A sugarcane endophytic Serratia marcescens strain and its application

Abstract

The invention discloses a sugarcane endophytic Serratia marcescens and its application, belonging to the technical field of agricultural microorganisms. The sugarcane endophytic Serratia marcescens is classified and named as Serratia marcescens ( Serratia marcescens )SM, which was deposited in the China Center for Type Culture Collection on October 18, 2023, with a deposit number of CCTCC NO: M 20231923, and a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The Serratia marcescens is isolated from sugarcane stems, and its living body can be used as a living body of Serratia marcescens or its metabolites, which have the following effects: (1) insecticide effect on armyworm larvae on sugarcane; (2) antibacterial effect on sugarcane white streak disease and sugarcane tip rot pathogens; (3) promoting the germination and growth of sugarcane.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, in particular to a sugarcane endophytic Serratia marcescens and application thereof. Background Art

[0002] Sugarcane is the most important sugar crop and an important low-carbon economic crop, accounting for 92% of my country's total sugar production. In addition, sugarcane is also used as an energy crop, which will play a significant role in people's pursuit of renewable energy. Therefore, it is of great significance to ensure the stable and increased production of sugarcane. However, sugarcane has long been threatened by various diseases and pests during its growth. At present, there are more than 120 kinds of sugarcane diseases and pests, and the prevention and control of sugarcane diseases and pests is mainly through chemical control. At the same time, the problems of pest resistance, environmental pollution and ecological damage caused by changes in farming systems, crop layout and climate are becoming increasingly serious, which has a serious impact on the yield and quality of sugarcane. Therefore, new prevention and control ideas and methods are urgently needed.

[0003] Mythimnaseparata (Walker), belonging to the family Noctuidae of the order Lepidoptera, is a typical long-distance migratory pest that can feed on more than 100 economic crops such as corn and rice. In my country's sugarcane-growing areas, intermittent outbreaks of armyworms have also had a serious impact on the healthy production of sugarcane. Sugarcane white streak disease is a bacterial vascular disease caused by Xanthomonas albilineans. It is common in major sugarcane-growing countries or regions around the world and causes great losses to sugarcane yield and sugar content. Sugarcane tip rot is a serious disease that mainly harms the tender leaves at the tip of the tip, and also infects the leaf sheaths and sugarcane stems. In the past, the disease occurred sporadically, but now it is becoming more and more serious, which has a certain impact on sugarcane yield and wind resistance.

[0004] As a new type of microbial resource, plant endophytes have become a research hotspot in the field of agriculture and forestry in recent years, and are also a resource library for green prevention and control of plant diseases and insect pests. With the in-depth study of plant endophytes, their species diversity and functional diversity have brought new ideas and methods for plant pest control. In addition, there are also considerable studies on sugarcane endophytes in promoting plant growth. At present, good applications have been achieved in medicinal plants; in terms of crops, corn, rice, etc. have also had relatively systematic research on endophytes, but research on sugarcane endophytes is relatively vacant.

[0005] In summary, screening a sugarcane endophytic strain with biological control effect is of great significance to alleviate the above problems. In view of this, the present invention is proposed. Summary of the invention

[0006] The present invention aims to provide an endophytic Serratia marcescens of sugarcane and multiple applications thereof, which has insecticidal effects on sugarcane armyworms and fall armyworm larvae; has antibacterial effects on sugarcane white streak disease and sugarcane tip rot pathogens; and also has the effect of promoting the germination and growth of sugarcane.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] A strain of endophytic Serratia marcescens in sugarcane, classified and named Serratia marcescens SM, was deposited in the China Center for Type Culture Collection on October 18, 2023, with the deposit number CCTCCNO: M20231923, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] The invention discloses an application of the above-mentioned sugarcane endophytic Serratia marcescens strain in preventing and controlling armyworm.

[0010] Application of the above-mentioned sugarcane endophytic Serratia marcescens in the preparation of insecticides.

[0011] The invention discloses an application of the sugarcane endophytic Serratia marcescens in inhibiting sugarcane white streak pathogen and sugarcane tip rot pathogen.

[0012] The above-mentioned sugarcane endophytic Serratia marcescens is used in the preparation of antibacterial and disease prevention drugs, and the bacteria inhibited are: sugarcane white streak pathogen and sugarcane tip rot pathogen.

[0013] The invention discloses an application of the sugarcane endophytic Serratia marcescens strain in promoting sugarcane germination.

[0014] The invention discloses an application of the sugarcane endophytic Serratia marcescens in preparing a sugarcane germination promoting drug.

[0015] A pesticide containing the above-mentioned endophytic Serratia marcescens in sugarcane.

[0016] A bacteriostatic and disease-preventing drug containing the above-mentioned endophytic Serratia marcescens in sugarcane.

[0017] A sugarcane germination-promoting drug contains the above-mentioned sugarcane endophytic Serratia marcescens.

[0018] The significant advantages of the present invention are:

[0019] The Serratia marcescens disclosed in the present invention is separated from sugarcane stems, and the biological preparation is prepared with the living body of Serratia marcescens or its metabolites as active ingredients, which is applied to the prevention and treatment of Lepidoptera armyworms, and the insecticidal property thereof can reach 46.7%. Moreover, the Serratia marcescens disclosed in the present invention has an inhibition rate of 70.47% on sugarcane white stripe pathogens and an inhibition rate of 57.20% on sugarcane tip rot pathogens. At the same time, it can also be used as an active ingredient for preparing biological fertilizers, improve the germination rate and germination index of sugarcane, and improve the yield and quality of sugarcane. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Colony (a) and bacterial cell (b) morphology of Serratia marcescens SM.

[0021] Figure 2 : Specific identification of Serratia marcescens SM.

[0022] Figure 3 :Survival curves of Serratia marcescens SM fermentation broth on oriental armyworm under different culture conditions. planktonic means shaking culture, static means static culture.

[0023] Figure 4 : Survival curve of Serratia marcescens SM fermentation broth supplemented with streptomycin and / or protease on oriental armyworm.

[0024] Figure 5 : Antibacterial plots of Serratia marcescens SM against sugarcane white streak pathogen (A) and sugarcane tip rot pathogen (B). DETAILED DESCRIPTION

[0025] The technical solution of the present invention is now specifically described in the form of examples, but it should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention. In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific examples. Preferred embodiments of the present invention are given in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0026] Example 1: Isolation, purification and identification of Serratia marcescens

[0027] 1. Isolation and purification of strains

[0028] The grinding and dilution plating method was used to isolate microorganisms in the healthy tissues of sugarcane plants. The stem tissue was cut from the sugarcane plant for pretreatment, and the soil particles attached to it were gently brushed off with a brush. Then, it was rinsed with tap water for 2 to 3 minutes to remove excess impurities on the surface, and the surface moisture was absorbed by filter paper. 2.0 g of pretreated sugarcane stems were soaked in 75% alcohol for 1 minute; then disinfected with 2% NaClO solution for 3 minutes; then rinsed with sterile water for 6 times to remove the disinfectant; finally, the residual moisture on the surface was absorbed with sterile filter paper. 100 μL of sterile water from the last rinse was taken and spread on the NA medium plate to verify the disinfection effect. The disinfected tissue sample was peeled with a sterilized scalpel and placed in a sterile mortar, and 9 mL of sterile water was added to it. It was ground into a homogenous slurry, and then diluted 10 times in a gradient, and a certain volume of appropriate gradient dilution was evenly spread on the NA solid culture plate and placed in a 28°C incubator for culture. Single colonies with different colony morphology were selected and purified multiple times to obtain pure culture of strain SM, which was preserved.

[0029] like Figure 1 As shown, the colonies of strain SM on the NA plate are milky white and round, slightly raised, and have a rough surface; the bacteria are straight rod-shaped, with blunt ends, and have obvious periflagellum and pili. The flagella are easily broken and can wander during the expansion process, and there are secretions around the bacteria that cover the bacterial surface.

[0030] 2. Strain Identification

[0031] (1) Homology analysis of 16S rDNA sequence of strain SM

[0032] The genomic DNA of strain SM was extracted using a bacterial genomic DNA extraction kit, and the extracted genomic DNA was used as a template to amplify 16S rDNA using two bacterial 16S rDNA universal primers.

[0033] ①27F: 5'-AGAGTTTGATCMTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'

[0034] The PCR system was 50 μL: 22 μL ddH2O, 25 μL 2×Ex Taq buffer, 1 μL 10 mM 27F primer, 1 μL 10 mM 1492R primer, and 1 μL template.

[0035] The PCR program was as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 90 seconds, for a total of 30 cycles; and complete extension at 72°C for 5 minutes.

[0036] The target fragment obtained by PCR amplification was sent to Sangon Biotech Co., Ltd. for sequencing analysis, and the obtained sequence is shown in SEQ ID NO. 1. The 16S rDNA sequence was compared with the 16S rDNA sequence of related strains through NCBI to analyze their homology. The results showed that the strain SM had 99.93% similarity with Serratia marcescens.

[0037] ②63F: 5'-CAGGCCTAACACATGCAAGTC-3', 1387R: 5'-GGGCGGWGTGTACAAGGC-3'

[0038] The PCR system was 50 μL: ddH2O 22 μL, 2×Ex Taq buffer 25 μL, 1 μL of 10 mM 63F primer, 1 μL of 10 mM 1387R primer, and 1 μL of template.

[0039] The PCR program was as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 90 seconds, for a total of 30 cycles; and complete extension at 72°C for 10 minutes.

[0040] The target fragment obtained by PCR amplification was sent to Sangon Biotech Co., Ltd. for sequencing analysis, and the obtained sequence is shown in SEQ ID NO. 2. By consulting the relevant international gene library in NCBI (National Center for Biotechnology Information), the 16S rDNA sequence was compared with the 16S rDNA sequence of related strains to analyze their homology. The results showed that the strain SM had a similarity of 99.77% with Serratia marcescens.

[0041] (2) Identification and analysis of strain-specific primers

[0042] The strain SM was identified using the Serratia marcescens-specific primers disclosed in the reference (Niu, Hongtao., et al. "The endophytic bacterial entomopathogen Serratia marcescens promotes plant growth and improves resistance against Nilaparvata lugens in rice." Microbiological research 256(2022):126956.).

[0043] F: 5'-GGGAGCTTGCTCCCCGG-3', R: 5'-AACGTCAATTGATGAACGTATTAAGT-3'. The PCR system was 50 μL: ddH2O 22 μL, 2×Ex Taq buffer 25 μL, 1 μL of 10 mM F primer, 1 μL of 10 mM R primer, and 1 μL of template. The PCR program was: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 20 seconds, for a total of 30 cycles; thorough extension at 72°C for 10 minutes.

[0044] The PCR amplification products were separated and identified by 2% agarose gel electrophoresis. Figure 2 As shown, 452 bp is the band size of the DNA fragment amplified by the specific primers.

[0045] Through the above-mentioned 16S rDNA analysis and specific primer amplification analysis, the strain SM isolated by the present invention was identified as Serratia marcescens. The strain SM was deposited in the China Center for Type Culture Collection on October 18, 2023, and was classified and named Serratia marcescens SM, with a deposit number of CCTCCNO: M20231923, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0046] Example 2: Screening of Serratia marcescens fermentation conditions

[0047] The present invention selects the optimal culture conditions for obtaining the fermentation liquid of Serratia marcescens by screening different culture conditions of Serratia marcescens, including regulating different culture times and culture states. We select the optimal culture conditions for obtaining the fermentation liquid by considering the insecticidal property of the fermentation liquid.

[0048] Serratia marcescens SM was inoculated into NB liquid culture medium and cultured at 28°C for 12-16 hours to obtain seed solution; the obtained seed solution was inoculated into fresh NB liquid culture medium at a 2% inoculum and then cultured under the following conditions:

[0049] 24D: incubate at 200 rpm, 28°C for 24 hours;

[0050] 24J: culture in a 28°C incubator for 24 hours;

[0051] 48D: incubate at 200 rpm, 28°C for 48 hours;

[0052] 48J: culture in a 28°C incubator for 48 hours;

[0053] 72D: incubate at 200 rpm, 28°C for 72 hours;

[0054] 72J: Culture in a 28°C incubator for 72 hours.

[0055] Uninoculated NB liquid medium was used as the control (CK).

[0056] The leaf immersion method was used. 1 cm × 1 cm cabbage leaves were cut and soaked in fermentation liquid for 30 seconds and then dried. 15 newly hatched Oriental armyworm larvae were picked and placed on the treated cabbage leaves and placed in a feeding cup. Three replicates were used for each treatment. Freshly treated cabbage leaves were replaced every day and the mortality rate was calculated. Figure 3 As shown, the fermentation broth of Serratia marcescens in group 72J had the highest insecticidal activity, reaching 46.7%.

[0057] Example 3: Determination of the insecticidal effect of Serratia marcescens fermentation broth on armyworm larvae

[0058] The Serratia marcescens SM was inoculated into NB liquid culture medium and cultured at 28°C for 12 to 16 hours to obtain seed liquid; the obtained seed liquid was inoculated into fresh NB liquid culture medium at a 2% inoculation amount and then cultured in a 28°C incubator for 72 hours to obtain fermentation liquid. Streptomycin and / or protease (Solebo Biotechnology Co., Ltd., item number P1120) were added to the fermentation liquid, and the insecticidal activity against the oriental armyworm was determined by the leaf immersion method according to Example 2. Figure 4 As shown, the addition of protease greatly weakened the insecticidal activity of the fermentation broth of Serratia marcescens SM, so the main insecticidal component in the fermentation broth is protein.

[0059] Example 4: Antibacterial effect of Serratia marcescens on sugarcane white streak pathogen and sugarcane tip rot pathogen Serratia marcescens SM was inoculated into NB liquid culture medium and cultured at 28°C for 12 to 16 hours to obtain seed liquid; the obtained seed liquid was inoculated into fresh NB liquid culture medium at an inoculum of 2% and then cultured in a 28°C incubator for 72 hours to obtain fermentation liquid; the fermentation liquid was diluted with pure water to OD 600 =0.6, obtain OD 600 =0.6 of Serratia marcescens SM fermentation broth.

[0060] First, prepare the culture medium for sugarcane white streak pathogen and sugarcane tip rot pathogen. The sugarcane white streak pathogen uses XaS culture medium: 20g sucrose, 5g bacterial peptone, 0.5g potassium dihydrogen phosphate, 0.25g magnesium sulfate heptahydrate, 0.05g sodium sulfate, 1L water, adjust pH to 7 and add 15g agar powder. The sugarcane tip rot pathogen uses PDA culture medium: directly use PDA solid culture medium and prepare according to the instructions. Inoculate 2% sugarcane white streak pathogen (Xanthomonas albicans) and sugarcane tip rot pathogen (Fusarium moniliforme) in their respective uncooled and solidified culture media, shake well and pour the plates in time. Add 5μl OD 600 = 0.6, and the fermentation liquid of Serratia marcescens SM was cultured in a 28°C incubator for 72 hours to observe the antibacterial effect. Figure 5 As shown in Table 1, Serratia marcescens has a significant antibacterial effect on sugarcane white streak pathogen and sugarcane tip rot pathogen.

[0061] Table 1 Inhibition rate of Serratia marcescens against pathogens

[0062]

[0063] Example 5: Preparation of bacterial fertilizer by Serratia marcescens

[0064] The Serratia marcescens SM was inoculated into NB liquid culture medium and cultured at 28°C for 12-16 hours to obtain seed solution; the obtained seed solution was inoculated into fresh NB liquid culture medium at a 2% inoculation amount and then cultured in a 28°C incubator for 72 hours to obtain fermentation solution; the fermentation solution was diluted with pure water to OD 600 =0.6, obtain OD 600 =0.6 of Serratia marcescens SM fermentation liquid, according to 3.5% of the weight of the organic fertilizer, added into the organic fertilizer, and the organic fertilizer and the fermentation liquid were evenly mixed to prepare the Serratia marcescens fertilizer.

[0065] Example 6: Effect of Serratia marcescens fermentation broth on the germination of sugarcane

[0066] The Serratia marcescens SM was inoculated into NB liquid culture medium and cultured at 28°C for 12-16 hours to obtain seed solution; the obtained seed solution was inoculated into fresh NB liquid culture medium at a 2% inoculation amount and then cultured in a 28°C incubator for 72 hours to obtain fermentation solution; the fermentation solution was diluted with pure water to OD 600 =0.6, obtain OD 600 =0.6 of Serratia marcescens SM fermentation broth.

[0067] Healthy sugarcane seeds were selected from the sugarcane field for growth promotion experiments. Three different sugarcanes were selected: black sugarcane, green sugarcane, and fruit sugarcane (provided by the National Sugarcane Engineering Technology Research Center of Fujian Agriculture and Forestry University), and 200 healthy seed stems with single bud segments of each variety were obtained. 100 seed stems with bud segments were randomly selected and immersed in OD 600 =0.6 of Serratia marcescens SM fermentation liquid, and another 100 bud stems were soaked in pure water as a control. After soaking for 24 hours, they were placed in a greenhouse with a temperature of 25-28°C, a humidity of 70%-90%, and a long daylight (16 hours of light / 8 hours of darkness) for conventional planting. The germination was observed and counted until no new germination occurred for 3 consecutive days. The results are shown in Table 2. The fermentation liquid of Serratia marcescens SM promoted the germination of the three varieties of sugarcane, and the germination rate was significantly improved. It also improved the germination index and germination potential of Zhongzhe 1211 and Guitang 58.

[0068] Table 2 Effect of Serratia marcescens on sugarcane germination

[0069]

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement modes. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A sugarcane endophytic Serratia marcescens strain, characterized by: The taxonomic name is Serratia marcescens ( Serratia marcescens )SM, was deposited in the China Center for Type Culture Collection on October 18, 2023, with the deposit number CCTCCNO: M 20231923, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the endophytic Serratia marcescens of sugarcane as claimed in claim 1 in controlling armyworms.

3. The use of endophytic Serratia marcescens in sugarcane in the preparation of pesticides according to claim 1, characterized in that: The insects killed are: sticky insects.

4. Use of the endophytic Serratia marcescens of sugarcane according to claim 1 in inhibiting sugarcane white streak pathogen and sugarcane tip rot pathogen.

5. The use of endophytic Serratia marcescens in sugarcane in the preparation of antibacterial and disease prevention drugs as claimed in claim 1, characterized in that: The inhibited bacteria are: sugarcane white streak pathogen and sugarcane tip rot pathogen.

6. Use of the endophytic Serratia marcescens of sugarcane as claimed in claim 1 in promoting germination of sugarcane.

7. Use of the endophytic Serratia marcescens of sugarcane as claimed in claim 1 in the preparation of a drug for promoting germination of sugarcane.

8. An insecticide, characterized in that: The invention contains the endophytic Serratia marcescens of sugarcane according to claim 1.

9. An antibacterial and disease-preventing drug, characterized in that: The invention contains the endophytic Serratia marcescens of sugarcane according to claim 1.

10. A sugarcane germination-promoting drug, characterized in that: The invention contains the endophytic Serratia marcescens of sugarcane according to claim 1.

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