Pseudomonas canavaninivorans and application thereof

Abstract

The application discloses a bacterium Pseudomonas canavaninivorans for preventing and treating diseases and promoting growth of Chinese rose, and application thereof. The bacterium has been preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 30656 and a classification name of Pseudomonas canavaninivorans. The bacterium is gram-negative, can produce protease, cellulase and siderophore, can dissolve phosphorus and fix nitrogen, and can resist salt and various heavy metal stresses. The bacterium can inhibit growth of Cylindrocarpon destructans, and can antagonize growth of other plant pathogens Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum and F. equiseti, has a wide antibacterial spectrum, and can be applied in a wide range. The bacterium can prevent and treat Chinese rose black spot and powdery mildew, and can promote growth of Chinese rose. The application provides a fine biocontrol strain for preventing and treating diseases of Chinese rose and promoting growth of Chinese rose, and has important significance for green prevention and control technology of diseases of Chinese rose and sustainable development of the Chinese rose industry.

Description

TECHNICAL FIELD

[0001] The present application relates to a Pseudomonas canavaninivorans LX5 capable of inhibiting the growth of the black spot disease pathogen Marssonina rosae, and other plant pathogens Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum and Fusarium equiseti, as well as its application. The strain has the ability to dissolve phosphorus, fix nitrogen, produce siderophores, proteases and cellulases, and is resistant to salt and heavy metal stress, and can prevent and control black spot disease and powdery mildew of roses, and can promote the growth of roses. BACKGROUND

[0002] Rosa is a common name for hybrid varieties of the Rosa genus formed by crossbreeding. It is not only one of the top ten traditional flowers in China, but also edible and can extract essential oil. During the cultivation of roses, they are often attacked by various diseases such as black spot disease, gray mold disease, powdery mildew, and anthracnose. Among them, black spot disease of roses is the most harmful to open field cultivation, and the pathogen is the facultative parasitic Ascomycota Diplocarpon rosae, and the asexual state is Marssonina rosae (alias Actinonema rosae). The diseased plant leaves show irregular black spots on the edges, and the leaves turn yellow and fall off at the later stage, and even the whole plant dies. The diseased plants have lower vitality and are more susceptible to other abiotic stresses such as frost and drought, which seriously restricts the development of the rose industry.

[0003] Currently, the prevention and control of black spot disease of roses in production mainly relies on chemical agents. Since black spot disease of roses is a multi-cycle disease, effective prevention of large-scale occurrence of black spot disease requires frequent application of chemicals such as carbendazim and prochloraz throughout the growing season. Long-term use of large amounts of chemical agents not only increases costs, but also pollutes the environment due to residual pesticides. In addition, the use of chemical agents can also cause directional selection of pathogenic fungi to produce drug resistance. With the proposal of the concept of "green plant protection", safe, low-toxic, long-acting, and less likely to produce drug-resistant biological control has received more and more attention. Therefore, the application of green and pollution-free beneficial microorganisms to prevent and control rose diseases is the direction of future development.

[0004] Pseudomonas bacteria are widely present in the environment and are one of the main beneficial bacteria currently applied to plant disease prevention and growth promotion, and have the advantages of wide distribution, rapid reproduction, easy colonization, and rich metabolic products. The main Pseudomonas bacteria reported to have disease prevention and growth promotion effects include P. fluorescens, P. aeruginosa, P. chlororaphis, P. brassicacearum, and P. putida. These Pseudomonas bacteria can improve the yield of tomatoes, cucumbers, soybeans, and lettuce through mechanisms such as increasing phosphate and iron dissolution, nitrogen fixation, and plant hormone production. They can also prevent plant diseases caused by Fusarium, Rhizoctonia, Pythium, and Botrytis cinerea through mechanisms such as producing antibacterial secondary metabolites and inducing resistance. Although Pseudomonas bacteria have been widely applied to plant growth promotion and disease prevention, there is no reported application of Pseudomonas bacteria in rose disease prevention and growth promotion. SUMMARY

[0005] The purpose of the present application is to provide a strain of bacteria that can inhibit the growth of rose pathogenic fungi Dissoconium rosarium and other four plant pathogenic fungi, and can dissolve phosphorus, fix nitrogen, produce siderophores, proteases and cellulases, and resist salt and heavy metal stress, as well as its application in rose black spot and powdery mildew prevention and growth promotion.

[0006] The rose disease prevention and growth promotion bacteria provided by the present application are Pseudomonas canavaninivorans LX5.

[0007] The Pseudomonas canavaninivorans LX5 has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on May 16, 2024, and the deposit number is CGMCC No. 30656. The Pseudomonas canavaninivorans LX5 is a gram-negative bacterium isolated from soil. The bacterial cells can form white colonies on LB medium, and the colonies are round with regular edges, smooth surface, and slight viscosity.

[0008] The bioinsecticide or microbial fertilizer containing the above Pseudomonas canavaninivorans LX5 as the active ingredient also falls within the protection scope of the present application.

[0009] Experiments prove that the bacteria capable of preventing and treating various diseases of Chinese rose and promoting growth in the application is Pseudomonas canavaninivorans LX5, which can effectively prevent and treat Chinese rose black spot and Chinese rose powdery mildew, and can effectively promote the growth of Chinese rose.

[0010] The bacteria with the disease-preventing and growth-promoting effect on Chinese rose in the application is Pseudomonas canavaninivorans LX5, which is isolated from soil. The plate antibacterial experiment shows that the strain has good antagonistic effect on Chinese rose black spot pathogen Diaporthe phaseolorum, and other plant pathogenic fungi Botrytis cinerea, Glomerella cingulata, Fusarium oxysporum and Fusarium equiseti. The strain can also resist salt and heavy metal stress, can produce protease, cellulase and siderophore, and can fix nitrogen, dissolve inorganic phosphorus and organic phosphorus. The in vitro leaf protection efficiency experiment shows that the strain has a disease control effect of 91.1% on Chinese rose black spot. The greenhouse experiment shows that the strain can also prevent and treat Chinese rose powdery mildew, and the disease control effect can reach 69.0%; the plant height of Chinese rose treated by the strain is increased by 8.7 cm compared with the control. The strain is expected to provide an environmentally friendly, simple and effective way for disease control and growth promotion of Chinese rose. The Pseudomonas canavaninivorans LX5 of the application is a strain with good disease-preventing and growth-promoting application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Pseudomonas canavaninivorans LX5 growth inhibition results on pathogenic bacteria.

[0012] Figure 2 Pseudomonas canavaninivorans LX5 disease control effect on Chinese rose black spot.

[0013] Figure 3 Pseudomonas canavaninivorans LX5 colony characteristics on protease, cellulase, siderophore, inorganic phosphorus and organic phosphorus dissolution, and nitrogen fixation detection plates.

[0014] Figure 4 Pseudomonas canavaninivorans LX5 growth on LB plates added with heavy metals and salt. 2+ Cadmium sulfate octahydrate 30 mg / L; Pb 2+ Lead nitrate 50 mg / L; Cu 2+ Copper sulfate 50 mg / L; Zn 2+ Zinc sulfate 150 mg / L; Na + Sodium chloride 800 mmol / L.

[0015] Biological material preservation

[0016] Accession No.: CGMCC No. 30656

[0017] Name: Pseudomonas canavaninivorans LX5

[0018] Taxonomic designation: Pseudomonas canavaninivorans

[0019] Whether alive: alive

[0020] Preservation time: May 16, 2024

[0021] Preservation agency: China General Microbiological Culture Collection Center, Beijing, China DETAILED DESCRIPTION

[0022] The methods in the following examples are routine methods unless otherwise specified.

[0023] Example 1, Screening of bacteria inhibiting the growth of Diaporthe phaseolorum and determination of antibacterial spectrum thereof

[0024] In order to screen bacteria against Diaporthe phaseolorum, 300 bacteria isolated from soil in the laboratory were inoculated in 2.5 mL centrifuge tubes containing 2 mL LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of distilled water, pH 7.0) and cultured at 30°C and 180 rpm overnight. Then, 1 mL of bacterial solution was transferred into three 100 mL LB medium bottles and cultured at 30°C and 180 rpm for 24 h. The collected bacterial solution was centrifuged at 10000 x g for 20 min, and the supernatant was filtered with a 0.22 μm filter. The supernatant was mixed with high-pressure sterilized PDA (400 g of potato, 40 g of sucrose, 40 g of agar, 1000 mL of distilled water) cooled to 45°C at a certain ratio, shaken well, and poured into a 9 cm diameter culture dish. After solidification, it was used. Diaporthe phaseolorum was cultured on PDA medium (200 g of potato, 20 g of sucrose, 20 g of agar, 1000 mL of distilled water) at 30°C for 30 days. A 0.5 cm diameter bacterial cake was taken from the edge of the colony with a puncher and placed in the center of the PDA plate mixed with bacterial supernatant. The PDA plate with sterile water was used as a control. Each treatment was repeated three times, and the colony diameter was measured after 30 days of culture at 30°C. The inhibition rate was calculated.

[0025] The antagonistic effect of the above-mentioned bacterial strains on the plant pathogenic fungi Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum and F. solani was determined by means of the flat plate confrontation method. The pathogenic fungi and the screened bacterial strain antagonizing Diaporthe phaseolorum were cultured according to the above-mentioned method. A pathogenic fungus cake with a diameter of 0.5 cm was placed in the center of a PDA culture medium, and 2 μL of a bacterial suspension to be tested was inoculated at a position 3 cm from the center point, with 3 inoculation points per culture dish and 3 repeats. A flat plate inoculated with sterile water was used as a control. The culture was incubated at 30°C for 7 days, and the colony diameter was measured, and the inhibition rate was calculated. The calculation formula was: inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / (colony diameter of the control group - cake diameter) x 100%

[0026] The screening results showed that the sterile metabolic solution of a bacterial strain LX5 could effectively inhibit the growth of the rose pathogenic fungus Diaporthe phaseolorum, with an inhibition rate of 86.9%. The bacterial strain had antagonistic effects on the plant pathogenic fungi Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum and F. solani (see Table 1). Figure 1

[0027] Table 1 Inhibition of the rose pathogenic fungus by the bacterial strain LX5

[0028]

[0029]

[0030] * Indicates a difference from the control at the P<0.05 level.

[0031] Example 2: Inhibition of spore germination and germ tube elongation of the rose black spot fungus by LX5

[0032] The rose black spot fungus was cultured according to the method of Example 1. The flat plate was washed with sterile water to collect the fungal conidia. The conidial suspension was filtered through sterile gauze, and then diluted with PDB (potato 200 g, sucrose 20 g, distilled water 1000 mL) until the conidial concentration was 1 x 10 5 The antagonistic bacterial strain LX5 was cultured according to the method of Example 1, and the sterile supernatant was collected. The conidial suspension of the rose black spot fungus 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 three times. After 14 days, the germination of 100 spores was observed and recorded, and the length of the germ tube was measured.

[0033] The results showed that, after 14 days of treatment, the germination rate of the conidia of the rose black spot fungus in the control group was 100%, and the length of the germ tube was 8.1 μm, while the germination rate of the conidia treated with the bacterial strain LX5 was only 35.47%, and the length of the germ tube was only 2.86 μm (Table 2).

[0034] ​Table 2L Inhibition of spore germination and germ tube elongation of Dispira rosae by LX5

[0035] Treatment Spore germination rate (%) Length of germ tube (μm) Control 100.0±0.0 8.10±3.13 LX5 35.3 ± 7.4 * ]] 2.86 ± 0.83 * ]]

[0036] * The difference between the test and the control was significant at the P < 0.05 level.

[0037] Example 3, Species identification of LX5 strain

[0038] The LX5 strain was identified by routine physiological and biochemical tests and 16S rDNA sequence analysis, and was confirmed to be Pseudomonas canavaninivorans. The physiological and biochemical characteristics and basic biological characteristics of the strain are shown in Table 3.

[0039] Table 3 Basic biological characteristics of LX5

[0040]

[0041]

[0042] Note: "+" indicates that the carbon source (or nitrogen source) can be utilized, and "-" indicates that the carbon source (or nitrogen source) cannot be utilized

[0043] The genomic DNA of the LX5 strain was used as a template, and bacterial 16S rDNA sequence universal primers were used as primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; reverse primer 1492R: 5'-TACGGGTACCTTGTTACGACTT-3'), and a product fragment of about 1.5 kb was amplified by PCR, and 1433 nucleotides were obtained by sequencing with 27F and 1492R (Sequence 1 in the sequence listing). The sequence has the highest homology with Pseudomonas canavaninivorans (Accession No.: NR_181810.1), reaching 99.93%.

[0044] According to the Handbook of Common Bacterial Identification, the physiological and biochemical characteristics of the LX5 strain are the same as those of Pseudomonas canavaninivorans, and combined with the above molecular identification, it is identified as Pseudomonas canavaninivorans. LX5 is named Pseudomonas canavaninivorans LX5. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) (address: No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, China) on May 16, 2024, and the preservation number is CGMCC NO. 30656.

[0045] Example 4, Determination of the efficacy of Pseudomonas canavaninivorans LX5 against rose disease

[0046] 1. Determination of the efficacy against rose black spot

[0047] Pseudomonas canavaninivorans LX5 was cultured according to the method in Example 1, and the concentration of the bacterial body was adjusted to 1 x 10 8 GFU / mL with sterile water. Healthy leaves of rose 'Yueyuefen' with uniform size were selected, surface sterilized with 75% alcohol for 2 min, and then washed with sterile water for 3 times. The surface water of the leaves was absorbed with sterile filter paper, and the leaves were soaked in the prepared LX5 bacterial suspension for 5 s, and then placed in a culture dish lined with double layers of filter paper, with 5 mL of sterile water added for moisturizing. Rose black spot was cultured according to the method in Example 1, and spores were eluted with sterile water, filtered with double layers of sterile gauze, and the spore suspension was diluted to 10 6 CFU / mL with sterile water. 15 μL of the pathogenic spore suspension was dropped on the center of the leaf surface, and the culture was incubated at 25°C with 12 h light / 12 h darkness. To maintain humidity, 5 mL of sterile water was added to the culture dish every 3 days after inoculation. There were 3 leaves per treatment, and 3 replicates, with sterile water as the control. The incidence was investigated according to the disease grading after 14 days of inoculation.

[0048] Disease grading standard for black spot in vitro leaf method:

[0049] Disease grade Classification standard 0 grade No symptoms 1 grade Visible lesions, no arthrospore disc and arthrospore 2 grade Visible lesions, arthrospore disc, no arthrospore 3 grade Visible lesions, arthrospore disc and a small amount of arthrospore 4 grade Lesion area larger than inoculation area, arthrospore disc and a small amount of arthrospore 5 grade Lesion area larger than inoculation area, a large amount of arthrospore disc and arthrospore

[0050] Note: The disease grade is divided into 0, 1, 2, 3, 4 and 5 grades according to the different symptoms and degrees of disease.

[0051] The control efficiency was calculated according to the following formula:

[0052] Disease incidence (%) = number of symptomatic leaves / number of test leaves x 100

[0053] Disease index = [∑(number of leaves at each disease grade x the disease grade)] / (total number of leaves investigated x the highest disease grade) x 100

[0054] Control efficiency (%) = (disease index of the control - disease index of the treatment) / disease index of the control x 100

[0055] The results showed that after 14 days of inoculation with rose black spot, the leaves in the control group all showed large areas of typical black spot lesions, while the symptoms of the leaves treated with LX5 bacterial suspension were significantly reduced, with a control efficiency of 91.1% (Table 4). Figure 3

[0056] Table 4, Determination of the efficacy of LX5 against rose black spot by in vitro leaf method​

[0057] Treatment Disease incidence (%) Disease index Control Sterile water 0.0±0.0b 0.0±0.0b / Sterile water + Disosphaera rosae 100.0±0.0a 100.0±0.0a / LX5 + Disosphaera rosae 22.2±11.1b 8.9±4.4b 91.1

[0058] Note: The same column data after different letters represent significant difference (P <0.05).

[0059] 2. The control effect of LX5 on powdery mildew of Rosa chinensis

[0060] The Rosa chinensis cuttings were planted in the soil with vermiculite: peat = 1:1, and were placed in a glass greenhouse at 22°C / 16°C 12h / 12h light / dark when the powdery mildew occurred seriously in the greenhouse. The LX5 bacterial suspension with a concentration of 1 x 10 8 GFU / mL was prepared according to the method in Example 1. The prepared LX5 bacterial suspension was uniformly sprayed on the surface of each leaf of the cuttings, and water and Kangpu 2000 times liquid were used as controls, 4 plants per treatment, 3 replicates. The disease incidence was observed 20 days after treatment. The grading standard was as follows: 0 level: no lesion; 1 level: lesion area accounted for less than 1 / 4 of the total leaf area of the whole plant; 2 level: lesion area accounted for 1 / 4 to less than 1 / 2 of the total leaf area of the whole plant; 3 level: lesion area accounted for 1 / 2 to less than 3 / 4 of the total leaf area of the whole plant; 4 level: lesion area accounted for more than 3 / 4 of the total leaf area of the whole plant. The control efficiency was calculated according to the following formula:

[0061] Disease incidence (%) = number of plants with symptoms / number of test plants x 100

[0062] Disease index = [∑(number of plants at each level x the disease level)] / (total number of plants surveyed x the highest disease level) x 100

[0063] Control efficiency (%) = (disease index of control - disease index of treatment) / disease index of control x 100

[0064] The test results were counted 20 days after inoculation, and the results showed that the disease index of the water control group reached 27.1. The disease index of the LX5 bacterial suspension pre-treatment group after inoculation of the pathogen was only 8.4, and the control efficiency reached 69% (Table 5). The control efficiency of the commonly used agent Kangpu 2000 times liquid was only 54.2%. The experimental results showed that pre-inoculation of LX5 could effectively prevent the occurrence of powdery mildew of Rosa chinensis.

[0065] Table 5 Greenhouse control effect of LX5 on powdery mildew of Rosa chinensis

[0066] Treatment Disease incidence (%) Disease index Control Sterile water 66.7a 27.1±4.2a / Sterile water + Disosphaera rosae 50.0a 12.4±3.5b 54.2 LX5 + Disosphaera rosae 33.3b 8.4±2.1b 69.0

[0067] Note: The same column data after different letters represent significant difference (P <0.05).

[0068] Example 4, P. canavaninivorans LX5 promotes growth of Rosa hybrida

[0069] P. canavaninivorans LX5 bacterial suspension with a concentration of 1 x 10 8 The cuttings of 'Yueyuefen' were planted according to the method of Example 3, and the plants with uniform growth were selected and sprayed with the prepared LX5 bacterial suspension on the surface of the leaves. Sterile water was used as a control. After 60 days, the growth of the plants was observed, and the plant height of Rosa hybrida was measured. The results showed that the plant height of 'Yueyuefen' treated with sterile water was 15.3 cm, while the plant height of the plants treated with P. canavaninivorans LX5 bacterial suspension was 24.0 cm, which was 8.7 cm higher than the control (Table 6).

[0070] Table 6 Promoting effect of LX5 on Rosa hybrida

[0071] Treatment Plant height (cm) Control 15.3±1.8 Sterile water 24.0 ± 1.3 * ]]

[0072] * Indicates a significant difference from the control at the P < 0.05 level.

[0073] Example 6, P. canavaninivorans LX5 can produce protease

[0074] Activated P. canavaninivorans LX5 was inoculated into LB liquid medium and shaken overnight at 28°C and 200 rpm. 10 μL of the culture was added to the protease detection medium plate (100 g of skim milk powder, 5 g of proteose peptone, 15 g of agar, and 1000 mL of distilled water) three times. After drying, it was cultured at 28°C for 2 days, and whether a transparent circle appeared was observed. The results showed that a clear hydrolysis transparent circle appeared around the colonies on the plate inoculated with P. canavaninivorans LX5 ( LX5 ), proving that P. canavaninivorans LX5 has the ability to produce protease and can decompose proteins in the medium.

[0075] Example 7, P. canavaninivorans LX5 can produce cellulase

[0076] Strain LX5 was cultured according to the method in Example 6, and 10 μL of the culture was added dropwise to a cellulase activity detection medium plate (10 g of proteose peptone, 10 g of yeast powder, 10 g of sodium carboxymethyl cellulose, 1 g of potassium dihydrogen phosphate, 5 g of sodium chloride, 15 g of agar, and 1000 mL of distilled water). The procedure was repeated three times. After drying in a clean bench, the plate was cultured at 28°C in the dark for 7 days, and whether a hydrolysis halo appeared around the colonies was observed. The results showed that the control plate had no change, but a hydrolysis halo appeared around the colonies of P. canavaninivorans LX5 on the cellulase activity detection medium plate (Fig. 4), indicating that P. canavaninivorans LX5 has the ability to produce cellulase. Figure 3

[0077] Example 8, P. canavaninivorans LX5 can produce siderophore

[0078] Strain LX5 was cultured according to the method in Example 6, and 10 μL of the culture was added dropwise to a siderophore-CAS agar medium plate (60.5 mg of chrome azurol S, 72.9 mg of cetyltrimethylammonium bromide, 2.645 mg of ferric chloride hexahydrate, 295.25 mg of sodium dihydrogen phosphate dihydrate, 1213.5 mg of sodium hydrogen phosphate dihydrate dodecahydrate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, 62.5 mg of sodium chloride, 9000 mg of agar, and distilled water to 1000 mL, pH 6.8±0.2). The procedure was repeated three times. After drying in a clean bench, the plate was cultured at 28°C in the dark for 3 days, and whether the color of the medium around the colonies changed from blue to orange was observed. The results showed that the control plate had no change, but a blue discoloration halo appeared around the colonies of P. canavaninivorans LX5 on the siderophore-CAS agar medium plate (Fig. 5), indicating that P. canavaninivorans LX5 has the ability to produce siderophore. Figure 3

[0079] Example 9, P. canavaninivorans LX5 can dissolve inorganic phosphorus and organic phosphorus

[0080] ​​The strain LX5 was cultured according to the method in Example 6, and 10 μL of the culture was dropped on an inorganic phosphorus culture medium (yeast powder 0.5 g, glucose 10 g, tricalcium phosphate 5 g, ammonium sulfate 0.5 g, magnesium sulfate 0.3 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate 0.03 g, ferrous sulfate 0.03 g, agar 15 g, and distilled water 1000 mL) for 3 times. After drying in a clean bench, the culture was incubated at 28°C in the dark for 7 days, and whether a hydrolysis halo appeared around the colonies was observed. Meanwhile, 10 μL of the culture was dropped on an organic phosphorus culture medium (glucose 10 g, calcium carbonate 1 g, ammonium sulfate 0.5 g, magnesium sulfate 0.3 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate 0.03 g, ferrous sulfate 0.03 g, lecithin 0.2 g, agar 15 g, and distilled water 1000 mL) for 3 times. After drying in a clean bench, the culture was incubated at 28°C in the dark for 7 days, and whether a hydrolysis halo appeared around the colonies was observed.

[0081] The results showed that a hydrolysis halo appeared around the colonies of P. canavaninivorans LX5 in the inorganic phosphorus culture medium Figure 3 , indicating that P. canavaninivorans LX5 could hydrolyze inorganic phosphorus. A weak hydrolysis halo appeared around the colonies of P. canavaninivorans LX5 in the organic phosphorus culture medium Figure 3 , indicating that P. canavaninivorans LX5 could hydrolyze organic phosphorus.

[0082] Example 10, P. canavaninivorans LX5 could fix nitrogen

[0083] The strain LX5 was cultured according to the method in Example 6, and 10 μL of the culture was dropped on an inorganic phosphorus culture medium (yeast powder 0.5 g, glucose 10 g, tricalcium phosphate 5 g, ammonium sulfate 0.5 g, magnesium sulfate 0.3 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate 0.03 g, ferrous sulfate 0.03 g, agar 15 g, and distilled water 1000 mL) for 3 times. After drying in a clean bench, the culture was incubated at 28°C in the dark for 7 days, and whether a hydrolysis halo appeared around the colonies was observed. Meanwhile, 10 μL of the culture was dropped on an inorganic phosphorus culture medium (yeast powder 0.5 g, glucose 10 g, tricalcium phosphate 5 g, ammonium sulfate 0.5 g, magnesium sulfate 0.3 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate 0.03 g, ferrous sulfate 0.03 g, agar 15 g, and distilled water 1000 mL) for 3 times. After drying in a clean bench, the culture was incubated at 28°C in the dark for 7 days, and whether a hydrolysis halo appeared around the colonies was observed.

[0084] The results showed that LX5 could grow normally on the Ashby medium, while the negative control Escherichia coli could not grow, indicating that the strain LX5 had the ability to fix nitrogen Figure 3 .

[0085] Example 12, P. canavaninivorans LX5 could tolerate multiple heavy metal and salt stresses

[0086] After sterilization, the LB medium at about 65℃ was added with the filtered heavy metal solution and carbendazim to the final concentration of 30mg / L of cadmium sulfate octahydrate, 50mg / L of lead nitrate, 50mg / L of copper sulfate and 150mg / L of zinc sulfate, respectively. Meanwhile, the LB plate with the final concentration of 800mmol / L NaCl was prepared. The bacteria were picked from the fresh LX5 colony with sterilized toothpick and streaked on the prepared LB medium plate with different heavy metals. The plate was cultured at 28℃ in dark for 7 days, and the growth of LX5 was observed.

[0087] The results showed that the strain LX5 could grow normally on the LB culture containing high concentration of cadmium, lead, copper or zinc, and could grow normally on the plate containing high concentration of sodium chloride. Figure 3 Figure 4 ) These results showed that the strain LX5 had the ability to tolerate multiple heavy metal ions and salt stress.

[0088] The above description is only illustrative and is not intended to limit the present application, and those skilled in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present application.

Claims

1. A disease-preventing and growth-promoting bacterium for roses, characterized by: Name is Pseudomonas canavaninivorans LX5 has the accession number CGMCC No. 30656 at the China General Microbiological Culture Collection Center.

2. The application of the rose disease-preventing and growth-promoting bacteria according to claim 1 in the prevention and control of plant diseases; wherein the plant disease is *Discocephalus roseae* (…). Marssonina roses Rose black spot disease and / or rose monofilament shell (caused by) Podosphere rag Powdery mildew caused by ) 3. The application of the rose disease-preventing and growth-promoting bacteria according to claim 1 in the preparation of biocontrol agents or microbial fertilizers targeting plant pathogens; wherein the plant pathogen is *Discocephalus roseae* (…). Marssonina roses ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), Botrytis cinerea ( Botrytis cinerea ), Horsetail ( Fusarium horsetail ) 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 canavaninivorans CGMCC No. 30656.

5. A microbial fertilizer for preventing and controlling plant diseases, characterized in that, The active ingredient of the microbial fertilizer is Pseudomonas canavaninivorans CGMCC No. 30656.

6. Pseudomonas canavaninivorans Applications of LX5 CGMCC No. 30656 in the production of proteases, cellulases, hemiphiles, phosphorus solubilizers, and / or nitrogen fixation.

7. Pseudomonas canavaninivorans Application of LX5 CGMCC No. 30656 in promoting rose growth.

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