *Serratia marcescens* S1 fermentation broth against *Amanita muscaria*. Figure 3 It was found that the lethality of *Serratia marcescens* S1 fermentation broth against *Fungiformis larvae* increased with prolonged treatment time and with increasing fermentation broth concentration. In the control group, most larvae survived for 6 days, but at 4.0 × 10⁻⁶... 9 cfu / mL, 4.0×10 8 cfu / mL, 4.0×10 7 cfu / mL, 4.0×10 6 cfu / mL, 4.0 × 10 5 Treatment with fermentation broth at cfu / mL resulted in lethalities of 96.67%, 93.33%, 90%, 90%, and 90% respectively on day 6.

[0026] Figure 4 The image shows a bioassay of *Serratia marcescens* S1 suspension against *Amanita muscaria*. Figure 4 It can be seen that the lethality of Serratia marcescens S1 bacterial suspension against larvae of the ... Figure 5 The image shows a bioassay of *Serratia marcescens* S1 fermentation supernatant against *Amanita muscaria*. Figure 5 It can be seen that most of the control group survived after 6 days of feeding, but the 4.0 × 10⁻⁶ group... 9 cfu / mL, 4.0 × 10 8 cfu / mL, 4.0×10 7 The three treatments (CFU / m³, CFU / m³, and CFU / m³) achieved mortality rates of 90%, 63.33%, and 66.67% respectively on day 6. In contrast, the mortality rate of the fermentation supernatant for larvae was weaker, reaching 4.0 × 10⁻⁶ on day 8. 9 cfu / mL, 4.0 × 108 cfu / mL, 4.0 × 10 7 cfu / mL, 4.0×10 6 cfu / mL, 4.0×10 5 The lethality rates for cfu / mL were only 26.67%, 20.00%, 20.00%, 6.67%, and 3.33%, respectively.

[0027] In conclusion, both the fermentation broth and bacterial suspension of Serratia marcescens S1 have a high mortality rate against the larvae of the fungus gnatula, with the fermentation broth showing better control effect.

[0028] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. The application of a strain of Serratia marcescens S1 in the preparation of agricultural pest control agents, characterized in that, The agricultural pest control agent is used to control *Heterophthalmus hygrophorus*, and the name of *Serratia marcescens* S1 is... Serratia marcescens S1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 2, 2022, with accession number GDMCC No:62756.

2. An application according to claim 1, characterized in that, The method for culturing *Serratia marcescens* S1 includes the following steps: Method 1: Solid culture method: Serratia marcescens diluted with sterile water was plated on LB solid medium and incubated in the dark at 28±1 ℃ for 48 h. Method 2: Liquid culture method: First, inoculate Serratia marcescens into LB liquid medium, and then place it on a shaker at 28±1℃, 80 rpm~180 rpm for 15 h~25 h.

3. The application according to claim 2, characterized in that, LB solid medium consists of the following components: 15-20 g agar, 10 g sodium chloride, 10 g tryptone, 5 g yeast extract, 1 L water, and the pH of the medium is 7.

0.

4. The application according to claim 2, characterized in that, LB liquid medium consists of the following components: 10 g sodium chloride, 10 g tryptone, 5 g yeast extract, 1 L water, and the pH of the medium is 7.

0.

5. An agricultural pest control agent, characterized in that, Contains *Serratia marcescens* S1 and / or its fermentation broth, wherein the name of *Serratia marcescens* S1 is... Serratia marcescens S1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 2, 2022, with accession number GDMCC No:62756.

6. The agricultural pest control agent according to claim 5, characterized in that, The concentration of *Serratia marcescens* S1 was 4.0 × 10⁻⁶. 5 cfu / mL~4.0×10 9 cfu / mL.

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