Serratia marcescens redj and its use in antagonizing rice blast

Abstract

The application discloses Serratia marcescans RedJ and application thereof in antagonizing rice blast. The Serratia marcescans RedJ can synthesize antagonistic fungal substances siderophore and chitinase, and has a good effect on treating rice infected by Magnaporthe oryzae. In the rice blast treatment experiment of rice Huaimiang No. 5, the lesion area reduction degrees of the bacterial suspensions of two concentrations (1x10 5 / mL and 1x10 9 / mL) are similar, and the average reduction is 38.3%; in the rice blast treatment experiment of rice Nanjing 9108, the average lesion area reduction degrees of the bacterial suspensions of two concentrations are 29.03% and 70.45% respectively, and the treatment effect of the high-concentration bacterial solution is better than that of the low-concentration bacterial solution. The Serratia marcescans RedJ strain provided by the application is a new strain for antagonizing and treating rice blast, provides a new strain resource for the prevention and treatment research of rice blast, and can be applied in the treatment of rice blast.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a RedJ strain of Serratia marcescens that antagonizes rice blast fungus. Background Technology

[0002] Rice blast, caused by *Magnaporthe oryzae*, is one of the most serious diseases in rice production, affecting rice-growing regions across China and the world, causing severe yield losses annually. Current control methods primarily focus on breeding resistant varieties, optimizing cultivation methods, developing chemical pesticides, and biological control. Breeding resistant rice varieties is the most economical, effective, and safe measure for controlling rice blast. However, due to the high mutation rate of *Magnaporthe oryzae*, the resistance of resistant varieties is not long-lasting, making it difficult to control rice blast solely through resistant varieties. Optimized cultivation methods only offer some protection before the onset of the disease. Chemical pesticides are currently the most important and efficient method for controlling rice blast. However, the irrational use of chemical pesticides can easily lead to resistance / resistance in *Magnaporthe oryzae*, cause environmental pollution and harm human health, and affect ecological balance and sustainable agricultural development. Therefore, the use of chemical pesticides is increasingly questioned. Biological control, which utilizes the interspecies relationships, involves using one organism to inhibit the growth or reproduction of another. Biological control has become a research hotspot for rice blast control due to its advantages such as high biocompatibility, safety for humans and animals, and low likelihood of pests developing resistance. Currently, many bacteria with inhibitory effects on rice blast fungus have been reported, mainly Bacillus and Pseudomonas bacteria. These bacteria primarily inhibit the growth of pathogenic microorganisms by secreting antagonistic small organic molecules (such as heparin, various antibiotics, and phytoalexins) and extracellular proteins or polypeptides (such as extracellular chitinases, proteases, and some antibacterial polypeptides), through antagonism, cell wall degradation, or induction of systemic resistance in plants. The effects of heparin-producing and chitinase-producing bacteria have been extensively studied and have yielded promising results.

[0003] Siderophores are a class of substances synthesized by microorganisms under low-iron conditions via a non-ribosomal pathway and specifically chelate Fe. 3+ Small molecular weight compounds, ferrophilic elements and Fe 3+ Its binding ability is very strong, and it can remove Fe from various water-soluble and insoluble compounds. 3+Some microorganisms can secrete siderophores to form siderophores-Fe chelates with iron ions in the surrounding environment. These chelates then transport iron into the microorganisms via specific transport mechanisms to meet their own growth needs, thereby reducing the iron concentration in the environment. This leads to the inability of some pathogenic microorganisms to grow and reproduce due to iron deficiency, thus controlling plant pathogens. Ben J. Duijff et al. discovered that *Pseudomonas fluorescens* can compete for Fe by secreting siderophores. 3+ It can inhibit the occurrence of Fusarium wilt in carnations and induce resistance in carnation plants to Fusarium. Arora et al. isolated rhizobia from the medicinal plant *Mucunapruriens* that can secrete heptaphilin and effectively inhibit peanut bud rot caused by *Macrophomina phaseolina*. Xu Yuquan et al. screened a *Pseudomonas* sp. JKD-2 strain with strong inhibitory ability against various plant pathogens, including *Oryza sativa*. In rice seedling stage experiments, it could effectively inhibit the inoculation infection of *Oryza sativa*. Further analysis of the characteristics and antibacterial activity of the heptaphilin secreted by this bacterium revealed that the heptaphilin secreted by this bacterium had a significant inhibitory effect on the growth of *Oryza sativa*. Ran Longxian et al. showed that *Pseudomonas fluorescens* WCS374r and *Pseudomonas putida* WCS358r inhibited the growth of *Botrytis cinerea* through competitive absorption of iron ions.

[0004] In addition, during their growth and development, microorganisms can synthesize and secrete enzymes that cause malformation of pathogenic fungal hyphae or inhibit spore germination, thereby suppressing fungal growth and development. For example, the bacteriocin surfactant peptides produced by *Bacillus licheniformis* can cause the germ tubes of *Bacillus oryzae* to swell and affect the formation of normal appressorium. The antifungal protein bacisubin produced by *Bacillus subtilis* has a strong antagonistic effect on the hyphal growth of fungi such as *M. grisea*, *Rhizoctonia solani*, and *B. cinerea*. Jia Shujuan et al. isolated an antimicrobial protein from *Bacillus subtilis* C-D6, which can inhibit the reproduction of *Bacillus oryzae* by affecting appressorium formation and normal germ tube growth. The *Bacillus subtilis* UKM1 screened by Ali et al. significantly inhibited the occurrence of rice blast under greenhouse conditions. Kumar et al. treated rice seeds with *Pseudomonas fluorescens* ABPf-1 isolated from the rice rhizosphere, which significantly reduced the incidence of rice blast. Yang Shuiying et al. used the plate confrontation method to study the inhibitory effect of the chitinase-producing bacterium CHB101 on *P. oryzae*, showing that this bacterium inhibits the growth of *P. oryzae* by hydrolyzing its cell wall and also has a strong inhibitory effect on various plant pathogenic fungi. Furthermore, Yang Xiaolu et al. found that the *Streptomyces* OsiSh-2 strain of actinomycetes can significantly inhibit the growth of *P. oryzae* hyphae, speculating that this may be related to the degradation of fungal cell walls by the chitinase and cellulase it synthesizes.

[0005] The diversity of bacteria and their varied metabolic types provide abundant resources for screening biocontrol bacteria. Besides the Bacillus and Pseudomonas genera, which have been extensively studied for inhibiting rice blast fungus, are there other superior genera / species that can antagonize it? Isolating and identifying other genera / species of biocontrol bacteria that inhibit rice blast will not only help to explore microbial resources for the biological control of rice blast but also contribute to improving the effectiveness of rice blast control. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a strain of Serratia marcescens Red J that antagonizes rice blast fungus.

[0007] Another object of the present invention is to provide the application of this Serratia marcescens.

[0008] Another object of the present invention is to provide RedJ chitinase derived from Serratia marcescens and its encoding gene.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A strain of Serratia marcescens RedJ was deposited on January 20, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21691.

[0011] A bacterium antagonistic to rice blast fungus was isolated from rice rhizosphere soil using the plate confrontation method. Figure 1 ), named RedJ, and then Gram-stained RedJ respectively. Figure 2 ), spore staining ( Figure 3 ) and physiological and biochemical analyses (fermentation experiment, indole experiment, VP experiment, methyl red experiment and starch hydrolysis experiment) (Table 1), etc. Further, the 16S rDNA sequence of RedJ was amplified by PCR using the universal primers B1 (5'-AACTGAAGAGTTTGATCCTGGCTC-3') and B2 (5'-TACGGTTACCTTGTTACGACTT-3'), and detected by agarose gel electrophoresis. Figure 4 Sequencing was then performed (SEQ ID NO.1). Blast sequence alignment and phylogenetic tree construction were then conducted. Figure 5 RedJ was identified as *Serratia marcescens*. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21691 on January 27, 2021.

[0012] Qualification by CAS plate method and SA medium colorimetric method ( Figure 6 The magnitude of RedJ's ability to synthesize heptapherin was analyzed by PCR and quantitative methods (Table 2). Furthermore, genes related to heptapherin synthesis or transport in the RedJ genome were amplified by PCR. Figure 7 The PCR amplification primers are shown in Table 3.

[0013] The bacterial agent prepared from Serratia marcescens RedJ.

[0014] As a preferred embodiment of the present invention, the bacterial agent is prepared by the following method: a single colony of *Serratia marcescens* Red J is inoculated into PDA liquid medium and cultured at 28-32°C with shaking at 180-200 rpm for 16-18 hours. The bacterial cells are centrifuged, washed with sterile water, and resuspended in sterile water to prepare a concentration of 1×10⁻⁶. 5 The bacterial agent is obtained by preparing a bacterial suspension with a concentration of more than 100 cells / mL.

[0015] The application of *Serratia marcescens* Red J in the preparation of chitinase.

[0016] The application of *Serratia marcescens* Red J in the prevention and control of rice blast.

[0017] The application of *Serratia marcescens* Red J in the preparation of products for the prevention and control of rice blast.

[0018] The application of the aforementioned inoculant in the prevention and control of rice blast.

[0019] The application of the aforementioned microbial agent in the preparation of products for the prevention and control of rice blast.

[0020] This study investigated the antagonistic effect of RedJ bacteria on rice blast fungus by treating the development of rice blast disease after inoculation with *Serratia marcescens* (RedJ) suspension. The rice varieties used in the inoculation experiment were Nanjing 9108 and Huaidao 5, both major varieties promoted in Jiangsu Province.

[0021] First, a rice seed germination test was conducted. The seeds were placed in a greenhouse with a temperature of 28℃, humidity of 90%, and alternating light and dark periods of 12 hours. They were then regularly sprayed with water and cultured for about two weeks before use.

[0022] Because the rice blast fungus strain 70-15 is prone to mutation, resulting in mycelial whitening and reduced sporulation capacity, this invention selects the rice blast fungus strain CH131, commonly used in basic research on rice blast, for rice inoculation experiments. After activation, rice blast fungus CH131 is transferred to a sporulation medium, and the spore concentration is adjusted to 10⁻⁶ using a hemocytometer. 5 Units / mL, ready for use;

[0023] After activation, *Serratia marcescens* RedJ was cultured in liquid and the bacterial cells were collected. The cells were washed twice with sterile water, and 1×10⁻⁶ cells were prepared by plate count method. 5 cells / mL and 1×10 9 Two bacterial suspensions of different concentrations (cFU / mL) are prepared for use.

[0024] First, a pre-prepared suspension of rice blast fungus CH131 spores was inoculated onto rice leaves using a small spray bottle. The leaves were then placed in a dark chamber at 28°C and high humidity (RH>95%) for 24 hours. Next, two concentrations of RedJ bacterial suspension were inoculated onto the rice leaves using the same spray bottle. The leaves were then removed and placed in a 28°C humid transparent chamber, sprayed with water daily to maintain a high-humidity environment. Samples were taken after 7 days to observe the incidence of rice blast. Three rice seedlings were used for each treatment, with sterile water as a control. Treatment results are shown below. Figure 12 , Figure 13 See Table 6.

[0025] The chitinase encoding gene from *Serratia marcescens* RedJ is selected from the chitinase encoding gene of chitinA1 shown in SEQ ID NO.2, the chitinase encoding gene of chitinA2 shown in SEQ ID NO.3, the chitinase encoding gene of chitinB shown in SEQ ID NO.4, and the chitinase encoding gene of chitinC shown in SEQ ID NO.5.

[0026] The chitinase derived from Serratia marcescens RedJ is selected from chitinA1 encoded by the gene shown in SEQ ID NO.2, chitinA2 encoded by the gene shown in SEQ ID NO.3, chitinB encoded by the gene shown in SEQ ID NO.4, and chitinC encoded by the gene shown in SEQ ID NO.5.

[0027] Amplification, sequencing, and bioinformatics analysis of chitinase genes in the RedJ genome:

[0028] Primers were designed based on chitinase genes reported in the database of *Serratia marcescens* (Table 4). Genes encoding chitinases in the RedJ genome, namely ChinA1, ChinA2, ChinB, and ChinC, were amplified by PCR and detected by 1% agarose gel electrophoresis. Figure 8 After gel purification, the cells were ligated into the pMD19-T vector (TaKaRa) and transformed into *E. coli* DH5α chemocompetent cells. Ampicillin-resistant plates were used for selection, and positive clones identified by colony PCR were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were assembled using Sequencher 4.5 software. The assembled sequences of chitinase genes ChinA1, ChinA2, ChinB, and ChinC are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5. The molecular weight and isoelectric point of the proteins encoded by chitinase genes ChinA1, ChinA2, ChinB, and ChinC were predicted online using Expasy software (Table 5). The conserved domains of the proteins encoded by chitinase genes ChinA1, ChinA2, ChinB, and ChinC were analyzed using the pfam online database. The results showed that they all encode a conserved chitinase 18 domain. Figure 9 ), belonging to the 18th family of glycosidases; using Phyre2 software, the tertiary structures of the proteins encoded by the chitinase genes ChinA1, ChinA2, ChinB, and ChinC were predicted. The results showed that their tertiary structures are all composed of three types of secondary elements: random coils, elongations, and α-helices, but their morphologies vary. Figure 10Chitinases ChiA1, ChiA2, and ChiB belong to the A subfamily of chitinase family 18, while chitinase ChiC belongs to the B subfamily of glycoside hydrolases family 18. Using the SignalP-5.0 online software, we predicted whether chitinases ChiA1, ChiA2, ChiB, and ChiC contained signal peptide sequences. The results showed that chitinases ChiA1 and ChiB do not encode a signal peptide, suggesting that ChiA1 and ChiB are non-secretory proteins. Chitinase ChiA2 contains a signal peptide, with a potential signal peptide break site between amino acid sequences 26 and 27 (AQA–AY) with a probability of 0.895, suggesting that chitinase ChiA2 is a secretory protein. Chitinase ChiC also contains a signal peptide, with a potential signal peptide break site between amino acid sequences 23 and 24 (AQA–AA) with a probability of 0.838, suggesting that chitinase ChiC is also a secretory protein.

[0029] Heterologous expression and enzyme activity assay of chitinase gene in RedJ genome:

[0030] Chitinase genes ChinA1, ChinA2, ChinB, and ChinC were constructed into the prokaryotic expression vector pGEX-6P-1 using a double enzyme digestion method. These vectors were then transformed into *E. coli* BL21(DE3) expression cells, plated on LB agar plates containing ampicillin resistance, and cultured at 37°C. After 12 h of shaking culture, positive clones were inoculated into 50 mL of induction expression medium at a 1:1000 ratio. When the OD600 reached 0.5-0.6, IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16°C for 16 h. The bacterial culture was then collected and lysed. The four proteins were purified using the Beyotime GST-tag purification kit and analyzed by SDS-PAGE. The results are shown in the figure below. Figure 11 The results showed that chitinases ChinA2, ChinB, and ChinC were soluble proteins, while chitinase ChinA1 was an inclusion body. The concentration of the purified enzymes was further determined using the BCA method, and each enzyme was diluted to 300 μg / ml. Enzyme activity was measured using the 3,5-dinitrosalicylic acid (DNS) method (Niu et al. 2017), and the activity was calculated using the formula y = 0.4036x - 0.04953 (R0). 2Enzyme activity was calculated using a 0.9956 μg / mL ratio, with PBS buffer as a control, and three replicates were performed. Chitinase ChinA1 was subjected to protein renaturation treatment before enzyme activity was measured. One unit of enzyme activity (U) was defined as the amount of enzyme required to generate 1 μmol of NAG in 1 min under the reaction conditions. The final enzyme activities of the four chitinases were as follows: chitinase ChinA2 activity was 0.73 U / μg; chitinase ChinB activity was 3.96 U / μg; chitinase ChinC activity was 6.28 U / μg; no activity was detected in the renaturated chitinase ChinA1.

[0031] The beneficial effects of this invention:

[0032] 1. The Serratia marcescens RedJ strain provided by this invention is a novel antagonistic strain against rice blast fungus. This strain can treat rice blast and reduce its development.

[0033] 2. The Serratia marcescens RedJ strain provided by this invention provides a new strain resource for the prevention and control of rice blast, and can be applied in the treatment of rice blast. Attached Figure Description

[0034] Figure 1 RedJ strain inhibited rice blast fungus strain 70-15 on PDA plates.

[0035] In a plate confrontation experiment between strain RedJ and rice blast fungus strain 70-15, the center of the plate showed the colony of rice blast fungus strain 70-15, while the red colony was that of strain RedJ. The presence of an inhibition zone around the RedJ colony indicates that RedJ can inhibit the growth of rice blast fungus strain 70-15.

[0036] Figure 2 This is a field of view of Gram staining results for bacteria RedJ (magnification 100*10).

[0037] Escherichia coli (A) and Staphylococcus aureus (B) were used as negative controls (red cells) and positive controls (blue-purple cells) for Gram staining, respectively. After staining, RedJ cells appeared red (C), indicating they are Gram-negative bacteria.

[0038] Figure 3 This is a field of view showing the staining results of RedJ spores of the fungus (magnification: 100*10).

[0039] Using Bacillus subtilis (A) as a positive control, which contains spores and is stained green, and the vegetative cells of Bacillus subtilis are stained red; Bacillus RedJ does not contain spores and its cells are stained red.

[0040] Figure 4 This is an agarose gel electrophoresis image of the 16S rDNA sequence amplification product of bacteria RedJ.

[0041] Lanes 1, 2, 3, and 4 contain the 16S rDNA PCR amplification products of bacterium RedJ, with a size of approximately 1500 bp. M is the standard molecular weight marker, and the band sizes from top to bottom are: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0042] Figure 5 It is the phylogenetic tree of the bacterium RedJ.

[0043] A neighbor-joining (NJ) phylogenetic tree (Bootstrap = 1000) was constructed using the 16S rDNA sequences of bacterium Red J and 14 other species of *Serratia*, with *Enterobacillus*, *Ensifer*, and *Bacillus* as outgroups. The red triangle represents bacterium Red J. Since Red J is most closely related to *Serratia marcescens*, it was identified as *Serratia marcescens*.

[0044] Figure 6 The ability of RedJ bacteria to synthesize hepatophilia was qualitatively determined using CAS plates.

[0045] The red color indicates RedJ colonies, and the presence of a clear zone around the colony indicates that RedJ bacteria can produce heptaphilin.

[0046] Figure 7 The agarose gel electrophoresis results of PCR amplification products of genes related to hematophilic synthesis or uptake in bacterium RedJ.

[0047] M represents the standard molecular weight marker, which, from top to bottom, are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Lanes 1-5 represent FEPA, FEPB, FEPC, FEPG, and FEPD, with band sizes of 2283bp, 1008bp, 825bp, 1056bp, and 1038bp, respectively. Figure 8 The PCR amplification products of chitinase genes ChinA1, ChinA2, ChinB, and ChinC in strain RedJ were obtained by agarose gel electrophoresis.

[0048] M is the standard molecular weight marker, and the band sizes from top to bottom are: 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. The size of ChinA1 is approximately 1700bp, ChinA2 is approximately 1300bp, ChinB is approximately 1500bp, and ChinC is approximately 1400bp.

[0049] Figure 9 These are the predicted results of conserved domains of proteins encoded by the chitinase genes ChiA1, ChiA2, ChiB, and ChiC.

[0050] Chitinases ChiA1, ChiA2, ChiB, and ChiC all encode a conserved Chitinase 18 domain, belonging to the 18th family of glycoside hydrolases.

[0051] Figure 10 These are the predicted three-dimensional structures of proteins encoded by the chitinase genes ChiA1, ChiA2, ChiB, and ChiC.

[0052] The tertiary structures of chitinases ChiA1, ChiA2, ChiB, and ChiC are all composed of three types of secondary elements: random coils, extensions, and α-helices, but their morphologies vary. Chitinases ChiA1, ChiA2, and ChiB belong to the A subfamily of the chitinase family 18, while chitinase ChiC belongs to the B subfamily of the glycoside hydrolase family 18.

[0053] Figure 11 These are SDS-PAGE results of heterologous expression of chitinases A1, A2, B, and C.

[0054] Chitinases ChiA1, ChiA2, ChiB, and ChiC were fused with GST-tag on the vector pGEX-6P-1 for expression. The band size of the chitinase ChiA1 fusion protein was approximately 89 kDa, and the band size of the ChiA2, ChiB, and ChiC fusion proteins was approximately 70 kDa.

[0055] Figure 12 The efficacy of RedJ bacterial suspension in treating rice blast disease on Nanjing 9108 rice variety.

[0056] Serratia marcescens RedJ showed good therapeutic effects against rice blast disease caused by blast fungus CH131 in the Nanjing 9108 rice variety. In treatment trials at two different bacterial concentrations, the average lesion area decreased by 70.45% and 29.03%, respectively. At a high concentration (1×10⁻⁶), the lesion area decreased even more significantly. 9 RedJ bacterial suspension (1 × 10⁻⁶ cells / mL) showed better therapeutic effects than low concentration (1 × 10⁻⁶ cells / mL). 5 (units / mL).

[0057] Figure 13 The efficacy of RedJ bacterial suspension in treating rice blast disease on Huai Dao 5 rice variety.

[0058] Serratia marcescens RedJ showed good therapeutic effect on rice blast caused by blast fungus CH131 in the Huai Dao 5 rice variety. The lesion area was reduced similarly after treatment with two different bacterial concentrations, with an average reduction of 38.3%.

[0059] Information on the preservation of biological materials

[0060] RedJ, classified as *Serratia marcescens*, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21691 on January 20, 2021. Detailed Implementation

[0061] The present invention will now be described in detail with reference to the accompanying drawings, tables, and specific embodiments.

[0062] Example 1

[0063] 1. Isolation and Identification of Microbial Strains

[0064] A bacterium antagonistic to rice blast fungus strain 70-15 was isolated from rice rhizosphere soil using the dilution plating method and named RedJ. Bacterium RedJ showed a significant inhibitory effect on rice blast fungus 70-15 on PDA solid medium. Figure 1 The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21691 on January 20, 2021.

[0065] 1.1 Gram staining

[0066] Following the methods described in *Microbiology Experiments*, Gram staining was performed on RedJ bacteria cultured to the logarithmic growth phase (16 hours). *Staphylococcus aureus* and *Escherichia coli* were used as positive and negative controls, respectively. The Gram staining results are shown below. Figure 2 The Gram staining reaction of the target bacterium RedJ was identical to that of Escherichia coli (a Gram-negative bacterium)—the cells turned red. Therefore, the target bacterium RedJ was identified as a Gram-negative bacterium. Furthermore, the cell morphology of the target bacterium RedJ was short rod-shaped.

[0067] 1.2 Spore staining

[0068] Following the methods described in *Microbiology Experiments*, spore staining was performed on RedJ bacteria cultured to the stationary phase (72 hours), with *Bacillus subtilis* used as a positive control. The spore staining results are shown below. Figure 3The positive control strain Bacillus subtilis can produce spores after 72 hours of culture. After spore staining, the spores were stained green, the vegetative cells were stained red, while the target strain RedJ was stained red. It is speculated that RedJ cannot produce spores after 72 hours of culture.

[0069] 1.3 Physiological and Biochemical Reactions

[0070] Following the methods outlined in "Experiments in Microbiology," the fermentation experiments of basic sugars (glucose, lactose, and sucrose), indole experiments, VP experiments, methyl red experiments, and starch hydrolysis experiments of strain RedJ were analyzed. The results are shown in Table 1.

[0071] Table 1. Results of physiological and biochemical analysis of RedJ.

[0072] Note: +: positive; -: negative; ND: not measured.

[0073] 1.4 Strain Identification

[0074] The 16S rDNA sequence of bacterium RedJ was amplified by PCR using universal primers B1 (5'-AACTGAAGAGTTTGATCCTGGCTC-3') and B2 (5'-TACGGTTACCTTGTTACGACTT-3'). The amplification conditions were: 95℃ for 3 min pre-denaturation, followed by 30 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 120 s, with a final extension at 72℃ for 10 min. PCR amplification system:

[0075]

[0076]

[0077] PCR amplification was performed using a Biometra TAdvanced 96SG PCR instrument. The PCR-amplified 16S rDNA was detected by 0.8% agarose gel electrophoresis and observed using a Tanon 2500 image analyzer. The PCR product length was approximately 1500 bp. The gel running distances of the replicate samples were consistent, and the bands were uniform. Figure 4 The obtained PCR products were sent to a sequencing company for sequencing and assembly. The 16S rDNA sequence of the obtained strain RedJ (see SEQ ID NO.1) was compared with nucleic acid data in GenBank using the BLAST program, and a phylogenetic tree was constructed using MEGA7 software. Figure 5The 16S rDNA sequences of *Enterobacillus*, *Ensifer*, and *Bacillus* were used as outgroups. The constructed phylogenetic tree showed that bacterium RedJ had the highest homology (99%) with *Serratia marcescens*, therefore RedJ was identified as *Serratia marcescens*.

[0078] Example 2. Determination of the ability of bacterium RedJ to synthesize hepatophiles

[0079] The CAS plate method and SA medium colorimetric method were used to qualitatively and quantitatively analyze the ability of RedJ bacteria to produce ferrophosphate, and genes related to ferrophosphate synthesis or transport in the RedJ genome were amplified by PCR.

[0080] 2.1 Qualitative Detection and Analysis

[0081] The RedJ bacterium was streaked onto LB agar and incubated at 28°C for 2 days. After incubation, single colonies of RedJ were inoculated onto CAS plates and incubated at 28°C for 6 days. The size of the clear zone around the colonies was observed and recorded. The colorimetric results qualitatively determined that RedJ has the ability to produce siderophores. RedJ can produce a clear zone on CAS solid medium. Figure 6 This indicates that it can produce heptaphilin.

[0082] 2.2 Quantitative Detection and Analysis

[0083] Single colonies of activated RedJ bacteria were inoculated into SA liquid medium, with no inoculation serving as a control. The medium was incubated at 28℃ and 150 rpm with shaking for 2 days. After incubation, the medium was centrifuged at 8000g at 4℃ for 20 min, and 10 mL of the supernatant was accurately aspirated and added to 10 mL of CAS detection solution. The mixture was thoroughly mixed and allowed to stand for 1 h. The absorbance (As630) was measured. The siderophore activity unit is (Ar–As) / Ar×100, where Ar is the absorbance value of the control group. The results are shown in Table 2.

[0084] Table 2. Quantitative determination of RedJ synthesized ferrophile.

[0085]

[0086] Note: The unit of siderophore activity is (Ar–As) / Ar×100, where Ar(CK) is the absorbance value of the control group and As(RedJ) is the absorbance value of the experimental group. As(RedJ) is greater than Ar(CK), indicating that the siderophore can be synthesized. The larger the absolute value of (Ar–As) / Ar×100, the stronger the ability to synthesize siderophores.

[0087] 2.3 PCR amplification of heptaphilic metabolism-related genes in the RedJ genome

[0088] Genes related to hemerophilic absorption and transport, namely FEPA, FEPB, FEPC, FEPG, and FEPD, were retrieved from the NCBI database, and primers (Table 3) were designed for PCR amplification. Amplification conditions were: 95℃ for 5 min pre-denaturation, 95℃ for 30 s, 50℃ for 30 s, 72℃ for 150 s for 30 cycles, followed by a 72℃ extension for 10 min. PCR amplification system:

[0089]

[0090] PCR amplification reactions were performed using a Biometra TAdvanced 96SG PCR instrument. PCR products were detected by 0.8% agarose gel electrophoresis and then observed using a Tanon 2500 image analyzer. The amplification results of the FEPA, FEPB, FEPC, FEPG, and FEPD genes are shown in the figure. Figure 7 The sizes are consistent with the expected results, with values ​​of 2283bp, 1008bp, 825bp, 1056bp, and 1038bp, respectively.

[0091] Table 3 Primer sequences for PCR amplification of heptaphilic metabolism-related genes

[0092]

[0093]

[0094] Example 3. Amplification, sequencing, heterologous expression, and enzyme activity assay of chitinase in the genome of bacterium RedJ.

[0095] Primers were designed based on the chitinase gene of Serratia marcescens reported in the database. The gene encoding chitinase in the genome of the RedJ bacterium was amplified by PCR, and the gene was cloned, sequenced, analyzed by bioinformatics, and heterologously expressed in Escherichia coli.

[0096] 3.1 Amplification, sequencing, and sequence feature analysis of the chitinase gene

[0097] Primers were designed based on conserved regions of chitinase genes reported in the NCBI database (Table 4) to amplify chitinase genes in the RedJ genome using PCR. The Serratia marcescens genome contains four chitinase genes: ChinA1, ChinA2, ChinB, and ChinC.

[0098] Table 4 Primer sequences used for PCR amplification of four chitinase genes.

[0099]

[0100] Note: Underlined lines in the table indicate the restriction sites of EcoRI (GAATTC), XhoI (CTCGAG), and BamHI (GGATCC).

[0101] The amplification conditions were: 95℃ for 5 min pre-denaturation, 95℃ for 30 s, 53℃ for 30 s, 72℃ for 120 s for 30 cycles, and 72℃ for 10 min extension. PCR amplification system:

[0102]

[0103] PCR amplification was performed using a Biometra TAdvanced 96SG PCR instrument. PCR products were detected by 0.8% agarose gel electrophoresis and then observed using a Tanon 2500 image analyzer. The amplified target bands were compared with markers; the chitinase gene ChinA1 was approximately 1700 bp, ChinA2 approximately 1300 bp, ChinB approximately 1500 bp, and ChinC approximately 1400 bp, all consistent with expectations. The results are shown in the table below. Figure 8 PCR amplification results were detected by 0.8% agarose gel electrophoresis, followed by gel purification and ligation into the pMD19-T vector (TaKaRa). The resulting cells were transformed into *E. coli* DH5α chemocompetent cells. Positive clones identified by ampicillin-resistant LB plates were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were assembled using Sequencher 4.5 software. The nucleotide sequences of the chitinase genes ChinA1, ChinA2, ChinB, and ChinC are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5. The full-length coding regions of the chitinase genes ChinA1, ChinA2, ChinB, and ChinC are 1692 bp, 1278 bp, 1500 bp, and 1443 bp, respectively. The physicochemical properties of their encoded proteins are shown in Table 5.

[0104] Table 5. Molecular weight and isoelectric point of proteins encoded by chitinase genes ChinA1, ChinA2, ChinB, and ChinC.

[0105]

[0106] Using the pfam online database, the conserved domains of proteins encoded by the chitinase genes ChinA1, ChinA2, ChinB, and ChinC were analyzed. The results showed that they all encode a conserved chitinase 18 domain. Figure 9), belonging to the 18th family of glycosidases; using Phyre 2 software, the tertiary structures of the proteins encoded by the chitinase genes ChinA1, ChinA2, ChinB, and ChinC were predicted. The results showed that their tertiary structures are all composed of three types of secondary elements: random coils, elongations, and α-helices, but their morphologies vary. Figure 10 Chitinases ChinA1, ChinA2, and ChinB belong to the A subfamily of chitinase family 18, while chitinase ChinC belongs to the B subfamily of glycoside hydrolases family 18. Using the online software SignalP-5.0, we predicted whether chitinases ChinA1, ChinA2, ChinB, and ChinC contained signal peptide sequences. The results showed that chitinases ChinA1 and ChinB do not encode a signal peptide, suggesting that ChinA1 and ChinB are non-secretory proteins. Chitinase ChinA2 contains a signal peptide, with a potential signal peptide break site between amino acid sequences 26 and 27 (AQA-AY) with a probability of 0.895, suggesting that chitinase ChinA2 is a secretory protein. Chitinase ChinC also contains a signal peptide. There is a potential signal peptide break site between amino acid sequence 23 and 24 (AQA to AA) with a probability of 0.838, suggesting that chitinase ChinC is also a secretory protein.

[0107] 3.2 Construction and Induction of Heterologous Expression Vector for Chitinase Gene

[0108] Recombinant pMD-19T plasmids containing the chitinase genes ChinA1, ChinA2, ChinB, and ChinC were extracted and digested with the corresponding restriction enzymes. The bands of the chitinase genes ChinA1, ChinA2, ChinB, and ChinC were then recovered via gel extraction and ligated into the corresponding double-digested linearized pGEX-6P-1 vectors (containing GST-tags) at a 3:1 ratio. The constructed expression vectors pGEX-6P-1-ChinA1, pGEX-6P-1-ChinA2, pGEX-6P-1-ChinB, and pGEX-6P-1ChinC were transformed into *E. coli* BL21(DE3) expression cells using chemical transformation. The cells were plated on LB agar plates containing ampicillin and incubated at 37°C. Positive clones identified by colony PCR were cultured for 12 h and then inoculated into 50 mL of induction expression medium at a 1:1000 ratio. When the OD600 reached 0.5-0.6, IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16℃ for 16 h. The bacterial culture was then collected and lysed. The four chitinase proteins were purified and analyzed by SDS-PAGE using the Beyotime Biotechnology GST-tag purification kit. The results are shown in the figure below. Figure 11 SDS-PAGE analysis showed that the chitinase genes ChinA1, ChinA2, ChinB, and ChinC were successfully induced and expressed. ChinA2, ChinB, and ChinC were soluble proteins, while ChinA1 was an inclusion body. Comparison with marker bands showed that the ChinA1 fusion protein band was approximately 89 kDa, while the bands of the ChinA2, ChinB, and ChinC fusion proteins were approximately 70 kDa.

[0109] 3.3 Determination of chitinase activity

[0110] Four chitinases were purified using the GST-tagged purification kit from Beyotime Biotechnology Co., Ltd. The concentrations of the purified chitinases were determined using the BCA method, and each enzyme was diluted to 300 μg / ml. Enzyme activity was measured using the 3,5-dinitrosalicylic acid (DNS) method (Niu et al. 2017), and the activity was calculated using the formula y = 0.4036x - 0.04953 (R0). 2 Enzyme activity was calculated using a 0.9956 μg / mL ratio, with PBS buffer as a control, and three replicates were performed. Chitinase ChinA1 was subjected to protein renaturation treatment before enzyme activity was measured. One unit of enzyme activity (U) was defined as the amount of enzyme required to generate 1 μmol of NAG in 1 min under the reaction conditions. The final enzyme activities of the four chitinases were: ChinA2 activity 0.73 U / μg; ChinB activity 3.96 U / μg; ChinC activity 6.28 U / μg; no activity was detected in the renaturated ChinA1.

[0111] Example 4. Activity assay of RedJ in treating rice blast

[0112] Preparing rice materials

[0113] This invention further verifies the antagonistic effect of RedJ bacteria on rice blast fungus by conducting a treatment experiment using a suspension of Serratia marcescens RedJ after rice blast fungus inoculation and subsequent disease development. The rice varieties selected for the inoculation experiment were Nanjing 9108 and Huaidao 5, both major varieties promoted in Jiangsu Province. First, a seed germination test was conducted. Healthy, plump rice seeds were selected, soaked in water, and placed in a dark, constant-temperature incubator at 37°C. The water was changed three times a day (morning, noon, and evening) to prevent the seeds from becoming smelly and rotting. Germination was carried out for about three days, until the seeds developed tender shoots suitable for sowing. The normally germinated seeds were evenly sown in plastic pots containing approximately 30 seeds per pot, with a 1:1 ratio of black soil to vermiculite. A layer of vermiculite was placed on the seed surface, and the seeds were thoroughly watered. The plastic pots were then placed in a large basin filled with water. Place it in a greenhouse with a temperature of 28℃, humidity of 90%, and alternating light and darkness for 12 hours. Spray water regularly for about 2 weeks before use.

[0114] Preparation of rice blast fungus spore suspension

[0115] Because the rice blast fungus strain 70-15 is prone to mutation, resulting in mycelial whitening and reduced sporulation capacity, this invention uses the commonly used rice blast fungus strain CH131 for rice inoculation experiments. Around 7 days into rice growth, the rice blast fungus mycelia were transferred to sporulation plates (SDC solid medium) and cultured in the dark at 28°C for approximately 3 days. Later, the surface mycelia were scraped off using sterilized 1.5 mL centrifuge tubes, and the plates were irradiated under black light for approximately 3 days. The spores on the sporulation plates were then brushed off with ultrapure water to prepare a spore suspension, and the concentration of the spore suspension was adjusted to 10 using a hemocytometer. 5 Quantity / mL, ready for use.

[0116] Preparation of RedJ bacterial suspension

[0117] Single colonies of *RedJ* bacteria were picked and inoculated into PDA liquid medium, and cultured at 30°C with shaking at 180 rpm for 18 h. The bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 5 min, washed twice with an equal volume of sterile water, and finally resuspended in sterile water. 1×10⁻⁶ colonies were prepared using the plate count method. 5 cells / mL and 1×10 9 Two concentrations of bacterial suspension per mL.

[0118] RedJ bacteria treatment experiment for rice blast

[0119] First, use a small spray bottle to inoculate rice leaves with a pre-prepared spore suspension of rice blast fungus CH131, and then place them in a dark chamber at 28℃ and high humidity (RH>95%) for 24 hours. Next, use a small spray bottle to inoculate rice leaves with a 1×10⁻⁶ spore suspension of fungus RedJ. 5 cells / mL and 1×10 9Rice blast disease was inoculated onto rice leaves using a bacterial suspension (50 μL Tween 20 was added to every 100 mL of bacterial suspension before inoculation to increase the adsorption capacity of the bacterial suspension on the rice leaves). The leaves were then placed in a 28℃ humid transparent box, sprayed with water daily to maintain a high-humidity environment. Samples were taken after approximately 7 days to observe the disease incidence. Three pots of rice seedlings were used for each treatment, with sterile water as a control. The treatment results (…) Figure 12 and Figure 13 As can be seen, the *Serratia marcescens* RedJ reported in this invention has a good therapeutic effect on rice blast caused by *Bacillus oryzae* CH131 in two rice varieties, Nanjing 9108 and Huaidao 5. Statistical analysis of the results showed that in the treatment experiment on Huaidao 5 rice, the lesion area reduced by the two concentrations of bacterial suspension was similar, with an average reduction of 38.3%; while in the treatment experiment on Nanjing 9108 rice, the lesion area reduced by the two concentrations of bacterial suspension by an average of 70.45% and 29.03% respectively (Table 6), with the high concentration (1×10⁻⁶) showing the greatest reduction. 9 RedJ bacterial suspension (1 × 10⁻⁶ cells / mL) showed better therapeutic effects than low concentration (1 × 10⁻⁶ cells / mL). 5 (Number of lesions / mL). Furthermore, the number of lesions on rice leaves sprayed with the biocontrol bacterium RedJ was also less than that in the control group (Number of lesions / mL). Figure 12 and Figure 13 ).

[0120] Table 6 shows the area of ​​rice blast lesions treated with RedJ bacterial solution on two rice varieties.

[0121]

Claims

1. A strain of Serratia marcescens ( Serratia marcescens RedJ was deposited on January 20, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21691.

2. The *Serratia marcescens* strain described in claim 1 (… Serratia marcescens The bacterial agent prepared by RedJ is characterized in that, The bacterial agent is prepared by the following method: a single colony of *Serratia marcescens* Red J as described in claim 1 is inoculated into PDA liquid medium and cultured at 28-32℃ with shaking at 180-200 rpm for 16-18 h. The bacterial cells are centrifuged, washed with sterile water, and resuspended in sterile water to prepare a concentration of 1×10⁻⁶. 5 The bacterial agent is obtained by preparing a bacterial suspension of more than one bacterial cell per mL.

3. The use of Serratia marcescens Red J as described in claim 1 in the preparation of chitinase.

4. The application of Serratia marcescens Red J as described in claim 1 in the prevention and control of rice blast.

5. The use of Serratia marcescens Red J as described in claim 1 in the preparation of products for the prevention and control of rice blast.

6. The application of the microbial agent according to claim 2 in the prevention and control of rice blast.

7. The use of the microbial agent according to claim 2 in the preparation of products for the prevention and control of rice blast.

8. The chitinase encoding gene derived from Serratia marcescens RedJ as described in claim 1, characterized in that... The gene encoding chitin A1 (SEQ ID NO.2), chitin A2 (SEQ ID NO.3), chitin B (SEQ ID NO.4), and chitin C (SEQ ID NO.5) are selected from the gene encoding chitin B.

9. The chitinase derived from Serratia marcescens Red J as described in claim 1, characterized in that... Chitin A1 encoded by the gene shown in SEQ ID NO.2, chitin A2 encoded by the gene shown in SEQ ID NO.3, chitin B encoded by the gene shown in SEQ ID NO.4, and chitin C encoded by the gene shown in SEQ ID NO.5.

Images