Serratia pumila strain for preventing and treating soybean root rot and application of serratia pumila strain

Abstract

The invention discloses a serratia pumila strain for preventing and treating soybean root rot and application of the serratia pumila strain. The serratia pumila strain G25-16 is separated from rhizosphere soil of soybeans and wheat, and the microbial preservation number of the serratia pumila strain G25-16 is CGMCC (China General Microbiological Culture Collection Center) No.34772; a plate confrontation experiment result shows that the strain has an obvious antagonistic effect on fusarium solani and fusarium malodosa; a control effect experiment result shows that the strain has a remarkable control effect on fusarium solani and fusarium malodosa; potted plant growth promotion experiments find that after soybeans are treated by the separated serratia pumila strain, the fresh weight and plant height of the soybeans are obviously improved compared with those of a contrast. The serratia pumila strain G25-16 separated by the invention has application prospects in the aspects of promoting soybean growth, preventing and treating soybean diseases and the like.

Description

Technical Field

[0001] This invention relates to biocontrol strains for plant diseases, and more particularly to Serratia pluvialis isolated from the rhizosphere soil of soybeans and wheat. Serratia plymouthica Serratia pluvialis and its application in the prevention and control of plant diseases and the promotion of plant growth belong to the field of Serratia pluvialis and its application. Background Technology

[0002] Soybean root rot is a widespread, highly damaging, and difficult-to-control global disease affecting both seedlings and mature plants. It primarily damages the root system, impairing water and nutrient absorption, leading to stunted growth, severely impacting soybean yield and quality, and causing significant economic losses.

[0003] Currently, soybean root rot is mainly controlled through chemical agents, screening for disease-resistant varieties, and biological control measures. Chemical control can effectively reduce the occurrence of plant diseases, but improper use can lead to problems such as pesticide residues and soil pollution.

[0004] Soybean root rot is caused by complex pathogens, and the breeding cycle for resistant varieties is long, resulting in a limited number of resistant varieties suitable for production. Therefore, using biological agents to control soybean root rot has advantages such as being green, ecological, and less prone to developing drug resistance, making it an important means of controlling soybean root rot. Summary of the Invention

[0005] One objective of this invention is to provide a strain of *Serratia pluvialis* isolated from the rhizosphere soil of soybeans and wheat. Serratia plymouthica ); The second objective of this invention is to provide a microbial preparation made from the strain of Serratia pluvialis.

[0006] The third objective of this invention is to apply the aforementioned Serratia pluvialis strain or microbial preparation to prevent and control soybean diseases and promote soybean growth.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include: One aspect of the present invention is to provide a strain of Serratia pluvialis (Serratia pluvialis). Serratia plymouthica G25-16, its microbial preservation number is: CGMCC No.34772; its classification name is: Serratia pluvialis. Serratia Plymouth The deposit date is June 5, 2025; the depositary institution is the China General Microbiological Culture Collection Center; the depositary address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Another aspect of the present invention is to provide a microbial preparation made from the aforementioned Serratia pluvialis G25-16.

[0009] 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.

[0010] 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.

[0011] In a preferred embodiment, the pathogen of the plant disease is preferably *Fusarium solani* (…). N. nightshade ) or Fusarium moniliforme ( F. odoratissimum The preferred plant disease mentioned is soybean root rot.

[0012] 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.

[0013] 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 plymouthica 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

[0014] Figure 1 This is a colony morphology diagram of Serratia marcescens strain G25-16.

[0015] Figure 2 Phylogenetic tree of Serratia procumbens strain G25-16 based on 16S-rDNA.

[0016] 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.

[0017] 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.

[0018] 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). Very fragrant The results of causing soybean root rot are shown in Figure (e), which shows the effect of G25-16 on the disease. Very fragrant The control efficacy of strain G25-16 against soybean infection is shown in Figure (f). Very fragrant The statistical results of the prevention and control efficacy. Detailed Implementation

[0019] 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.

[0020] culture medium 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.

[0021] 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.

[0022] 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.

[0023] CMC culture medium: CMC-Na 15.0 g, NH4H2PO4 1.0 g, K&N 1.0 g, MgSO4·7H2O 0.5 g, yeast extract 1.0 g, the remainder being distilled water, sterilized at 121℃ for 20 min.

[0024] Example 1: Isolation and Identification of Serratia pluvialis strain G25-16 1. Strain Collection and Isolation Strain G25-16 isolation: Soybean and wheat rhizosphere soils were obtained from Xinjiang. After sieving, 5 g of soil was taken and added to 45 mL of sterile water. The mixture was incubated at 30℃ and 180 rpm for 30 min, then allowed to stand for 30 min. 1 mL of the supernatant was added to 9 mL of sterile water to obtain strain G25-16. -1 Dilute the solution, mix thoroughly by pipetting, and continue diluting until you obtain 10. -2 10 -3 10 -4 10 -5 10 -6 Diluent. Take 100 μL of each dilution and spread it onto an LA plate, then invert it and incubate overnight at 30°C.

[0025] Single colonies of different colors and morphologies were picked up with a pipette tip and added to 500 μL of LB broth medium, and incubated at 30°C and 200 rpm for 12 h. The bacterial suspension was then diluted and spread onto LA medium, and cultured at 30°C and 200 rpm for 12 h to produce single colonies. Single colonies were then picked up again until a uniform color and morphology were observed on the LA medium. Figure 1 Store at -80°C in 50% glycerin.

[0026] 2. Identification of Serratia marcescens strain G25-16 Single colonies were picked and added to 1 mL of LB broth medium and incubated at 30°C and 200 rpm for 12 h. Genomic DNA was extracted from rhizosphere bacteria using a bacterial genomic DNA extraction kit. Primers were used to detect 27-F (5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID No. 1)) and 1492... -The 16S-rDNA gene sequence was amplified using R (5'-AAGGAGGTGATCCAGCC-3' (SEQ ID No. 2)). The PCR amplification program was 95℃ for 3 min; 95℃ for 15 s; 60℃ for 45 s; 72℃ for 90 s; 72℃ for 10 min; 35 cycles. The PCR product was sequenced to obtain the sequence. The sequencing results were compared with the data from the National Center for Biotechnology Information (NCBI) gene bank (https: / / www.ncbi.nlm.nih.gov / ), and the final nucleotide sequence of the 16S-rDNA is shown in SEQ ID No. 3.

[0027] To clarify the species and closely related strains of strain G25-16, a phylogenetic tree was constructed using 16S-rDNA and the top 30 16S-rDNA sequences with the highest identification rate (see [link to phylogenetic tree]). Figure 2 Strain G25-16 was found to be related to... S. plymouthica 4Rx13 (GCF-000176835.2) is the most closely related strain, with 99.42% homology. Based on morphological characteristics, strain G25-16 was preliminarily identified as *Serratia pluvialis*. Serratia plymouthica ).

[0028] Example 2: Preparation of fermentation broth for Serratia pluvialis strain G25-16 A single colony of *Serratia pluvialis* strain G25-16 was inoculated into 500 μL of LB broth and incubated at 28°C and 200 rpm for 12 h. Then, 500 μL of the bacterial culture was inoculated into 500 mL of LB broth and fermented at 28°C and 200 rpm for 18 h, adjusting the OD value. 600 Up to 1, for backup.

[0029] Experimental Example 1: Plate confrontation test between Serratia pluvialis strain G25-16 and the tested pathogenic fungus. 1. Experimental Methods A single colony of Serratia pluvialis strain G25-16 was inoculated into 500 μL of LB broth and incubated at 28°C and 180 rpm for 18 h for later use.

[0030] The in vitro antibacterial activity of the tested pathogenic fungi against rhizosphere bacteria was investigated. The antagonistic effect was determined using a two-point confrontation method. A 9 mm perforator was used to cut a piece from a PDA plate, and the test bacterial solution was inoculated 2 cm from the center of the fungal cake. An uninoculated fungal cake served as a control. The plates were incubated upside down at 28°C in complete darkness for 7 days. The diameter of the pathogen was determined using a cross-measurement method.

[0031] 2. Experimental Results The results of the plate standoff test are shown in Table 1. Figure 3As 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.

[0032] Serratia procumbens G25-16 pairs N. solani The inhibition rate was 55.61%; Serratia marcescens G25-16 was effective against... F. most fragrant The antibacterial rate was 54.44%.

[0033] Table 1. Plate inhibition effect of Serratia marcescens strain G25-16 against Fusarium oxysporum, the pathogen of soybean root rot. Experiment Example 2: Potted Plant Growth Promotion Experiment of Serratia puchengensis Strain G25-16 1. Experimental Methods 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.

[0034] 2. Experimental Results 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 ).

[0035] Experiment Example 3: Potted Plant Efficacy Test of Serratia marcescens strain G25-16 1. Test materials and test methods 1.1 Test pathogenic fungi Fusarium solani N. solani (CX3-4) and Fusarium putrefaction Very fragrant (ZP3-6).

[0036] 1.2 Test Methods 1.2.1 Method for determining the pathogenicity of the tested fungi Inoculation of soybean seedlings at one node with pathogenic fungi N. solani (CX3-4) and F. odoratissimum (ZP3-6) Symptoms appeared 45 days later. The disease index was calculated as follows: The mycelial discs of the tested pathogenic fungal strain were inoculated into CMC medium and cultured at 28°C and 200 rpm for 14 days. Mycelia were removed by filtration using a 70 μm Cell Strainer. The spore count was observed under a microscope and adjusted to 10-1. 7 1 spore / mL. 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, a needle was used to pierce the soil five times at a 45° angle from 1 cm above the soil surface. Each soybean seedling was inoculated with 3 mL of the tested strain's spore solution. Forty-five days after inoculation, the disease incidence rate was calculated, and the pathogenicity of the tested fungus was calculated according to the disease index standards (Table 2) and the disease index formula.

[0037] Table 2 Grading Standards for Soybean Root Rot

[0038] The disease index is calculated using the following formula:

[0039] 1.2.2 Pot test to determine the control efficacy of Serratia pluvialis strain G25-16 Test strains N. solani and F. odoratissimum The mycelial discs were inoculated into CMC medium and cultured at 28°C and 200 rpm for 14 days. Mycelia were then removed by filtration. The spore count was calculated under a microscope, and the count was adjusted to 10-1. 7 1 spore / mL, for later use. Inoculate 3 mL of the prepared G25-16 fermentation broth into one-node soybean seedlings. 24 h later, inoculate each soybean seedling with 3 mL of the test strain spore solution. Use soybeans inoculated only with the test strain as a positive control, and soybeans inoculated with neither bacterial solution nor spore solution as a blank control. Calculate the control efficacy according to the following formula.

[0040]

[0041] 2. Experimental Results 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. nightshade (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 ).

[0042] The disease index and efficacy test results are shown in Table 3.

[0043] Table 3. Efficacy determination of Serratia pluvialis strain G25-16.

[0044] 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. most fragrant 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, Its microbial preservation number is: CGMCC No.34772.

2. A microbial preparation made from Serratia pluvialis G25-16 as described in claim 1.

3. The application of Serratia pluvialis G25-16 as described in claim 1 in the prevention and control of plant diseases; wherein the plant disease is soybean root rot.

4. The application of the microbial agent according to claim 2 in the prevention and control of plant diseases; wherein the plant disease is soybean root rot.

5. The application according to claim 3 or 4, characterized in that, The pathogen causing the plant disease is Fusarium solani (…). N. solani ) or Fusarium moniliforme ( F. odoratissimum ).

6. The application of Serratia pluvialis G25-16 as described in claim 1 in promoting soybean growth.

7. The application of the microbial preparation according to claim 2 in promoting soybean growth.

8. The application according to claim 6 or 7, characterized in that, The promotion of soybean growth includes increasing soybean fresh weight and plant height; wherein, the fresh weight refers to the weight of the entire plant.

Images