Siamese bacillus, microbial liquid and preparation method and application thereof

Abstract

The present invention discloses a strain of Bacillus siamensis, named KD50, which was deposited in the China Center for Type Culture Collection on March 10, 2023, with a deposit number of CCTCC NO: M 2023289. The present invention also discloses a microbial bacterial liquid containing the Bacillus siamensis and a preparation method thereof, and discloses the application of the biological bacterial liquid in inhibiting the growth of plant fungal pathogens and a method for preventing and treating rapeseed root knot disease using the microbial bacterial liquid. The Bacillus siamensis of the present invention and the microbial bacterial liquid containing the strain can inhibit the growth of a variety of plant fungal pathogens, have a significant effect on plant prevention and treatment of fungal pathogenic diseases, and provide a new direction for preventing and treating plant diseases. The Bacillus siamensis of the present invention and the microbial bacterial liquid containing the strain have a good inhibitory effect on rapeseed root knot disease, and the prevention and control effect reaches more than 85.7%.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a strain of Bacillus siamensis, a microbial bacterial liquid, and a preparation method and application thereof. Background Art

[0002] Microbial control refers to a control method that inhibits the activity of pathogens and improves plant resistance through competition, antagonism, induced resistance and growth promotion under the action of beneficial microorganisms. It has the advantages of being non-toxic, harmless, pollution-free and not easy to produce drug resistance. It has become a hot research field in biological control of plant diseases to replace chemical agents in recent years.

[0003] Plant diseases have always been an important factor threatening the healthy production of agriculture. Rapeseed clubroot first occurred in southern Europe and the Mediterranean region, and has now become a global disease of rapeseed. According to research reports, rapeseed clubroot is a type of soil-borne disease caused by infection with Plasmodiophora brassica. The pathogen mainly harms the roots of rapeseed, forming finger-shaped or spindle-shaped tumors of varying sizes on the main roots and lateral roots. In recent years, clubroot has become more serious year by year in Sichuan, Hubei, Zhejiang, Jiangsu, Anhui and other provinces, the main winter rapeseed producing areas in the Yangtze River Basin of my country, resulting in a serious decline in rapeseed yield and quality, which has seriously restricted the healthy development of my country's rapeseed industry.

[0004] Bacillus siamensis is a Gram-positive bacterium with the characteristics of fast growth, strong reproduction ability, strong stress resistance and easy cultivation. A large number of studies have shown that Bacillus siamensis has a strong antagonistic ability to a variety of plant pathogenic microorganisms, among which it has an inhibitory effect of more than 80.0% on Fusarium oxysporum and an inhibition efficiency of 85.3% on Alternaria tenuissima. At the same time, it shows good control effects on Colletotrichum capsici, Sclerotium rolfsii, Pseudomonas solanacearum and Botryosphaeria dothidea, but whether it is suitable for the control of rapeseed clubroot remains to be studied. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a siam bacillus, a microbial bacterial liquid and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0007] A strain of Bacillus siamensis, named KD50, was deposited in the China Center for Type Culture Collection on March 10, 2023, with a deposit number of CCTCC NO:M 2023289.

[0008] Based on a general inventive concept, the present invention also provides a microbial liquid comprising the above-mentioned Bacillus siamensis.

[0009] Based on a general inventive concept, the present invention also provides a method for preparing the above-mentioned microbial liquid, comprising the following steps:

[0010] (1) inoculating Siamese Bacillus on LB solid medium and performing activation treatment to obtain activated Siamese Bacillus;

[0011] (2) inoculating the activated Siamese Bacillus on a seed culture medium for cultivation to obtain Siamese Bacillus seed liquid;

[0012] (3) Inoculating the Siamese Bacillus subtilis seed liquid into the fermentation medium and culturing it on a shaking table to obtain a Siamese Bacillus subtilis suspension, i.e., the microbial bacterial liquid.

[0013] In the above-mentioned preparation method, preferably, in step (1), the activation treatment refers to constant temperature cultivation at 28°C to 30°C for 22 to 24 hours.

[0014] In the above preparation method, preferably, in step (2), the components of the seed culture medium include: 1.5-2.5% soybean meal, 0.5-1.5% yeast powder and 0.5-1.5% magnesium sulfate heptahydrate;

[0015] The activated Siamese Bacillus is inoculated on the seed culture medium for cultivation: the liquid volume is 50-60%, the inoculation amount is 1%-2%, the culture temperature is 35-37°C, and the culture is shaken at 170-190 rpm for 18-22 hours.

[0016] In the above preparation method, preferably, in step (3), the components of the fermentation medium include: 1.5-2.5% soybean meal, 0.5-1.5% yeast powder and 0.5-1.5% magnesium sulfate heptahydrate;

[0017] The temperature of the shaking incubator is 35-37°C, the rotation speed is 170-190 rpm, and the incubation time is 22-24 hours.

[0018] Based on a general inventive concept, the present invention also provides a use of the above-mentioned Bacillus siamese or the above-mentioned microbial liquid or the microbial liquid prepared by the above-mentioned preparation method in inhibiting the growth of plant fungal pathogens.

[0019] In the above application, preferably, the fungal pathogen includes at least one of Plasmodiophorabrassica, Sclerotinia sclerotiorum, Pythium aphanidermatum, Marssonina mali, and Rhizoctonia solani Kola.

[0020] The above application preferably includes irrigating the above microbial solution or the microbial solution prepared by the above preparation method into the soil of the plant.

[0021] Based on a general inventive concept, the present invention also provides a method for preventing and controlling rapeseed clubroot, which comprises irrigating the soil of rapeseed plants with the above-mentioned microbial solution or the microbial solution prepared by the above-mentioned preparation method.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The Siamese Bacillus and the microbial liquid containing the strain of the present invention can inhibit the growth of a variety of plant fungal pathogens, have a significant effect on preventing and controlling fungal pathogenic diseases in plants, and provide a new direction for preventing and controlling plant diseases.

[0024] (2) The siam bacillus and the microbial liquid containing the strain of the present invention have a good inhibitory effect on rapeseed clubroot disease, and the control effect reaches more than 85.7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a colony plate morphology diagram of Bacillus siamensis KD50 in Example 1 of the present invention.

[0026] Figure 2 This is a phylogenetic tree analysis diagram of Bacillus siamensis KD50 in Example 1 of the present invention.

[0027] Figure 3 This is the antagonistic result of Bacillus subtilis KD50 against plant pathogens in Example 1 of the present invention.

[0028] Figure 4 The results are as follows: ... Bacillus siamensis KD50 in Example 1 of the present invention is used to prevent and treat rapeseed clubroot. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0030] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0032] The bacterial culture medium used in the following examples is as follows:

[0033] Bacillus siamese KD50 seeds and fermentation medium: 2.0% soybean meal, 1.0% yeast powder, 1.0% magnesium sulfate heptahydrate, pH 7.3±0.2, high pressure sterilization (121°C, 30min).

[0034] LB solid medium (g / L): 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar powder, dissolve in water, and finally make up to 1000 mL, pH 7.0±0.2, and sterilize under high pressure (121°C, 30 min).

[0035] PDA solid culture medium (g / L): 6 g potato powder, 20 g glucose, and 20 g agar, dissolve in water, and finally make up to 1000 mL, pH 5.6±0.2, and sterilize under high pressure (121°C, 30 min).

[0036] Embodiment 1:

[0037] 1. Isolation and screening of bacteria

[0038] At the Gaoqiao rapeseed base demonstration garden of the Hunan Academy of Agricultural Sciences, the surface layer of the rapeseed soil was removed with a small sampling shovel, and 100 g of soil from a depth of 2-10 cm was put into a sterile kraft paper bag. The collection site and time were numbered and recorded, and the bags were brought back to the laboratory for isolation and purification of the strains.

[0039] Take 0.1 g of sample with a sterile spoon and pour it into a 1.5 mL centrifuge tube containing 0.9 mL of LB liquid medium. Incubate at 30°C and 100 rpm for 30 min, then let stand for 20 min and dilute to 10 by 10-fold gradient dilution. -5 Gradient, take 100 μL of the gradient dilution, evenly spread it on LB medium, pick a single colony for bacterial purification, the KD50 positive colony morphology is as follows Figure 1 shown.

[0040] 2. Identification of bacteria

[0041] The morphological, cultural, physiological and biochemical characteristics of the strain are as follows:

[0042] The strain is a Gram-positive aerobic bacterium. After culturing on LB solid medium for 3 days, the strain is milky white with a slightly raised center and a moist and transparent surface, which can be easily picked up by an inoculation loop. The strain can liquefy gelatin and reduce nitrates. The starch hydrolysis test and (VP) test are both positive, while the hydrogen sulfide test, methyl red test, and indole test are all negative.

[0043] The 16S rRNA gene of the strain was amplified, and the gene sequence (nucleotide sequence of the 16S rRNA gene) obtained after the PCR product was sequenced is shown in SEQ ID NO: 1:

[0044]

[0045] Comparison analysis was performed in NCBI to clarify the taxonomic status of the strain. The sequencing results were compared with BLAST on the NCBI website, and the results showed that the strain had a similarity of 99.93% with Bacillus siamensis, and its morphological characteristics and physiological and biochemical properties were closest to Bacillus siamensis. The phylogenetic tree of strain KD50 was constructed using MEGA6.0. The phylogenetic tree results are shown in Figure 2 Comprehensive analysis of 16S rRNA gene sequencing, morphological characteristics, physiological and biochemical characteristics, and phylogenetic tree showed that the strain could be classified as Bacillus siamensis.

[0046] The strain was named Bacillus siamense KD50 and was deposited in the China Center for Type Culture Collection on March 10, 2023. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2023289.

[0047] 3. Antagonism experiment of Bacillus siamensis KD50 against plant pathogens

[0048] The plate confrontation method was used: Sclerotinia sclerotiorum, Pythium aphanidermatum, Marssonina mali, and Rhizoctonia solani Kola were inoculated in the center of the PDA solid culture medium, and then the strain Bacillus siamensis KD50 was inoculated 2 cm away from the center of the culture medium by the cross method; the control group was inoculated with pathogens but not with Bacillus siamensis KD50, and cultured at 28°C. After the pathogens in the control group covered the plate, the width of the inhibition zone was measured, and each group was treated 6 times. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter * 100%.

[0049] Table 1 Inhibitory effect of Bacillus siamensis KD50 on plant pathogens

[0050]

[0051]

[0052] Table 1 shows the statistical results of the inhibition rate of Bacillus siamensis KD50 against plant pathogens. Figure 3This is a plate confrontation test record of KD50 against plant pathogens. The analysis results show that Bacillus siamensis KD50 can effectively inhibit the growth of rapeseed sclerotinia, corn stalk rot, apple brown spot and tobacco target spot. The inhibition effects on the corresponding pathogenic microorganisms Sclerotinia sclerotiorum, Pythiumaphanidermatum, Marssonina mali and Rhizoctonia solaniKola are 100%, 80.6%, 80.4% and 78.8% respectively, and can be used in the field of microbial control technology.

[0053] 4. Preparation method of Bacillus siamensis microbial culture liquid

[0054] (1) Activation of bacterial strains: Bacillus siamensis KD50 was inoculated on LB solid medium and cultured at 28° C. for 22 h to obtain activated Bacillus siamensis KD50;

[0055] (2) Fermentation seed liquid culture: The activated Bacillus siamensis KD50 was inoculated on the seed culture medium, with a liquid volume of 50%, an inoculum amount of 1%, a culture temperature of 35° C., and a shaking culture of 180 rpm for 20 h to obtain a Bacillus siamensis KD50 seed liquid;

[0056] (3) Fermentation suspension culture: The seed liquid of Bacillus siamensis KD50 was inoculated into the fermentation medium, and the culture was shaken at 180 rpm in a shaker at 37°C for 22 h to obtain a suspension of Bacillus siamensis KD50, i.e., a microbial culture liquid, which contained 3.5 billion CFU / mL of effective viable bacteria of Bacillus siamensis KD50.

[0057] 5. Application of microbial liquid containing Bacillus siamensis KD50

[0058] After the rapeseed seeds were sterilized and germinated, the rapeseed seedlings were transplanted into 10×10 cm square pots filled with nutrient medium, with one seed planted in each pot. One week after transplanting, the following treatments were performed:

[0059] Treatment 1: Root irrigation was used to add a final concentration of 10 6Inoculate rapeseed with dormant spores of Brassica rapa at a concentration of 100 / mL, and evenly water each pot with 10 mL; at the same time, dilute the microbial liquid (i.e., the fermented suspension of Bacillus siamensis prepared above) 100 times with sterile water and then water the roots, 10 mL per pot;

[0060] Treatment 2: Root irrigation was used to add a final concentration of 10 6 Inoculate rapeseed with dormant spores of Brassica rapa at a concentration of 100 / mL, and evenly irrigate each pot with 10 mL; at the same time, irrigate the roots with 50% carbendazim wettable powder (200 times diluted), 10 mL per pot;

[0061] Control: The final concentration of 10 6 Inoculate rapeseed with dormant spores of Brassica rapa at a concentration of 100 mL / mL, and evenly irrigate each pot with 10 mL of the solution. At the same time, irrigate the roots with sterile water at a concentration of 10 mL / pot.

[0062] Each treatment was replicated in 5 pots.

[0063] Rapeseed clubroot disease is divided according to the Kugimali (1999) grading standard: Grade 0: normal root system, no disease symptoms; Grade 1: no tumors on the main root, and a small number of small tumors on less than 1 / 3 of the lateral roots; Grade 2: clubroot forms on the main root or tumors on 1 / 3 to 2 / 3 of the lateral roots; Grade 3: larger tumors form on the main root or more than 2 / 3 of the lateral roots.

[0064] The disease index of rape clubroot was calculated and the pot control effect of Bacillus siamensis fermented suspension was analyzed. The results are shown in Table 2 and Figure 4 As shown, disease index = ∑ (number of diseased plants × corresponding level) / (total number of plants surveyed × highest level) × 100.

[0065] Table 2 Effect of different treatments on rapeseed clubroot in potted plants

[0066]

[0067] From Table 2 and Figure 4 The experimental results show that the disease index of rapeseed clubroot disease using Bacillus siamensis KD50 fermentation suspension (treatment 1) was significantly reduced by 13.4 compared with that using thiabendazim (treatment 2). The control effect of Bacillus siamensis fermentation suspension on rapeseed clubroot disease reached 85.7%, and the control effect of thiabendazim treatment (71.4%) was significantly improved.

[0068] The above test results show that Bacillus siamensis KD50 suspension can be used as an effective biological control agent for rapeseed clubroot disease.

Claims

1. A method for preventing and treating rape clubroot, characterized in that: A microbial solution containing Siamese Bacillus or a microbial solution prepared by the following preparation method is irrigated into the soil of rapeseed plants, wherein the Siamese Bacillus is named KD50, deposited in the China Center for Type Culture Collection on March 10, 2023, with a deposit number of CCTCC NO: M2023289, and the preparation method comprises the following steps: (1) inoculating Siamese Bacillus on LB solid culture medium and performing activation treatment to obtain activated Siamese Bacillus; (2) inoculating the activated Siamese Bacillus on a seed culture medium for cultivation to obtain Siamese Bacillus seed liquid; (3) The Siamese Bacillus subtilis seed liquid is inoculated into a fermentation medium and cultured on a shaking table to obtain a Siamese Bacillus subtilis suspension, that is, to prepare a microbial bacterial liquid.

2. The method for preventing and treating rape clubroot according to claim 1, characterized in that: In step (1), the activation treatment refers to constant temperature cultivation at 28°C to 30°C for 22 to 24 hours.

3. The method for preventing and treating rape clubroot according to claim 1, characterized in that: In step (2), the components of the seed culture medium include: 1.5-2.5% soybean meal, 0.5-1.5% yeast powder and 0.5-1.5% magnesium sulfate heptahydrate; During the cultivation process of inoculating the activated Siamese Bacillus into the seed culture medium: the liquid volume is 50~60%, the inoculation amount is 1%~2%, the culture temperature is 35~37°C, and the shaking culture is carried out at 170~190 rpm for 18~22 hours.

4. The method for preventing and treating rape clubroot according to claim 1, characterized in that: In step (3), the fermentation medium comprises: 1.5-2.5% soybean meal, 0.5-1.5% yeast powder and 0.5-1.5% magnesium sulfate heptahydrate; The shaking incubation temperature is 35~37℃, the rotation speed is 170~190 rpm, and the incubation time is 22~24h.

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