Bacillus velezensis F3A for promoting growth of Lanzhou lily and preventing and treating fusarium wilt, wettable powder and application of bacillus velezensis F3A

Abstract

The invention relates to the technical field of biology, in particular to application of bacillus velezensis F3A and wettable powder to prevention and treatment of Lanzhou lily fusarium wilt and application of the bacillus velezensis F3A and wettable powder to promotion of Lanzhou lily growth, the bacillus velezensis F3A is preserved in China General Microbiological Culture Collection Center on December 21, 2020, the preservation number is CGMCC No.21518, and the preservation number is CGMCC No.21518; the bacterial inhibition rate of the bacillus velezensis F3A on the Lanzhou lily wilt pathogenic bacterium fusarium tripterygium reaches 75.23%, and fermentation liquor effectively inhibits mycelial growth and spore germination of the fusarium tripterygium and can be stably colonized at the root system and bulb of the Lanzhou lily. The invention discloses a formula of bacillus velezensis F3A wettable powder. The bacillus velezensis F3A wettable powder disclosed by the invention has a relatively good growth promoting effect on Lanzhou lilium brownii, can be used for effectively preventing and treating fusarium wilt, and has a relatively good market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of plant disease biological control technology and microbial agent preparation, and particularly to application of Bacillus velez F3A and its wettable powder in preventing and controlling Lanzhou lily wilt disease and promoting the growth of Lanzhou lily. Background Art

[0002] The Lanzhou lily is a cultivated variety of the Sichuan Lanzhou lily, with a cultivation history of over 140 years. Lanzhou lilies have large bulbs, a sweet taste, high sugar content, low crude fiber, and delicate flesh. They are rich in starch, vitamins, pectin, and other nutrients, possessing high nutritional value and health benefits. In recent years, Lanzhou lily prices have continued to rise, and production has expanded to surrounding areas, with the cultivated area increasing year by year. Furthermore, environmental constraints have resulted in insufficient time between lily crop rotations, leading to an increasing incidence of Fusarium wilt caused by the fungus. This has severely impacted the yield and quality of Lanzhou lilies, hindering the healthy development of the Lanzhou lily industry.

[0003] Fusarium wilt is one of the main causes of Lanzhou lily wilt. Fusarium primarily invades the plant through wounds in the bulb or roots, causing the root tissue and base to brown and rot, then slowly spreads to the surrounding area. The bulb surface then slowly sinks, turns brown, and rots, and the scales detach from the base, leading to poor stem and root growth. The plant exhibits noticeably stunted growth and slows growth. Above-ground leaves gradually turn yellow and wither from the bottom up, eventually leading to plant wilt and death. Chemical control is currently the most commonly used measure for Lanzhou lily wilt, but long-term improper use has resulted in severe environmental pollution, leading to soil compaction and a decrease in beneficial soil microorganisms. Furthermore, pesticide residues on crops pose a serious health risk. Furthermore, because Lanzhou lilies are endemic and minor crops, there are currently no pesticides specifically registered for the prevention and treatment of Lanzhou lily wilt. The Bacillus Velez F3A and its wettable powder described in this patent can be registered as a microbial agent or a microbial pesticide. By dressing seeds, it can not only effectively prevent and control the occurrence of Lanzhou lily wilt disease, but also increase the abundance of beneficial microorganisms in the soil, alleviate continuous cropping obstacles, and has good market application prospects. Summary of the Invention

[0004] The primary purpose of the present invention is to provide a Bacillus Velez F3A that can prevent and control Lanzhou lily wilt disease and promote the growth of Lanzhou lily.

[0005] The inventors screened the biocontrol bacteria already available in the research group and obtained the most potent antagonistic strain, Bacillus velez F3A, with an inhibition rate of 75.23%. The Bacillus velez F3A was deposited with the General Microbiology Center of the China Culture Collection of Microorganisms on December 21, 2020, under the deposit number CGMCC No. 21518. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The contact number is 010-64807355.

[0006] A second object of the present invention is to provide a method for inhibiting the mycelial growth and spore germination of Fusarium oxysporum, the causative agent of Lanzhou lily wilt disease. The causative agent of Lanzhou lily wilt disease is Fusarium oxysporum, and the Bacillus velez F3A can stably colonize the roots and bulbs of Lanzhou lilies.

[0007] The third object of the present invention is to provide a formulation of a wettable powder of Bacillus Velez F3A.

[0008] Preferably, the formula of Bacillus Velez F3A wettable powder is: 10 11 CFU / mL bacterial powder 10%, diatomaceous earth carrier 76%, dispersant sodium lignin sulfonate 4.8%, wetting agent polyethylene glycol 7.2%, protective agent sodium acetate 2%.

[0009] The fourth object of the present invention is to provide the use of Bacillus Velez F3A wettable powder in promoting the growth of Lanzhou lily and preventing and controlling Lanzhou lily wilt disease.

[0010] Preferably, the Bacillus Velez F3A wettable powder can promote the growth of the above-ground parts and underground roots of Lilium lanzhouensis.

[0011] Preferably, the method for using the Bacillus Velez F3A wettable powder is as follows: before planting, the Bacillus Velez F3A wettable powder is diluted 10 times with water and mixed with seeds, and then sowed.

[0012] Compared with the prior art, the Bacillus Velez F3A, wettable powder and application thereof for promoting growth of Lanzhou lily and preventing wilt disease of the present invention have the following advantages:

[0013] (1) The Bacillus velezensis F3A of the present invention has a good inhibitory effect on the pathogenic bacteria of Lanzhou lily wilt, Fusarium thunbergii, with an inhibition rate of 75.23% on mycelium and an inhibition rate of 87.60% on spore germination in the fermentation liquid.

[0014] (2) The present invention provides a wettable powder of Bacillus velezensis F3A that can stably colonize lily bulbs and roots, promoting the growth of the aerial parts and root systems of Lanzhou lilies.

[0015] (3) Bacillus Velez F3A wettable powder can increase the abundance of beneficial microorganisms in the soil around the roots of Lanzhou lily, has a high preventive effect on Lanzhou lily wilt disease, and effectively alleviates the obstacles of continuous cropping.

[0016] (4) The product of the present invention has been optimized through pilot fermentation, with the optimal fermentation medium (corn flour 15 g / L, soybean flour 7 g / L, NaCl 8 g / L), low production cost, simple usage method, and good market development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Antagonistic effects of different biocontrol bacteria against lily wilt pathogen: a: Bacillus velezensis F3A; b: Bacillus amyloliquefaciens; c: Bacillus tequila SY89; d: Paenibacillus polymyxa YF; e: Serratia prausnitzii MM; f: Bacillus gelatinosa; g: Bacillus megaterium; h: control.

[0018] Figure 2 This is the effect of Bacillus Velez F3A fermentation broth on the germination of pathogen spores.

[0019] Figure 3 Confocal laser scanning microscopy images of root and bulb colonization by Bacillus Velez F3A at different stages.

[0020] Figure 4 This is the in vitro effect of Bacillus Velez F3A on Lanzhou lily.

[0021] Figure 5 This is the potted effect of Bacillus Velez F3A on Lanzhou lily. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0023] Example 1: Screening of biocontrol bacteria that effectively inhibit the wilt pathogen of lily of Lanzhou

[0024] The biocontrol effect of Bacillus velez F3A ( Bacillus velezensis ), Bacillus tequila SY89 ( Bacillus tequilensis ), Bacillus megaterium ( Bacillus megaterium ), Serratia pumilia MM ( Serratia plymuthica )、Paenibacillus polymyxa YF( Paenibacillus polymyxa ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) and Bacillus jelly-like ( Bacillus mucilaginosus ), with the Lanzhou lily wilt pathogen Fusarium thunbergii ( Fusarium. tricinctum ) was used as the indicator bacteria, and the best biocontrol bacteria against Lanzhou lily wilt disease were screened by double confrontation method.

[0025] The test results showed that Bacillus Velez F3A had the strongest antibacterial effect on Fusarium oxysporum f. trilineatum, reaching 75.23%. Therefore, Bacillus Velez F3A was selected as the biocontrol agent for lily wilt disease (Table 1, Figure 1 The strain Bacillus velezensis F3A was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms and named Bacillus velezensis. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 21518. The contact number is 010-64807355.

[0026] Table 1 Determination of the inhibition rate of biocontrol bacteria Biocontrol bacteria Inhibition rate (%) Bacillus megaterium 23.41±0. 10d Bacillus amyloliquefaciens 74.55±0.06a Paenibacillus polymyxa 68. 18±0.06b Bacillus tequila 71.36±0.02ab Bacillus jelly 43.64±0.08c Bacillus Velez 75.23±0.04a Serratia prausnitzii 66. 14±0.00b CK 0 Note: Different letters indicate significant differences among treatment levels (p<0.05).

[0027] Example 2: Effect of Bacillus Velez F3A fermentation broth on pathogen hyphae and spore germination

[0028] After the mycelium was treated with the fermentation broth of Bacillus Velez F3A, the pathogen mycelium underwent obvious deformation, such as swelling and breaking, under an optical microscope; under an electron microscope, the pathogen mycelium shrank, swollen, and broke, while the mycelium of the control group appeared relatively intact and smooth. After treatment with the sterile filtrate of Bacillus Velez F3A, the number of spores that germinated gradually decreased with the increase of the sterile filtrate concentration. When the sterile filtrate was diluted 10 times, most of the spores failed to germinate and no germ tubes were produced. The spore germination rate was 11.61%, and the inhibition rate was 87.60% (Table 2, Figure 2 ).

[0029] Table 2 Inhibitory effects of different concentrations of biocontrol bacteria fermentation broth on pathogen spore germination Fermentation filtrate dilution multiple Germination rate (%) Inhibition rate of Bacillus velez F3A (%) CK 93.59±0.03a 0.00 100 times 87.73±0.02b 6.26±0.02c 50 times 84. 18±0.03b 10.06±0.03c 20 times 73.65±0.04c 21.31±0.05b 10 times 11.61±0.03d 87.60±0.03a Note: Different letters indicate significant differences among treatment levels (p<0.05)

[0030] Example 3: Colonization of Bacillus velez F3A in Lanzhou lily bulbs

[0031] After disinfection, the bulbs of Lilium lanzhouensis were planted in sterile soil. After emergence, 100 mL of fluorescently labeled F3A-GFP (10 8CFU / mL) fermentation broth, with 100 mL of water as a blank control. Lanzhou lily bulb samples were randomly collected 5, 10, 15, and 20 days after inoculation and observed using a confocal laser scanning microscope (CLSM Olympus FV3000) at an excitation wavelength of 488 nm. The experimental results showed that 10 days after inoculation, the green fluorescence intensity of the roots and bulbs reached a maximum, indicating that GFP-F3A had clearly colonized the roots and bulb surfaces of the Lanzhou lily. After 15 days of inoculation, GFP-F3A was observed to have penetrated into the roots and bulbs of the Lanzhou lily and then stabilized, indicating that Bacillus Velez F3A had stably colonized the bulbs of the Lanzhou lily for a long time ( Figure 3 ).

[0032] Example 4: Preparation method of Bacillus Velez F3A wettable powder

[0033] After activation of Bacillus velezensis strain F3A on NA plates, it was inoculated into LB liquid medium using an inoculation loop on a sterile operating table and shaken at 28°C and 180 rpm for 24 hours to prepare seed liquid. The seed liquid was inoculated into a 500 L fermenter at a 3% inoculum volume and fermented in an optimized optimal medium (corn flour 15 g / L, soybean flour 7 g / L, NaCl 8 g / L). The fermentation liquid was spray-dried to prepare 10 11 CFU / mL mother powder. Add a certain amount of carrier, dispersant, wetting agent, stabilizer, protective agent, etc. to the mother powder and mix them to make wettable powder. Through indoor screening of carriers, adjuvants and protective agents, we finally determined 10 10 The formula of the wettable powder of Bacillus velezensis strain F3A is: 10 CFU / mL 11 CFU / mL bacterial powder 10%, diatomaceous earth carrier 76%, dispersant sodium lignin sulfonate 4.8%, wetting agent polyethylene glycol 7.2%, protective agent sodium acetate 2%.

[0034] Example 5: Growth-promoting effect of Bacillus Velez F3A wettable powder on Lanzhou lily

[0035] After lily bulbs emerged from planting, the treated group was irrigated with 50 mL of the 100-fold diluted WP of Bacillus Velez F3A prepared in Example 4, while the control group was irrigated with the same amount of sterile water. Plant height, root length, and stem diameter were measured 30 days later. The results showed that compared with the control, plant height, root length, and stem diameter increased by 29.90%, 53.87%, and 8.38%, respectively, in the lily treated with the WP of Bacillus Velez F3A prepared in Example 4. This indicates that the WP of Bacillus Velez F3A has a significant growth-promoting effect on lilies (Table 3).

[0036] Table 3 Evaluation of the growth-promoting effect of Bacillus velez F3A on Lilium lanzhouensis plants Plant height (cm) Root length (cm) Stem diameter (cm) Control <![CDATA[34.00±1.50 b ]]> <![CDATA[9.47±1.07 b ]]> <![CDATA[0.60±0.05 b ]]> Bacillus Velez <![CDATA[44.17±2.35 a ]]> <![CDATA[14.57±2.06 a ]]> <![CDATA[0.65±0.04 a ]]> Growth promotion rate (%) 29.90% 53.87% 8.38% Note: Different letters indicate significant differences among the groups.

[0037] Example 6: In vitro control effect of Bacillus Velez F3A wettable powder on Lanzhou lily wilt

[0038] Uniformly sized bulbs of Lilium dasyphyllum were disinfected with 30% sodium hypochlorite and rinsed with sterile water. The bulbs were then placed in a Petri dish with sterilized, moistened cotton wool in the center. A control group and a treatment group were set up. In the control group, a sterile inoculation needle was used to slightly puncture the bulb's surface in the middle, and 1 mL of a pathogen spore suspension was added dropwise. In the treatment group, 1 mL of a 10-fold dilution of the Bacillus Velez F3A wettable powder prepared in Example 4 was first added to the puncture site. After 48 hours, 1 mL of the pathogen spore suspension was added. All treatments were incubated in the dark at 28°C. Disease indexes were calculated, and control efficacy was calculated.

[0039] The results of the in vitro test showed that the lesions of the Lanzhou lily bulbs in the control group were large and rotten, accompanied by the growth of hyphae, while the lesions of the group treated with Bacillus Velez F3A WP were smaller, with a control efficiency of 80.09% (Table 4, Figure 4 ).

[0040] Table 4 In vitro control efficacy of Bacillus Velez F3A wettable powder against Lanzhou lily wilt disease deal with Disease index (%) Prevention effect (%) CK 1.26 - Bacillus Velez F3A wettable powder 6.33 80.09

[0041] Example 7: Potted control effect of Bacillus Velez F3A wettable powder on Lanzhou lily wilt disease

[0042] Lily bulbs of uniform size were selected and disinfected, then rinsed three times with sterile water. The treatment group was treated with a 10-fold dilution of Bacillus Velez F3A wettable powder, while the control group was treated with sterile water and planted in sterile soil. After seedlings emerged, the treatment and control groups were irrigated with 20 mL of a 1×10 5 CFU / mL of pathogen spore suspension was used, and the disease index was calculated after 30 days to calculate the prevention effect.

[0043] The results of the potted plant test showed that 30 days after inoculation with the pathogen, the aboveground parts of the control Lanzhou lily plants showed obvious yellowing, the bulbs rotted, and the root system was small, with an incidence rate of 100%; the aboveground parts of the treated group grew normally, with some bulbs having small lesions, and the incidence rate control efficiency was 76.35% (Table 5, Figure 5 ).

[0044] Table 5 Control efficacy of Bacillus Velez F3A wettable powder against Lanzhou lily wilt in potted plants deal with Incidence rate (%) Disease index (%) Prevention effect (%) Bacillus Velez F3A wettable powder 22.66 2.72 76.35 CK 100.00 11.50 -

[0045] Example 8: Field control effect of Bacillus Velez F3A wettable powder on Lanzhou lily wilt

[0046] A field trial was conducted in 2023-2024 on a small plot of continuously cropped land in Chaihejia, Lintao County, Jiangsu Province. Two treatments were used: the wettable powder of Bacillus Velez F3A prepared in Example 4 and a control of pure water, with three replicates for each treatment. During Lanzhou lily cultivation, seeds in the treatment group were treated with Bacillus Velez F3A wettable powder (2 kg of the agent per 100 kg of Lanzhou lily bulbs), while seeds in the control group were treated with pure water. During the peak growth period, when field wilt was most prevalent, three random surveys were conducted, with 30 plants at each site. The incidence rate and disease index were calculated, and the control efficacy was calculated.

[0047] The results of the field test showed that the Lanzhou lilies treated with Bacillus Velez F3A wettable powder grew healthily, with a disease incidence rate of 6.50%, much lower than that of the control. The prevention effect against lily wilt disease reached 78.26%, which has good prospects for promotion and application (Table 6).

[0048] Table 6 Field control efficacy of Bacillus Velez F3A wettable powder against Lanzhou lily wilt deal with Incidence rate (%) Disease index (%) Control effect (%) Bacillus Velez F3A wettable powder 6.50 3.30 78.26 comparison 30.25 15.18 -

[0049] The above embodiments are preferred implementations of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the present invention are included in the protection scope of the present invention.

Claims

1. Bacillus velezinoffii ( Bacillus velezensis ) Application of F3A or its fermentation liquid in preventing and treating Lanzhou lily wilt disease and in promoting plant growth, characterized in that: The Bacillus Velez F3A was deposited in the General Microbiology Center of the China Culture Collection Administration on December 21, 2020, with the deposit number CGMCC No. 21518. The storage address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The contact number is: 010-64807355.

2. The use according to claim 1, characterized in that The pathogen of Lanzhou lily wilt disease is Fusarium thunbergii ( Fusarium tricinctum ).

3. The Bacillus Velezii of claim 1 ( Bacillus velezensis ) F3A or its fermentation broth inhibits the three-line Fusarium ( Fusarium tricinctum ) Application in mycelial growth and spore germination.

4. The use according to claim 1, wherein The formula of the described Bacillus Velez F3A wettable powder is: 10 11 CFU / mL bacterial powder 10%, diatomaceous earth carrier 76%, dispersant sodium lignin sulfonate 4.8%, wetting agent polyethylene glycol 7.2%, protective agent sodium acetate 2%.

5. The wettable powder of Bacillus Velez F3A according to claim 4, wherein The application in promoting the growth of Lanzhou lily and preventing and treating wilt disease.

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