A biocontrol bacterium QHL1 for red root disease of rubber tree and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a rubber tree red root disease biocontrol bacterium QHL1 and its application. The rubber tree red root disease biocontrol bacterium QHL1 of the present invention has a broad-spectrum antibacterial activity and is effective against rubber tree red root disease bacteria. 、 Rhizoctonia solani 、 Rice seedling pathogen, eggplant wilt pathogen 、 Tomato early blight, wheat fusarium, wheat root rot, tobacco black shank pathogen, tobacco brown spot pathogen 、 Corn leaf spot fungus and the like have good antagonistic effects and are potential biological agents for preventing and treating rubber tree red root disease, rice seedling disease, eggplant wilt and the like, and the rubber tree red root disease biocontrol bacteria of the present invention also have good ability to produce protease, cellulase, β-glucanase, amylase and siderophore, etc. The rubber tree red root disease biocontrol bacteria QHL1 of the present invention has broad development space and good development and application prospects, and provides candidate resources for the prevention and treatment of rubber tree red root disease and other plant diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a rubber tree red root disease biocontrol bacterium QHL1 and an application thereof. Background Art

[0002] The pathogen of rubber tree red root disease is Ganoderma pseudoferreum (Wakef.) Over.etsteinm, has many hosts and a long incubation period, which can reach several years in the soil. Once the disease occurs, it spreads rapidly in the soil. If it is not treated in time, the mortality rate of rubber trees can reach 100%. At present, the prevention and control of rubber tree red root disease mainly rely on mechanical excavation and chemical control. Since rubber tree red root disease mainly harms the roots or root necks of rubber trees, when obvious symptoms appear on the aboveground parts, the infection is already very serious. Mechanical excavation is costly and can only be used to control the spread of the disease after the root disease occurs, which will seriously affect the yield of rubber plantations. Chemical control production mainly relies on chemical agents such as tridemorph and methyl thiophanate. Due to the increasing prominence of various problems caused by chemical control, people have gradually shifted their research direction to biological control. Biocontrol bacteria control is one of the important means of biological control. Discovering biocontrol bacteria with control effects on rubber tree red root disease is currently a research hotspot for the prevention and control of rubber tree red root disease, which plays an important role in promoting the prevention and control of rubber tree red root disease. Summary of the invention

[0003] The invention aims to overcome the deficiencies in the prior art and provide a rubber tree red root disease biocontrol bacterium QHL1 and an application thereof.

[0004] The first aspect of the present invention is to provide a rubber tree red root disease biocontrol bacterium QHL1, named: Bacillus Velez QHL1 ( Bacillus velezensis QHL1), deposited in the China Center for Type Culture Collection (CCTCC) on June 11, 2024, with the deposit number CCTCC NO: M 20241191.

[0005] The second aspect of the present invention is to provide a fermentation broth of the rubber tree red root disease biocontrol bacterium QHL1 as described in the first aspect of the present invention.

[0006] Furthermore, the preparation method of the above fermentation broth is: inoculate single colonies of the strain into NB culture medium respectively, and culture in a shaking incubator at 28°C and 180 rpm to obtain the broth.

[0007] The third aspect of the present invention is to provide a preparation containing the rubber tree red root disease biocontrol bacterium QHL1 described in the first aspect of the present invention or the fermentation broth described in the second aspect of the present invention.

[0008] The fourth aspect of the present invention is to provide the rubber tree red root disease biocontrol bacterium QHL1 as described in the first aspect of the present invention, or the fermentation liquid as described in the second aspect of the present invention, or the preparation as described in the third aspect of the present invention in the preparation of antagonistic rubber tree red root disease bacteria Ganoderma pseudoferreum Application in preparations.

[0009] The fifth aspect of the present invention is to provide the rubber tree red root disease biocontrol bacterium QHL1 as described in the first aspect of the present invention, or the fermentation liquid as described in the second aspect of the present invention, or the preparation as described in the third aspect of the present invention in the preparation of the rubber tree red root disease biocontrol bacterium QHL1 Ganoderma pseudoferreum Application of preparations to treat diseases caused by it.

[0010] The sixth aspect of the present invention is to provide the rubber tree red root disease biocontrol bacterium QHL1 as described in the first aspect of the present invention, or the fermentation liquid as described in the second aspect of the present invention, or the preparation as described in the third aspect of the present invention for preparing antagonistic rice sheath blight fungi. Rhizoctonia solani、 and / or Bakanae oryzae Fusarium moniliforme , and / or Fusarium oxysporum f. Fusarium oxysporum、 and / or Alternaria sinensis Alternaria solan , and / or Gibberella graminearum Fusarium graminearum , and / or Pseudomonas aeruginosa Bipolaris sorokiniana , and / or Phytophthora nicotianae Phytophthora nicotianae , and / or tobacco brown spot pathogen Alternaria alternat、 and / or Bipolaris maydis Bipolaris maydis Application in preparations.

[0011] The seventh aspect of the present invention is to provide the rubber tree red root disease biocontrol bacterium QHL1 as described in the first aspect of the present invention, or the fermentation liquid as described in the second aspect of the present invention, or the preparation as described in the third aspect of the present invention in the preparation of controlling rice sheath blight fungi. Rhizoctonia solani、 and / or Bakanae oryzae Fusarium moniliforme , and / or Fusarium oxysporum f. Fusarium oxysporum、 and / or Alternaria sinensis Alternaria solan , and / or Gibberella graminearum Fusarium graminearum , and / or Pseudomonas aeruginosa Bipolaris sorokiniana , and / or Phytophthora nicotianae Phytophthora nicotianae , and / or tobacco brown spot pathogen Alternaria alternat、 and / or Bipolaris maydis Bipolaris maydis Application of preparations for diseases caused by it.

[0012] The eighth aspect of the present invention is to provide the use of the rubber tree red root disease biocontrol bacterium QHL1 as described in the first aspect of the present invention in the preparation of a preparation for producing protease, and / or cellulase, and / or β-glucanase, and / or amylase, and / or siderophore.

[0013] The rubber tree red root disease biocontrol bacterium QHL1 disclosed by the invention has a broad-spectrum antibacterial activity and is effective against rubber tree red root disease bacteria. Ganoderma pseudoferreum、 Rhizoctonia solani Rhizoctonia solani、 Bakanae oryzae Fusarium moniliforme Fusarium wilt Fusarium oxysporum、 Alternaria blight Alternaria solan , Gibberella graminearum Fusarium graminearum , Triticum aestivum Bipolaris sorokiniana , Phytophthora nicotianae Phytophthora nicotianae , Tobacco brown spot pathogen Alternaria alternat、 Bipolaris maydis Bipolaris maydis The present invention has good antagonistic effects and is a potential biological agent for preventing and treating red root disease of rubber tree, rice seedling disease, eggplant wilt, etc., and the rubber tree red root disease biocontrol bacteria of the present invention also has good ability to produce protease, cellulase, β-glucanase, amylase and siderophore, etc. The rubber tree red root disease biocontrol bacteria QHL1 of the present invention has broad development space and good development and application prospects, and provides candidate resources for the prevention and treatment of rubber tree red root disease and other plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These are the results of antagonistic screening of strain QHL1.

[0015] Figure 2 This is the plate culture status of strain QHL1.

[0016] Figure 3 This is an electron micrograph of strain QHL1.

[0017] Figure 4 For strain QHL1 based 16S rRNA Phylogenetic tree of gene sequence alignment results.

[0018] Figure 5 For strain QHL1 based gyrA Phylogenetic tree of gene sequence alignment results.

[0019] Figure 6 The results of biological characteristics determination of strain QHL1.

[0020] Figure 7 This is the broad-spectrum antibacterial result of strain QHL1.

[0021] The strain QHL1 described in the present invention is Bacillus Velez subsp., named as Bacillus Velez subsp. QHL1 ( Bacillus velezensisQHL1), deposited in China Center for Type Culture Collection (CCTCC) on June 11, 2024, with the deposit number CCTCC NO: M 20241191, located at Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0023] The bacterial culture medium used in the following examples is as follows:

[0024] NA solid medium (g / L): 3 g beef extract, 5 g polypeptone, 10 g sucrose, 1 g yeast powder, 15 g agar powder, dissolve in water and make up to 1000 mL, adjust pH to 7.0-7.2, and sterilize by high pressure (121°C, 20 min).

[0025] NB liquid medium (g / L): 3 g beef extract, 5 g polypeptone, 10 g sucrose, 1 g yeast powder, dissolve in water and make up to 1000 mL, adjust pH to 7.0-7.2, and sterilize by high pressure (121°C, 20 min).

[0026] PDA solid culture medium (g / L): 200 g potato, 20 g glucose, 15 g agar, add water to dissolve, and finally make up to 1000 mL, pH 7.0-7.2, and sterilize by high pressure (121°C, 20 min).

[0027] LA medium: tryptone 10 g, sodium chloride 10 g, yeast extract 5 g, agar 18 g, deionized water 1000 mL.

[0028] Skim milk agar medium: 15 g skim milk powder, 15 g agar, 1000 mL distilled water.

[0029] Cellulose Congo red medium: 1 g potassium dihydrogen phosphate, 2 g ammonium sulfate, 0.5 g magnesium sulfate heptahydrate, 2 g CMC-Na, 0.5 g sodium chloride, 0.4 g Congo red, 15 g agar, and 1000 mL distilled water.

[0030] Siderophore assay medium (CAS). First, prepare CAS A solution: 1mmol / L CAS (chrome azurol), 4mmol / L CTMAB (hexadecyltrimethylammonium bromide), 0.1mmol / L FeCl3·6H2O. Then prepare CAS B solution: 0.1mol / L phosphate buffer, pH 7.0, and then prepare CAS C solution: 2g sucrose, 3g acid hydrolyzed casein, 20mL 1mmol / L MgSO4, 1mL 1mmol / L CaCl2, 18-20g agar powder, pH 7.0. Sterilize CAS A solution, B solution, and C solution at 115℃ for 15min respectively. When the temperature drops to about 60℃, add 5mL CAS A solution and 0.5mL CAS B solution to 100mL CAS C test plate and mix well.

[0031] Amylase detection medium: 2 g soluble starch, 10 g beef extract, 10 g glucose, 7 g peptone, 18 g agar, and 1000 mL deionized water.

[0032] β-Glucanase detection medium: casein 5g, yeast extract 2.5g, glucose 1g, agar 18g, 1% CMC, deionized water 1000mL.

[0033] Example 1 Obtaining the rubber tree red root disease biocontrol bacterium QHL1

[0034] 1. Sample source

[0035] On May 6, 2023, in the rubber forest of the third team of the Danzhou Experimental Field of the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. The lateral roots of the rubber trees were collected by digging the soil surface 10 cm away from the trunk, and 100g of root samples were collected at each point. Three samples were collected from each plot, and the time, location and type of sampling were recorded. The collected samples were stored at 4℃ for bacterial isolation.

[0036] 2. Screening of strains

[0037] The rubber tree samples were rinsed with running water for 30 min, and the surface moisture was wiped dry with sterile gauze. Rinse with sterile water three times in the clean bench, and dry with sterile filter paper. Then the surface was sterilized with 75% ethanol (1 min) and 2% NaClO solution (5 min) in turn. After rinsing with sterile water, the root segments were cut into small pieces of tissue of 1-2 cm using a sterilized scalpel, and inoculated into LA medium in the shape of a "pin". Five groups of experiments were repeated for each medium. The inoculated plates were placed in a constant temperature incubator at 28°C for culture. When obvious colonies were observed on the culture medium, single colonies with different morphologies were inoculated into new culture medium with an inoculation needle or inoculation loop for strain purification. The purified strains were stored at 4°C. At the same time, in order to ensure that the isolated bacteria were endophytes of rubber trees, two control groups were set up. One group used sterilized uncut tree roots to print on the plate; the other group used sterile water from the last rinse to spread on the plate. If no microorganisms grow on the plates of the two control groups, it can be determined that endophytes are obtained.

[0038] The single colonies of the strain were inoculated into NB medium and cultured in a shaker at 28 °C and 180 rpm for 12 h to obtain the fermentation broth. After that, the concentration of the fermentation broth was adjusted to OD 600 =2.0, set aside. Take the rubber tree red root pathogen ( Ganoderma pseudoferreum ) on the PDA medium. When the hyphae have grown all over the plate, use a 5 mm hole puncher to punch out the bacterial block on the plate. Take the bacterial cake, with the hyphae facing down, and inoculate it in the center of a new PDA plate. Use a 5 mm hole puncher to punch holes symmetrically on the left and right of the bacterial cake at equal distances (20 mm). Add 30 ul of the strain fermentation liquid into the hole. At the same time, the plate inoculated with NB medium and rubber tree red root disease bacterial cake was used as the control (CK). Place it in an incubator at 28°C, observe and record the antibacterial results after 7 days of incubation. Relative inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - 5) × 100%. In the formula, the unit of each colony diameter is "mm"; "5" is the initial diameter of the treated colony.

[0039] Finally, it was found that strain QHL1 showed strong inhibitory activity against rubber tree red root pathogen ( Figure 1 ), the relative inhibition rate was 88.46%, indicating that strain QHL1 had a significant antagonistic effect on rubber tree red root pathogen. After streaking culture on LA medium for 2 days, the colonies of strain QHL1 were milky white, round or oval, with a rough surface and wrinkled edges. Gram-positive bacteria ( Figure 2 The bacterial cell is short rod or oval, with a size of (1~3) μm×(0.5~0.8) μm ( Figure 3 ).

[0040] Example 2 Strain QHL116S rRNA Genes and gyrA Gene

[0041] The genomic DNA of strain QHL1 was extracted using the Omega Becterial DNA Kit. The extracted genomic DNA was used as a template and the bacterial 16S rDNA gene primers: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGGCTACCTTGTTACGACTT-3' were used for PCR amplification and sequencing. The result is shown in SEQ ID No. 1. DNA Star was used to analyze the sequencing results, and BLAST comparison was performed on the NCBI website to determine the species of closely related bacterial strains. The Neighbor-Joining phylogenetic tree constructed based on the 16S rRNA gene sequence comparison results with Bacillus cereus ATCC 14579 (NR 074540.1) as the outer branch is shown in Figure 4 shown.

[0042] Using primers for housekeeping genes of Bacillus velez: GyrA -F 5'-CAGTCAGGAAATGCGTACGTCCTT-3' and GyrA -R 5'-CAAGGTAATGCTCCAGGCATTGCT-3', using the extracted DNA as a template, PCR amplification was performed to obtain the target fragment. The PCR reaction system is:

[0043] Table 1 Ex-Taq polymerase chain reaction system

[0044] Component Volume Template DNA 2 μL -F 1 μL -R 1 μL Ex Taq 25 μL <![CDATA[ddH2O]]> 21 μL

[0045] Reaction conditions: 94℃ pre-denaturation for 10min, 94℃ denaturation for 1min, 55℃ annealing for 1min, 72℃ extension for 1min, 72℃ pre-extension for 10min, 4℃ storage, a total of 30 cycles. The PCR stock solution was sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing. The sequencing results were analyzed using DNA Star and BLAST comparison was performed on the NCBI website to determine the species of closely related bacteria. gyrA The gene is shown in SEQ ID NO. 2. gyrA The gene sequence comparison results are Bacillus mojavensis The Neighbor-Joining phylogenetic tree constructed with DSMZ 9205 (AY212986.1) as an outbranch is shown in Figure 5 The results showed that strain QHL1 and Bacillus velezensisThe similarity is the highest, and it is preliminarily determined to be Bacillus bacteria.

[0046] Based on the above test results, combined with morphological, cultural and biological characteristics, the strain QHL1 was identified as Bacillus Velezii and named: Bacillus Velezii QHL1 ( Bacillus velezensis QHL1), deposited in China Center for Type Culture Collection (CCTCC) on June 11, 2024, with the deposit number CCTCC NO: M 20241191, located at Wuhan University, Wuhan, China.

[0047] Example 3 Determination of biological characteristics of strain QHL1

[0048] The plate method was used to determine the activity of strain QHL1 in producing extracellular enzymes such as protease, cellulase, β-glucanase, and amylase, as well as the secretion of siderophore.

[0049] (1) Protease assay method:

[0050] Use a 5mm hole puncher to punch two holes symmetrically at 20mm from the center of the skim milk agar plate. Take 10μL of the fermentation liquid of the strain to be tested that has been cultured with NB for 24 hours and slowly inoculate it into one of the holes. Inoculate NB in ​​the other hole as a control. Place the plate in a 28℃ incubator for 3 days. If a transparent circle can be formed around the colony, the strain can produce protease; measure and record the diameter of the transparent circle.

[0051] (2) Cellulase assay method: Inoculate strain QHL1 onto a CMC plate according to the above method. After placing it in a 28°C incubator for 3 days, pour an appropriate amount of Congo red solution (1 mg / mL) onto the plate and let it stand for staining for 15 min. Then rinse it several times with sodium chloride solution (1 mol / L) and observe whether there is a yellow halo around the colony. If there is, it indicates that the strain can produce cellulase. Measure and record the diameter of the halo.

[0052] (3) Amylase assay method: Test whether the strain has amylase activity. Inoculate strain QHL1 on starch hydrolysis medium and culture at 28°C for 5-7 days. Add iodine solution that does not contain mycelium around the colony for detection. If a transparent circle is produced, it is positive. Measure and record the diameter of the transparent circle.

[0053] (4) Siderophore determination method: Inoculate QHL1 onto a CAS plate according to the method in (1). After placing it in a 28°C incubator for 3 days, observe whether a light yellow halo appears around the colony. If so, it indicates that the strain can produce siderophore. Measure and record the diameter of the halo.

[0054] (5) Determination of β-glucanase: Inoculate QHL1 onto a glucan detection medium plate according to the method in (1). After placing it in a 28°C incubator for 3 to 4 days, observe whether there is a transparent circle around the colony. If there is, it indicates that the strain can produce β-glucanase. Measure and record the diameter of the halo.

[0055] The results are as follows Figure 6 The strain QHL1 has a strong ability to secrete proteases, with a transparent zone diameter of 28.74 mm, and also has a strong ability to secrete cellulase (halo diameter 25.53 mm) and β-glucanase (transparent zone diameter 24.58 mm). It has the results of secreting amylase (transparent zone diameter 16.05 mm) and siderophore (halo diameter 11.32 mm).

[0056] Example 4 Determination of the antagonistic spectrum of strain QHL1

[0057] The single colonies of the strain were inoculated into NB medium and cultured in a shaker at 28 °C and 180 rpm for 12 h to obtain the fermentation broth. After that, the concentration of the fermentation broth was adjusted to OD 600 =2.0, spare.

[0058] The rice sheath blight pathogen was isolated by the filter paper confrontation culture method. Rhizoctonia solani、 Bakanae oryzae Fusarium moniliforme Fusarium wilt Fusarium oxysporum、 Alternaria blight Alternaria solan , Gibberella graminearum Fusarium graminearum , Triticum aestivum Bipolaris sorokiniana , Phytophthora nicotianae Phytophthora nicotianae , Tobacco brown spot pathogen Alternaria alternat、 Bipolaris maydis Bipolaris maydis Cultivate on NA medium respectively. When the hyphae cover the entire plate, use a 5mm aperture puncher to punch out the bacterial blocks on the plate. Take one of the bacterial cakes, with the hyphae facing up, and inoculate it in the center of a new NA plate. Use a 5mm puncher to punch holes symmetrically at equal distances (20 mm) on the left and right of the bacterial cake, and add 30ul of the fermentation liquid of the test strain QHL1 into the holes. Use the inoculated NB medium and the bacterial cake of the pathogen to be tested as the control (CK). Each treatment was repeated 3 times and cultured in a 28 ℃ incubator. After 5 days, observe and record the antibacterial phenomenon. The results are shown in Figure 7 The relative inhibition rates of strain QHL1 against various pathogens are shown in Table 2. Strain QHL1 has good antagonism against these nine pathogenic fungi.

[0059] Table 2 Antibacterial effect of strain QHL1 on eight plant pathogenic fungi

[0060] Pathogens Latin name Relative antibacterial rate (%) Rhizoctonia solani 92.36±9.65a Bakanae oryzae 74.63±10.32d Fusarium wilt 82.47 ±7.85c Alternaria blight 85.94 ±7.69bc Gibberella graminearum 41.87±6.75e Triticum sphaeroides 90.52±6.54ab Phytophthora nicotianae 94.36 ±8.45a Tobacco brown spot pathogen 88.64 ±9.64b Bipolaris maydis 90.25 ±5.78ab

[0061] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A rubber tree red root disease biocontrol bacterium QHL1, characterized in that, Name: Bacillus Velez QHL1 ( Bacillus velezensis QHL1), deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20241191.

2. the fermentation liquid of rubber tree red root disease biocontrol bacterium QHL1 as claimed in claim 1, is characterized in that, The method for preparing the fermentation broth comprises the following steps: inoculating single colonies of the strain into NB culture medium respectively, and culturing in a shaking incubator at 28°C and 180 rpm to obtain the fermentation broth.

3. A preparation, characterized in that Contains the rubber tree red root disease biocontrol bacterium QHL1 according to claim 1 or the fermentation liquid according to claim 2.

4. The rubber tree red root disease biocontrol bacterium QHL1 as claimed in claim 1, or the fermented liquid as claimed in claim 2, or the preparation as claimed in claim 3 in the preparation of antagonistic rubber tree red root disease bacterium Ganoderma pseudoferreum Application in preparations.

5. The rubber tree red root disease biocontrol bacterium QHL1 as claimed in claim 1, or the fermented liquid as claimed in claim 2, or the preparation as claimed in claim 3 in the preparation of the control rubber tree red root disease bacterium Ganoderma pseudoferreum Application of preparations to treat diseases caused by.

6. The rubber tree red root disease biocontrol bacterium QHL1 according to claim 1, or the fermentation liquid according to claim 2, or the preparation according to claim 3 in the preparation of antagonistic rice sheath blight fungi Rhizoctonia solani, and / or Bakanae oryzae Fusarium moniliforme , and / or Fusarium oxysporum f. Fusarium oxysporum, and / or Alternaria sinensis Alternaria solan , and / or Gibberella graminearum Fusarium graminearum , and / or Pseudomonas aeruginosa Bipolaris sorokiniana , and / or Phytophthora nicotianae Phytophthora nicotianae , and / or tobacco brown spot pathogen Alternaria alternat, and / or Bipolaris maydis Bipolaris maydis Application in preparations.

7. The rubber tree red root disease biocontrol bacterium QHL1 according to claim 1, or the fermentation liquid according to claim 2, or the preparation according to claim 3 in the preparation of preventing and controlling rice sheath blight Rhizoctonia solani, and / or Bakanae oryzae Fusarium moniliforme , and / or Fusarium oxysporum f. Fusarium oxysporum, and / or Alternaria sinensis Alternaria solan , and / or Gibberella graminearum Fusarium graminearum , and / or Pseudomonas aeruginosa Bipolaris sorokiniana , and / or Phytophthora nicotianae Phytophthora nicotianae , and / or tobacco brown spot pathogen Alternaria alternat, and / or Bipolaris maydis Bipolaris maydis Application of preparations for diseases caused by it.

8. Use of the rubber tree red root disease biocontrol bacterium QHL1 as claimed in claim 1 in preparing a preparation for producing protease, and / or cellulase, and / or β-glucanase, and / or amylase, and / or siderophore.

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