A soil bacillus and its application in preventing and controlling rice sheath blight

Abstract

The present invention discloses a soil Paenibacillus terrae and its application in the prevention and treatment of rice sheath blight, belongs to the technical field of rice sheath blight prevention and treatment, and solves the problem that the prevention and treatment of rice sheath blight in the prior art is not safe and environmentally friendly. The soil Paenibacillus terrae provided by the present invention is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO: 29689. The soil Paenibacillus terrae provided by the present invention can inhibit the growth of Rhizoctonia solani and can prevent and treat rice sheath blight. The present invention provides an effective prevention and treatment method for the green prevention and treatment of rice sheath blight, the prevention and treatment process is safe and environmentally friendly, the antibacterial mechanism is unique, and the pathogenic bacteria are not easy to develop drug resistance.

Description

Technical Field

[0001] The invention relates to the technical field of rice sheath blight prevention and control, and more specifically to an application of soil Paenibacillus in rice sheath blight prevention and control. Background Art

[0002] Rice has extremely high nutritional value and is the most important food source in my country. The high quality and high yield of rice plays an extremely important role in ensuring food security and people's living standards. However, diseases such as rice blast, bacterial blight and sheath blight seriously affect the growth of rice, restricting the improvement of rice quality and yield. Rice sheath blight may occur from the seedling stage to the end of the growth period due to infection by pathogens, resulting in the inability of rice to produce headings normally, and in severe cases, the whole plant may die. The pathogen of rice sheath blight is Rhizoctonia solani, which belongs to the order of Basidiomycetes in the sexual stage, has a wide host range and is highly saprophytic. In recent years, climate change, excessive use of nitrogen fertilizers, and the prevalence of short-stem and intensive planting methods have led to the increasing harm of rice sheath blight.

[0003] Traditional prevention and control of rice sheath blight is mainly based on chemical control. However, chemical control is somewhat polluting, and long-term use can easily cause drug resistance in the pathogen, resulting in reduced prevention and control efficiency. Summary of the invention

[0004] The invention provides an application of soil Paenibacillus in the prevention and treatment of rice sheath blight, so as to solve the problem of low prevention and treatment efficiency of rice sheath blight in the prior art.

[0005] In a first aspect, the present invention provides a soil Paenibacillus terrae, wherein the soil Paenibacillus terrae is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC NO: 29689.

[0006] In a second aspect, the present invention provides a method for fermenting soil Bacillus, wherein the soil Bacillus is placed in NB culture medium and fermented at 25-28° C. and 150-180 rpm for 1-2 days.

[0007] In combination with the second aspect, the present invention provides a soil Bacillus fermentation liquid obtained by applying the fermentation method.

[0008] In combination with the first aspect and the second aspect, the present invention provides use of the soil Paenibacillus or the fermentation liquid in inhibiting Rhizoctonia solani.

[0009] Specifically, the soil Paenibacillus or the fermentation liquid is used to prevent and control rice sheath blight.

[0010] Specifically, the concentration of the soil Bacillus fermentation liquid is 1×10 8 -2×10 8 cfu / mL.

[0011] Specifically, the soil Paenibacillus fermentation liquid is applied to the rice leaf sheath or rice leaf pillow from the four-leaf stage to the end of tillering stage of rice.

[0012] In a third aspect, the present invention provides a bacterial agent for preventing and controlling rice sheath blight, wherein the bacterial agent comprises the soil Paenibacillus or the soil Paenibacillus fermentation liquid.

[0013] Specifically, the bacterial agent includes microbiologically acceptable auxiliary materials; optionally, the auxiliary materials include one or a combination of surfactants, stabilizers, antifreeze agents, thickeners, disintegrants, plant growth regulators, and carriers.

[0014] The present invention provides an effective prevention and control method for green prevention and control of rice sheath blight. The plate confrontation test verifies that the soil Paenibacillus Jnb805 provided by the present invention has an inhibitory effect on the pathogenic bacteria Rhizoctonia solani of rice sheath blight, and the inhibition rate reaches more than 80%. The soil Paenibacillus Jnb805 provided by the present invention can prevent and control rice sheath blight by inhibiting Rhizoctonia solani. The prevention and control process is safe and environmentally friendly, the antibacterial mechanism is unique, and the pathogen is not easy to develop drug resistance.

[0015] The present invention shows through in vitro inoculation experiments and greenhouse control efficacy experiments that soil Bacillus Jnb805 can effectively inhibit the lesion area and lesion length of rice sheath blight in both in vitro and greenhouse control efficacy determinations, indicating that soil Bacillus Jnb805 can effectively inhibit the infection of rice sheath blight pathogens, which is of great significance for the green prevention and control of rice sheath blight.

[0016] The invention provides a culture medium formula, culture conditions and application parameters of soil Paenibacillus fermentation liquid, provides materials for biological control of rice sheath blight, and provides new ideas and theoretical basis for further development of biocontrol agents.

[0017] The deposit information of soil Bacillus provided by the present invention is as follows:

[0018] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0019] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0020] Deposit date: January 19, 2024;

[0021] Deposit number: CGMCC NO: 29689;

[0022] Taxonomic nomenclature: Paenibacillus terrae Jnb805;

[0023] Latin name: Paenibacillus terrae. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The identification results of soil Bacillus Jnb805 provided in the embodiment of the present invention, wherein a is a colony morphology diagram, b is a Gram staining result diagram, and c is a flagella staining result diagram.

[0026] Figure 2 A phylogenetic tree based on the 16S rRNA gene provided in an embodiment of the present invention.

[0027] Figure 3 The results of the plate confrontation experiment between soil Bacillus Jnb805 and Rhizoctonia solani provided in the embodiments of the present invention, wherein the left column is the CK group, the right column is the Jnb805 group, the first row is the initial screening experiment results, and the second row is the re-screening experiment results.

[0028] Figure 4 The experimental results of the effects of soil Bacillus Jnb805 on the hyphae morphology and nucleus of Rhizoctonia solani provided in the embodiments of the present invention, wherein the left column is the CK group, the right column is the Jnb805 group, the first row is the hyphae morphology observation results, and the second row is the hyphae nucleus staining results.

[0029] Figure 5 The results of the greenhouse protection experiment of soil Bacillus sp. Jnb805 against rice sheath blight provided in the embodiments of the present invention are shown.

[0030] Figure 6 The statistical results of the lesion length of the greenhouse protection experiment of the soil Bacillus sp. Jnb805 on rice sheath blight provided by the embodiment of the present invention are shown, wherein the ordinate represents the lesion length.

[0031] Figure 7 The results of the in vitro inoculation experiment of soil Bacillus sp. Jnb805 on rice sheath blight provided in the embodiments of the present invention.

[0032] Figure 8The statistical results of the percentage of the lesion area in the in vitro inoculation experiment of rice sheath blight with soil Paenibacillus Jnb805 provided in the embodiment of the present invention are shown, wherein the ordinate represents the percentage of the lesion area. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In order to solve the problem of low efficiency in the prevention and control of rice sheath blight in the prior art, this embodiment provides a soil Paenibacillus and its application in the prevention and control of rice sheath blight, and the soil Paenibacillus (P.terrae) is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO: 29689. The embodiment of the present invention provides an effective prevention and control method for green prevention and control of rice sheath blight, the prevention and control process is safe and environmentally friendly, the antibacterial mechanism is unique, and the pathogens are not easy to develop drug resistance.

[0035] The embodiment of the present invention provides a soil Paenibacillus fermentation liquid. The soil Paenibacillus is fermented in NB medium; the fermentation temperature is 25-28° C., and the fermentation is carried out at a rotation speed of 150-180 rpm for 1-3 days to obtain the soil Paenibacillus fermentation liquid.

[0036] The soil Paenibacillus provided by the embodiment of the present invention can inhibit the growth of Rhizoctonia solani, has a unique antibacterial mechanism and a good antibacterial effect.

[0037] Preferably, in the embodiment of the present invention, soil Paenibacillus or soil Paenibacillus fermentation liquid is applied to rice, and the concentration of soil Paenibacillus fermentation liquid is 1×10 8 -2×10 8 cfu / mL, sprayed on rice leaf sheaths or leaf pillows from the fourth leaf stage to the end of tillering. Experiments have shown that it can effectively inhibit the infection of Rhizoctonia solani on rice and effectively prevent the spread of rice sheath blight.

[0038] Experiments have shown that the soil Paenibacillus or soil Paenibacillus fermentation liquid provided by the embodiments of the present invention has a significant inhibitory effect on rice sheath blight, providing materials for biological control of rice sheath blight and providing new ideas and theoretical basis for the further development of biocontrol agents.

[0039] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0040] Example 1

[0041] This example provides a strain isolation and verification experiment of soil Bacillus Jnb805.

[0042] 1 g of soil (collected from the garlic rhizosphere in Zouzhuang Town, Xuzhou City, Jiangsu Province) was placed in a conical flask filled with 99 mL of sterile water, shaken in a shaker at 180 rpm for 30 min, and the supernatant was taken and diluted with sterile water to 10 -2 Up to 10 -4 Gradient, take 10 -2 , 10 -3 and 10 -4 Spread 200 μL of the gradient soil dilution solution on NA solid culture medium, put it in an incubator at 28°C and culture it upside down for 3 days, observe the colonies of different colors, glossiness, sizes and types on the plate and mark them, use an inoculation loop to pick up the marked colonies for streak purification culture, wait for single colonies to grow on the streaked plate, pick up the single colony and put it into NB culture medium, and store it at 4°C after shaking successfully.

[0043] The isolated strain was streaked on NA medium for 2 days to form circular colonies with a diameter of 1-2 mm. Figure 1 The colony morphology shown in a is Figure 1 As can be seen from a in the figure, the single colony of the obtained strain after cultivation can show a nipple-like protrusion in the middle, with a moist and transparent surface, thick bacterial moss, and easy to pick; the colony was subjected to a Gram staining experiment, and the following was obtained: Figure 1 From the staining results shown in b, it can be seen that the isolated bacteria are Gram-positive bacteria; the flagella staining experiment was performed on the colonies, and the following Figure 1 From the results shown in c, it can be seen that the isolated bacteria have peritrichous flagella.

[0044] The sequencing result of the isolated strain 16s RNA is shown in SEQ ID No: 1.

[0045] SEQ ID No: 1 is as follows:

[0046] CTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCCATGAGA

[0047] CTGGGATAACTACCGGAAACGGTAGCTAATACCGGATACATCCTTTCCCTG

[0048] CATGGGGAGGGGAGGAAAGACGGAGCAATCTGTCACTGATGGATGGGCC

[0049] TGCGGCGCATTAGCTAGTTGGTGGGGTAATGGCCTACCAAGGCGACGATG

[0050] CGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGC

[0051] CCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAA

[0052] AGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAA

[0053] AGCTCTGTTGCCAGGGAAGAACGTCTTGTAGAGTAACTGCTACAAGAGT

[0054] GACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCG

[0055] GTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCG

[0056] CGCAGGCGGCTCTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGGG

[0057] TCGCACTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATT

[0058] CCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGAACACCAGTGGCG

[0059] AAGGCGACTCTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGG

[0060] AGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGC

[0061] TAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGC

[0062] ATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGAC

[0063] GGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCG

[0064] AAGAACCTTACCAGGTCTTGACATCCCCCTGACCGGTCTAGAGATAGACC

[0065] TTTCCTTCGGGACAGGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCG

[0066] TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGCTT

[0067] AGTTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAGACTGCCGGTGACAA

[0068] ACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTG

[0069] GGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAAGAGCGA

[0070] TCTGGAGCGAATCCTAGAAAAGCCGGTCTCAGTTCGGATTGCAGGCTGC

[0071] AACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCC

[0072] GCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGA

[0073] GAGTTTACAACACCCGAAGTCGGTGGGGTAACCCGCAAGGGAGCCAGCC

[0074] Based on such as Figure 2The phylogenetic tree constructed from the known strains with the closest homology relationship of the 16S rRNA gene pair shown in the figure shows that the bacteria isolated in this example belong to the same species as the soil Bacillus, and is named as the soil Bacillus Jnb805.

[0075] The isolated soil Paenibacillus Jnb805 was tested for physiological and biochemical indicators, and the results are shown in Table 1, where "+" represents positive and "-" represents negative.

[0076] Table 1 Biochemical test results of soil Bacillus Jnb805

[0077]

[0078]

[0079] As shown in Table 1, the soil Bacillus Jnb805 isolated in this example can utilize glucose, raffinose, trehalose and malonate, hydrolyze starch, liquefy gelatin, produce siderophore, produce β-galactosidase, release potassium, and utilize organic and inorganic phosphorus.

[0080] Example 2

[0081] This example provides a plate confrontation experiment between soil Bacillus sp. Jnb805 and Rhizoctonia solani and an experiment on the hyphae inhibition effect.

[0082] The fermentation liquid of the soil Bacillus Jnb805 isolated and obtained in Example 1 was prepared, and the fermentation medium formula was: by mass ratio, 1.0% glucose, 1.7% beef extract, 0.4% calcium chloride, and the rest was distilled water, the pH was adjusted to 7.0, the inoculation ratio of the soil Bacillus Jnb805 strain was 3%, the fermentation temperature was 28°C, and the culture was carried out at a speed of 150 rpm for 2 days to obtain the soil Bacillus Jnb805 strain fermentation liquid; according to application requirements, the fermentation temperature can be between 25-30°C, the rotation speed can be between 120-180 rpm, and the culture time can be selected between 1-3d, which is not specifically limited in the present invention.

[0083] The pathogen of rice sheath blight, Rhizoctonia solani AG-1 (preserved in the Molecular Plant Pathology Laboratory, College of Plant Protection, Jilin Agricultural University) was used as the target pathogen for a plate confrontation primary screening experiment. The specific steps were as follows: two symmetrical points were punched on the PDA culture medium used for primary screening in the Jnb805 group, 50 μL of the prepared fermentation liquid of the soil Bacillus Jnb805 strain was dripped into one well, and a R. solani AG-1 bacterial cake with a diameter of 0.5 cm was added to the other well; two symmetrical points were punched on the PDA culture medium used for primary screening in the CK group, 50 μL of the fermentation liquid not inoculated with the soil Bacillus Jnb805 strain was dripped into one well, and a R. solani AG-1 bacterial cake with a diameter of 0.5 cm was added to the other well, and the plates were cultured at 28°C for 2 days, and the inhibition zone and inhibition rate were measured. The screened strains were subjected to a plate confrontation rescreening experiment, and the specific steps were as follows: holes were punched in the middle of the two groups of PDA culture media used for rescreening and R.solani AG-1 cakes with a diameter of 0.5 cm were placed in the holes, 50 μL of the prepared soil Bacillus Jnb805 strain fermentation liquid was dripped at two points around the cake of the PDA culture medium of the Jnb805 group used for rescreening, and 50 μL of the fermentation liquid of the uninoculated soil Bacillus Jnb805 strain was dripped at two points around the cake of the PDA culture medium of the CK group used for rescreening, and the cultures were cultured at 28°C for 2 days, the inhibition zones and inhibition rates were measured, the mycelial morphology of the two groups was observed under an electron microscope, and a small amount of strains were picked for DAPI staining.

[0084] The results of the plate confrontation primary screening and rescreening experiments are as follows Figure 3 As shown by Figure 3 It can be seen that the antibacterial rate of the screened soil Bacillus Jnb805 against R.solani AG-1 reached more than 80%, and the inhibitory effect was good. Figure 4 As shown in the first row of the figure, it can be seen that after Jnb805 treatment, the hyphae showed abnormal changes, with increased hyphae branches and twisted tips; the DAPI staining results are shown in Figure 4 As shown in the second row in the figure, it can be seen that after being treated with Jnb805, no obvious nucleus was found in the mycelium, indicating that soil Bacillus Jnb805 can inhibit mycelial growth and destroy the nucleus.

[0085] Example 3

[0086] This example provides a greenhouse protection effect experiment of soil Bacillus Jnb805.

[0087] Thirty NIP rice plants in the greenhouse at the end of tillering stage with the same growth conditions were selected and randomly divided into two groups, namely, the Jnb805 group and the CK group. A toothpick with R.solani AG-1 hyphae was inserted into the third leaf pulvinus from top to bottom of the Jnb805 group rice, and a toothpick with NB culture medium was inserted into the same position of the CK group rice. The treated parts of the two groups of rice were wrapped with plastic wrap, and the plastic wrap was removed after 24 hours. The two groups of rice were sprayed with the fermentation liquid of the soil Bacillus Jnb805 strain prepared in Example 2. The fermentation liquid was used per plant in a single dose of 50 mL and a concentration of 1.5×10 8 cfu / mL, and photos were taken 15 days after the fermentation liquid was sprayed and the length of the lesions in each group was measured.

[0088] The results are as follows Figure 5 and Figure 6 As shown, Figure 5 There are three representative plants in each group. Figure 6 The statistical results are the average of the statistical results of 15 plants in each group. Figure 5 and Figure 6 It can be seen that the lesion length of rice treated by spraying with the fermentation liquid of soil Bacillus Jnb805 strain was significantly lower than that of the CK group, indicating that soil Bacillus Jnb805 can significantly inhibit rice sheath blight caused by R. solani AG-1 infection.

[0089] Example 4

[0090] This example provides an in vitro inoculation experiment of soil Bacillus Jnb805.

[0091] Select four-leaf stage greenhouse NIP rice with the same growth conditions, cut the third leaf from top to bottom, cut the middle 6 cm leaf sample of each leaf, cut 30 leaves, and randomly divide them into two groups, namely Jnb805 group and CK group. Put the leaf samples of Jnb805 group into 50 mL of soil Paenibacillus Jnb805 strain fermentation liquid prepared in Example 2, and the fermentation liquid concentration is 1.5×10 8 cfu / mL, soaked for 1h, and the leaf samples of CK group were placed in the same dose of NB medium. After soaking, a bacterial cake of R.solani AG-1 with a diameter of 1cm was inoculated in the middle of each group of leaf samples. After culturing for 72h at 28℃ and 80% humidity, photos were taken and the lesion area was counted using Image J.

[0092] The results are as follows Figure 7 and Figure 8 As shown, Figure 7 For each group of three representative leaves, Figure 8 The statistical results are the average values ​​of the statistical results of 15 leaves in each group. Figure 7 and Figure 8 It can be seen that after soaking in the fermentation liquid of soil Bacillus Jnb805 strain, the lesion area of ​​rice leaves was significantly smaller than that of the CK group, indicating that soil Bacillus Jnb805 can significantly inhibit the infection of R.solani AG-1 to rice.

[0093] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0094] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An application of soil Paenibacillus or soil Paenibacillus fermentation liquid in preventing and controlling rice sheath blight, characterized in that: The soil Bacillus ( Paenibacillusterrae ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC NO: 29689; The preparation method of the soil Paenibacillus fermentation liquid is as follows: the soil Paenibacillus is placed in NB culture medium, and fermented at 25-30° C. and 120-180 rpm for 1-3 days to obtain the soil Paenibacillus fermentation liquid.

2. The use according to claim 1, characterized in that: The bacterial concentration of the soil Bacillus fermentation liquid is 1×10 8 -2×10 8 cfu / mL.

3. The use according to claim 1, characterized in that: The soil bacillus fermentation liquid is applied to the rice leaf sheath or the rice leaf pillow from the four-leaf stage to the end of tillering stage of the rice.

4. A bacterial agent for preventing and controlling rice sheath blight, characterized in that: The bacterial agent comprises the soil Bacillus or soil Bacillus fermentation liquid according to claim 1.

5. The bacterial agent according to claim 4, characterized in that The bacterial agent includes microbiologically acceptable excipients.

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