Fluorescent pseudomonas for preventing black spot disease of rosa and promoting growth of rosa and application thereof
By using Pseudomonas fluorescens LX6, the environmental pollution and drug resistance problems caused by chemical control of rose black spot disease have been solved, achieving high efficiency of biological control and promoting rose growth.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING UNIV OF AGRI
- Filing Date
- 2024-09-01
- Publication Date
- 2026-06-05
AI Technical Summary
Current technologies for controlling rose black spot disease rely on chemical agents, leading to environmental pollution and pathogen resistance. There is a lack of effective biological control methods.
Pseudomonas fluorescens LX6 was used to inhibit the production of ironophiles and cellulase by Diplosporium rosenbergii, the pathogen of rose black spot disease, and other plant pathogens. It also promotes the growth and flowering of roses by dissolving phosphorus, fixing nitrogen, and tolerating salt and heavy metal stress.
It effectively prevents and controls black spot and powdery mildew in roses, promotes rose growth and flowering, with control efficacy of 93.3% and 76.8% respectively, and increases plant height and number of flowers.
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Abstract
Description
Technical Field
[0001] This invention relates to a fluorescent pseudomonad that controls rose black spot disease and promotes rose growth, and its application. Specifically, it relates to a fluorescent pseudomonad (Pseudomonas fluorescens) LX6 that can inhibit the growth of rose pathogens such as *Discocephalus roseus*, *Colletotrichum gloeosporioides*, *Rhizoctonia solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium*, and its application. Background Technology
[0002] Rose is a general term for hybrid varieties formed from the hybridization of plants in the genus *Rosa*. It is not only one of China's ten traditional famous flowers, but also edible and used for extracting essential oils. During the cultivation of roses, they are often attacked by various diseases such as black spot, powdery mildew, and anthracnose. Among these, rose black spot is the most serious, causing damage to roses grown in open fields. The pathogen is *Diplocarpon rosae*, a facultative parasitic ascomycete, with its asexual form being *Marssonina rosae* (synonym *Actinonema rosae*). Infected plants develop irregular black spots on the leaf edges. In later stages, the leaves turn yellow and fall off, sometimes leading to the death of the entire plant. Infected plants also exhibit reduced vitality and are more susceptible to other abiotic stresses such as frost and drought, severely hindering the development of the rose industry.
[0003] Currently, the control of rose black spot disease in production mainly relies on chemical agents. Since rose black spot disease is a multi-cycle disease, effective prevention of large-scale outbreaks requires frequent application of pesticides throughout the growing season, such as carbendazim and iprodione. Long-term use of large quantities of chemical agents not only increases costs but also pollutes the environment with pesticide residues. Furthermore, the use of chemical agents can lead to targeted selection of pathogens, resulting in drug resistance. With the emergence of the concept of "green plant protection," safe, low-toxicity, long-lasting, and less likely to induce drug resistance biological control has received increasing attention. Therefore, applying green and pollution-free beneficial microorganisms to control rose diseases is the future direction of development.
[0004] The genus *Pseudomonas* comprises numerous species and is widely distributed in the environment. It is one of the main beneficial bacteria currently used for plant disease prevention and growth promotion, possessing advantages such as wide distribution, rapid reproduction, easy colonization, and abundant metabolites. Currently reported *Pseudomonas* species with disease prevention and growth promotion effects include *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, *Pseudomonas spp.*, *Pseudomonas brassicae*, and *Pseudomonas putida*. These *Pseudomonas* species can increase the yield of various plants such as tomatoes, cucumbers, soybeans, and lettuce through mechanisms such as enhancing phosphate and iron solubility, nitrogen fixation, and the production of plant hormones. They can also control plant diseases caused by various pathogens such as *Fusarium*, *Rhizoctonia solani*, *Pythium*, and *Botrytis cinerea* through mechanisms such as producing antibacterial secondary metabolites and inducing resistance. Although *Pseudomonas* are widely used in plant growth promotion and disease prevention, there are currently no reports of their application in the prevention and promotion of disease and growth in roses. Summary of the Invention
[0005] The purpose of this invention is to provide a bacterium that inhibits the growth of rose pathogens such as *Diplosporium rosenbergii* and other plant pathogens such as *Anthracnose collodion*, *Rhizoctonia solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium*, and is capable of phosphorus solubilization, nitrogen fixation, production of ironophiles and cellulase, and tolerance to salt and heavy metal stress, and its application in the prevention and control of rose black spot and powdery mildew, and in promoting growth and flowering.
[0006] The bacterium for controlling rose diseases and promoting growth provided by this invention is *Pseudomonas fluorescens* LX6. *Pseudomonas fluorescens* LX6 was deposited on May 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCC No. 30657. *Pseudomonas fluorescens* LX6 was isolated from soil and is a Gram-negative bacterium. On LB agar, the bacteria form milky-white colonies that are round, with regular edges, smooth, flat, opaque, and moist.
[0007] Biological agents or microbial fertilizers containing the active ingredient of Pseudomonas fluorescens LX6 are also within the scope of protection of this invention.
[0008] Experiments have shown that the bacterium Pseudomonas fluorescens LX6, which can prevent and control various diseases of roses and promote their growth, is effective in preventing and controlling rose black spot disease, while also effectively promoting rose growth and flowering.
[0009] The bacterium of this invention, *Pseudomonas fluorescens* LX6, isolated from soil, exhibits growth-promoting and disease-preventing effects on roses. Plate inhibition experiments showed that this strain inhibits the growth of *Discodilobacter rosenbergii*, the pathogen of rose black spot disease. This strain is also tolerant to salt and heavy metal stress, and possesses properties such as dissolving inorganic and organic phosphorus, producing hematophilic acid and cellulase, and nitrogen fixation. In vitro leaf efficacy tests showed that this strain achieved a control efficacy of 93.3% against rose black spot disease. Greenhouse experiments showed that this strain achieved a control efficacy of 76.8% against rose powdery mildew. Rose plants treated with this strain were 9.2 cm taller than the control, and each plant produced 0.5 more flowers than the control. The acquisition of this strain is expected to provide an environmentally friendly, simple, and effective method for rose disease control and growth promotion. *Pseudomonas fluorescens* LX6 of this invention is a strain with promising applications in disease prevention and growth promotion. Attached Figure Description
[0010] Figure 1 The results show the inhibition of Pseudomonas fluorescens LX6 on Diplosporium rosenbergii and other plant pathogens.
[0011] Figure 2 To determine the efficacy of Pseudomonas fluorescens LX6 against black spot disease in roses.
[0012] Figure 3 To determine the colony characteristics of Pseudomonas fluorescens LX6 on cellulase, organic and inorganic phosphorus dissolution, ironophilic, and nitrogen fixation detection plates.
[0013] Figure 4 The growth of Pseudomonas fluorescens LX6 on LB agar plates supplemented with heavy metals and salts. Pb 2+ Lead nitrate 50 mg / L; Cd 2+ Cadmium sulfate octahydrate 30 mg / L; Cu 2+ Copper sulfate 50 mg / L; Zn 2+ Zinc sulfate 150 mg / L; Na + Sodium chloride 800 mmol / L.
[0014] Preservation of biological materials
[0015] Accession number: CGMCC No. 30657
[0016] Name: Pseudomonas fluorescens LX6
[0017] Classification and nomenclature: Pseudomonas fluorescens
[0018] Survival status: Survival
[0019] Preservation period: May 16, 2024
[0020] Preservation Institution: China General Microbiological Culture Collection Center, China Association for the Preservation and Management of Microbial Cultures, Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Detailed Implementation
[0021] Unless otherwise specified, the methods described in the following embodiments are conventional methods.
[0022] Unless otherwise specified, all percentages in the following examples are by mass percentages.
[0023] Example 1: Screening of bacteria that inhibit the growth of black spot fungus in roses
[0024] To screen for bacteria antagonizing the rose black spot fungus *Discocephalus rosea*, 300 bacterial strains previously isolated from soil and preserved in our laboratory were inoculated into 2.5 mL centrifuge tubes containing 2 mL of LB medium (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, diluted to 1000 mL with distilled water, pH 7.0). After incubation overnight at 30°C and 180 rpm, 1 mL of the bacterial culture was transferred to three bottles containing 100 mL of LB medium and incubated at 30°C and 180 rpm for 24 h. The collected bacterial culture was centrifuged at 10000 × g for 20 min, and the supernatant was filtered through a 0.22 μm filter. The supernatant was mixed with autoclaved and cooled PDA (400 g potato, 40 g sucrose, 40 g agar, diluted to 1000 mL with distilled water) in equal proportions, shaken well, and poured into 9 cm diameter petri dishes. The mixture was allowed to solidify before use. The pathogen *Discocele rosenbergii*, the pathogen causing black spot disease of roses, was cultured on PDA medium (200g potato, 20g sucrose, 20g agar, and distilled water to a final volume of 1000mL) at 30℃ for 30 days. Mycelial discs with a diameter of 0.5cm were taken from the edge of each colony using a punch and placed in the center of a PDA plate containing bacterial supernatant. PDA plates containing sterile water served as a control. Each treatment was repeated three times, and cultured at 30℃ for 30 days. The colony diameter was measured, and the inhibition rate was calculated. The formula was: Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / (Coronary diameter of control group - Diameter of mycelial disc) × 100%
[0025] Screening results showed that a sterile metabolic broth of bacterial strain LX6 could effectively inhibit the growth of *Diplostomum villosum*, a pathogen of roses, with an inhibition rate of 72.5%. Figure 1 (and Table 1).
[0026] Table 1. Inhibitory effect of strain LX6 on rose pathogens.
[0027]
[0028] * This indicates a difference between the control and the control at the P<0.05 level.
[0029] Example 2: LX6 inhibits spore germination and germ tube elongation of rose black spot fungus.
[0030] Following the method described in Example 1, *Dioscorea rosea* was cultured. The plates were rinsed with sterile water, and fungal conidia were collected. The spore suspension was filtered through sterile gauze, and then diluted with PDB (200g potato, 20g sucrose, and distilled water to a final volume of 1000mL) until the spore concentration reached 1×10⁻⁶. 5 Spores / mL. Antagonistic bacteria LX6 were cultured according to the method described in Example 1, and the sterile supernatant was collected. The spore suspension of *Discochaeta spp.* and the sterile supernatant were mixed at a volume ratio of 1 / 10 and cultured at 28°C and 180 rpm. Sterile water was used as a control, and each treatment was repeated in triplicate. After 14 days, the germination of 100 spores per treatment was observed and recorded, and the length of the germ tube was measured.
[0031] The results showed that after 14 days of treatment, the germination rate of *Diplosporium rosenbergii* spores in the control group was 100%, and the germ tube length was 8.1 μm, while the germination rate of spores treated with strain LX6 was only 37.0%, and the germ tube length was only 0.48 μm (Table 2).
[0032] Table 2. Inhibitory effect of LX6 on spore germination and germ tube elongation of Diplospermia rosenbergii.
[0033] deal with Spore germination rate (%) Germ tube length (μm) Comparison 100.0±0.0 8.10±3.13 LX6 <![CDATA[37.0±5.5 * ]]> <![CDATA[0.48±0.83 * ]]>
[0034] * This indicates a difference between the control and the control at the P<0.05 level.
[0035] Example 3: Determination of LX6's antagonism against other plant pathogenic fungi
[0036] The plate confrontation method was used to determine whether LX6 had an antagonistic effect on the plant pathogenic fungi *Anthracis colloidalis*, *Rhizoctonia solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium*. Two pathogenic fungi and the bacterial strain to be screened were cultured according to the above method. A 0.5 cm diameter fungal disc was placed in the center of PDA medium, and 2 μL of the bacterial suspension to be tested was inoculated 3 cm from the center. Three inoculations were performed per petri dish, with three replicates. Plates inoculated with sterile water served as controls. The culture was carried out at 30℃ for 7 days, and the colony diameter was measured to calculate the inhibition rate. The calculation formula was: Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / (Coronary diameter of control group - Diameter of fungal disc) × 100%
[0037] LX6 exhibits antagonistic effects against plant pathogens *Anthracis colloidalis*, *Rhizoctonia solani*, *Fusarium oxysporum*, and *Fusarium equisetifolium*, with inhibition rates of 33.8%, 33.8%, 40.9%, and 32.5%, respectively. Figure 1 (and Table 3).
[0038] Table 3. Inhibitory effect of strain LX6 on rose pathogens.
[0039]
[0040]
[0041] * This indicates a difference between the control and the control at the P<0.05 level.
[0042] Example 3: Species identification of LX6 strain
[0043] LX6 strain was identified as *Pseudomonas fluorescens* using routine physiological and biochemical methods and 16S rDNA sequence analysis. The physiological, biochemical, and basic biological characteristics of this strain are shown in Table 4.
[0044] Table 4 Basic biological characteristics of LX6
[0045]
[0046] Note: "+" indicates that a carbon source (or nitrogen source) can be utilized, and "-" indicates that a carbon source (or nitrogen source) cannot be utilized.
[0047] Using the genomic DNA of strain LX6 as a template, and universal primers for bacterial 16S rDNA sequences (27F: 5'-AGAGTITGATCCTGGCTCAG-3'; reverse primer 1492R: 5'-TACGGGTACCTTGTTACGACTT-3'), a product fragment of approximately 1.5 kb was amplified by PCR. Sequencing with 27F and 1492R yielded 1414 nucleotides (Sequence 1 in the sequence listing). BLAST alignment of this sequence with NCBI showed that the 16S rDNA sequence of LX6 shared 99.93% homology with that of Pseudomonas fluorescens strain PS15 (accession number: OR485143.1).
[0048] According to the *Handbook of Common Bacteria Identification*, strain LX6 exhibits the same physiological and biochemical characteristics as *Pseudomonas fluorescens*. Based on the aforementioned molecular and biochemical identifications, it was identified as *Pseudomonas fluorescens*. LX5 was named *Pseudomonas fluorescens* LX6. This strain was deposited on May 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 30657.
[0049] Example 4: Efficacy test of P. fluorescens LX6 against rose diseases
[0050] 1. Efficacy test for control of black spot disease in roses
[0051] P. fluorescens LX6 was cultured according to the method described in Example 1, and the bacterial concentration was adjusted to 1×10⁶ using sterile water. 8 GFU / mL. Select healthy leaves of the 'Monthly Powder' rose variety of uniform size, disinfect them with 75% alcohol for 2 minutes, and rinse them three times with sterile water. Blot the leaves dry with sterile filter paper and immerse them in the prepared LX6 bacterial suspension for 5 seconds. Place them in a petri dish lined with double-layered filter paper, adding 5 mL of sterile water to each dish to maintain humidity. Culture the rose black spot pathogen according to the method in Example 1, add sterile water to wash away the spores, filter through double-layered sterile gauze, and dilute the spore suspension to 10 g / mL with sterile water. 6 CFU / mL. A 15 μL suspension of pathogenic spores was dropped onto the center of the leaf surface and incubated at 25°C for 12 h / 12 h light / dark. To maintain humidity, 5 mL of sterile water was added to the culture dish every 3 days after inoculation. Three leaves were treated per batch, with three replicates, using sterile water as a control. Disease incidence was assessed 14 days after inoculation according to disease severity grading.
[0052] Black spot disease grading criteria using detached leaf method:
[0053] Disease level Grading Standards Level 0 Asymptomatic Level 1 Lesions appear, but there are no conidiophores or conidia. Level 2 Visible lesions are present, with conidiophores but no conidia. Level 3 Visible lesions appear, with conidiophores and a small number of conidia. Level 4 The lesion area is larger than the inoculated area, and contains conidiophores and a small number of conidia. Level 5 The lesions were larger than the inoculated area and contained numerous conidiophores and conidia.
[0054] Note: The severity of the disease is classified into five levels: 0, 1, 2, 3, 4, and 5, based on the different symptoms and degrees of the disease.
[0055] Calculate the control efficiency using the following formula:
[0056] Incidence rate (%) = Number of symptomatic leaves / Number of tested leaves × 100
[0057] Disease index = [∑(number of diseased leaves at each level × disease severity)] / (total number of leaves surveyed × highest disease severity) × 100
[0058] Efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100
[0059] The results showed that 14 days after inoculation with rose black spot fungus, the leaves of the control group all developed large areas of typical black spot lesions. Pretreatment with LX6 bacterial suspension significantly reduced the symptoms, achieving a control efficacy of 93.3%. Figure 2 (and Table 5).
[0060] Table 5. Determination of the control efficacy of LX6 against black spot disease in roses using the detached leaf method.
[0061] deal with Incidence rate (%) Disease index Preventive efficacy (%) sterile water 0.0±0.0b 0.0±0.0b / Sterile water + Diplospermia ulmoides 100.0±0.0a 100.0±0a / LX6+ Rose Disc Diplosporum 22.2±11.1b 6.7±6.7b 93.3
[0062] Note: Different letters after the data in the same column indicate significant differences (P<0.05).
[0063] 2. Efficacy test for controlling powdery mildew in roses
[0064] 'Monthly Powder' cuttings were planted in a vermiculite:peat moss = 1:1 soil mixture and placed in a glass greenhouse at 22℃ / 16℃ for 12h / 12h light / dark conditions when powdery mildew was severe. A concentration of 1×10⁻⁶ was prepared according to the method in Example 1. 8 GFU / mL *P. fluorescens* LX6 bacterial suspension. The prepared LX6 bacterial suspension was evenly sprayed onto the surface of each leaf of the cuttings. Water and a 2000-fold dilution of Kangpu (a Chinese herbal medicine) were used as controls. Each treatment consisted of 4 plants and 3 replicates. Disease incidence was observed 20 days after treatment. The grading criteria were: Grade 0: No lesions; Grade 1: Lesions covering less than 1 / 4 of the total leaf area; Grade 2: Lesions covering 1 / 4 to less than 1 / 2 of the total leaf area; Grade 3: Lesions covering 1 / 2 to less than 3 / 4 of the total leaf area; Grade 4: Lesions covering 3 / 4 or more of the total leaf area. The control efficiency was calculated using the following formula:
[0065] Incidence rate (%) = Number of symptomatic plants / Number of tested plants × 100
[0066] Disease index = [∑(number of diseased plants at each level × disease level)] / (total number of plants surveyed × highest disease level) × 100
[0067] Efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100
[0068] Twenty days after inoculation, the experimental results were analyzed. The results showed that all plants in the water control group had lesions, with a disease index of 27.1. After pretreatment with LX6 bacterial suspension, the disease index after inoculation with the pathogen was only 6.3, achieving a control efficacy of 76.8% (Table 6). In contrast, treatment with a commonly used pesticide, Kangpu 2000-fold dilution, only achieved a control efficacy of 54.2%. The experimental results indicate that pre-inoculation with LX6 can effectively prevent the occurrence of powdery mildew in roses.
[0069] Table 6. Greenhouse control efficacy of LX6 against powdery mildew in roses
[0070] deal with Incidence rate (%) Disease index Preventive efficacy (%) Water comparison 66.7±8.3a 27.1±4.2a / Kangpu 2000 times dilution 50.0±14.4ab 12.4±3.5b 54.2 LX6 25.0±14.4b 6.3±3.6b 76.8
[0071] Note: Different letters after the data in the same column indicate significant differences (P<0.05).
[0072] Example 4: Determination of P. fluorescens LX6's effect on promoting rose growth and flowering.
[0073] The culture and preparation of a concentration of 1×10 were carried out according to the method in Example 1. 8 A GFU / mL suspension of *P. fluorescens* LX6 was prepared. *Rosebud* cuttings were planted according to the method described in Example 3. Plants with uniform growth were selected, and the prepared LX6 suspension was evenly sprayed onto the leaf surface of the cuttings, with sterile water as a control. Growth was observed after 60 days, and the plant height was measured. The results showed that the plant height of *Rosebud* treated with sterile water was 15.3 cm, while the plant height treated with *P. fluorescens* LX6 suspension was 24.5 cm, significantly higher than the control (Table 7). The average number of flowers per plant treated with LX6 suspension was 0.8, while the control group averaged only 0.3 flowers per plant (Table 7). These results indicate that *P. fluorescens* LX6 can promote rose growth and flowering.
[0074] Table 7. LX6 promotes rose growth and flowering
[0075] deal with Plant height (cm) Number of flowers / plant Comparison 15.3±1.8 0.3±0.2 LX6 <![CDATA[24.5±3.8 * ]]> <![CDATA[0.8±0.2 * ]]>
[0076] * This indicates a difference between the control and the control at the P<0.05 level.
[0077] Example 5: P. fluorescens LX6 can produce cellulase.
[0078] Activated *P. fluorescens* LX6 was inoculated into LB broth and incubated overnight at 28°C and 200 rpm with shaking. 10 μL of the culture was added dropwise to a cellulase activity assay plate (10 g peptone, 10 g yeast extract, 10 g sodium carboxymethyl cellulose, 1 g potassium dihydrogen phosphate, 5 g sodium chloride, 15 g agar, and distilled water to a final volume of 1000 mL), repeated three times. After drying in a clean bench, the plates were incubated upside down at 28°C in the dark for 7 days, observing for the appearance of hydrolysis halos.
[0079] The results showed that hydrolysis halos appeared around the colonies on cellulase activity assay plates inoculated with P. fluorescens LX6 (e.g., ...). Figure 3 As shown in the figure, this indicates that P. fluorescens LX6 has the ability to produce cellulase.
[0080] Example 6: P. fluorescens LX6 can dissolve both inorganic and organic phosphorus.
[0081] Strain LX6 was cultured according to the method in Example 5. 10 μL of the culture solution was added dropwise to an inorganic phosphorus medium plate (0.5 g yeast extract, 10 g glucose, 5 g tricalcium phosphate, 0.5 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g potassium chloride, 0.3 g sodium chloride, 0.03 g manganese sulfate, 0.03 g ferrous sulfate, 15 g agar, 1000 mL distilled water), and this was repeated three times. After drying in a clean bench, the plates were incubated upside down at 28°C in the dark for 7 days, and the presence of hydrolysis halos was observed. Simultaneously, 10 μL of the culture solution was inoculated onto an organic phosphorus medium plate (10 g glucose, 1 g calcium carbonate, 0.5 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g potassium chloride, 0.3 g sodium chloride, 0.03 g manganese sulfate, 0.03 g ferrous sulfate, 0.2 g lecithin, 15 g agar, and distilled water to a final volume of 1000 mL), and this was repeated three times. After drying in a clean bench, the samples were incubated upside down at 28°C in the dark for 7 days to observe whether hydrolysis halos appeared.
[0082] The results showed that hydrolysis halos appeared around P. fluorescens LX6 colonies in inorganic phosphorus media, indicating that P. fluorescens LX6 can hydrolyze inorganic phosphorus. Weak hydrolysis halos also appeared around P. fluorescens LX6 colonies in organic phosphorus media (e.g., ...). Figure 3 As shown in the figure, this indicates that P. fluorescens LX6 can hydrolyze organophosphorus compounds.
[0083] Example 7: P. fluorescens LX6 can produce heparin.
[0084] Following the method described in Example 5, strain LX6 was cultured. 10 μL of the culture solution was added dropwise to a ferrophilic CAS agar plate (Crazin S 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg, diluted to 1000 mL with distilled water, pH 6.8 ± 0.2). This was repeated three times. After drying in a clean bench, the plates were incubated upside down at 28°C in the dark for 3 days. The color of the medium around the colonies was observed to change from blue to orange-yellow. The results showed that a blue fading halo appeared around the colonies of *P. fluorescens* LX6 (e.g., ...). Figure 3 As shown in the figure, this indicates that P. fluorescens LX6 has the ability to produce ferrophiles.
[0085] Example 8: P. fluorescens LX6 is capable of nitrogen fixation.
[0086] Strain LX6 was cultured according to the method described in Example 5. 10 μL of the culture solution was added dropwise to Assab nitrogen-fixing medium (0.2 g magnesium sulfate, 5 g calcium carbonate, 0.2 g potassium dihydrogen phosphate, 10 g mannitol, 0.1 g calcium sulfate, 0.2 g sodium chloride, 15 g agar, and distilled water to a final volume of 1000 mL). After drying in a clean bench, the medium was incubated upside down at 28°C in the dark for 7 days. Colony formation was observed, and each treatment was repeated three times. If colonies appeared, a single colony was subcultured twice more. Strains that could still grow stably on Assab nitrogen-fixing medium in the third generation indicated nitrogen-fixing ability. *Escherichia coli* was used as a negative control.
[0087] The results showed that LX6 could grow normally on Assumption medium, while the negative control Escherichia coli could not grow, indicating that strain LX6 has nitrogen-fixing ability. Figure 3 ).
[0088] Example 9: P. fluorescens LX6 can withstand various heavy metal and salt stresses.
[0089] Sterilized LB medium, cooled to approximately 65°C, was supplemented with filtered, sterilized heavy metal solutions to final concentrations of 30 mg / L chromium chloride, 50 mg / L copper, 50 mg / L lead, and 150 mg / L zinc. LB agar plates with a final concentration of 800 mmol / L NaCl were also prepared. Bacterial cells were picked from freshly cultured LX6 colonies using sterile toothpicks and streaked onto the prepared LB agar plates containing the different heavy metals. The plates were incubated at 28°C in the dark for 7 days, and the growth of LX6 cells was observed.
[0090] The results showed that strain LX6 was able to grow normally in LB cultures containing high concentrations of cadmium, copper, lead, or zinc. Figure 4 And it can be used in a solution containing 800 mmol / L Na. 2+ Normal growth on the plate ( Figure 4 These results demonstrate that strain LX6 has the ability to tolerate various heavy metal ions and salt stress.
[0091] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A strain of *Pseudomonas fluorescens*, characterized in that, The name is *Pseudomonas fluorescens* ( Pseudomonas fluorescens LX6, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 30657.
2. The application of the fluorescent Pseudomonas as described in claim 1 in the prevention and control of plant diseases; wherein the plant disease is rose black spot disease caused by Diplostomum rosenbergii.
3. The use of the fluorescent Pseudomonas as described in claim 1 in the preparation of biocontrol agents or microbial fertilizers targeting plant pathogenic fungi and bacteria; wherein the plant pathogenic fungi are *Discocephalus rosea* (…). Marssonina rosae ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Horsetail ( Fusarium equiseti ) and Fusarium oxysporum ( Fusarium oxysporum ).
4. A biocontrol agent for preventing and controlling plant diseases, characterized in that, The active ingredient of the biocontrol agent is *Pseudomonas fluorescens* (…). Pseudomonas fluorescens LX6, with accession number CGMCC No. 30657.
5. A microbial fertilizer for preventing and controlling plant diseases, characterized in that, The active ingredient in the microbial fertilizer is *Pseudomonas fluorescens* (…). Pseudomonas fluorescens LX6, with accession number CGMCC No. 30657.
6. The biocontrol agent according to claim 4 or the microbial fertilizer according to claim 5, characterized in that, The plant disease mentioned is black spot disease of roses caused by Diplostomum rosaemonii.
7. *Pseudomonas fluorescens* ( Pseudomonas fluorescens Application of CGMCC No. 30657 in phosphorus solubilization, nitrogen fixation, iron production, and cellulase production.
8. *Pseudomonas fluorescens* ( Pseudomonas fluorescens Application of CGMCC No. 30657 in promoting rose growth and flowering.