Serratia plymuthica strain for controlling soybean root rot and use thereof

Abstract

The application discloses a Serratia plymuthica strain for preventing and treating soybean root rot and application thereof. A Serratia plymuthica strain G25-16 is isolated from the rhizosphere soil of soybean and wheat, and the microbial preservation number of the strain is CGMCC No.34772; the results of a plate confrontation experiment show that the strain has obvious antagonistic effects on Fusarium solani and Fusarium retusum; the results of a prevention and treatment experiment show that the strain has significant prevention and treatment effects on the Fusarium solani and the Fusarium retusum; and the results of a pot planting growth promotion experiment show that after the soybean is treated by the isolated Serratia plymuthica strain, the fresh weight and the plant height of the soybean are obviously improved compared with a control. The isolated Serratia plymuthica strain G25-16 has application prospects in promoting the growth of soybean and preventing and treating soybean diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to a plant disease biocontrol strain, in particular to a Serratia plymuthica strain isolated from the rhizosphere soil of soybean and wheat, Serratia plymuthica and its application in preventing and treating plant diseases and promoting plant growth, belonging to the field of Serratia plymuthica and its application. BACKGROUND

[0002] Soybean root rot is a worldwide disease with wide distribution, serious harm, multiple pathogenic bacteria and difficult prevention and treatment, which can infect seedlings and adult plants. Soybean root rot mainly damages the root system of plants, thereby affecting the absorption of water and nutrients by the root system, causing plant growth retardation, seriously affecting the yield and quality of soybean, and causing huge economic losses.

[0003] At present, in the process of soybean planting, the main prevention and treatment measures for soybean root rot are chemical agents, screening of disease-resistant varieties and biological control. Chemical agent control can effectively reduce the occurrence of plant diseases, but improper use will cause problems such as pesticide residues and soil pollution.

[0004] The pathogenic bacteria of soybean root rot are complex, the breeding cycle of disease-resistant varieties is long, and there are few disease-resistant varieties available for production. Therefore, using biological bacteria to prevent and treat soybean root rot has the advantages of being green, ecological and not easy to produce drug resistance, and is an important means of preventing and controlling soybean root rot. SUMMARY

[0005] One of the purposes of the present application is to provide a Serratia plymuthica strain isolated from the rhizosphere soil of soybean and wheat, Serratia plymuthica ;

[0006] The second purpose of the present application is to provide a microbial preparation prepared from the Serratia plymuthica strain.

[0007] The third purpose of the present application is to apply the Serratia plymuthica strain or the microbial preparation to prevent and treat soybean diseases and promote soybean growth.

[0008] In order to achieve the above purposes, the main technical solutions adopted by the present application include:

[0009] One aspect of the present application is to provide a Serratia plymuthica strain G25-16, the microbial preservation number of which is CGMCC No.34772, the taxonomic name of which is Serratia plymuthica Serratia plymuthica . Serratia plymuthica The preservation time is June 5, 2025, the preservation unit is the General Microbiological Center of the Chinese Microorganism Strain Preservation Management Committee, and the preservation address is No.3, North Chenxi Road, Yard 1, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences.

[0010] Another aspect of the present invention is to provide a microbial preparation made from the aforementioned Serratia pluvialis G25-16.

[0011] Those skilled in the art can prepare various conventional microbial preparations from the Serratia pumila G25-16 provided by this invention using conventional methods for preparing microbial preparations. These are all technical means well known to those skilled in the art.

[0012] Another aspect of the present invention is to apply the aforementioned Serratia pluvialis G25-16 or Serratia pluvialis G25-16 microbial preparation to the prevention and control of plant diseases.

[0013] In a preferred embodiment, the pathogen of the plant disease is preferably *Fusarium solani* (…). N. solani ) or Fusarium moniliforme ( F. odoratissimum The preferred plant disease mentioned is soybean root rot.

[0014] Another aspect of the present invention is to apply the aforementioned Serratia puchengensis G25-16 or Serratia puchengensis G25-16 microbial preparation to promote soybean growth; wherein, the promotion of soybean growth is preferably to increase the fresh weight and plant height of wheat, wherein the fresh weight refers to the weight of the entire plant.

[0015] This invention isolated *Serratia pluvialis* from the rhizosphere soil of soybeans and wheat, and ultimately screened out a strain G25-16 that can prevent and control plant diseases and promote plant growth. Molecular biological and morphological identification confirmed that strain G25-16 is indeed *Serratia pluvialis*. Serratia plymuthica Experiments have shown that strain G25-16 has a significant control effect on soybean root rot caused by fungi, and can increase the fresh weight and plant height of soybeans. Compared with existing biocontrol resources for soybean root rot, the Pucheng Serratia strain G25-16 can not only effectively control the pathogen, but also promote the growth of soybeans. Attached Figure Description

[0016] Figure 1 This is a colony morphology diagram of Serratia marcescens strain G25-16.

[0017] Figure 2 Phylogenetic tree of Serratia procumbens strain G25-16 based on 16S-rDNA.

[0018] Figure 3 For Serratia marcescens strain G25-16 N.solani and F. odoratissimum The results of plate antagonism and inhibition rate were shown; Figure (a) shows the plate confrontation results of strain G25-16, and Figure (b) shows the inhibition rate of strain G25-16.

[0019] Figure 4 This study investigated the growth-promoting effects of Serratia puchengensis strain G25-16 on soybean. Figure (a) shows the phenotypic observation results of the effect of strain G25-16 on soybean plant height, Figure (b) shows the statistical results of the effect of strain G25-16 on soybean plant height, Figure (c) shows the statistical results of the effect of strain G25-16 on soybean root length, Figure (d) shows the statistical results of the effect of strain G25-16 on soybean fresh weight, and Figure (e) shows the statistical results of the effect of strain G25-16 on soybean root weight.

[0020] Figure 5 The results show the efficacy of Serratia marcescens strain G25-16 in controlling the disease; Figure (a) shows... N. solani The result of causing soybean root rot, Figure (b) shows the effect of G25-16 on soybean root rot. N. solani The control efficacy of strain G25-16 against soybean infection is shown in Figure (c). N. solani The statistical results of the control efficacy experiment on diseased plants are shown in Figure (d). F.odoratissimum The results of causing soybean root rot are shown in Figure (e), which shows the effect of G25-16 on the disease. F.odoratissimum The control efficacy of strain G25-16 against soybean infection is shown in Figure (f). F.odoratissimum The statistical results of the prevention and control efficacy. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments or test examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments or test examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0022] culture medium

[0023] LA medium (solid): 1% tryptone, 0.5% yeast extract, 1% sodium chloride, 2% agar, the remainder being distilled water, pH adjusted to 7.0±0.1, and then sterilized at 121℃ for 15 min.

[0024] LB broth medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, the remainder being distilled water, pH adjusted to 7.0±0.1, and then sterilized at 121℃ for 15 min.

[0025] PDA medium: 0.6% potato starch, 2% glucose, 2% agar, the remainder being distilled water, pH adjusted to 5.6±0.2, and then sterilized at 115℃ for 20 min.

[0026] CMC medium: CMC-Na 15.0 g, NH4H2PO4 1.0 g, K2N 1.0 g, MgSO4·7H2O 0.5 g, yeast extract 1.0 g, the rest is distilled water, sterilized at 121°C for 20 min.

[0027] Example 1 Isolation and identification of Serratia proteamaculans strain G25-16

[0028] 1 Strain collection and isolation

[0029] Isolation of strain G25-16: The rhizosphere soil of soybean and wheat was collected in Xinjiang region. The soil was sieved, 5 g of the soil was added to 45 mL of sterile water, shaken at 30°C for 30 min at 180 rpm, and then allowed to stand for 30 min. 1 mL of supernatant was added to 9 mL of sterile water to obtain 10 -1 dilution liquid, which was mixed by blowing and then diluted, and the process was repeated to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 dilution liquid. 100 μL of each dilution was spread on LA plates and incubated overnight at 30°C in an inverted incubator.

[0030] Single colonies of different colors and shapes were picked with a gun tip into 500 μL of LB broth medium and incubated at 30°C at 200 rpm for 12 h. The bacterial solution was diluted and spread on LA medium, and incubated at 30°C at 200 rpm for 12 h to obtain single colonies, which were then picked until a uniform color and shape appeared on the LA medium Figure 1 . The colonies were stored in 50% glycerol at -80°C.

[0031] 2 Identification of Serratia proteamaculans strain G25-16

[0032] Single colonies were picked into 1 mL of LB broth medium and incubated at 30°C at 200 rpm for 12 h. The genomic DNA of the rhizosphere bacteria was extracted using a bacterial genomic DNA extraction kit. The primers 27-F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1)) and 1492 -R (5'-AAGGAGGTGATCCAGCC-3' (SEQ ID No. 2)) to amplify 16S-rDNA gene sequence. The PCR amplification procedure was 95℃ 3 min; 95℃ 15 s; 60℃ 45 s; 72℃ 90 s; 72℃ 10 min; 35 cycles. The sequence of PCR product was obtained by sequencing. The sequencing results were compared with the data results of the National Center for Biotechnology Information (NCBI) gene library (https: / / www.ncbi.nlm.nih.gov / ). The final 16S-rDNA nucleotide sequence is shown in SEQ ID No. 3.

[0033] To determine the species and genus of strain G25-16 and its close strains, the phylogenetic tree was established using the top 30 16S-rDNA with the highest identify (see Figure 2 ). It was found that strain G25-16 was most closely related to Serratia plymuthica S. plymuthica 4Rx13 (GCF-000176835.2) with a homology of 99.42%. Combined with morphological characteristics, strain G25-16 was preliminarily identified as Serratia plymuthica (Serratia plymuthica). Serratia plymuthica

[0034] Example 2 Preparation of Serratia plymuthica strain G25-16 fermentation broth

[0035] Serratia plymuthica strain G25-16 single colony was inoculated into 500 μL LB broth medium, and cultured at 28℃, 200 rpm for 12 h. 500 μL bacterial solution was inoculated into 500 mL LB broth medium, and cultured at 28℃, 200 rpm for 18 h. The OD 600 was adjusted to 1 for standby.

[0036] Test Example 1 Plate confrontation test of Serratia plymuthica strain G25-16 and test pathogenic fungi

[0037] 1 Test method

[0038] Serratia plymuthica strain G25-16 single colony was inoculated into 500 μL LB broth medium, and cultured at 28℃, 180 rpm for 18 h for standby.

[0039] The test pathogenic fungi were subjected to in vitro antibacterial activity experiment of rhizosphere bacteria. The antagonistic effect of the bacteria was detected by two-point confrontation method. A 9 mm puncher was used to cut from the PDA plate, and the test bacteria solution was inoculated at a distance of 2 cm from the center of the bacteria cake. The bacteria cake without inoculation of bacteria was used as a control. Inverted culture, no light, 28℃ culture for 7 days. The pathogenic fungi diameter was measured by cross measurement method.

[0040] 2 Test results​

[0041] The results of the plate standoff test are shown in Table 1. Figure 3 As shown, Serratia pluvialis G25-16 exhibits antimicrobial activity against the tested pathogenic fungi on agar plates. N. solani and F. odoratissimum Both have significant antagonistic effects.

[0042] Serratia procumbens G25-16 pairs N. solani The inhibition rate was 55.61%; Serratia marcescens G25-16 was effective against... F. odoratissimum The antibacterial rate was 54.44%.

[0043] Table 1. Plate inhibition effect of Serratia marcescens strain G25-16 against Fusarium oxysporum, the pathogen of soybean root rot.

[0044]

[0045] Experiment Example 2: Potted Plant Growth Promotion Experiment of Serratia puchengensis Strain G25-16

[0046] 1. Experimental Methods

[0047] Wood ash and vermiculite were mixed in a 1:1 ratio, and one seed of New Soybean No. 26 was sown in each pot. When the soybeans reached the first node stage, 5 mL of Serratia pluvialis strain G25-16 fermentation broth was inoculated into each pot using the root drenching method, with sterile water used as a negative control. The soybeans were cultured at 25℃ under 16 h of light and 8 h of darkness. Plant height, root length, fresh weight, and root weight were measured 10 days after inoculation.

[0048] 2. Experimental Results

[0049] The results showed that soybeans treated with fermentation broth of *Serratia marcescens* strain G25-16 from Pucheng did not show significant differences in root weight and root length compared to the control (CK). However, soybeans treated with fermentation broth of *Serratia marcescens* strain G25-16 from Pucheng showed an average increase in fresh weight of approximately 0.52 g; soybeans treated with strain G25-16 showed an average increase in plant height of approximately 4.25 cm (see...). Figure 4 ).

[0050] Experiment Example 3: Potted Plant Efficacy Test of Serratia marcescens strain G25-16

[0051] 1. Test materials and test methods

[0052] 1.1 Test pathogenic fungi

[0053] Fusarium solani N. solani (CX3-4) and Fusarium putrefaction F.odoratissimum (ZP3-6).

[0054] 1.2 Test Methods

[0055] 1.2.1 Pathogenicity test of the test pathogenic fungi

[0056] Inoculation of the one-node soybean seedlings with the pathogenic fungi N. solani (CX3-4) and F. odoratissimum (ZP3-6), and 45 days later, the disease index was calculated according to the following formula:

[0057] The agar plugs of the test pathogenic fungi strains were inoculated into CMC medium, and cultured at 28°C, 200 rpm for 14 days. The mycelium was removed by filtration with a 70 μm Cell Strainer. The spore number was calculated under a microscope, and the spore number was adjusted to 10 7 The wood ash and vermiculite were mixed at a ratio of 1:1, and one seed of new soybean 26 was sown in each pot. The soybean was cultured to the one-node stage, and 3 mL of the spore solution of the test strain was inoculated into each soybean seedling by puncturing 5 times at an angle of 45° downward from 1 cm above the soil with a needle tip. The incidence rate was calculated 45 days after inoculation, and the pathogenicity of the test pathogenic fungi was calculated according to the disease index standard (Table 2) and the disease index formula.

[0058] Table 2 Classification standard of soybean root rot

[0059]

[0060] The disease index was calculated according to the following formula:

[0061]

[0062] 1.2.2 Potting test for the control effect of Serratia plymuthica strain G25-16

[0063] The agar plugs of the test strains N.solani and F. odoratissimum were inoculated into CMC medium, and cultured at 28°C, 200 rpm for 14 days. The mycelium was removed by filtration. The spore number was calculated under a microscope, and the spore number was adjusted to 10 7 spores / mL for standby. The one-node soybean seedlings were inoculated with 3 mL of the prepared G25-16 fermentation broth. 24 hours later, 3 mL of the spore solution of the test strain was inoculated into each soybean seedling. The soybean inoculated only with the test strain was used as a positive control, and the soybean inoculated neither with the bacterial broth nor with the spore solution was used as a blank control. The control effect was calculated according to the following formula.

[0064]

[0065] 2 Test results

[0066] The control efficacy results showed that after colonization of Serratia marcescens strain G25-16 inoculation with pathogens was effective. N. solani (CX3-4) or F. odoratissimum (ZP3-6) will cause the area of ​​brown lesions at the base of soybean stems to be generally smaller than that of soybeans that have only been exposed to the pathogen. N. solani (CX3-4) or F. odoratissimum The area of ​​brown lesions treated with (ZP3-6) was larger, and the root system was generally more developed than that of the root system inoculated only with the pathogen (see...). Figure 5 ).

[0067] The disease index and efficacy test results are shown in Table 3.

[0068] Table 3. Efficacy determination of Serratia pluvialis strain G25-16.

[0069]

[0070] Inoculation with pathogens only N. solani The disease index of the positive control (CX3-4) was 41.67, and only the pathogen was inoculated. F. odoratissimum The disease index of the positive control (ZP3-6) was 46.67, while the treatment group (Serratia marcescens strain G25-16 colonized and then inoculated with pathogens) had a higher disease index. N. solani (CX3-4) or F. odoratissimum The disease indices of soybean seedlings (ZP3-6) were 21.67 and 23.33, respectively, indicating that the *Serratia marcescens* strain G25-16 was effective against... N. solani and F. odoratissimum It has significant prevention and control effects, with control efficacy of 48.00% and 50.01%, respectively.

Claims

1. A strain of Serratia procyonii ( Serratia plymuthica G25-16, characterized in that, The microbial preservation number is CGMCC No. 34772.

2. A microbial preparation prepared from the Serratia plymuthica G25-16 of claim 1.

3. The Serratia plymuthica G25-16 of claim 1 for use in the prevention of plant diseases, wherein the plant disease is soybean root rot.

4. The microbial preparation of claim 2 for use in the prevention of plant diseases, wherein the plant disease is soybean root rot.

5. Use according to claim 3 or 4, characterized in that, The pathogenic bacteria of the plant disease is Fusarium solani pisi or Fusarium reticulatum.

6. The use of Proteus mirabilis G25-16 of claim 1 in promoting soybean growth, which is increasing fresh weight and plant height of soybean; wherein, The fresh weight refers to the weight of the whole plant.

7. Use of the microbial preparation of claim 2 in promoting soybean growth, said promoting soybean growth is increasing fresh weight and plant height of soybean; wherein, The fresh weight refers to the weight of the whole plant.

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