A strain of Paenibacillus polymyxa and its applications

Abstract

The present invention provides a strain of Paenibacillus polymyxa and its application. The deposit number of the Paenibacillus polymyxa strain BJ-2 of the present invention is CGMCC No. 24114. The Paenibacillus polymyxa strain BJ-2 of the present invention has a good antagonistic effect on a variety of grape pathogenic fungi. The fermentation liquid of the strain can achieve a therapeutic effect of 82.04% on grape canker artificially inoculated on detached green branches. In addition, the fermentation liquid of strain BJ-2 has a significant promoting effect on the growth of plants such as cucumbers and radishes. The Paenibacillus polymyxa of the present invention can be applied to grape diseases, especially the biological control of grape branch diseases and the promotion of plant growth.

Description

Technical Field

[0001] The invention relates to a Paenibacilus polymyxa strain and application thereof in preventing and controlling plant diseases such as grape branch diseases, belonging to the technical field of biological control of plant diseases. Background Art

[0002] Grape is an important economic crop in my country and has a long history of cultivation in my country. According to FAO statistics, as of 2021, my country's grape cultivation area ranks second in the world, and grapes and related industries account for a large proportion of my country's agricultural production. However, grape cultivation and production are often threatened by various diseases. Currently, 71 grape-related diseases have been reported worldwide, and disease problems have become one of the key factors restricting the healthy development of the grape industry. Grape trunk diseases are a general term for vascular diseases that harm grape trunks, roots, etc. Since they were first reported in the 1970s, they have been found to be widely distributed in major grape-growing areas around the world (VARELA CP, REDONDO V, COSTAS D, AGUIN O, MANSILLA P. Fungi associated with grapevine trunk diseases in nursery-produced Vitis vinifera plants. Phytopathologia Mediterranea, 2019, 57(3):407-424.). Grape stem diseases are characterized by a wide variety of pathogens, complex symptoms, unclear patterns of occurrence, and relatively basic research on their pathogenesis. To date, effective prevention and control methods are lacking in the field. Currently, chemical control is still the primary method for grape stem diseases, but in actual production, there is still a lack of effective pesticides for these diseases. Furthermore, the use of chemical pesticides can easily cause environmental pollution and pesticide residues. Therefore, the use of biological control methods to control grape stem diseases has become a hot topic in research on the prevention and control of these diseases (GRAMAJE D, -TORRES JR, SOSNOWSKI M R. Managing grapevine trunk diseases with respect to etiology and epidemiology: Current strategies and future prospects. Plant Disease, 2018, 102(1): 1-28.). Therefore, adhering to the concepts of environmental protection, health and sustainable agricultural development, clarifying the inhibitory effect of antagonistic microorganisms on grape trunk disease fungi and developing new and environmentally friendly and efficient control products are of great significance for the practical application of biological control.

[0003] Paenibacilus spp. are widely distributed in nature and are one of the dominant species in soil and plant microecology. They have strong stress resistance, rich antibacterial substances, and good growth-promoting effects. Some strains have been commercialized. Paenibacilus polymyxa is an important plant growth-promoting rhizobacteria (PGPR), which has been shown to be effective against rice sheath blight (KAVITHA S, SENTHILKUMAR S, GNANAMA NICKAM S, et al. Isolation and partial characterization of antifungal protein from Bacilus polymyxa strain VLB16. Process Biochemistry, 2005, 40(10): 3236-3243.), pepper anthracnose (DENG Yang. Structural identification of antifungal substance of Paenibacillus polymyxa JSa-9 and its biocontrol application research in wheat. Nanjing: Nanjing Agricultural University, 2012.), and Fusarium wilt (ZHAI Y, ZHU JX, TAN TM, et al. Isolation and characterization of antagonistic Paenibacilus polymyxa HX-140 and its biocontrol potential against Fusarium wilt of cucumber). Paenibacilus polymyxa has a strong inhibitory effect on a variety of plant pathogenic fungi, including phytopathogenic fungi (seedlings. BMC Microbiology, 2021, 21:75). The U.S. Environmental Protection Agency (EPA) has recognized it as a commercially applicable microorganism (Grady EN, Macdonald J, Liu L, et al. Current knowledge and perspectives of Paenibacilus: A review. Microbial Cell Factories, 2016, 15:203). my country's Ministry of Agriculture and Rural Affairs has also listed it as a Class I fungus exempt from safety certification. Currently, there are no reports on the role of Paenibacilus polymyxa in the prevention and control of grapevine stem diseases. Therefore, exploring the role of Paenibacilus polymyxa in the prevention and control of grapevine stem diseases is expected to provide an effective measure for the prevention and control of grapevine stem diseases, including these. Summary of the Invention

[0004] The present invention aims to provide a strain of Paenibacilus polymyxa BJ-2 and its uses. The strain has a strong inhibitory effect on pathogens that cause grapevine stem diseases, and its use effectively prevents and controls grapevine stem diseases, thereby reducing the use of chemical pesticides, alleviating environmental pollution, and achieving sustainable agricultural development.

[0005] The Paenibacilus polymyxa of the present invention was collected and separated from the diseased tissue of grape branch diseases in Huailai County, Zhangjiakou City, Hebei Province. It was classified and named Paenibacilus polymyxa, named BJ-2, and was deposited in the China General Microorganism Culture Collection Center (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on December 16, 2021, with the deposit number CGMCC No. 24114.

[0006] A strain with broad-spectrum inhibitory activity against grape pathogenic fungi was isolated from diseased grape stem tissue samples collected from the Tumu grape base in Huailai County, Zhangjiakou City, Hebei Province. Based on 16S rRNA molecular analysis and physicochemical properties, the strain was identified as Paenibacilus polymyxa and named Paenibacilus polymyxa BJ-2. Its physiological characteristics are as follows:

[0007] The Paenibacilus polymyxa BJ-2 of the present invention does not produce pigment on LB culture medium, and its colonies are milky white, opaque, round or nearly round, with smooth edges and an oily surface. The Paenibacilus polymyxa BJ-2 has the physiological and biochemical characteristics of being Gram-positive and capable of producing cellulase and amylase.

[0008] The polymyxa bacillus of the present invention has antibacterial activity against grape pathogenic fungi, including Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Diaporthe sojae, Coletotrichum viniferum, Botrytis cinerea, Neopestalotiopsis sp., Coniela vitis, and Dactylonectria macrodidyma.

[0009] The present invention also provides a biological pesticide, which contains the Paenibacillus polymyxa BJ-2.

[0010] The present invention further provides a microbial fertilizer, which contains the Paenibacillus polymyxa BJ-2.

[0011] The use of the polymyxa bacillus or the antibacterial active substances produced by it in the preparation of biological pesticides and / or biological fertilizers having antagonistic activity against plant pathogenic fungi and / or promoting plant growth also falls within the scope of protection of the present invention.

[0012] The present invention also provides application of the Paenibacillus polymyxa in preventing and controlling plant pathogenic fungi.

[0013] The plant pathogenic fungi are grape pathogenic fungi, preferably one or more of Lasiodiplodiatheobromae, Botryosphaeria dothidea, Diaporthe eres, Diaporthe sojae, Coletotrichum viniferum, Botrytis cinerea, Neopestalotiopsis sp., Coniela vitis and Dactylonectria macrodidyma.

[0014] The application of the Paenibacillus polymyxa in promoting the growth of plant seedlings also falls within the protection scope of the present invention.

[0015] In the application, preferably, the method for promoting the growth of plant seedlings is to dilute the polymyxa fermentation liquid of claim 1 to 1×10 8 -2×10 8 CFU / mL concentration, root irrigation of plants or soaking of seeds for germination before sowing; the plants are preferably cucumbers or radishes.

[0016] The Paenibacilus polymyxa BJ-2 strain of the present invention has antibacterial activity against grapevine pathogenic fungi and a broad antibacterial spectrum. Strain BJ-2 exhibits the best inhibitory effect against GG-6, a strain of Diaporthesojae that causes grapevine vine blight, with an inhibition rate of 67%, and significantly inhibits the growth of Diaporthesojae. Strain BJ-2 also exhibits the poorest antibacterial activity against PTP038, a strain of Neopestalotiopsis sp. that causes grapevine branch blight, with an inhibition rate of 44%. BJ-2's inhibition rates against five other major grapevine fungal diseases ranged from 54% to 65%. Furthermore, the fermentation broth of strain BJ-2 has a significant preventive and therapeutic effect on detached green grape branches and stem diseases on field grapes, and significantly promotes the growth of plants such as cucumbers and radishes. Strain BJ-2 has excellent application prospects in biological preparations and is of great significance for the biological control of grapevine fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Colony characteristics of strain BJ-2

[0018] Figure 2 Amylase test results of strain BJ-2

[0019] Among them, left: Escherichia coli JM109, right: strain BJ-2.

[0020] Figure 3 Protease detection results of strain BJ-2

[0021] Among them, left: Escherichia coli JM109, right: strain BJ-2.

[0022] Figure 4 Cellulase test results of strain BJ-2

[0023] Among them, left: Escherichia coli JM109, right: strain BJ-2.

[0024] Figure 5 Phylogenetic tree based on 16S rDNA

[0025] Figure 6 Effects of Different Treatments on Chlorophyll Content in Seedlings of Bacterial Strain BJ-2

[0026] Treatment 1 was the clear water control, and treatment 2 was 2×10 8 CFU / mL of BJ-2 fermentation broth, treatment 3 was 2×10 7 CFU / mL of BJ-2 fermentation broth, treatment 4 was 2×10 6CFU / mL of BJ-2 fermentation broth.

[0027] Figure 7 Effects of Different Treatments of BJ-2 on Fresh Weight of Cucumber Seedlings

[0028] Treatment 1 was the clear water control, and treatment 2 was 2×10 8 CFU / mL of BJ-2 fermentation broth, treatment 3 was 2×10 7 CFU / mL of BJ-2 fermentation broth, treatment 4 was 2×10 6 CFU / mL of BJ-2 fermentation broth.

[0029] Figure 8 Effects of Different Treatments of Bacterial Strain BJ-2 on the Dry Weight of Cucumber Seedlings

[0030] Treatment 1 was the clear water control, and treatment 2 was 2×10 8 CFU / mL of BJ-2 fermentation broth, treatment 3 was 2×10 7 CFU / mL of BJ-2 fermentation broth, treatment 4 was 2×10 6 CFU / mL of BJ-2 fermentation broth.

[0031] Figure 9 Effects of Different Treatments of Bacterial Strain BJ-2 on the Seedling Index of Cucumber Seedlings

[0032] Treatment 1 was the clear water control, and treatment 2 was 2×10 8 CFU / mL of BJ-2 fermentation broth, treatment 3 was 2×10 7 CFU / mL of BJ-2 fermentation broth, treatment 4 was 2×10 6 CFU / mL of BJ-2 fermentation broth.

[0033] Figure 10 Effects of different concentrations of fermentation broth of strain BJ-2 on the stem length of radish seedlings.

[0034] Figure 11 Effects of different concentrations of fermentation broth of strain BJ-2 on root length of radish seedlings.

[0035] Figure 12 Effects of different concentrations of fermentation broth of strain BJ-2 on the fresh weight of radish seedlings.

[0036] Figure 13 Effects of different concentrations of fermentation broth of strain BJ-2 on the dry weight of radish seedlings. DETAILED DESCRIPTION

[0037] Example 1. Acquisition and identification of strain BJ-2

[0038] The strain BJ-2 was isolated from diseased grape stem tissues showing symptoms of grape stem disease in Huailai County, Zhangjiakou, Hebei Province. The isolation method was tissue separation, which was as follows: (1) Remove the bark of the diseased grape stems collected in the field, and cut a 5mm thick section from the diseased and healthy junction of the stem tissue. 2 The tissue blocks were disinfected with 2% sodium hypochlorite for 2 minutes, 70% ethanol for 30 seconds, and rinsed with sterile water 3 times. Sterilized filter paper was placed on the tissue blocks to dry. After the water was absorbed, the tissue blocks were placed on PDA plates. Each PDA plate was placed with 4 to 5 tissue blocks and sealed with sealing film. (2) Cultured at 25°C in the dark for 3 days. After 3 days, the colony growth was observed and counted, and the corresponding separation ratio was calculated. (3) In the process of isolating pathogenic fungi, a bacterial strain was found to have a good antagonistic effect on pathogenic fungi. It was purified by streaking on LB culture medium plates to obtain a pure culture of bacteria. After identification, a strain with antagonistic effect on pathogenic fungi was obtained and named BJ-2. The colony morphology of strain BJ-2 on LB plates is as follows: Figure 1 As shown, the colonies are milky white and opaque, round or nearly round, with smooth edges and an oily surface.

[0039] The physiological and biochemical characteristics of strain BJ-2 were studied with reference to the Manual of Systematic Identification of Common Bacteria, and molecular identification of strain BJ-2 was performed based on the 16S rRNA sequence.

[0040] 1 Physiological and biochemical characteristics of strain BJ-2

[0041] 1.1 Detection of amylase

[0042] After culturing the strain to be tested in LB solid culture for 3 days, pick a single colony and spot-inoculate it on amylase detection medium (1g soluble starch, 5g peptone, 5g glucose, 5g NaCl, 5g beef extract, 15g agar). Incubate at 28℃ for 24-48h and observe whether there is a transparent circle. If there is, it means that the strain has the ability to produce amylase.

[0043] Test results: Figure 2 As shown, there is no obvious transparent zone around the colony of the tested strain BJ-2, indicating that the strain BJ-2 does not produce amylase and has no starch hydrolyzing enzyme activity.

[0044] 1.2 Protease detection

[0045] After culturing the strain to be tested in LB solid medium for 3 days, pick a single colony and spot-inoculate it on protease detection medium (peptone 10.0 g, NaCl 5.0 g, CaCl2 0.1 g, skim milk 100.0 mL, agar 15 g), and culture it at 28°C for 24-48 hours. Observe for the presence of a clear zone. If there is a clear zone, it indicates that the strain has the ability to produce protease.

[0046] Test results: Figure 3 As shown, no transparent zone is produced around the strain BJ-2, indicating that it cannot produce protease and has no proteolytic enzyme activity.

[0047] 1.3 Cellulase detection

[0048] After culturing the strain to be tested in LB solid medium for 3 days, pick a single colony and spot-inoculate it on cellulase assay medium (half LB for carbon and nitrogen source, plus 0.2% sodium carboxymethyl cellulose). Incubate at 28°C for 24-48 hours. Stain with 1g / L Congo red for 1 hour. Wash and soak twice in NaCl solution for 30 minutes each time to elute. Observe for the presence of a clearing zone; if present, the strain is capable of producing cellulase.

[0049] Test results: Figure 4 As shown, a transparent zone can be produced around the BJ-2 strain, indicating that the strain can produce cellulase and has cellulose hydrolase activity.

[0050] 2 Molecular identification of strain BJ-2

[0051] 2.1 Extraction of genomic DNA

[0052] The preserved bacterial liquid was spread on a plate and cultured at 25°C for 3 days. The bacteria were scraped off with an inoculating loop and washed out with LB liquid culture medium. The bacterial genomic DNA was extracted using the bacterial genomic DNA rapid extraction kit from Beijing Bomade Gene Technology Co., Ltd.

[0053] 2.2 PCR amplification

[0054] Using genomic DNA from strain BJ-2 as a template, PCR amplification was performed using universal bacterial 16S rDNA primers 27f and 1492r. The primer sequences were: 27f: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492r: 5'-TACCTTGTTACGACTT-3'.

[0055] The PCR reaction system was (50 μL): 25 μL 2× Taq PCR Master Mix, 2 μL DNA template, 2 μL 27f, 2 μL 1492r, and 19 μL ddH2O.

[0056] PCR amplification reaction procedure: pre-denaturation at 95°C for 3 min, followed by denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 90 s, 35 cycles, and finally extension at 72°C for 8 min, and termination at 12°C.

[0057] The PCR amplification product was detected by electrophoresis, and the fragment length was approximately 1500 bp.

[0058] 2.3 Sequence analysis and molecular identification

[0059] The PCR product was verified by 1% agarose gel electrophoresis and then sent to Beijing Biomed Gene Technology Co., Ltd. for sequencing. The resulting sequence was compared with sequences in the GenBank database on the NCBI website to identify the strain with the highest similarity to the sequenced 16S rDNA sequence. The results showed that the 16S rDNA sequence of strain BJ-2 shared 99.58% similarity with that of Paenibacilus polymyxa strain P2-5, preliminarily identifying it as a closely related species of Paenibacilus polymyxa.

[0060] The sequencing results of the PCR amplified fragment of the 16S rDNA sequence of the BJ-2 strain are shown in Sequence 1 in the sequence listing.

[0061] According to the gene fragment sequence, MEGA software was used to construct phylogenetic trees based on 16S rDNA sequence using ML method, such as Figure 5 As shown, the results showed that strain BJ-2 was clustered with the standard strain of Paenibacilus polymyxa IAM 13419 (GenBank accession number: D16276.1). Based on this, strain BJ-2 was identified as Paenibacilus polymyxa. Strain BJ-2 was deposited in the China General Microbial Culture Collection (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on December 16, 2021, with the deposit number CGMCC No. 24114.

[0062] Example 2. Determination of antibacterial activity of strain BJ-2

[0063] The antagonistic activity of Paenibacilus polymyxa strain BJ-2 against grapevine pathogenic fungi was tested using a stand-off culture method with 17 strains of 9 grapevine pathogenic fungi (Table 1, strains in the table were collected and isolated and purified in the field and all were identified by conventional strain identification). 5 mm microporous holes were used to cut the antagonistic activity of Lasiodiplodia theobromae, Botryosphaeria dothidea, Diaporthe eres, Diaporthesojae, Coletotrichum viniferum, Botrytis cinerea, Neopestalotiopsis sp., Coniela vitis, and Dactylonectria spp., which had been cultured on PDA medium at 25°C for 3-7 days. The bacterial cake at the edge of the macrodidyma colony was placed in the center of the PDA plate. At the same time, 4 μL of the cultured fermentation liquid (strain BJ-2 was cultured on LB solid medium at 37°C for 2 to 3 days, and then a single colony was picked and placed in LB liquid medium and shaken at 37°C at 200 rpm / min for 8 to 10 hours) was dripped onto a 6 mm diameter filter paper and placed about 1 cm from the edge of the plate. Four filter paper pieces were placed on one plate, and the same amount of clean water was dripped onto the filter paper as a control. The plate was cultured in a 28°C incubator for 5 days. The radius (D) of the pathogen hyphae growth relative to the strain BJ-2 and the width of the inhibition zone (d) were measured to calculate the inhibition rate. Each treatment was repeated 3 times. The results are shown in Table 2:

[0064] Inhibition rate (%) = (control colony radius - treated colony radius) / control colony radius × 100%

[0065] As shown in Table 3, Paenibacilus polymyxa BJ-2 exhibited varying degrees of antifungal activity against various grapevine fungal diseases. It exhibited the greatest inhibitory effect against GG-6, a strain of Diaporthe sojae that causes grapevine vine blight, with an inhibition rate of 67.16%, and significantly inhibited the growth of Diaporthe sojae. BJ-2 also exhibited the weakest antifungal activity against PTP038, a strain of Neopestalotiopsis sp., with an inhibition rate of 44.59%. BJ-2 exhibited inhibition rates exceeding 50% against the other five grapevine fungal pathogens, ranging from 54% to 65%.

[0066] Table 1 17 grape pathogenic fungi tested

[0067]

[0068] Table 3 Antibacterial activity of strain BJ-2 against 17 grape pathogenic fungi

[0069]

[0070]

[0071] Example 3. Effect of strain BJ-2 on the prevention and treatment of grape canker

[0072] 3.1 Preparation of fermentation broth

[0073] Preparation of BJ-2 fermentation broth: A single colony of strain BJ-2 was picked with a sterilized toothpick and inoculated into 10 mL of LB liquid medium. The culture was shaken at 25°C and 180 rpm for 12 h, then poured into a flask containing 100 mL of fermentation medium and shaken at 25°C and 180 rpm for 3 days to obtain the fermentation broth of strain BJ-2 (1.08 × 10 9 CFU / mL), and 1×10 6 CFU / mL, 1×10 7 CFU / mL and 1×10 8 The fermentation broths were stored at 4°C with three concentration gradients of CFU / mL.

[0074] 3.2 Test methods

[0075] 3.2.1 Therapeutic effect of strain BJ-2 on grape canker pathogen GX-5-5 on fresh branches in vitro

[0076] Healthy, fresh, semi-lignified, green branches of the current-year Summer Black grape variety were collected from the Xiangyi Vineyard in Beiwu, Shunyi. The leaves were removed and the surface was cleaned with clean water, then wiped and disinfected with 75% alcohol wipes, rinsed with sterile water, and air-dried. A 4.0 mm diameter, 1.0 mm deep wound was made in the middle of the internode of the branch using a sterile 4.0 mm diameter punch. The epidermis was peeled off, and a previously cultured pathogen strain was evenly punched into a cake using a 4.0 mm punch. The cake was placed over the wound using a sterile toothpick, and the inoculated area was wrapped with short pieces of Parafilm. The cake was then inserted into a nutrient pot filled with vermiculite. Blank PDA plates served as controls. Branches sprayed with BJ-2 fermentation broth, inoculated with bacteria and immersed in sterile water, and inoculated with blank PDA and immersed in sterile water served as controls. Three concentrations of BJ-2 fermentation broth, supplemented with 1 / 1000th of Tween, were evenly sprayed onto the branches. A 2000-fold dilution of Bacillus subtilis was used as a control. Seven branches were inoculated per treatment, with three replicates. After inoculation, the treated branches were placed in a temperature-controlled inoculation chamber and incubated for two days at a relative humidity of 90%. Thereafter, the temperature was maintained at 25°C and the humidity at 60%. Seven to ten days after inoculation, the disease incidence of each treatment was investigated, and the lesion length was measured.

[0077] 3.2.2 Data Statistics and Analysis

[0078] The incidence and lesion length of each treatment were measured 7-10 days after inoculation, and the data were statistically analyzed using SPSS 18 software. The calculation formula is as follows:

[0079] Control effect (%) = [(control lesion length - treatment lesion length) / (control lesion length - fungus cake diameter)] × 100%

[0080] 3.3 Results

[0081] The experimental results of the therapeutic and control effects of three concentrations of BJ-2 fermentation liquid and 2000 times of Bacillus subtilis on grape canker pathogen GX-5-5 are shown in Figure 10. 6 The control effect of BJ-2 fermentation broth with CFU / mL concentration was significantly higher than that of fermentation broths with other concentrations of BJ-2 and control agents, which was 82.04%, indicating that BJ-2 fermentation broth has basically met the product requirements.

[0082] Table 4 Control effect of strain BJ-2 on GX-5-5

[0083]

[0084] Example 4. Growth-promoting effect of BJ-2 fermentation liquid on cucumber seedlings

[0085] 4.1 Materials and Methods

[0086] 4.1.1 Preparation of BJ-2 fermentation broth

[0087] Seed solution preparation: Use a 250 mL Erlenmeyer flask to fill it with 100 mL of sterilized LB liquid medium, pick a single colony of BJ-2 and inoculate it into the LB liquid medium, and culture it at 25°C and 180 rpm / min with shaking for 12 h.

[0088] Fermentation culture: BJ-2: Pour the seed liquid into 100 mL of fermentation medium (sucrose 15 g / L, peptone 15 g / L, MgSO4 5 g / L, CaCl2 3 g / L; pH 6.8) in a 500 mL Erlenmeyer flask with an inoculum size of 10%. Incubate at 25°C and 180 rpm / min for 72 h to obtain fermentation broth.

[0089] 4.1.2 Growth-promoting effect of strain BJ-2 fermentation broth on cucumber seedlings

[0090] The root irrigation method was used to determine the growth-promoting effect of the fermentation liquid of strain BJ-2 on cucumber seedlings. Cucumber seeds were treated with 3% NaClO for 1 minute, rinsed three times with sterile water, and then soaked in warm water for 6 hours. Cucumber seeds with similar germination status were selected and sown in pots (14.5 cm in diameter and 12.0 cm in height) filled with peat nutrient medium. After emergence, healthy plants were retained. 10 CFU / mL of BJ-2 fermentation broth was diluted to 2×10 8 CFU / mL, 2×10 7 CFU / mL and 2×10 6 Three concentrations of BJ-2 fermentation broth (CFU / mL) were used. After the cucumber seedlings grew one true leaf, 100 mL of each of the different concentrations of BJ-2 fermentation broth was used to irrigate the roots. An equal amount of sterile water was used as a blank control. Two root irrigation treatments were performed, 7 days apart; each treatment consisted of seven pots, with three replicates. The pots were incubated in a greenhouse at (25 ± 2)°C.

[0091] 4.1.3 Data Statistics and Analysis

[0092] On the 28th day after the first root irrigation, the fresh and dry weights of the aboveground parts and roots were measured and recorded. Chlorophyll content was also measured using a SPAD-502 chlorophyll meter. Statistical analysis of the data was performed using SPSS 25 software.

[0093] 4.2 Results

[0094] The results of chlorophyll content determination showed that the three concentrations of BJ-2 fermentation liquid had a significant promoting effect on the chlorophyll content of cucumber, which was significantly better than the blank control ( Figure 6 ), which increased by 7.40%, 5.76% and 7.50% respectively compared with the blank control, and there was no significant difference among the three concentrations of fermentation broth treatments.

[0095] The results of cucumber root fresh weight determination showed that 2×10 8 The root fresh weight of the treatment with BJ-2 fermentation liquid with CFU / mL was 3.04 g, which was significantly higher than that of the other two concentrations and the blank control treatment ( Figure 7 ), which increased by 36.68% compared with the blank control, indicating that 2×10 8 CFU / mL of BJ-2 fermentation liquid has a significant promoting effect on the growth of cucumber roots. 8 The dry weights of the aboveground part and roots treated with BJ-2 fermentation liquid with CFU / mL were 3.86g and 0.84g, respectively, which were significantly higher than those of the other concentrations of fermentation liquid and the blank control group ( Figure 8 ), the aboveground dry weight and root dry weight increased by 17.81% and 74.39% respectively compared with the blank control, indicating that 2×10 8 CFU / mL of BJ-2 fermentation liquid has a significant promoting effect on the growth of cucumber rhizomes.

[0096] The results of seedling index showed that 2×10 8 The cucumber seedling index treated with BJ-2 fermentation liquid with CFU / mL was 0.607, which was significantly higher than that of the other two concentrations of fermentation liquid ( Figure 9 ), indicating 2×10 8 The CFU / mL of BJ-2 fermentation liquid had a significant promoting effect on the growth of the aboveground part and roots of cucumber. Overall, BJ-2 fermentation liquid had a promoting effect on the growth of cucumber seedlings, and the seedling index could reach 0.607.

[0097] Example 5. Growth-promoting effect of BJ-2 fermentation liquid on radish seedlings

[0098] 5.1 Materials and Methods

[0099] 5.1.1 Preparation of BJ-2 fermentation broth

[0100] The method is the same as 4.1.1 of Example 4.

[0101] 5.1.2 Growth-promoting effect of strain BJ-2 fermentation liquid on radish seedlings

[0102] Radish seeds were soaked in 3% NaClO for 1 minute for surface disinfection, and then washed three times with sterile water. The seeds were placed in a culture dish, soaked with sterile water, and then placed in a 26°C incubator for germination for 12 hours. 9 CFU / mL of BJ-2 fermentation broth, and diluted them to 1×10 8 , 1×10 7 , 1×10 6, 1×10 5 The seeds were placed in different concentrations of BJ-2 fermentation broth and placed in a 26°C incubator for 2 hours. Sterile water was used as a control group. Finally, radish seeds with relatively consistent germination status were selected and placed in a seed germination box (13cm×19cm×12cm) lined with two sterile filter papers. Each seed germination box contained 20 seeds and 9mL of the previously diluted fermentation broth of different concentrations was added. An equal amount of sterile water was added as a control. Each treatment was repeated 3 times. The germination box was placed in an artificial culture room with a light:dark ratio of 12h:12h and cultured at 25°C. The same dose of sterile water and fermentation broth of different concentrations were added every 1 day.

[0103] 5.1.3 Data Statistics and Analysis

[0104] The stem length, root length and fresh weight of the seedlings under each treatment were measured after 4, 7 and 10 days, and the data were analyzed using SPSS 25 software.

[0105] 5.2 Results

[0106] The results of the effect of BJ-2 fermentation liquid on the stem growth of radish seedlings showed that 1×10 8 The CFU / mL of BJ-2 treatment reached 3.18 cm and 3.13 cm on the 7th and 10th day, respectively, which were significantly different from those of other treatments and increased by 62.17% ( Figure 10 The results showed that there was no significant difference in the effects of the four concentrations of BJ-2 fermentation liquid on the root length of radish seedlings at 7d and 10d. 8 The longest root length was 13.42 cm ( Figure 11 ); The results of the effect of BJ-2 fermentation liquid on the fresh weight of radish seedlings showed that there was no significant difference in the effect of the four concentrations of BJ-2 fermentation liquid on the fresh weight of radish seedlings on the 7th and 10th days, but on the 10th day, it was significantly higher than the control group, with the fresh weights being 1.02g, 0.96g, 0.82g and 0.80g respectively ( Figure 12 ); The results of the effect of BJ-2 fermentation liquid on the dry weight of radish seedlings showed that at the 10th day, 1×10 8 The dry weight of the BJ-2 fermentation liquid treatment with CFU / mL reached 0.045g, which was significantly increased compared with other treatments ( Figure 13 ).

[0107] The results showed that compared with the water control treatment, BJ-2 fermentation liquid had a significant effect on promoting the stem length growth of radish seedlings, but had no significant effect on the root length growth. 8CFU / mL of BJ-2 fermentation liquid had a significant promoting effect on the stem length, fresh weight and dry weight of radish seedlings, which increased by 62.17%, 50% and 60.71% respectively compared with the water control. 8 CFU / mL of fermentation broth has a more obvious growth-promoting effect on radish.

[0108] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific, they are intended to be illustrative rather than restrictive of the present invention. Those skilled in the art will readily appreciate that numerous modifications, variations, and improvements can be made without departing from the spirit and scope of the appended claims, all of which fall within the scope of protection of the present invention.

Claims

1. A polymyxa bacillus ( Paenibacillus polymyxa ), characterized in that, The strain of Paenibacillus polymyxa is named Paenibacillus polymyxa BJ-2 and has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 24114.

2. The polymyxa bacillus according to claim 1, characterized in that The Paenibacillus myxobacterium BJ-2 can secrete and produce enzymes, including cellulase.

3. The Paenibacillus polymyxa according to claim 1, characterized in that The Paenibacillus myxobacterium BJ-2 has antagonistic activity against grape pathogenic fungi.

4. The Paenibacillus polymyxa according to claim 3, characterized in that The grape pathogenic fungus is Diplodia theobromin Lasiodiplodia theobromae , Botrytis cinerea Botryosphaeria dothidea 、Sweet cherry shell Diaporthe eres , soybean shell Diaporthe sojae 、 Colletotrichum viniferum Botrytis cinerea Botrytis cinerea 、 Neopestalotiopsis sp.、 Coniella vitis and Dactylonectria macrodidyma One or more of .

5. A biological pesticide, characterized in that: The biological pesticide contains the Paenibacillus polymyxa according to claim 1.

6. A microbial fertilizer, characterized in that: The microbial fertilizer contains the Paenibacillus polymyxa according to claim 1.

7. Use of the polymyxa bacillus according to claim 1 in the preparation of a biopesticide and / or biofertilizer having antagonistic activity against plant pathogenic fungi and / or a growth-promoting effect on plants; the plant pathogenic fungus is Diplodia theobromin Lasiodiplodia theobromae , Botrytis cinerea Botryosphaeria dothidea 、Sweet cherry shell Diaporthe eres , soybean shell Diaporthe sojae , Colletotrichum thunbergii Colletotrichum viniferum Botrytis cinerea Botrytis cinerea 、 Neopestalotiopsis sp.、 Coniella vitis and Dactylonectria macrodidyma One or more of; the plant is cucumber or radish.

8. Use of the polymyxa bacillus according to claim 1 in preventing and treating plant pathogenic fungal diseases; the plant pathogenic fungus is Diplodia theobromin Lasiodiplodia theobromae , Botrytis cinerea Botryosphaeria dothidea 、Sweet cherry shell Diaporthe eres , soybean shell Diaporthe sojae , Colletotrichum thunbergii Colletotrichum viniferum Botrytis cinerea Botrytis cinerea 、 Neopestalotiopsis sp.、 Coniella vitis and Dactylonectria macrodidyma One or more of; the plant is cucumber or radish.

9. Use of the Paenibacillus polymyxa according to claim 1 in promoting the growth of plant seedlings; the plant is cucumber or radish.

10. The use according to claim 9, characterized in that The method for promoting the growth of plant seedlings is to dilute the polymyxa fermentation liquid of claim 1 to 1×10 8 -2×10 8 CFU / mL concentration, irrigate the roots of plants or soak the seeds for germination before sowing.

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