Lactic acid paenibacillus X2T3 and application thereof

Abstract

The invention relates to a lactic acid paenibacillus X2T3, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20242885 (short for CCTCC; the address is China Center for Type Culture Collection in Wuhan University, Wuhan City, Hubei Province), and the preservation date is December 23, 2024. Compared with the prior art, the lactic acid paenibacillus X2T3 provided by the invention can be used for effectively antagonizing ralstonia solanacearum. When being used for planting plants represented by hot peppers, the compound fertilizer can obviously reduce the morbidity of bacterial wilt and also can obviously promote the growth of the plants. The paenibacillus lactis X2T3 disclosed by the invention has a wide application prospect in the aspects of preventing and treating plant bacterial diseases and preparing an antibacterial growth promoting agent capable of preventing diseases.

Description

Technical Field

[0001] The present invention relates to the fields of plant disease control and microbial technology, and in particular to the application of Paenibacillus lactis X2T3. Background Art

[0002] Bacterial wilt is a bacterial disease caused by Ralstonia solanacearum (also known as Burkholderia solanacearum, Pseudomonas solanacearum, Ralstonia solanacearum), which seriously harms a variety of crops. Ralstonia solanacearum is mainly transmitted through rainwater, irrigation water, farming operations, etc., and can also be transmitted through insects such as the Colorado potato beetle. The pathogen invades through wounds or natural orifices at the roots of plants, multiplies and spreads in the vascular bundles, resulting in plant diseases.

[0003] Bacterial wilt appears during the growth of crops such as peppers, eggplants, tomatoes, peppers, potatoes, and peanuts. After a crop is infected with bacterial wilt, the stems of the diseased plants become hollow. When cutting open the diseased plants, the fiber tube tissues inside will turn brown. Squeezing the base of the horizontally cut diseased plants forcefully, white liquid will flow out from the cut. The young tissues at the top of the upper part of the plant (such as young leaves, tender shoots, and newly emerging leaves) wilt. Before the leaves turn yellow and wither, the plants will quickly dehydrate, turn green and wither, and wilt. Under dry and high-temperature conditions, the plants will wither and die within a few days, having a huge negative impact on the quality and yield of agricultural crops.

[0004] At present, spraying pesticides of the chemical fungicide type is the most commonly used and direct means for controlling bacterial wilt in agricultural crops. However, the extensive use of pesticides will cause the pesticides to remain on the surface of agricultural crops, or penetrate into the soil, water, and air, which may lead to problems related to food safety and environmental safety. Moreover, the long-term use of a single fungicide can cause Ralstonia solanacearum to develop resistance, and the control effect of the fungicide decreases. In order to achieve the control purpose, farmers increase the dosage and frequency of pesticide application, ultimately forming a vicious cycle and further exacerbating the problems of food safety and environmental safety. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a Paenibacillus lactis X2T3, which can effectively control bacterial wilt in peppers, and in particular has a good antagonistic effect against the pathogenic bacterium Ralstonia solanacearum, achieving the purpose of preventing and controlling the occurrence of diseases.

[0006] The specific technical solution is as follows:

[0007] A Bacillus parafirmus X2T3, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242885 (abbreviation: CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province, China), and the deposit date is December 23, 2024.

[0008] Compared with the prior art, the present invention provides a Bacillus parafirmus X2T3, which can effectively antagonize Ralstonia solanacearum. When used in the planting of plants represented by peppers, it can significantly reduce the incidence of bacterial wilt, and at the same time can significantly promote plant growth. The Bacillus parafirmus X2T3 of the present invention has broad application prospects in the prevention and control of plant bacterial diseases and in the preparation of antibacterial and growth-promoting agents that can prevent diseases.

[0009] In some embodiments, the Bacillus parafirmus X2T3 is applied to promote plant growth.

[0010] In some embodiments, the Bacillus parafirmus X2T3 is applied to inhibit the growth of Ralstonia solanacearum.

[0011] In some embodiments, the Bacillus parafirmus X2T3 is applied to prevent and control plant diseases.

[0012] Further, the plant disease is a plant disease caused by Ralstonia solanacearum.

[0013] Further, the plant diseases include one or more of bacterial wilt of pepper, bacterial wilt of eggplant, bacterial wilt of tomato, bacterial wilt of pepper, bacterial wilt of potato, and bacterial wilt of peanut.

[0014] The present invention also provides an antibacterial and growth-promoting agent, which includes Bacillus parafirmus X2T3, and the Bacillus parafirmus X2T3 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20242885.

[0015] Further, the concentration of the Bacillus parafirmus X2T3 is 1.0×10 4 cfu / ml to 1.0×10 10 cfu / ml.

[0016] Further, the concentration of the Bacillus parafirmus X2T3 is 5.0×10 4 cfu / ml to 1.0×10 6 cfu / ml.

[0017] The present invention also provides a method for preventing and controlling bacterial wilt of plants: the antibacterial and growth-promoting agent is used to irrigate the roots of plants, the irrigation amount is 5-50 ml per plant, and it is irrigated once every 3-8 days for at least 3 consecutive times.

[0018] The present invention also provides a method for promoting plant growth: the antibacterial and growth-promoting agent is used to irrigate the roots of plants, the irrigation amount is 5-50 ml per plant, and it is irrigated once every 3-8 days for at least 3 consecutive times. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a colony morphology diagram of Bacillus laevolacticus X2T3 described in the present invention after being cultured on an LB solid medium for 48 h;

[0020] Figure 2 It is a PCR amplification nucleic acid electrophoresis diagram of the 16S rDNA sequence of Bacillus laevolacticus X2T3 described in the present invention; the left band is the nucleic acid standard band, which are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom in sequence; the right is the band of the PCR amplification product;

[0021] Figure 3 It is a phylogenetic tree constructed using the 16S rDNA sequence of Bacillus laevolacticus X2T3 described in the present invention.

[0022] Figure 4 It is a result diagram of the antibacterial effect of the fermentation supernatant of Bacillus laevolacticus X2T3 described in the present invention against Ralstonia solanacearum; among them, the left figure is the result of the antibacterial experimental group, and 20 μl of the fermentation supernatant of Ralstonia solanacearum is spotted in each well; the right figure is the result of the antibacterial control group, and 20 μl of sterile water is spotted in each well;

[0023] Figure 5 It is an effect diagram of the prevention and control of pepper bacterial wilt by Bacillus laevolacticus X2T3 described in the present invention; among them, the left figure is the incidence results of the prevention and control blank control group, prevention and control negative control group, and prevention and control experimental group in sequence; the right figure is the disease index results of the prevention and control blank control group, prevention and control negative control group, and prevention and control experimental group in sequence;

[0024] Figure 6 It is an effect diagram of promoting the growth of peppers by Bacillus laevolacticus X2T3 described in the present invention; among them, the left plant is the growth promotion control group, and the right plant is the growth promotion experimental group. DETAILED DESCRIPTION OF THE INVENTION

[0025] In order to avoid a series of safety problems caused by pesticides, the present invention controls bacterial wilt of crops through the method of biological antagonism, thereby reducing or even avoiding the use and introduction of pesticides and enriching the means of preventing bacterial wilt. Biological antagonism refers to the struggle of mutual inhibition, mutual exclusion, and even mutual killing among microorganisms. In the soil environment with extremely complex and diverse microorganisms, various microorganisms compete for limited nutrient resources. In order to obtain sufficient resources to maintain their own survival and reproduction, some microorganisms have evolved the ability to inhibit the growth of other microorganisms. For example, certain bacteria in the soil can secrete antibacterial substances with antibacterial activity such as antibiotics and bacteriocins to inhibit or kill other bacteria competing for resources around them, which is the antagonistic effect among microorganisms. The antibacterial substances secreted by these bacteria may have the ability to inhibit the growth of plant pathogens including Ralstonia solanacearum. At the same time, a bacterium that can inhibit the growth of plant pathogens and originally exists in the soil may be more likely to colonize in the farmland soil environment, continuously play an inhibitory role on plant pathogens, and even co-evolve with them to maintain the balance of the microecology, having great potential for preventing bacterial diseases. Therefore, the present invention selects to isolate antagonistic strains of Ralstonia solanacearum in the soil.

[0026] Furthermore, in order to avoid the introduction of new diseases by the bacteria that inhibit the growth of Ralstonia solanacearum, the present invention selects to isolate strains from healthy and disease-free farmland soil and selects non-pathogenic strains among them. Finally, the present invention selects a strain of Paenibacillus lacticus, which has good antibacterial ability against Ralstonia solanacearum, and this Paenibacillus lacticus is a common soil bacterium without pathogenicity, so it can be used as an antibacterial agent against Ralstonia solanacearum. In addition, when the present invention attempts to use Paenibacillus lacticus X2T3 on crops, it is also found that it can promote the growth of crops, so this strain can also be used as a plant growth promoter or a microbial organic fertilizer.

[0027] The following combines the accompanying drawings and specific embodiments to further detail the Paenibacillus lacticus X2T3 of the present invention and its application method for preventing bacterial wilt of plants by introducing the isolation and purification method of Paenibacillus lacticus X2T3, the liquid culture method, the antibacterial effect of Paenibacillus lacticus X2T3 on Ralstonia solanacearum in an agar plate, the prevention and control effect of Paenibacillus lacticus X2T3 on bacterial wilt in pepper plants, and the growth promotion effect of Paenibacillus lacticus X2T3 on pepper plants.

[0028] Isolation and purification method of Paenibacillus lacticus X2T3

[0029] The isolation and purification method of the Paenibacillus lacticus X2T3 strain described in the present invention is as follows:

[0030] S101 Soil sampling and treatment: Weigh 10 g of soil samples, where the soil samples are from the soil of pineapple plantations in Xuwen County, Zhanjiang City, Guangdong Province; then, in a laminar flow hood, add 90 ml of sterile water to the soil samples, place them on an oscillator and shake for 60 min to evenly disperse the soil samples in the diluent to form a soil suspension; after the soil is dispersed, aspirate 100 μl of the soil suspension into 900 μl of sterile water to obtain a 10-fold dilution, and then sequentially dilute 10-fold to obtain 10 2 -fold dilution, 10 3 -fold dilution, 10 4 -fold dilution, 10 5 -fold dilution, 10 6 -fold dilution, and the whole process is carried out in a laminar flow hood.

[0031] S102 Strain isolation: Take 100 μl of the 10-fold dilution, 10 2 -fold dilution, 10 3 -fold dilution, 10 4 -fold dilution, 10 5 -fold dilution, 10 6 -fold dilution and spread them on LB solid medium respectively, then place the culture plates in an incubator at 37 °C for 1 - 2 days of constant temperature culture until single colonies grow on the solid medium.

[0032] S103 Strain purification: After the culture is completed, according to the colony growth situation, pick one by one the single colonies with different shapes, colors, sizes, etc. from the LB solid medium with single colonies, then carry out streak plating on clean LB solid medium respectively, and then place them in an incubator at 37 °C for 2 - 3 days of constant temperature culture until single colonies grow on the culture plates. The grown single colonies are the isolated Bacillus sporolactis X2T3 strain.

[0033] S104 Strain preservation: Pick the purified Bacillus sporolactis X2T3 strain from the LB agar solid culture plate and inoculate it into a 500 ml conical flask containing 200 ml of LB liquid medium, and culture it for 48 h under the condition of constant temperature shaking culture at 37 °C and 200 rpm to obtain a seed solution. The seed solution and glycerol are filled into tubes and mixed according to a volume ratio of 1:4 to obtain a glycerol seed solution of Bacillus sporolactis X2T3 with a concentration of 1.0×10 7 - 1.0×10 8 cells / ml. After making marks on the tube walls, store them at -20 °C or store them for a long time at -80 °C to complete strain preservation.

[0034] The morphological diagram of the isolated Bacillus sporolactis X2T3 in this example after 48 h of culture on the LB agar solid culture plate is as shown in Figure 1 The colonies are grayish-white circles, with a soft texture and a smooth surface.

[0035] Among them, the preparation method of LB solid medium is as follows: Weigh 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, and 3.75 g of agar powder, stir and dissolve in distilled water, adjust the pH to 7.2 - 7.4 and make up the volume to 1000 ml, dispense into conical flasks, and perform autoclaving at 121 °C and 101 KPa for 20 min. Pour the sterilized medium into petri dishes in a laminar flow bench before it solidifies. About 20 ml of medium is poured into each petri dish. After the medium cools and solidifies, LB solid medium is obtained.

[0036] The preparation method of LB liquid medium is as follows: Weigh 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, stir and dissolve in distilled water, adjust the pH to 7.2 - 7.4 and make up the volume to 1000 ml, dispense into conical flasks, and perform autoclaving at 121 °C and 101 KPa for 20 min.

[0037] Liquid culture method of Bacillus sporothermodurans X2T3

[0038] Activation of strain S201: Inoculate the glycerol seed liquid of Bacillus sporothermodurans X2T3 stored at -20 °C in step S104 into LB liquid medium at a ratio of 1:100 (volume ratio), and perform shaking culture at a constant temperature of 37 °C and a shaking speed of 200 rpm for 48 h to obtain activated bacterial liquid.

[0039] Liquid culture of S202: Inoculate the activated bacterial liquid obtained in step S201 into 500 ml of LB liquid medium at a ratio of 1:100 (volume ratio), and then perform constant temperature shaking culture at a constant temperature of 37 °C and a shaking speed of 180 rpm - 220 rpm for 24 h - 48 h to obtain fermentation broth. In this example, the shaking speed is preferably 200 rpm and the culture time is preferably 48 h. Identification of Bacillus sporothermodurans X2T3

[0040] S301 Extract bacterial genomic DNA:

[0041] Extract genomic DNA using the Omega Bacterial DNA Kit (D3350-01). First, take 2 ml of the glycerol seed solution described in step S104 and place it in a sterile 2 ml centrifuge tube. Centrifuge at 12,000 rpm for 2 min, discard the supernatant and retain the pellet. Then, add 100 μl of 1×TE Buffer to the pellet, vortex to mix evenly, add 10 μl of lysozyme and mix well, incubate at 37 °C for 10 min. Add 100 μl of BTL Buffer and 20 μl of proteinase K, mix well, incubate at 55 °C for 1 h, and mix by oscillation three times in the middle. Add 5 μl of RNase A enzyme, mix well, let it stand at room temperature for 5 min, then centrifuge at 10,000 rpm for 2 min. Transfer 200 μl of the supernatant to a new sterile 1.5 ml centrifuge tube. Add 200 μl of BTL Buffer, mix well, and incubate at 65 °C for 10 min. Add 200 μl of absolute ethanol, vortex to mix evenly, transfer all the samples to the adsorption column, centrifuge at 10,000 rpm for 2 min, discard the supernatant and the adsorption column, and place the adsorption column in a new collection tube. Add 500 μl of HBC Buffer to the adsorption column, centrifuge at 10,000 rpm for 2 min, discard the supernatant. Add 700 μl of DNA Wash Buffer to the adsorption column, centrifuge at 10,000 rpm for 2 min, discard the supernatant, and repeat twice. Place the empty adsorption column back into the collection tube and centrifuge at 10,000 rpm for 2 min. Add 30 μl - 50 μl of Elution Buffer (preheated at 65 °C) to dissolve the DNA pellet in the adsorption column to obtain bacterial genomic DNA, and store it at -20 °C for standby.

[0042] S302 Amplify the 16S rDNA sequence by polymerase chain reaction (PCR):

[0043] Using the genomic DNA obtained in step S301 as a template and Eubac27F and Eubac1492R as primers for PCR amplification, the PCR reaction system (50 μl) includes: 0.25 μl of TaKaRa LA Taq (5 U / μl), 2.5 μl of 10×LA Taq Buffer II (Mg 2+Plus), 4 μl dNTPs Mixture (2.5 mM Each), 1 μl genomic DNA, 0.5 μl upstream primer Eubac27F (10 μM), 0.5 μl downstream primer Eubac1492R (10 μM), 16.25 μl ddH2O. Among them, the sequence of the upstream primer Eubac27F is: 5’-agagtttgat cctggctcag-3’ (SEQ ID NO: 1); the sequence of the downstream primer Eubac1492R is: 5’-ggttaccttg ttacgactt-3’ (SEQ ID NO: 2).

[0044] The PCR reaction program is as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and the processes of denaturation, annealing, and extension are repeated 30 times; extension at 72 °C for another 10 min, and the PCR amplification product is stored at 4 °C.

[0045] Perform nucleic acid electrophoresis on the S303 PCR product

[0046] Take 5 μl of the PCR product obtained in step S302 and load it on a 1.2% agarose gel, and perform nucleic acid electrophoresis at a voltage of 120 V for 25 min. The electrophoresis results are as Figure 2 shown. The length of the 16S rDNA sequence amplified using the genomic DNA of Bacillus sporolactis X2T3 obtained in the present invention as a template is approximately 1500 bp.

[0047] Sequence the 16S rRNA sequence

[0048] Take 30 μl of the PCR product prepared in step S302 and send it to Guangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequence determination. The sequencing results show that the length of the 16S rDNA sequence of the Bacillus sporolactis strain described in the present invention is 1450 bp, and the specific sequence is shown in SEQ ID NO: 3.

[0049] The 16S rDNA sequence (SEQ ID NO: 3) of the Paenibacillus lactic strain obtained by sequencing was input into NCBI for BLAST alignment. It was found that its sequence similarity with Paenibacillus lactis strain LG2 (Accession No KF607091.1) was 97.33%, and it was highly similar but not completely identical to most sequences in the 16S rDNA barcode database of Paenibacillus lactic. To further confirm the bacterial species, sequences with relatively high similarity to the SEQ ID NO: 3 sequence and the 16S rDNA sequences of some other common strains were downloaded. The software MEGA11 was used to construct a phylogenetic tree based on SEQ ID NO: 3 and the downloaded 16S rDNA segment sequences by the Neighbor-Joining Method (NJ method). The results are as Figure 3 shown. According to the phylogenetic tree, the strain obtained in the present invention clusters with other Paenibacillus lactis on the same branch. Based on the sequence similarity of 16S rDNA and the results of the phylogenetic tree, it is determined that this strain belongs to the Paenibacillus lactis species. Therefore, the strain in the present invention is named Paenibacillus lactis X2T3.

[0050] Antibacterial effect of the fermentation supernatant of Paenibacillus lactis X2T3 against Ralstonia solanacearum

[0051] S401 Obtaining the fermentation supernatant: The fermentation broth obtained in step S202 was centrifuged at 4°C and 8000 rpm for 10 min to remove the precipitate, and the supernatant was collected to obtain the fermentation supernatant of Paenibacillus lactis X2T3.

[0052] S402 Preparation of the Ralstonia solanacearum bacterial solution: Dip the glycerol seed solution of Ralstonia solanacearum and streak it on a solid medium plate, and place it in a biochemical incubator at 30°C for constant temperature culture for 2 - 3 days. After single colonies grow, pick a single colony and inoculate it into LB + C liquid medium, and perform constant temperature shaking culture at 30°C and a shaking speed of 200 rpm for 48 h to obtain the Ralstonia solanacearum bacterial solution.

[0053] Among them, Ralstonia solanacearum was purchased from the Guangdong Provincial Microbial Culture Collection Center, GDMCC No: 1.1561.

[0054] Verification of the antibacterial effect: The antibacterial effect of the fermentation supernatant on *Ralstonia solanacearum* was detected by the method of punching holes on a plate. Take 250 ml of prepared LB+C solid medium, heat and melt it, then cool it to about 40 °C. Add 10 ml of the *Ralstonia solanacearum* bacterial liquid obtained in step S402, shake well and pour it into an empty plate, and wait for it to cool and solidify. Punch holes on the solid LB+C plate containing *Ralstonia solanacearum* after cooling and solidification. Each hole is 2 cm away from the center of the plate, and the hole diameter is 8 mm, with a total of three holes. The verification experiment is divided into an antibacterial experimental group and an antibacterial control group. The antibacterial experimental group aspirates 20 μl of the fermentation supernatant obtained in S202 and drops it into each hole of the plate, that is, the experiment is repeated three times technically. After the operation, it is placed in a biochemical incubator and incubated at a constant temperature of 30 °C for 2 days, and observe whether an antibacterial circle is produced and measure the size of the antibacterial circle. The experimental method of the antibacterial control group is the same as that of the experimental group, except that 20 μl of sterile water is dropped into each hole.

[0055] The results are as Figure 4 shown: An antibacterial circle with an average diameter of 18 mm was produced on the plate of the antibacterial experimental group ( Figure 4 left figure), and no antibacterial circle was produced in the antibacterial control group ( Figure 4 right figure). The above results indicate that the fermentation supernatant of the lactic acid spore-forming bacillus X2T3 has the ability to inhibit the growth of *Ralstonia solanacearum*, that is, the lactic acid spore-forming bacillus X2T3 has an antibacterial effect on *Ralstonia solanacearum* and has the potential to be used in the preparation of antibacterial agents against *Ralstonia solanacearum*. In addition, a little lactic acid spore-forming bacillus X2T3 grew on the holes of the antibacterial experimental group. This is a small amount of lactic acid spore-forming bacillus X2T3 bacteria remaining in the inoculated fermentation supernatant, which indicates that even in an environment containing a large amount of *Ralstonia solanacearum*, the lactic acid spore-forming bacillus X2T3 can still survive and at the same time produce an inhibitory effect on *Ralstonia solanacearum*, which further indicates that the lactic acid spore-forming bacillus X2T3 has the potential to prevent and control bacterial wilt caused by *Ralstonia solanacearum*.

[0056] Among them, the preparation method of the LB+C solid medium is as follows: Weigh 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 5 g of glucose, and 3.75 g of agar powder, stir and dissolve in distilled water, adjust the pH to 7.0 - 7.2 and make the volume up to 1000 ml, then divide and pour it into conical flasks, and carry out high-pressure steam sterilization at 121 °C and 101 KPa for 20 min. Pour the sterilized medium into petri dishes in a laminar flow hood before solidification. About 20 ml of the medium is poured into each petri dish. After the medium cools and solidifies, the LB+C solid medium is obtained.

[0057] The preparation method of LB+C liquid medium is as follows: Weigh 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, and 5 g of glucose, stir and dissolve them in distilled water, adjust the pH to 7.0 - 7.2 and make up the volume to 1000 ml, dispense it into conical flasks, and perform autoclaving at 121 °C and 101 KPa for 20 min.

[0058] Control effect of Bacillus sporolactis X2T3 on bacterial wilt of pepper

[0059] Preparation of S501 antibacterial growth promoter: Take the activated bacterial liquid of Bacillus sporolactis X2T3 obtained in S201, inoculate the activated bacterial liquid obtained in S201 into LB liquid medium at a ratio of the volume of the activated bacterial liquid to LB liquid medium of 1:100, and perform constant temperature shaking fermentation culture at 37 °C and 200 rpm until the bacterial density of Bacillus sporolactis X2T3 reaches 1.0×10 7 cfu / ml - 1.0×10 8 cfu / ml to obtain the antibacterial growth promoting concentrate. Dilute the obtained antibacterial growth promoting concentrate by 100 - 200 times to obtain the antibacterial growth promoter. In this example, it is preferably to dilute the antibacterial growth promoting concentrate by 100 times to obtain the antibacterial growth promoter.

[0060] Inoculation of S502 bacterial wilt pathogen: Select 90 potted pepper seedlings with a seedling age of 6 weeks, divide them into three groups: a control blank control group, a control negative control group, and a control experimental group, with 30 plants in each group. Inject 10 μl of the bacterial wilt pathogen into each pepper seedling in the control negative control group and the control experimental group. The inoculation method is to use a syringe for one-time injection at the root and stem part of the pepper seedling. The control blank control group injects an equal amount of sterile water in the same way. Among them, the preparation method of the bacterial wilt pathogen is as follows: Dip the glycerol seed liquid of Ralstonia solanacearum, streak it on a solid medium plate, place it in a biochemical incubator and culture it at a constant temperature of 30 °C for 2 - 3 days. After single colonies grow, pick a single colony and inoculate it into LB+C liquid medium, and perform constant temperature shaking culture at 30 °C and a shaking speed of 200 rpm until the density of Ralstonia solanacearum reaches 1.0×10 6 cfu / ml - 1.0×10 7 cfu / ml to obtain the bacterial wilt pathogen.

[0061] S503 irrigation administration: After completing step S502, immediately use the antibacterial growth promoter to irrigate and apply it to the roots of the pepper seedlings in the control experimental group. The irrigation amount for each pepper seedling is 20 ml, and irrigate once every 5 days for 3 consecutive times. The control negative control group and the control blank control group directly irrigate the roots with an equal amount of clear water in the same way.

[0062] S504 Result Statistics: 20 days after applying the bacterial solution, observe the disease incidence of pepper leaves, rhizomes and stems, and calculate and compare the incidence and disease index of bacterial wilt in three groups. The results are as Figure 5 shown. The difference in the incidence of bacterial wilt between the prevention and treatment experimental group and the negative control group for prevention and treatment was 81.99%, that is, the incidence of bacterial wilt in the prevention and treatment experimental group was significantly lower than that in the negative control group for prevention and treatment, and it was only slightly higher than that in the blank control group without significant increase. The difference in the disease index of bacterial wilt between the prevention and treatment experimental group and the negative control group for prevention and treatment was 53. The disease index of bacterial wilt in the prevention and treatment experimental group was significantly lower than that in the negative control group for prevention and treatment, and there was no significant increase compared with the blank control group for prevention and treatment. In summary, using the antibacterial and growth-promoting agent of the present invention can effectively prevent bacterial wilt of pepper seedlings.

[0063] Among them, the basis for judging whether a pepper seedling is diseased is as follows: the young tissues (such as young leaves, tender shoots, and new leaves) at the top of the upper part of the pepper seedling show wilting, but still remain green. At the same time, it may also be accompanied by hollow stems. After the stem is cut horizontally, it can be observed that the fiber tube tissue turns brown. When the base of the horizontally cut pepper seedling stem is squeezed hard, white liquid flows out from the cut.

[0064] The calculation formula for the incidence of each group of pepper seedlings is:

[0065] The judgment standard for the disease level is: The disease level is judged according to the proportion of the number of diseased leaves of a plant in the total number of leaves of the plant. When the proportion of the number of diseased leaves of a plant in the total number of leaves of the plant is successively (0, 10%], (10%, 20%], (20%, 30%], (30%, 40%], (40%, 50%], (50%, 60%], (60%, 70%], (70%, 80%], (80%, 90%], (90%, 100%], the representative values of the disease levels of the plant are successively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the representative value of the highest disease level is 10.

[0066] The calculation formula for the disease index is: Disease index = 100×∑(number of plants at each disease level × representative value of that disease level) / (number of plants surveyed × representative value of the highest disease level)

[0067] The above results show that the antibacterial and growth-promoting agent containing Paenibacillus lacticus X2T3 can effectively reduce the incidence of bacterial wilt of pepper seedlings and effectively reduce the severity of bacterial wilt, and has an effective and positive prevention and treatment effect on bacterial wilt.

[0068] Growth-promoting effect of Paenibacillus lacticus X2T3 on pepper seedlings

[0069] S601 Drug administration by irrigation: Select 60 healthy potted pepper seedlings at the seedling age of 6 weeks, and divide them into a growth promotion experimental group and a growth promotion control group, with 30 plants in each group. Use the antibacterial growth promoter obtained in step S501 to irrigate the roots of the pepper plants in the experimental group, applying 20 ml per plant, once every 5 days, and continuously irrigate for 3 times. The control group does not apply the antibacterial growth promoter, but irrigates with an equal amount of clear water according to the same method.

[0070] S602 Result statistics: On the 14th day after the last drug administration, collect the data of pepper root length, stem length, stem diameter, fresh weight, and dry weight for statistical analysis. The results are as Figure 6 shown in Table 1. The average root length, stem length, stem diameter, fresh weight, and dry weight data of the growth promotion experimental group are all higher than those of the growth promotion control group. The growth trend of the plants in the growth promotion experimental group is overall better than that of the plants in the growth promotion control group, indicating that the antibacterial growth promoter described in the present invention has an effective effect on promoting the growth of pepper seedlings.

[0071] Table 1 Growth promotion effect of Bacillus laevolacticus X2T3 on pepper

[0072]

[0073] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0074] 1. The antibacterial growth promoter containing Bacillus laevolacticus X2T3 can significantly reduce the incidence and severity of bacterial wilt in pepper seedlings, and has a good control effect on bacterial wilt. Using this antibacterial growth promoter to control bacterial wilt can greatly reduce the use of chemical pesticides and improve food safety and environmental safety issues.

[0075] 2. Bacillus laevolacticus X2T3 is a soil bacterium, which is easy to colonize and survive in the soil environment, and is a non-pathogenic bacterium, which is friendly to the environment and crops.

[0076] 3. The antibacterial growth promoter formula containing Bacillus laevolacticus X2T3 is simple, the preparation method is simple, no special equipment is required, no harsh culture conditions are required, and the production cost is low.

[0077] 4. Bacillus laevolacticus X2T3 can promote the growth of crops represented by pepper seedlings. It is a natural non-chemical growth promoter and can be used as an additive and added to organic fertilizers.

[0078] 5. After the antibacterial growth promoter containing Bacillus laevolacticus X2T3 is applied to crops or soil, Bacillus laevolacticus X2T3 colonized on crops and in the soil can adapt to environmental changes through natural evolution and co-evolve with pathogenic bacteria to maintain the balance and health of the soil microecology, that is, the antibacterial growth promoter containing Bacillus laevolacticus X2T3 can play a long-term role.

[0079] In the embodiments of the present invention, the experimental methods without specific conditions are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. All common chemical reagents used in the embodiments are commercially available products. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0080] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A Bacillus parafirmus X2T3, characterized in that: The Bacillus parafirmus X2T3 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242885 and the deposit date of December 23, 2024.

2. Use of the Bacillus parafirmus X2T3 according to claim 1 for inhibiting the growth of Ralstonia solanacearum.

3. Use of the Bacillus parafirmus X2T3 according to claim 1 for preventing and treating plant diseases and / or promoting plant growth.

4. The application according to claim 3, characterized in that: The plant disease is a plant disease caused by Ralstonia solanacearum.

5. The application according to claim 4, wherein: The plant diseases include one or more of bacterial wilt of pepper, bacterial wilt of eggplant, bacterial wilt of tomato, bacterial wilt of pepper, bacterial wilt of potato, and bacterial wilt of peanut.

6. An antibacterial growth promoter, characterized in that: The antibacterial and growth-promoting agent includes the Bacillus parafirmus X2T3, which is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242885.

7. The antibacterial growth promoter according to claim 6, wherein: The concentration of the Bacillus parafirmus X2T3 is 1.0×10 4 cfu / ml to 1.0×10 10 cfu / ml.

8. The antibacterial growth promoter according to claim 7, wherein: The concentration of the Bacillus parafirmus X2T3 is 5.0×10 4 cfu / ml to 1.0×10 6 cfu / ml.

9. A method for controlling bacterial wilt of plants, characterized in that: Irrigate the roots of plants with the antibacterial and growth-promoting agent according to any one of claims 6-8, with an irrigation amount of 5-50 ml per plant, once every 3-8 days, and irrigate continuously for at least 3 times.

10. A plant growth promotion method, characterized in that: Irrigate the roots of plants with the antibacterial and growth-promoting agent according to any one of claims 6-8, with an irrigation amount of 5-50 ml per plant, once every 3-8 days, and irrigate continuously for at least 3 times.

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