Bacillus subtilis for plant biological control and application thereof

Abstract

The invention discloses bacillus subtilis LXN-1, and the bacillus subtilis LXN-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC.NO 31697. The bacillus subtilis LXN-1 is a bacillus subtilis LXN-1 strain. The bacillus subtilis LXN-1 has an inhibition effect on various plant pathogenic bacteria, the inhibition rate on tomato botrytis cinerea can reach 61.5%, the inhibition rate on tobacco brown spot caused by alternaria longibrachiata can reach 72%, the bacillus subtilis LXN-1 also has a virus resistance effect, the inhibition rate on tobacco TMV viral plaque can reach 95%, and the bacillus subtilis LXN-1 has a good application prospect. In addition, the bacillus subtilis LXN-1 also has the potential of being compounded with pesticide bactericides with iprodione, trifloxystrobin, difenoconazole, pyraclostrobin, copper oxychloride and copper hydroxide as main components for use, and has market popularization and application values, and absorption of the plants to nutrient elements can be enhanced, and the growth of the plants can be promoted, and the bacillus subtilis LXN-1 also has the potential of being compounded with the pesticide bactericides with the iprodione, the trifloxystrobin, the difenoconazole, the pyraclostrobin, the copper oxychloride and the copper hydroxide as main components.

Description

Technical Field

[0001] The invention belongs to the field of food processing, and in particular relates to Bacillus subtilis for plant biological control and application thereof. Background Art

[0002] During the growth and development process, plants will be attacked by pests and affected by adverse growth conditions, which will interfere with their normal physiological activities, show morbidity or even death, and cause economic or ecological losses. This phenomenon is called plant disease. The pests that cause plant diseases mainly include fungal diseases, bacterial diseases and viral diseases. In order to reduce the economic losses caused by plant diseases, various plant disease prevention and control methods have emerged in an endless stream, which can be mainly summarized into four categories, including physical control, disease-resistant breeding control, chemical pesticide control and biological control. For most plant diseases, chemical pesticides are still the first choice of farmers. However, with the extensive use of chemical pesticides, negative problems such as ecological destruction, environmental pollution, and excessive pesticide residues in agricultural products have become increasingly serious. The use of safe, efficient and environmentally friendly biological control has received more and more attention, and will inevitably replace chemical pesticides as a new choice for plant disease control.

[0003] Bacteria are the most widely distributed microorganisms in nature. Biocontrol bacteria with good effects have fast reproduction ability, strong environmental adaptability and the ability to produce a variety of metabolites, playing an important role in biological control. At present, Bacillus subtilis has been the most widely studied bacteria that have been successfully used to control plant diseases. It can colonize in the rhizosphere, body surface or body of plants, compete with pathogens for their growth environment, and can secrete related metabolites to inhibit the growth of pathogens. It can also induce plants to produce resistance to resist the infection of pathogens. Studies have found that there are many types of Bacillus subtilis screened, and the control effects on different pathogens and different plants are not the same. For example: Bacillus subtilis B29 has a significant control effect on cucumber wilt; strain XG-1 can deform the hyphae of watermelon wilt fungus and has a strong inhibitory effect on the germination of its spores; R-11 has a good control effect on wheat sheath blight caused by Rhizoctonia graminearum.

[0004] There are many types of Bacillus subtilis, and their control effects on different pathogens and plants are different. To better apply them to biological control, it is necessary to evaluate and study different Bacillus subtilis and find superior strains with broad-spectrum effects in biological control. Summary of the invention

[0005] The purpose of the present invention is to provide a new strain, which has a broad-spectrum antibacterial and anti-viral effect, can be used for the prevention and treatment of various plant diseases, and also has the effects of nitrogen fixation, phosphorus dissolution and potassium dissolution, and IAA production, and can be used to promote plant growth.

[0006] The present invention provides a Bacillus subtilis LXN-1, which is deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC) on August 22, 2024, with a deposit number of CGMCC.NO31697, a classification name of Bacillus subtilis, and a deposit address of: General Microbiology Center of China Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides the use of the aforementioned Bacillus subtilis LXN-1 in preparing pesticides for preventing and controlling plant diseases and / or plant growth regulators for promoting plant growth.

[0008] Furthermore, the pesticide has the function of inhibiting plant pathogens; the pathogens include Agrobacterium tumefaciens, Pseudomonas syringae, Alternaria longibotium, Botrytis cinerea, Rice blast, Sclerotinia sclerotiorum, Fusarium graminearum, Pythium aphanidermatum, Phytophthora sojae and / or Anthracnose.

[0009] Furthermore, the pesticide has the function of resisting plant viruses; the plant viruses include tobacco mosaic virus TMV.

[0010] Furthermore, the plant growth regulator has the functions of fixing nitrogen, dissolving phosphorus and potassium and / or producing IAA.

[0011] Furthermore, the plants include Arabidopsis thaliana, tomato and tobacco.

[0012] The present invention also provides a pesticide for preventing and treating plant diseases, and its preparation raw materials include Bacillus subtilis LXN-1 and / or its fermentation liquid.

[0013] The present invention also provides a plant growth regulator for promoting plant growth, and its preparation raw materials include Bacillus subtilis LXN-1 and / or its fermentation liquid.

[0014] The present invention also provides a method for preparing the aforementioned pesticide or plant growth regulator, comprising the following steps:

[0015] The aforementioned Bacillus subtilis LXN-1 is inoculated into a Bacillus subtilis liquid culture medium for cultivation, the culture solution is centrifuged, and the supernatant and / or bacterial cells are collected to obtain the product.

[0016] Furthermore, the liquid culture medium is LB liquid culture medium.

[0017] Furthermore, the temperature is 32-37° C., the rotation speed is 100-300 r / min, the time is 12-36 h, the pH value of the LB liquid culture medium is 7, and the amount of LB liquid culture medium in each 250 mL container is 25-50 ml.

[0018] Finally, the present invention provides a use of the aforementioned Bacillus subtilis LXN-1 and / or its fermentation liquid in combination with a chemical fungicide in the preparation of pesticides for preventing and controlling plant diseases.

[0019] Furthermore, the chemical fungicide includes any one of iprodione, trifloxystrobin, difenoconazole, pyraclostrobin, pyraclostrobin, pyraclostrobin, and copper hydroxide.

[0020] The "nitrogen fixation" mentioned in the present invention refers to "biological nitrogen fixation", which is the process in which microorganisms convert nitrogen (N2) in the atmosphere into ammonia (NH3) or other nitrogen compounds that can be directly absorbed and utilized by plants.

[0021] The "dissolving phosphorus and potassium" mentioned in the present invention refers to the process in which microorganisms, through their biological action, convert insoluble phosphorus and potassium compounds into soluble phosphorus and potassium that can be directly absorbed and utilized by plants.

[0022] The term "IAA production" used in the present invention refers to the synthesis of indoleacetic acid by the microorganism itself.

[0023] The Bacillus subtilis LXN-1 of the present invention has an inhibitory effect on various plant pathogens (Agrobacterium tumefaciens, Pseudomonas syringae, Alternaria longifolia, Botrytis cinerea, Magnaporthe oryzae, Sclerotinia sclerotiorum, Fusarium graminearum, Pythium aphanidermatum, Phytophthora sojae and Colletotrichum), wherein the inhibition rate of tomato Botrytis cinerea can reach 61.5%, the inhibition rate of tobacco brown spot disease caused by Alternaria longifolia can reach 72%, and it also has the effect of resisting viruses, and the inhibition rate of tobacco TMV virus lesions can reach 95%, and it can also be compounded with pesticide fungicides with iprodione, fluopicolide·trifloxystrobin, difenoconazole, pyraclostrobin, spring thunder·copper and copper hydroxide as main components to enhance plant resistance to diseases, and at the same time, it can also enhance plant absorption of nutrient elements, play the role of nitrogen fixation, phosphorus and potassium dissolution, and IAA production to promote plant growth. It has market promotion and application value.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Colony morphology

[0027] Figure 2Gram staining of strains; AP: Gram staining results of strains SK0-SK15;

[0028] Figure 3 Electrophoresis diagram of strain DNA and PCR amplification products; A. Electrophoresis diagram of strain DNA; B. Electrophoresis diagram of strain PCR amplification products; Figure 4 Phylogenetic tree

[0029] Figure 5 Inhibitory effect of supernatants on bacterial pathogens; A. Inhibitory effect of supernatants on GV3101; B. Inhibitory effect of supernatants on PstDC3000; C. Statistical analysis of the inhibitory effect on GV3101; D. Statistical analysis of the inhibitory effect on PstDC3000;

[0030] Figure 6 SK0 has an inhibitory effect on Alternaria longipes; CK1 represents H 2 O treatment;

[0031] Figure 7 The antibacterial effect of the supernatant on detached leaves; A. The inhibitory effect of the supernatant on Botrytis cinerea on tomato leaves; B. The inhibitory effect of the supernatant on Alternaria longipes on tobacco leaves; C. The effect of the supernatant on the diameter of the tomato gray mold lesions; D. The effect of the supernatant on the diameter of the tobacco brown spot lesions; abc indicates significant differences at the P﹤0.05 level;

[0032] Figure 8 Detection of antibacterial substance-related genes; 1-6 represent Iturin A, Iturin B, Iturin D, Fengycin B, Fengycin D, Surfactin 110, respectively;

[0033] Fig. 9 The effect of culture time on bacterial growth and inhibition rate;

[0034] Fig.10 Effects of culture temperature on bacterial growth and inhibition rate

[0035] Fig.11 Effect of pH on bacterial growth and inhibition rate

[0036] Fig.12 Effect of liquid volume on bacterial growth and antibacterial rate

[0037] Fig.13 The effect of culture temperature on the antibacterial effect of the supernatant and the effect of pH on the antibacterial effect of the supernatant;

[0038] Fig.14 Effect of UV irradiation time on the antibacterial effect of supernatant and effect of protease on the antibacterial effect of supernatant

[0039] Fig.15 The effect of storage time on the antibacterial effect of the supernatant;

[0040] Fig.16 The supernatant activated the phosphorylation of MAPKs;

[0041] Fig.17 DAB staining method for detecting H in plant tissues 2 O 2 accumulation (A. tobacco; B. Arabidopsis; C. tomato);

[0042] Fig.18 The supernatant stimulated callose accumulation in Arabidopsis thaliana;

[0043] Fig.19 The effect of supernatant on tobacco's TMV resistance (A. tobacco plants; B. tobacco leaves; C. number of lesions on tobacco leaves; abc indicates significant difference at P﹤0.05 level;

[0044] Fig. 20 Nitrogen fixation ability of Bacillus subtilis

[0045] Fig.21 Phosphate and potassium solubilization ability of Bacillus subtilis

[0046] Fig. 22 The ability of Bacillus subtilis to produce IAA;

[0047] Fig.23 Determination of growth indexes of tobacco treated with Bacillus subtilis; A. Growth of tobacco plants; B. Determination of growth indexes; abc indicates significant difference at P﹤0.05 level

[0048] Fig.24 Effects of pesticides and fungicides on the growth of Bacillus subtilis SK0 DETAILED DESCRIPTION

[0049] Example 1 Preparation of the pesticide for preventing and controlling plant diseases of the present invention

[0050] 1) Inoculate Bacillus subtilis LXN-1 into LB solid medium and place in a 37°C incubator for 12 hours. Pick out a single colony and add it to 50 mL of LB liquid medium. Incubate at 37°C in a shaker at 200 r / min for 24 hours as the Bacillus subtilis seed liquid;

[0051] 2) Take the seed solution obtained in step 1) and inoculate it into a 250 mL conical flask containing 50 mL of LB medium with a pH value of 7, and culture it for 24 hours at a 1% inoculum, 37°C, and 200 r / min. Alternatively, centrifuge it in a low-temperature centrifuge at 8,000 r / min for 10 minutes, and take the supernatant or bacteria.

[0052] Example 2 Preparation of the plant growth regulator for promoting plant growth of the present invention

[0053] 1) Inoculate Bacillus subtilis LXN-1 into LB solid medium and place in a 37°C incubator for 12 hours. Pick out a single colony and add it to 50 mL of LB liquid medium with a pH value of 7. Incubate at 37°C with a shaker at 200 r / min for 24 hours as the Bacillus subtilis seed liquid;

[0054] 2) Take the seed solution obtained in step 1) and inoculate it into a 250mL conical flask containing 50mL of LB liquid culture medium with a pH value of 7, and culture it for 36 hours at a 2% inoculation amount, 35°C, and 300r / min, or further centrifuge it at 8,000r / min for 10min, and take the supernatant or bacteria.

[0055] Example 3 Preparation of the pesticide for preventing and controlling plant diseases of the present invention

[0056] 1) Inoculate Bacillus subtilis LXN-1 into LB solid medium and place in a 37°C incubator for 12 hours. Pick out a single colony and add it to 50 mL of LB liquid medium with a pH value of 7. Incubate at 37°C with a shaker at 200 r / min for 24 hours as the Bacillus subtilis seed liquid;

[0057] 2) The seed liquid is mixed with a chemical pesticide diluted according to the concentration recommended in the instructions at a volume ratio of 1:10 to 1:1000 to obtain the product; the chemical pesticide is any one of iprodione, fluopicolide·trifloxystrobin, difenoconazole, pyraclostrobin, pyraclostrobin, and copper hydroxide.

[0058] The beneficial effects of the present invention are further illustrated in the following experimental examples.

[0059] Experimental Example 1 Study on the Control of Plant Diseases by Bacillus subtilis

[0060] 1. Isolation and identification of Bacillus subtilis

[0061] 1 Experimental Materials

[0062] 1.1 Test strains

[0063] The strains isolated from the soil and the Bacillus subtilis agents purchased from the manufacturer were named and numbered, as shown in Table 1 for details.

[0064] Table 1. Source of strains

[0065]

[0066] 2 Experimental methods

[0067] 2.1 Isolation of strains from soil and pesticides

[0068] Use the soil dilution method to isolate microorganisms in the soil. The specific steps are as follows: weigh 1g of soil sample and put it into a conical bottle containing 9mL of sterile water. Oscillate it at 200r / min at 37℃ for about 2h. Use a pipette to draw 0.1mL of soil suspension and add it to an EP tube containing 0.9mL of sterile water. Oscillate the EP tube to mix it thoroughly. Repeat this process to dilute the suspension step by step to 10 -6 . Take 100 μL of the suspension and spread 10 -5 and 10 -6 The dilution was used to isolate the bacteria in the soil. Three replicates were set for each concentration until a single colony was clearly grown on the culture medium. A single colony was picked and continued to be isolated and purified until it was completely purified and stored in a small tube for screening. The isolation and purification of the Bacillus subtilis inoculant purchased by the manufacturer is similar to the soil dilution method.

[0069] 2.2 Identification of Bacillus subtilis strains

[0070] 2.2.1 Morphological identification

[0071] The Bacillus subtilis strains in Table 1 were streaked onto LB plates, cultured in a 37°C incubator for 12 h, and their colony morphology was observed and recorded. Single colonies were picked and Gram-stained.

[0072] 2.2.2 Molecular biological analysis and identification

[0073] 2.2.2.1 Identification of strains by 16S rDNA PCR

[0074] Pick different single colonies of Bacillus subtilis and extract total DNA. Use the extracted strain DNA as a template, use the universal primers of bacteria, and use 16S rDNA as the amplification region for PCR amplification. The primer sequences are: 27-F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492-R: 5'-GGTTACCTTGTTACGACTT-3';

[0075] The amplified product was verified by PCR using agarose gel electrophoresis. The PCR product was purified and recovered, and the purified 16S r DNA fragment was sequenced by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

[0076] 2.2.2.2 Construction of strain phylogenetic tree

[0077] After sequencing, the 16S rDNA sequence of the strain to be tested was compared with all known sequences in GenBank using BLAST. A phylogenetic tree was constructed using MEGA7 software to determine its phylogenetic position.

[0078] 3 Results Analysis

[0079] 3.1 Identification of strains

[0080] 3.1.1 Identification of morphological characteristics

[0081] The 16 strains were streaked on LB plates and cultured overnight in a 37°C incubator to observe their colony morphology. Figure 1 The single colonies of the 16 isolated strains growing on the plate surface are irregular circles, opaque, and some strains will have wrinkles and protrusions on the surface after a long period of culture, with irregular edges and no pigment. The 16 strains were gram-stained and their morphology was observed under an optical microscope. The results are as follows Figure 2 From the Gram staining results, it was found that 16 strains were stained blue-purple. Combined with the Gram staining principle, it showed that the isolated strains were all Gram-positive bacteria, and their shapes could be clearly seen as rods, so it was determined that the 16 strains were Gram-positive bacilli.

[0082] 3.1.2 Identification of molecular biological characteristics

[0083] The genomic DNA of 16 strains was extracted by preparing cell lysis buffer using the SDS method, and then detected by agarose gel electrophoresis. The results showed that the genomic DNA of each bacteria was successfully extracted ( Figure 3 A). Using bacterial DNA as a template, 16S rDNA was amplified by PCR using bacterial universal primers. The PCR amplification product was detected by agarose gel electrophoresis. The results showed that the length of the amplified gene fragment was about 1,500 bp, which was consistent with the expected target band size ( Figure 3 B).

[0084] It is known that in bacterial classification studies, when the homology of the 16S rDNA sequence of the strain is as high as 95%, it can be considered to belong to the same genus, and when the sequence homology is as high as 97% or more, it can be considered to belong to the same species. The PCR product was purified and sent to a biological company for sequencing to obtain the 16S rDNA sequence. The sequence was compared by BLAST on the NCBI website. Combined with bacterial classification studies, it was found that most of the homologous sequences obtained by comparing the 16 strains were Bacillus, and the homology with Bacillus subtilis was the highest. The phylogenetic tree was constructed using the neighbor-joining method using the software. It can be seen that the SK0 strain is closely related to a variety of standard strains Bacillius subtilius strains, with a similarity of up to 99%. The remaining strains are similar to related Bacillius subtilius strains at 98%-100%, further proving that the isolated strain is Bacillus subtilis ( Figure 4). It can also be seen from the figure that the genus Bacillus to which Bacillus subtilis belongs is distantly related to the introduced exogenous gray mold and has a low similarity.

[0085] The 16 strains isolated were identified as Bacillus subtilis, among which the SK0 strain was named Bacillus subtilis LXN-1 for deposit in the patent procedure, and was deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC) on August 22, 2024, with the deposit number CGMCC.NO 31697, and the classification name was: Bacillus subtilis. The deposit address is: General Microbiology Center of China Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0086] 2. Study on the antibacterial effect of Bacillus subtilis fermentation supernatant

[0087] 1 Experimental Materials

[0088] 1.1 Test strains

[0089] 1.1.1 Sources of biocontrol bacteria

[0090] A total of 16 strains of SK0 to SK15 were isolated

[0091] 1.1.2 Sources of pathogenic microorganisms

[0092] The pathogenic microorganisms used are shown in Table 2 below.

[0093] Table 2 Sources of pathogenic microorganisms

[0094]

[0095] 1.2 Plant materials

[0096] Tobacco Sansheng tobacco seeds were a gift from Professor Yang Hui of the College of Agriculture, Sichuan Agricultural University and Professor Yang Song of Guizhou University. Arabidopsis wild-type seeds were stored in the laboratory of the Botany Department of Sichuan Agricultural University in Ya'an, Sichuan Province. Tomato "Jinpeng No. 1" seeds were purchased from Xi'an Jinpeng Seedling Co., Ltd.

[0097] 1.3 Preparation of seed solution and fermentation supernatant

[0098] Different Bacillus subtilis were inoculated into LB solid culture medium (from Sangon Biotech (Shanghai) Co., Ltd.), and placed in a 37°C incubator for 12 hours. The cultured single colony was picked and added to 50mL of LB liquid culture medium (from Sangon Biotech (Shanghai) Co., Ltd.), and cultured at 37°C shaker at 200r / min for 24 hours as Bacillus subtilis seed liquid, and the seed liquid was stored for later use. Different Bacillus subtilis seed liquids were inoculated into a conical flask containing 50mL of LB culture medium, cultured for 24 hours at an inoculation amount of 1%, 37°C, and 200r / min, and centrifuged at 8,000r / min in a low-temperature centrifuge for 10 minutes to obtain the supernatant, and the supernatant was stored for later use.

[0099] 2 Experimental methods

[0100] 2.1 Antibacterial effect of supernatant on plates

[0101] 2.1.1 Supernatant inhibits bacterial pathogens

[0102] For bacterial pathogens, the filter paper method was used for the test, that is, 200 μL of bacterial indicator bacteria was aspirated under sterile conditions and evenly spread on the LB solid culture medium plate. After the indicator bacteria solution was dried, a circular sterile filter paper with a diameter of 5 mm was placed on the culture medium. 10 μL of the reserved Bacillus subtilis supernatant was aspirated with a pipette and added to the center of the circular paper. 25 μg / mL ampicillin and sterile water were used as controls, and the cells were cultured overnight at 37°C. The antibacterial effect was observed, the inhibition zone was measured, and the inhibition rate was calculated; inhibition rate (%) = inhibition zone diameter - 5 mm / inhibition zone diameter × 100%.

[0103] 2.1.2 Supernatant inhibits fungal pathogens

[0104] First, the pathogenic fungi were inoculated on a PDA culture medium plate and cultured in a 22°C incubator for 7 days for activation, until the mycelium covered the plate. The antibacterial effect can be determined by the plate pair method and the punching method in the antibacterial experiment. The pathogens cultured for 7 days were punched into a 7mm cake and inoculated in the center of the PDA plate. A hole was punched around it 2 cm away from the strain and inoculated with Bacillus subtilis strains and blank controls respectively. For further screening, experiments were conducted on both sides of the PDA plate, with one side being the pathogen cake and the other side being inoculated with Bacillus subtilis. A blank control group was added, and three replicates were set for each strain, and cultured at 25°C for 5 days. When the colonies of the control group covered the culture dish, the radius of the inhibition zone was recorded; inhibition rate (%) = (inhibition zone radius × 2-7mm) / (inhibition zone radius × 2) × 100%.

[0105] 2.2 Antibacterial effect of supernatant on isolated plants

[0106] Select leaves with similar growth conditions from plants with similar growth conditions and wash them with sterile water. Take a sterile culture dish with filter paper, add 4 mL of sterile water to each culture dish, soak the filter paper, put the washed leaves on the filter paper of the culture dish, and wrap each petiole with moist cotton. Use the supernatant of Bacillus subtilis with better antibacterial activity in the previous test to spray the above leaves, and sterile water is used as the control group. Then inoculate each leaf with a pathogenic fungal block with a diameter of 7 mm. All culture dishes are placed in a 25°C plant culture room for culture. After three days, observe the disease condition of each leaf and record the diameter of the lesion.

[0107] 2.3 Detection of antibacterial substance genes in supernatant

[0108] The Bacillus subtilis strains with good antibacterial effects were cultured on LB medium, and then the strain DNA was extracted. Then, the extracted bacterial DNA was used as a template to design specific primers for related antibacterial substances Iturin, Fengycin and Surfactin to perform PCR amplification to detect the expression of antibacterial substance genes in the bacteria. The primer sequences are shown in Table 3.

[0109] Table 3 Antibacterial substance gene primer sequences

[0110]

[0111] 3 Results and analysis

[0112] 3.1 Antibacterial effect of supernatant on plates

[0113] 3.1.1 Inhibitory effect of supernatant on bacterial pathogens

[0114] The 16 isolated strains were subjected to a filter paper antibacterial experiment using GV3101 and Pseudomonas syringae pv.tomato DC3000 as pathogen indicator bacteria to preliminarily screen strains with antibacterial effects. Figure 5 The results showed that SK0, SK2, SK3, SK9, SK10, SK12 and SK14 had the most obvious antibacterial effect on GV3101, with the maximum antibacterial diameter reaching 1.99 cm. SK1, SK4, SK5, SK6, SK7, SK8, SK11 and SK13 had less antibacterial effect, and SK15 had no inhibitory effect on GV3101; while SK0, SK2, SK3, SK10 and SK14 had good antibacterial effect on PstDC3000, with the maximum antibacterial diameter reaching 2.11 cm. SK7, SK8, SK9, SK11, SK12 and SK15 had less antibacterial effect, and the other strains had no antibacterial effect.

[0115] 3.1.2 Inhibitory effect of supernatant on fungal pathogens

[0116] The 16 isolated strains were subjected to filter paper antibacterial experiments using 8 common plant fungi as pathogenic indicator bacteria to screen strains with antifungal effects. Due to the large number of fungi tested, only the inhibition results of strain SK0 on Alternaria longipes are shown below ( Figure 6 ), it can be clearly observed from the figure that, compared with the CK treatment group, an obvious inhibition zone was produced around the SK0 treatment group, and the inhibition rate of the fungus reached 76.6%, indicating that the SK0 strain had a significant inhibitory effect on the growth of Alternaria longiflora.

[0117] The inhibitory effects of 16 strains on 10 test pathogenic indicator bacteria, including 2 bacterial pathogens, were counted (Tables 4-5, data in the table are the mean number of dead spots ± standard deviation and the mean number of dead spots ± standard error; "-" represents no inhibitory effect; abc represents significant difference at the P﹤0.05 level). Among them, for fungal pathogens, strains SK0, SK2, SK3, SK7, SK10, and SK14 all had inhibitory effects on Alternaria longifolia, Botrytis cinerea, Magnaporthe oryzae, and Sclerotinia sclerotiorum, among which SK0 had inhibitory effects on all pathogenic fungi.

[0118] Table 4 Bacillus subtilis antibacterial effect detection ①

[0119]

[0120] Table 5 Bacillus subtilis antibacterial effect detection ①

[0121]

[0122]

[0123] 3.2 Antibacterial effect of supernatant on isolated plants

[0124] The above-screened 6 strains, SK0, SK2, SK3, SK7, SK10, and SK14, were cultured and their supernatants were obtained for in vitro plant antibacterial experiments to further verify their antibacterial effects. Figure 7 As shown, in H 2 When the detached leaves treated with H0 were already obviously diseased, the treatment groups sprayed with the supernatants had almost no disease, and only the plants treated with SK2 had slight disease but not serious. Among them, the diameter of the lesions after SK0 treatment was smaller than that of H0. 2 The O treatment group had the most obvious reduction, with the lesion diameter being only 0.7 cm, and the inhibition rate of tomato gray mold could reach 61.5%, and the inhibition rate of tobacco brown spot disease could reach 72% ( Figure 7C, D). This indicates that Bacillus subtilis SK0, SK2, SK3, SK 7, SK10, and SK14 all have good inhibitory effects on Botrytis cinerea and Alternaria longibotia inoculated on detached leaves, among which SK0 is the best.

[0125] 3.3 Gene detection of antimicrobial substances in supernatant

[0126] In order to preliminarily explore the antibacterial mechanism of six Bacillus subtilis strains, namely SK0, SK2, SK3, SK7, SK10 and SK14, the genes related to known antibacterial substances were detected. Figure 8 ) found that SK0, SK3, and SK14 were able to amplify the target fragments of Iturin A, Iturin B, Iturin D, Fengycin D, and Surfactin110 with clear and bright bands, indicating that their antibacterial effects may depend on the production of iturin, fengycin, and surfactin; IturinA and Iturin D in SK2 and SK7 strains showed faint bright bands, while SK10 could not amplify the corresponding bands, indicating that their antibacterial effects may not depend on these common antibacterial substances detected.

[0127] The above experiments show that among the 16 strains SK0 to SK15, 6 strains, namely SK0, SK2, SK3, SK7, SK10 and SK14, showed inhibitory effects on some pathogenic fungi and bacteria in the experiment, among which SK0 had inhibitory effects on all pathogenic fungi and bacteria used in the experiment (Agrobacterium tumefaciens, Pseudomonas syringae, Alternaria longifolia, Botrytis cinerea, Rice blast, Sclerotinia sclerotiorum, Fusarium graminearum, Pythium aphanidermatum, Phytophthora sojae and Colletotrichum).

[0128] 3. Study on the antibacterial activity of Bacillus subtilis fermentation supernatant

[0129] 1 Experimental Materials

[0130] 1.1 Test strains

[0131] The SK0, SK2, SK3, SK7, SK10, and SK14 strains were isolated and screened.

[0132] 1.2 Preparation of seed solution and supernatant

[0133] Different Bacillus subtilis were inoculated into LB solid medium and cultured at 37°C for 12 hours. A single cultured colony was picked and added to 50 mL of LB liquid medium, and cultured at 37°C at 200 r / min for 24 hours as the Bacillus subtilis seed liquid. The Bacillus subtilis seed liquid was centrifuged at 8,000 r / min for 10 minutes in a low-temperature centrifuge to obtain the supernatant, and the supernatant was stored for later use.

[0134] 1.3 Preparation of protease solution

[0135] Weigh the powders of trypsin and pepsin respectively, add them to an appropriate amount of sterilized water to make the final concentration of the solution 1 mg / mL, filter and sterilize using a 0.22 μm filter, and store for later use.

[0136] 2 Experimental methods

[0137] 2.1 Effects of culture conditions on bacterial growth and antibacterial activity

[0138] 2.1.1 Cultivation time

[0139] Different Bacillus subtilis seed liquids were inoculated into a conical flask containing 50 mL LB medium and cultured at 1% inoculum, 37°C, and 200 r / min for 12 h, 24 h, 36 h, 48 h, and 72 h. The bacterial liquid was collected at each time point and the OD of the bacterial liquid was measured. 600 The antibacterial activity of the supernatant of Bacillus subtilis was determined using GV3101 as the pathogen indicator bacteria.

[0140] 2.1.2 Culture temperature

[0141] Different Bacillus subtilis seed liquids were inoculated into a conical flask containing 50 mL LB medium, and the inoculation amount was 1%, 37°C, and 200 r / min for 24 hours. The culture temperature was set to 25°C, 28°C, 30°C, 37°C, and 40°C, and the OD of the bacterial liquid was measured at each temperature. 600 Values ​​and antibacterial activity of the supernatant.

[0142] 2.1.3 pH

[0143] Different Bacillus subtilis seed solutions were added to 50 mL LB medium with initial pH values ​​of 3, 4, 5, 6, 7, 8, 9, and 10, respectively, with an inoculation amount of 1%, and cultured for 24 h under the same conditions as above. The OD of the bacterial solution in each conical flask was measured. 600 Values ​​and antibacterial activity of the supernatant.

[0144] 2.1.4 Liquid volume

[0145] Add different Bacillus subtilis seed liquids to new LB medium, and set the liquid volume to 20mL, 30mL, 50mL, 70mL, and 90mL in 250mL conical flasks, with an inoculation volume of 1%, and culture for 24h under the same conditions as above. Determine the OD of the bacterial liquid in each conical flask 600 Values ​​and antibacterial activity of the supernatant.

[0146] 2.2 Study on the stability of antibacterial substances of Bacillus subtilis

[0147] 2.2.1 Temperature stability of antimicrobial substances

[0148] The obtained supernatants were treated at different temperatures of 37°C, 45°C, 60°C, 95°C, and 121°C for 1 h. The supernatant treated at 37°C was used as a control. An antibacterial experiment was carried out with GV3101 as the pathogenic indicator bacteria. The antibacterial diameter was measured and the antibacterial rate was calculated.

[0149] 2.2.2 Stability of antimicrobial substances to acids and bases

[0150] The obtained supernatant was adjusted to different pH values ​​(pH gradient was 4, 5, 6, 7, 8, 9, 10) with 1 mol / L HCl and NaOH, respectively. After equilibration for 1 h, an antibacterial test was performed to measure the antibacterial diameter and calculate the antibacterial rate.

[0151] 2.2.3 Stability of antimicrobial substances to UV

[0152] The obtained supernatant was poured into an uncovered sterile culture dish, placed in a sterile clean bench, and irradiated with ultraviolet light. The irradiation times were 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min, respectively. The supernatant irradiated for 0 min was used as the control group for antibacterial experimental tests, the antibacterial diameter was measured, and the antibacterial rate was calculated.

[0153] 2.2.4 Stability of antimicrobial substances to proteases

[0154] The supernatant was mixed with pepsin and trypsin at a concentration of 1 mg / mL in equal amounts, placed in a 37°C water bath for 1 hour, and then placed in a 95°C metal bath for 10 minutes to inactivate the enzymes. After cooling to room temperature, the inhibitory ability of the Bacillus subtilis supernatant on the indicator bacteria was determined, and the untreated Bacillus subtilis supernatant was used as the control group. The antibacterial experiment was carried out to measure the antibacterial diameter and calculate the antibacterial rate.

[0155] 2.2.5 Stability of antimicrobial substances over storage time

[0156] The obtained supernatant was stored in a refrigerator at 4°C for 5, 10, 15, 30, 60 and 90 days. The unstored supernatant was used as a control for antibacterial experimental tests, and the antibacterial diameter was measured and the antibacterial rate was calculated.

[0157] 3 Results and analysis

[0158] 3.1 Effects of culture conditions on bacterial growth and antibacterial activity

[0159] 3.1.1 Effect of culture time on bacterial growth and antibacterial activity

[0160] The culture time can affect the growth of Bacillus subtilis strains and the production of antibacterial substances. In the range of 12-24 hours, the six strains continued to grow and reached a stable period in about 24 hours; in the range of 36-72 hours, the growth of the six strains showed a downward trend, the growth was relatively slow, and the bacteria gradually died ( Fig. 9 The antibacterial effect of antibacterial substances increased in 12-24h, but the production speed was slow, and the antibacterial effect gradually decreased during the subsequent growth period. Among them, only SK7 reached the highest antibacterial effect at 36h, and the other five strains had the best antibacterial effect at 24h ( Fig. 9 ). Combining the growth time and the antibacterial effect, it was finally determined that the optimal culture time of the screened Bacillus subtilis was 24h, and the production of antibacterial substances was the highest under this culture time.

[0161] 3.1.2 Effect of culture temperature on bacterial growth and antibacterial activity

[0162] Bacillus subtilis can sense changes in external temperature and adjust its own metabolism, changing the synthesis direction of metabolites, thereby affecting the synthesis of metabolites within the bacteria. Therefore, culture temperature is an important factor affecting the growth of strains and the production of antibacterial substances. Fig.10 As shown in the figure: when the culture temperature is in the range of 25-37℃, the growth and antibacterial activity of the six Bacillus subtilis strains all show an upward trend, and at 37-40℃, the growth and antibacterial activity of the strains show a downward trend. Therefore, the optimal culture temperature is 37℃, which ensures the best growth and antibacterial effect of Bacillus subtilis.

[0163] 3.1.3 Effect of pH on bacterial growth and antibacterial activity

[0164] Different pH values ​​will affect the growth of Bacillus subtilis and its ability to produce active substances. Inappropriate acid-base conditions are not conducive to the growth of the strain, which in turn affects its ability to produce secondary metabolites. Fig.11 As shown in the figure: when the pH is 4, only SK0 and SK10 can grow, but they have no antibacterial effect. As the acidity of the culture medium decreases, the growth and antibacterial effect of the six Bacillus subtilis are increasing; when the culture medium is alkaline, as the alkalinity increases, the antibacterial ability and growth and reproduction ability of the six Bacillus subtilis are weakened. When the pH reaches 10, except for the SK7 strain, the other strains have growth ability but no antibacterial activity. Therefore, the optimal pH is 7, under which the growth and reproduction ability of the six strains and the inhibition rate of pathogens are the highest.

[0165] 3.1.4 Effect of liquid volume on bacterial growth and antibacterial activity

[0166] During the strain culture process, the amount of liquid will affect the amount of dissolved oxygen, thus affecting the growth of Bacillus subtilis and the synthesis of its secondary metabolites. The growth of Bacillus subtilis is an aerobic process, so the amount of dissolved oxygen is also an important factor in the growth of the strain. Fig.12 It can be seen that when 50 mL of culture medium is placed in a 250 mL conical flask, the six strains of Bacillus subtilis grow most vigorously and have the strongest antibacterial activity. When the culture medium in the conical flask is less than or greater than 50 mL, the growth and antibacterial effect of the strain will be weakened. Therefore, 50 mL is selected as the optimal liquid volume.

[0167] 3.2 Study on the stability of antibacterial substances of Bacillus subtilis

[0168] 3.2.1 Temperature stability of antibacterial substances

[0169] Temperature has a certain effect on the antibacterial effect of the fermentation supernatant. When treated at 37°C as the control, the antibacterial activity of the supernatants of SK0, SK10, and SK14 strains remained at about 70%, but after being treated at 60°C-95°C, the antibacterial effect of the supernatants decreased to a certain extent, and the relative antibacterial capacity was about 53%; the antibacterial activity of the supernatants of SK2, SK3, and SK7 strains remained at about 60% when treated at 37°C, and after being treated at 60°C-95°C, the antibacterial effect showed a downward trend, and the relative antibacterial capacity was about 50%; after the supernatants of the six strains were treated at 121°C, the antibacterial activity decreased significantly, and except for the SK7 strain, which completely lost its antibacterial activity, the antibacterial activity of the remaining strains remained at about 20% ( Fig.13 ). This indicates that the antibacterial substances in the supernatant have a certain tolerance to temperature (37℃-95℃) and are sensitive to high temperatures.

[0170] 3.2.2 Stability of antibacterial substances to acid and alkali

[0171] like Fig.13 As shown in the figure, the antibacterial rates of the supernatants of the six strains changed significantly after being treated with different pH values. Taking pH 7 as the control treatment, it was found that the antibacterial rates of the six strains decreased after acid-base treatment. The antibacterial changes were more obvious during acid treatment, and the antibacterial activity decreased significantly. There was no antibacterial activity at pH 4, indicating that the antibacterial substances of Bacillus subtilis were sensitive to acid. The antibacterial effect also decreased after alkali treatment. The SK7 strain had no antibacterial activity at pH 10, and the antibacterial activity of the other strains remained at 30%-40%. This shows that the antibacterial substances produced by the six strains are sensitive to strong acid and strong alkali substances.

[0172] 3.2.3 Stability of antibacterial substances to UV

[0173] After testing, it was found that the antibacterial effect of the six strains of Bacillus subtilis showed a non-obvious downward trend after UV irradiation treatment for different periods of time. The antibacterial activity of the SK0, SK10, and SK14 strains remained at around 65%, and the antibacterial activity of the SK2, SK3, and SK7 strains remained at around 55% ( Fig.14 ), indicating that the antibacterial substances of Bacillus subtilis have a certain tolerance to ultraviolet rays.

[0174] 3.2.4 Stability of antibacterial substances to proteases

[0175] The supernatant was treated with pepsin and trypsin, and it was found that the antibacterial ability of the six strains decreased to a certain extent. Fig.14 As shown in the figure: after being treated with pepsin, SK0, SK10, and SK14 strains still had an antibacterial rate of about 60%, and SK2, SK3, and SK7 strains maintained an antibacterial rate of about 50%; after being treated with trypsin, the antibacterial ability of the six strains decreased more significantly, about 55% for SK0, SK10, and SK14 strains and about 45% for SK2, SK3, and SK7 strains. The above results show that the antibacterial substances produced by the six strains have a certain stability against proteases, and the substances that produce antibacterial effects include proteins and non-proteins.

[0176] 3.2.5 Stability of antibacterial substances over storage time

[0177] The effects of different storage times on the stability of antibacterial substances were tested. Fig.15 It can be seen that the antibacterial rate of the supernatant of the six strains did not change much during the storage time of 0-90 days, with only a small decrease, indicating that the antibacterial substances in Bacillus subtilis are relatively stable, but will subsequently degrade with the increase of storage time.

[0178] From the above experiment, it can be known that the optimal culture conditions and the optimal conditions for producing antibacterial substances of 6 strains of Bacillus subtilis, SK0, SK2, SK3, SK7, SK10 and SK14, are as follows: the strain seed liquid is inoculated in LB liquid medium for fermentation, and the conditions are temperature 37°C, liquid volume 50mL, culture time 24h, pH value 7. The antibacterial substances obtained by fermentation are sensitive to high temperature and strong acid, but relatively stable to temperature, ultraviolet irradiation time and protease treatment, and have good storage stability, and are not easily degraded in short-term storage. These strains with good antibacterial activity all show excellent physical and chemical properties.

[0179] 4. Study on immune induction of Bacillus subtilis supernatant

[0180] 1 Experimental Materials

[0181] 1.1 Test strains

[0182] Same as “1.1 Test strains” in “III. Study on the antibacterial activity of Bacillus subtilis fermentation supernatant”.

[0183] 1.2 Preparation of supernatant

[0184] Same as “1.2 Preparation of seed liquid and supernatant” in “III. Study on the antibacterial activity of Bacillus subtilis fermentation supernatant”.

[0185] 1.3 Plant materials

[0186] Same as “1.2 Plant materials” in “II. Study on the antibacterial effect of Bacillus subtilis fermentation supernatant”.

[0187] 2 Experimental methods

[0188] 2.1 Plant cultivation

[0189] 2.1.1 Cultivation of soil materials

[0190] (1) Arabidopsis thaliana cultivation: Prepare nutrient soil: perlite: vermiculite = 3:1:1, mix well, sterilize in a high pressure steam sterilizer at 121℃ for 20min, then evenly plant the vernalized Arabidopsis thaliana seeds on the surface of the sterilized soil, cover with a transparent plastic cover and cultivate in an artificial climate chamber. Remove the plastic cover after the seeds germinate. The environmental conditions of the artificial climate chamber are light intensity of 5,500lx, humidity of 60%, temperature of 22℃, and 12h light / 12h dark conditions for 4 weeks.

[0191] (2) Tobacco cultivation: Prepare as above and sterilize at high temperature. Sow the sterilized tobacco seeds evenly on the soil surface, cover with a transparent plastic cover and place in an artificial climate chamber. Remove the cover after the tobacco seeds germinate (about 5 days), transplant the seedlings to new nutrient soil and place in a plant room. Cultivate under the same conditions as above for 3-8 weeks. Adjust the cultivation time according to different experimental requirements.

[0192] (3) Tomato cultivation: Prepare as above and sterilize at high temperature. Sow the sterilized tomato seeds on the soil surface, four seeds per small pot. Cover with a transparent plastic cover and place in a plant room. Remove the cover after the seeds germinate (about 5 days), transplant the seedlings to new nutrient soil and place in a plant room. Culture conditions are as above and culture for 3-5 weeks.

[0193] 2.1.2 Cultivation of sterile seedlings

[0194] (1) Arabidopsis seedling culture: Arabidopsis seeds were sterilized in a clean bench and sterilized with 75% anhydrous ethanol for 5 min. The seeds and ethanol were thoroughly mixed, and the supernatant was removed by instant centrifugation. The seeds were then cleaned once with anhydrous ethanol, inverted and centrifuged to remove the supernatant, and appropriate amount of sterile water was added to mix thoroughly and wash four times. Finally, an appropriate amount of sterile 0.1% Agar was added to suspend the seeds. After vernalization for 2 days, Arabidopsis seeds were aseptically planted on 1 / 2 MS culture medium, sealed with sealing film, and placed in a light incubator for 14 days under 12 h light / 12 h dark conditions per day.

[0195] (2) Cultivation of sterile tobacco seedlings: Tobacco seeds were sterilized in a clean bench according to the same sterilization steps as above. The sterilized tobacco seeds were spotted on MS solid culture medium and cultured in a light incubator.

[0196] 2.2 Determination of immune-related indices

[0197] 2.2.1 MAPK phosphorylation detection

[0198] 2.2.1.1 Plant total protein extraction

[0199] The specific method is as follows: (1) In a sterile six-well plate experiment, add an appropriate amount of 1 / 2MS liquid culture medium to each well to submerge the plants, pick Arabidopsis seedlings cultured for 14 days from the 1 / 2MS solid culture medium, seal the plate and place it in a light incubator for 24 hours. Use 3μmol / L flg22 as a positive control, use the water treatment group as a blank control, and use the supernatant of SK0, SK2, SK3, SK7, SK10, and SK14 strains to dilute to 10 -2 For the experimental group, after 15 minutes of treatment, Arabidopsis seedlings were picked out with tweezers, wrapped in tin foil, and immediately frozen in liquid nitrogen; (2) the seedlings frozen in liquid nitrogen were ground into powder and added with 200 μL protein extraction buffer, quickly vortexed and mixed, and temporarily placed on ice; (3) 95℃ metal bath for 5 minutes, cooled and centrifuged at 13000r / min for 10 minutes, the supernatant was taken into a new 1.5mL centrifuge tube and stored at -20℃; (4) the total protein concentration of the sample was determined using a micro-spectrophotometer (Nanodrop 2000).

[0200] 2.2.1.2 SDS-PAGE separation and phosphorylated antibody Western-Blot

[0201] The specific method is as follows: (1) prepare a 12% SDS-PAGE protein gel and load the above-extracted protein solution onto the gel; (2) stop electrophoresis when the target protein band reaches the bottom of the separation gel, cut the gel and place it in transfer buffer; (3) cut the PVDF membrane, soak it in methanol for 10 seconds, install the transfer system according to the "sponge-membrane-gel-sponge" model, and perform electrotransfer at 120V and 2.5A for 7 minutes; (4) after the transfer is completed, rinse it with TBST buffer and place the membrane in blocking solution for 1.5 hours; (5) completely immerse the membrane in the primary antibody solution for incubation and shake it in a horizontal shaker at 4°C overnight; (6) wash the membrane with TBST for 3 times, 8 minutes each time, immerse the membrane in the secondary antibody solution, incubate it at room temperature for 1 hour, and wash the membrane with TBST buffer for 4 times; (7) use absorbent paper to absorb the residual buffer on the membrane, add ultra-sensitive ECL chemiluminescent developer, develop it on an imager and take pictures.

[0202] 2.2.2H 2 O 2 accumulation

[0203] DAB staining was used to detect H 2 O 2 The accumulation method is as follows: (1) Select 4-week-old Arabidopsis, tobacco, and tomato seedlings with the same growth trend, dilute the supernatant of SK0, SK2, SK3, SK7, SK10, and SK14 strains to 10 -2 3 h after the experimental spraying, the leaves were cut and placed in a culture dish with 3 μmol / L flg22 and water as controls; (2) 2 mg / mL DAB dye solution was added to the culture dish to completely submerge the plant leaves; (3) the dye treatment was carried out overnight in the dark; (4) the dye solution was aspirated, and a decolorizing solution (acetic acid: ethanol = 1:3) was added to decolorize the leaves in a 95°C water bath; (5) the leaves were observed for H 2 O 2 Build up the situation and take photos.

[0204] 2.2.3 Accumulation of callose

[0205] The specific method is as follows: (1) Select 3-4 week old plants in good growth condition, select 4 leaves of similar size from each plant, and inject H 2 O and 300nmol / L flg22 as controls, the strain supernatant was diluted to 10 -2 , and then injected. Treatment for 16 hours; (2) Cut the treated leaves and place them in a fixative (ethanol: acetic acid = 3:1) for 6 hours; (3) Wash the fixed leaves three times with sterile water, then add 50% ethanol and dry them in a dry oven at 65°C, repeating twice; (4) Wash the ethanol and stain the leaves with aniline blue dye, and stain them in the dark for 4 hours; (5) Observe the deposition of callose using a fluorescence microscope and take pictures.

[0206] 2.2.4 Bacillus subtilis induces antiviral effects in tobacco

[0207] 2.2.4.1 Preparation of tobacco viruses

[0208] Tobacco mosaic virus TMV was passaged and stored on common tobacco NC89. Weigh 1 g of fresh virus-infected leaves, add 20 mL of PBS buffer to fully grind and extract the virus, filter the residue with gauze, and the filtrate is set aside.

[0209] 2.2.4.2 Induced resistance studies

[0210] Using the leaf inoculation method, select 5-7 weeks old tobacco plants that are well grown and uniform in size. Dilute the six strains into 10 -2 The supernatant was sprayed on the whole leaves of tobacco, with water as the control. After 24 hours of treatment, the leaves were inoculated with TMV virus solution containing a small amount of quartz sand by friction and cultured in a plant room. This was repeated 3 times, and the number of dead spots was recorded 3 days after inoculation.

[0211] 3 Results and analysis

[0212] 3.1 Effects of supernatant on plant MAPK phosphorylation

[0213] H 2 O was used as a blank control, flg22 as a positive control, and the supernatants of the six strains were used as experimental groups. After 15 minutes of treatment, the Arabidopsis plants were collected and total protein was extracted to detect the effects of each treatment on MAPK phosphorylation. Fig.16 As shown in the figure, the SK0, SK3, and SK10 treatment groups had obvious phosphorylation activation compared with the water treatment group, but the intensity was lower than that of the flg22 treatment, indicating that SK0, SK3, and SK10 activated immunity dependent on the MAPK pathway. However, the detection found that SK2, SK7, and SK14 failed to activate MAPK phosphorylation, indicating that these strains may not have immune-inducing effects or exert immune-inducing effects through pathways that are not dependent on the MAPK cascade reaction.

[0214] 3.2 Effect of supernatant on plant H 2 O 2 Accumulation detection

[0215] DAB staining was used to detect H 2 O 2 The results showed that compared with the negative control water treatment, the tobacco leaves treated with the supernatant of strains SK0, SK3, and SK10 and the positive control flg22 had more obvious brown spots, SK7 and SK14 had a small amount of brown spots, and SK2 was not completely decolorized and no obvious brown spots were found ( Fig.17A). In Arabidopsis, the SK0 and SK14 strains had obvious brown spots, while the other treatment groups had weaker spots ( Fig.17 B). In tomato leaves, except for the SK2 strain treatment, there was no brown deposit, while the other treatment groups had obvious deposits ( Fig.17 C). This indicates that Bacillus subtilis SK0, SK3, SK7, SK10, and SK14 can induce H 2 O 2 accumulation.

[0216] 3.3 Effect of supernatant on plant callose accumulation

[0217] Four-week-old Arabidopsis leaves were treated with 300 nmol / L flg22 and water for 16 hours, and then stained with aniline blue. The accumulation of callose in the leaves was observed under a fluorescence microscope. The results showed that after the supernatant of SK0, SK3, SK7, SK10, and SK14 strains was treated, the fluorescent spots were clearly distributed after the leaves were stained, while no fluorescence was observed in the SK2 and water treatment samples ( Fig.18 ).

[0218] 3.4 Study on the supernatant's resistance to tobacco viruses

[0219] According to the results of the active oxygen and callose experiments, SK0, SK3, SK7, SK10, and SK14 strains were selected for virus resistance experiments. The results showed that the supernatants of the selected strains could enhance the resistance of tobacco plant leaves to TMV ( Fig.19 ), compared with the control group, the leaf spots caused by TMV were significantly inhibited after the tobacco was treated with the supernatant. It can be clearly seen from the figure that the virus spots in the water treatment group almost covered the second round of leaves, causing them to wilt, while the leaves in the strain treatment group can still be clearly seen to be upright ( Fig.19 A). Among them, after the SK0 strain was treated, the tobacco produced the least lesions, and its inhibition rate could reach 95%. The inhibition rates of the remaining strains after treatment could also reach 90% ( Fig.19 C).

[0220] The above experiments show that the 6 strains of SK0, SK2, SK3, SK7, SK10 and SK14, except for SK2, can significantly increase the H 2 O 2 The content of SK0 can stimulate the plant to produce disease resistance and induce the accumulation of callose on the leaf surface to prevent the invasion of pathogens. It has a significant effect in resisting the spread of tobacco mosaic virus TMV in thaliana, tomato and tobacco, among which the SK0 strain has the best effect.

[0221] 5. Study on the growth-promoting effect of Bacillus subtilis supernatant

[0222] 1 Experimental Materials

[0223] 1.1 Test strains

[0224] The SK0, SK3, SK7, SK10, and SK14 strains with antibacterial and immune-inducing abilities screened out in “IV. Study on immune-inducing effects of Bacillus subtilis supernatant” were selected for growth promotion studies.

[0225] 1.2 Preparation of supernatant

[0226] Same as “1.2 Preparation of seed solution and supernatant” in “III. Study on the antibacterial activity of Bacillus subtilis fermentation supernatant”.

[0227] 2 Experimental methods

[0228] 2.1 Detection of nitrogen fixation performance of supernatant

[0229] The nitrogen fixation activity of each strain was determined based on its growth in nitrogen-free medium. 10 μL of the fermentation liquid obtained by activating each strain was pipetted onto the nitrogen-free medium and placed in a 37°C incubator. After culturing for 5 days, the size of the transparent circle was observed and measured. Each treatment was repeated 3 times.

[0230] 2.2 Phosphorus and potassium dissolving performance test of supernatant

[0231] Absorb different Bacillus subtilis fermentation broths into potassium-solubilizing and phosphate-dissolving medium, place them in a 37°C incubator for culture, and observe whether there is a white transparent circle at the edge of the inoculated colony. The presence of a transparent circle means that the bacteria has the ability to dissolve phosphate. Observe and measure the size of the transparent circle.

[0232] 2.3 Detection of IAA production capacity of supernatant

[0233] Preparation of IAA standard curve: Prepare 10mg / L, 20mg / L, 30mg / L, 40mg / L and 50mg / L IAA standard solutions with Salkowski colorimetric reagent in a 1:1 ratio, and place at room temperature in the dark for 30 minutes. 530 The absorbance was measured using an ultraviolet spectrophotometer.

[0234] IAA content determination: The selected strains were inoculated into LB liquid medium for activation. Centrifuged at 4000r / min for 10min, 0.5mL of supernatant was added with an equal amount of Salkowski colorimetric reagent, and the OD 530 The absorbance of the strain was measured and the IAA production of the strain was obtained according to its standard curve.

[0235] 2.4 Experiment on plant growth of supernatant

[0236] After the tobacco seeds were immersed in the fermentation liquid of the strain for 24 hours, they were sown in a flower pot filled with sterilized soil. The tobacco with 4-5 leaves of consistent growth was selected and transplanted into a 10cm×10cm flower pot and cultured at 25℃. After the plant growth stabilized, the roots were irrigated with fermentation liquid every 6 days. After 20 days, the growth of the seedlings was observed and recorded, and the indicators such as the maximum leaf length, maximum leaf width, plant height, aboveground dry weight and underground dry weight of the tobacco were recorded.

[0237] 3 Results and Analysis

[0238] 3.1 Detection of nitrogen fixation performance of supernatant

[0239] The nitrogen fixation test was carried out with strains SK0, SK3, SK7, SK10 and SK14. The results showed that after 5 days of cultivation, strains SK3 and SK14 produced obvious transparent circles in the culture medium, indicating that these two strains have nitrogen fixation. The transparent circle of strain SK0 was smaller and less transparent ( Fig. 20 ), while the rest of the Bacillus subtilis had no nitrogen fixation ability. Among them, the diameter of the transparent zone of the SK14 strain was measured to be 1.3 cm (Table 6), indicating that Bacillus subtilis can grow on nitrogen-free medium because it obtains nitrogen from the air through biological nitrogen fixation.

[0240] Table 6 Test of nitrogen fixation ability of strains

[0241]

[0242] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate significant differences at the P﹤0.05 level by Student's test; "-" indicates that no transparent circle is produced.

[0243] 3.2 Phosphorus and potassium dissolving performance test of supernatant

[0244] In addition to nitrogen fixation, Bacillus subtilis can also promote plant growth by solubilizing phosphorus and potassium to enhance the absorption of these essential elements by plants. Fig.21 As shown in Table 7, among the tested strains, only SK0 and SK3 strains have the ability to dissolve phosphate and potassium, with the maximum transparent zone diameters reaching 1.18 cm and 1.25 cm, respectively.

[0245] Table 7 Phosphate-dissolving and potassium-dissolving ability test of strains

[0246]

[0247] Note: The data in the table are the mean ± standard error of the diameter of the transparent circle. The lowercase letters after the data indicate significant differences at the P﹤0.05 level by Student's test; "-" means no transparent circle is produced.

[0248] 3.3 Detection of IAA production capacity of supernatant

[0249] In addition to enhancing the absorption of nutrients by plants, Bacillus subtilis can also promote plant growth by producing IAA. After testing, SK0, SK3, and SK14 strains have the ability to produce IAA. According to their standard curves, we can know that different strains have different abilities to produce IAA, but the yield is about 20μg / mL ( Fig. 22 ).

[0250] 3.4 Effect of supernatant on plant growth promotion

[0251] The experimental results show that Bacillus subtilis can promote plant growth. Fig.23 As shown in the figure, compared with the CK treatment, the plant stem height, leaf length, fresh weight, dry weight and other indicators counted after 20 days of cultivation were significantly improved after being treated with SK0, SK3, SK7, SK10 and SK14 strains, indicating that these five strains have the ability to promote tobacco growth.

[0252] The above experiments show that the five strains SK0, SK3, SK7, SK10 and SK14 can not only inhibit pathogens and resist viruses, but also improve the growth performance of plants.

[0253] VI. Study on the Effect of Pesticides and Fungicides on the Growth of Bacillus subtilis

[0254] 1 Experimental Materials

[0255] 1.1 Test strains

[0256] The SK0 strain was selected to explore the effects of chemical pesticides on the growth state of Bacillus subtilis.

[0257] 1.2 Seed solution preparation

[0258] Bacillus subtilis SK0 was inoculated into LB solid medium and cultured at 37°C for 12 h. A single cultured colony was picked and added to 50 mL of LB liquid medium and cultured at 37°C with a shaker at 200 r / min for 24 h as the seed liquid of Bacillus subtilis SK0.

[0259] 1.3 Pesticides and fungicides

[0260] Mancozeb (active ingredient content: 80%, wettable powder, Shandong Jiaqi Biotechnology Co., Ltd.), pyraclostrobin (active ingredient content: 25%, suspension, Henan Hans Crop Protection Co., Ltd.), difenoconazole (active ingredient content: 60%, water dispersible granules, Qingdao Audis Biotechnology Co., Ltd.), copper hydroxide (active ingredient content: 46%, water dispersible granules, Shanghai Yuelian Biotechnology Co., Ltd.), quinoline copper (active ingredient content: 33.5%, suspension, Shandong United Linong Biotechnology Co., Ltd.) Technology Co., Ltd.), allicin (limited ingredient content: 5%, microemulsion, Chengdu Xinchaoyang Crop Science Co., Ltd.), Chunlei·Copper Oxide (Kasugamycin content 2%, Copper Oxide content 45%, wettable powder, Shanghai Hulian Biopharmaceutical (Xiayi) Co., Ltd.), Iprodione (active ingredient content: 500g / L, suspension concentrate, Suzhou FMC Plant Protection Agents Co., Ltd.), Fluopyram·Trifloxystrobin (fluopyram 21.5%, trifloxystrobin 21.5%, suspension concentrate, Bayer CropScience (China) Co., Ltd.).

[0261] 2 Experimental methods

[0262] Use sterile water to dilute various pesticides and fungicides to the concentration recommended in the instructions, and mix the prepared seed solution with the diluted pesticides and fungicides at a ratio of 1:1000, 1:500, 1:100, 1:50, and 1:10 respectively as the treatment group. The control group is a mixture of seed solution and sterile water, which is placed at room temperature for 1 hour, and then 10 μL is pipetted from it with a pipette and spotted on LB culture medium, which is placed in a 37°C incubator for 24 hours to observe the colony status.

[0263] 3 Results and analysis

[0264] Depend on Fig.24 It can be seen that the colony morphology and size of the groups treated with iprodione, fluopicolide·trifloxystrobin, difenoconazole, pyraclostrobin, pyraclostrobin·copper oxalate and copper hydroxide were almost the same as those of the control group, indicating that these pesticides and fungicides had no effect on the growth of Bacillus subtilis SK0; the colonies of the groups treated with mancozeb and quinoline copper had almost no growth compared with the control group, indicating that these two pesticides and fungicides had a serious inhibitory effect on the growth of Bacillus subtilis SK0.

[0265] The above experiments show that Bacillus subtilis SK0 cannot be used in combination with pesticide fungicides whose main ingredients are mancozeb and quinoline copper, but has the potential to be used in combination with pesticide fungicides whose main ingredients are iprodione, fluopicolide·trifloxystrobin, difenoconazole, pyraclostrobin, pyraclostrobin·copper oxadiazine and copper hydroxide.

[0266] In summary, the subtilis SKO obtained by the present invention screening has an inhibitory effect on various plant pathogens (Agrobacterium tumefaciens, Pseudomonas syringae, Alternaria longifolia, gray mold, rice blast fungus, sclerotinia, Fusarium graminearum, icterus fusarium, soybean phytophthora and anthrax), wherein the inhibition rate of tomato gray mold can reach 61.5%, the inhibition rate of tobacco brown spot caused by Alternaria longifolia can reach 72%, and it also has the effect of resisting viruses, and the inhibition rate of tobacco TMV virus lesions can reach 95%, and it can also enhance the absorption of nutrients by plants, play nitrogen fixation, dissolve phosphorus and potassium, produce IAA, to promote the growth of plants, its fermented liquid can be used as a pesticide for preventing and treating plant diseases, and can be used as a regulator for promoting plant growth. And subtilis SKO can also be compounded with pesticide bactericides with iprodione, flutoxin·trifloxystrobin, difenoconazole, pyraclostrobin, spring thunder·copper and copper hydroxide as main components, to enhance plant resistance to diseases. The advantages of Bacillus subtilis SK0 in plant protection make it valuable for market promotion and application. It has now been named Bacillus subtilis LXN-1 for the preservation of patent procedures so that it can be obtained by the public and applied in actual plant production.

Claims

1. A Bacillus subtilis, characterized in that: It is Bacillus subtilis LXN-1 which is deposited in China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC.NO 31697.

2. Use of the Bacillus subtilis LXN-1 according to claim 1 in the preparation of pesticides for preventing and controlling plant diseases and / or plant growth regulators for promoting plant growth.

3. The use according to claim 2, characterized in that: The pesticide has the function of inhibiting plant pathogens; the pathogens include Agrobacterium tumefaciens, Pseudomonas syringae, Alternaria longibotia, Botrytis cinerea, Rice blast fungus, Sclerotinia sclerotiorum, Fusarium graminearum, Pythium aphanidermatum, Phytophthora sojae and / or Anthracnose.

4. The use according to claim 2, characterized in that: The pesticide has the function of resisting plant viruses; the plant viruses include tobacco mosaic virus TMV.

5. The use according to claim 2, characterized in that: The plant growth regulator has the functions of fixing nitrogen, dissolving phosphorus and potassium and / or producing IAA.

6. The use according to claim 2, wherein the plants include Arabidopsis thaliana, tomato and tobacco.

7. A pesticide or plant growth regulator comprising the Bacillus subtilis LXN-1 according to claim 1 and / or its fermentation liquid.

8. A method for preparing the pesticide or plant growth regulator according to claim 7, characterized in that: The steps include: The Bacillus subtilis LXN-1 of claim 1 is inoculated into a Bacillus subtilis liquid culture medium for culture, the culture solution is centrifuged, and the supernatant and / or bacteria are collected to obtain the product.

9. The method according to claim 8, characterized in that: The liquid culture medium is LB liquid culture medium. The culture conditions include: temperature 32-37°C, rotation speed 100-300 r / min, time 12-36h, pH value of LB liquid culture medium 7, and LB liquid culture medium volume of 25-50 ml per 250 ml container.

10. Use of the Bacillus subtilis LXN-1 and / or its fermentation liquid in combination with a chemical fungicide in the preparation of a pesticide for preventing and controlling plant diseases according to claim 1, characterized in that: The chemical fungicide includes any one of iprodione, fluopicolide·trifloxystrobin, difenoconazole, pyraclostrobin, pyraclostrobin, pyraclostrobin·copper oxalate, and copper hydroxide.

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