Biocontrol strain F32 for preventing and treating litchi downy blight and application of biocontrol strain F32

Abstract

The invention discloses pseudomonas for preventing and treating peronophythora litchii, a biocontrol microbial inoculum prepared from the pseudomonas and application of the biocontrol microbial inoculum. The classification name of the pseudomonas oryzae F32 for preventing and treating the litchi downy mildew is Pseudomonas oryzae F32, the strain preservation number is GDMCC No.65232, the preservation unit name is Guangdong Microbial Culture Collection Center, the address is 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou City, and the preservation time is October 8, 2024. The pseudomonas oryzae F32 can be used for preventing and treating the litchi downy mildew. The pseudomonas oryzae F32 is used for preparing a microbial inoculum. The pseudomonas biocontrol microbial agent provided by the invention can effectively control the occurrence of peronophythora litchii, has an inhibition effect on the growth of pathogenic bacteria of peronophythora litchii, and shows an excellent biocontrol effect in an in-vitro fruit experiment.

Description

Technical Field

[0001] The invention relates to the field of agricultural microorganisms, and in particular to a pseudomonas for preventing and controlling litchi downy blight, a biocontrol agent prepared therefrom and applications thereof. Background Art

[0002] Litchi (Litchi chinensis Sonn.) is a widely planted economic crop in tropical and subtropical areas. It is deeply loved by consumers because of its rich nutrition and delicious taste. Litchi downy mildew is currently the most serious disease in the production, storage and transportation of litchi, which often causes litchi fruit rot, yield reduction and quality reduction, and can cause 10% to 30% yield loss. In epidemic years, it can cause 80% yield loss, causing huge economic losses to fruit farmers.

[0003] At present, the prevention and control of litchi diseases relies on chemical control, and chemical control is the main means of preventing and controlling litchi downy mildew. Common and effective chemical agents include dimethomorph, myclobutanil and thiophanate-methyl, which have good field control effects. However, studies have shown that due to the lack of understanding of the relevant technologies for safe use of pesticides by some fruit farmers, there are problems such as the use of a single type of pesticide, the rare use of rotation of pesticides, and unreasonable mixing, which have led to the emergence of resistance to litchi downy mildew and the increasing potential threat to the environment and human health. Therefore, it is urgent to explore a green, efficient and safe prevention and control method. However, as a relatively green and safe prevention and control method at present, biological control measures are relatively less studied and have certain drawbacks, such as being restricted by a specific ecological environment and the risk of invasion of alien organisms caused by the introduction of alien organisms. Therefore, the need for a localized biological control method is an important research direction in the field of biological control.

[0004] Pseudomonas is a genus of Gram-negative bacteria widely distributed in nature. They are known for their strong adaptability and metabolic diversity. Pseudomonas is widely used in bioremediation, agriculture and biotechnology. For example, CN202411116602.3 discloses a strain of rhizophagous Pseudomonas, which is widely used in the prevention and treatment of kiwifruit bacterial canker, and CN202411021564.3 discloses a strain of rapeseed Pseudomonas, which has a preventive effect on cruciferous crops and a conditioning effect on diseased soil. CN202411041658.7 discloses a photosynthetic bacterium called Rhodopseudomonas palustris, which can be used for the purification of medium and low-pollution water. However, different biocontrol bacteria usually control specific microorganisms, and no relevant reports on the use of Pseudomonas in the prevention and treatment of litchi downy blight have been found. Summary of the invention

[0005] The purpose of the present invention is to provide a pseudomonad isolated from litchi peel in view of the shortcomings of current chemical control and biological control of litchi downy blight.

[0006] Another object of the present invention is to provide a biocontrol agent prepared by the biocontrol bacteria.

[0007] Another object of the present invention is to provide applications of the biocontrol bacteria and the biocontrol agent.

[0008] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] A strain of Pseudomonas oryzihabitans F32 for controlling litchi downy mildew, with a classification name of Pseudomonas oryzihabitans, a strain collection number of GDMCC No.65232, a preservation unit name of Guangdong Provincial Microbiological Culture Collection Center, an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation time of October 8, 2024.

[0010] The bacterial agent is prepared by the Pseudomonas oryzihabitans F32.

[0011] As a preferred embodiment of the present invention, the seed liquid of Pseudomonas oryzihabitans F32 is inoculated into liquid LB culture medium, and the culture is placed in a constant temperature oscillator at 28° C. and 200 r / min for 8-12 hours to obtain a fermentation liquid as the bacterial agent.

[0012] As a preferred embodiment of the present invention, the seed liquid of Pseudomonas oryzihabitans F32 is inoculated into a liquid LB medium, and the medium is placed in a constant temperature oscillator at 28°C and 200 r / min to culture for 8-12 hours to obtain a fermentation liquid, the fermentation liquid is filtered, and the bacterial bodies are resuspended with sterile water to obtain a bacterial suspension as a bacterial agent.

[0013] The application of Pseudomonas oryzihabitans F32 in preventing and controlling litchi downy mildew.

[0014] The invention discloses an application of the Pseudomonas oryzihabitans F32 in preparing a product for preventing and treating litchi downy mildew.

[0015] The bacterial agent is used in preventing and controlling litchi downy mildew.

[0016] As a preferred embodiment of the present invention, the bacterial agent is used to inhibit the mycelial growth of litchi downy mildew, inhibit the germination of sporangium of litchi downy mildew and / or inhibit the germination of zoospores of litchi downy mildew, and improve the prevention effect of litchi downy mildew on litchi detached fruits and leaves.

[0017] As a preferred embodiment of the present invention, the bacterial agent is used to inhibit the growth of litchi downy mildew hyphae, inhibit the germination of sporangium of litchi downy mildew and / or inhibit the germination of zoospores of litchi downy mildew, and improve the prevention effect of litchi downy mildew on litchi detached fruits and leaves.

[0018] The bacterial agent is used in preparing an in vitro litchi preservative or a product for preventing and treating in vitro litchi downy mildew.

[0019] The above-mentioned biocontrol agent can be used in various growth stages of litchi to prevent and control litchi downy mildew by spraying the biocontrol agent on litchi leaves or peels.

[0020] The present invention has the following two advantages:

[0021] (1) The biocontrol agent provided by the present invention is composed of Pseudomonas, which is isolated from litchi peel, to avoid the invasion of foreign species and the environmental constraints of biocontrol agents. At the same time, as a biocontrol agent, it does not cause environmental pollution and drug resistance problems caused by chemical drugs.

[0022] (2) The biocontrol agent provided by the present invention has simple culture conditions and low price, is easy to industrialize and produce, can be widely promoted and applied, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 F32 early colony map

[0024] Figure 2 F32 late colony map

[0025] Figure 3 Effects of F32 on the hyphal growth of Downy Phytophthora

[0026] A: The inhibition rate of the three components of F32 on litchi downy mildew; Figure 3 B: Schematic diagram of the confrontation between the three components of F32 and litchi downy mildew on a plate; Note: Different letters indicate significant differences at P < 0.05.

[0027] Figure 4 Effects of F32 on the Sporangium Germination of Downy Phytophthora

[0028] A, 6h co-culture, germination rate and inhibition rate of conidia of litchi under the three-component treatment of F32; B, 9h co-culture, germination rate and inhibition rate of conidia of litchi under the three-component treatment of F32; C, Schematic diagram of conidia germination. Different letters indicate significant differences at P < 0.05.

[0029] Figure 5 Effects of F32 on the germination of zoospores of Downy Phytophthora

[0030] A, 6h co-culture, under the three-component treatment of F32, the zoospore germination rate and inhibition rate of litchi downy mildew; B, 9h co-culture, under the three-component treatment of F32, the zoospore germination rate and inhibition rate of litchi downy mildew; C, schematic diagram of zoospore germination. Different letters indicate significant differences at P < 0.05.

[0031] Figure 6 Disease classification standards for litchi fruits and leaves

[0032] A: Schematic diagram of fruit disease severity; B: Schematic diagram of leaf disease severity

[0033] Figure 7 Analysis on the biocontrol effect of F32 on frost blight of litchi fruit in vitro

[0034] A: Analysis of the protection effect of 72hip on fruits B: Analysis of the protection effect of 82hip on fruits Different letters indicate significant differences at P < 0.05.

[0035] Figure 8 Analysis on the biocontrol effect of F32 on frost disease of litchi leaves in vitro

[0036] A: Analysis of leaf protection effect B: Schematic diagram of leaf disease severity. Different letters indicate significant differences at P < 0.05.

[0037] Biological sample storage information

[0038] Pseudomonas oryzihabitans F32, classification name is Pseudomonas oryzihabitans, strain collection number is GDMCC No.65232, the preservation unit name is Guangdong Microbiological Culture Collection Center, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation time is October 8, 2024. DETAILED DESCRIPTION

[0039] Example 1 Isolation and screening of biocontrol bacteria

[0040] (1) Culture medium preparation: LB solid culture medium formula (10 g sodium chloride, 10 g tryptone, 5 g yeast powder, 15 g agar powder, dilute to 1 L with distilled water, adjust pH ≈ 7.2) was sterilized at 121°C for 20 min, and R2A solid culture medium formula (0.5 g / L yeast powder, 0.25 g / L casein peptone, 0.25 g / L meat peptone, 0.5 g / L hydrolyzed casein, 0.5 g / L glucose, 0.5 g / L starch, 0.3 g pyruvic acid, pH = 7.2) was sterilized at 121°C for 20 min.

[0041] (2) Strain isolation: Weigh 3 g of litchi 'Fei Zi Xiao' peel, place in a sterile mortar, add 27 mL of 0.85% sterile sodium chloride (containing sterile glass beads) for grinding, shake on a shaker at 180 rpm for 30 min, and let stand for 5 min. Filter with sterile cotton cloth to obtain 10 -1 of bacterial suspension.

[0042] (3) Dilution and coating: Take 0.1 mL of the supernatant and dilute it in 900 μL of sterile 0.85% NaCl. Mix well to obtain a 10-2 sample dilution. Repeat this process to obtain a 10-2 sample dilution. -3 , 10 -4 , 10-5, 10 -6 Dilution. Take 10 -4 , 10 -5 , 10 -6 Add 100 μL of three dilutions to R 2 A (yeast powder 0.5g / L, casein peptone 0.25g / L, meat peptone 0.25g / L, hydrolyzed casein 0.5g / L, glucose 0.5g / L, starch 0.5g / L, pyruvic acid 0.3g, pH = 7.2) solid culture medium plate, spread evenly with a spreader until completely absorbed, each gradient was repeated 3 times, and inverted culture was carried out at 28°C for 48-72h. Observation results: separation and purification of the strain was obtained.

[0043] (4) Identification and sequencing: Pick a single colony from the purified and stored bacteria and inoculate it into LB liquid culture medium to prepare a fermentation broth of beneficial bacteria, and take 1.5 mL for later use. Collect bacterial cells by centrifugation and extract them according to the AxyPrep Bacterial Genomic DNA Mini Kit. Take 5 μL of the extracted DNA and perform electrophoresis on a 0.8% agarose gel, and select a marker as a reference based on the predicted size of the target fragment. After completion, move the gel to a UV transilluminator for observation. If the band is bright and clear and meets the predicted size, it can be stored at -20°C for later use. Use the extracted bacterial DNA as a template and use universal primers to amplify the 16S rRNA gene. The primer sequences are: U8-27 (F): 5'-AGAGTTTGATC (AC) TGGCTCAG-3'; L1494-1514 (R): 5'-CTACGG (AG) TACCTTGTTACGAC-3'. The reaction system was: 25μLMix, 2μ primer F, 2μL primer R, 2μL template DNA and 19μL ultrapure water. The reaction procedure was: pre-denaturation at 95℃ for 3min; then denaturation at 95℃ for 10s, annealing at 56℃ for 40s, extension at 72℃ for 1min, 28 cycles; finally, post-extension at 72℃ for 5min. Take 5μL of product, add 1μL loading buffer and mix well, perform 1.0% agarose gel electrophoresis, voltage 120v, electrophoresis 25min, soak EB for 15min, use BIO-RAD gel system imaging system to observe the target band; observe the band, send the bright and clear amplification product that matches the expected size to Shanghai Shenggong Reagent Company for sequencing. The original sequence was compared by BLAST on NCBI, and the strain with the highest homology was selected. F32 was identified as Pseudomonas oryzihabitans.

[0044] Example 2 Preparation of three components of biocontrol bacteria, pathogenic fungus hyphae, sporangium and zoospores

[0045] Preparation of biocontrol bacteria fermentation broth: Use a toothpick to pick up an activated single colony and put it into 2 mL of liquid LB medium, place it in a constant temperature oscillator at 30°C and 180 r / min and culture it for 12 hours to obtain seed liquid, then take 1 mL of seed liquid and add it to 19 mL of liquid LB medium, place it in a constant temperature oscillator at 30°C and 200 r / min and culture it for 8 hours to obtain fermentation broth.

[0046] Preparation of biocontrol bacteria suspension: Divide the prepared fermentation broth into small portions and place them flat at 4°C, centrifuge at 8000r / min for 10min, discard the supernatant, resuspend the wet bacteria in sterile water, adjust the concentration and measure OD600 with an enzyme marker to obtain 0.9-1.1.

[0047] Preparation of biocontrol bacteria supernatant: Place the prepared fermentation broth in aliquots at 4°C, centrifuge at 8000r / min for 10min, and filter the supernatant with a sterilized 0.22μm filter membrane.

[0048] Activation of pathogens: Take a plate of litchi downy blight strain cultured in the dark at 26°C, use a sterilized yellow gun tip to take the fresh bacterial cake from the outermost layer of the plate, inoculate it into the center of a CJA (200 g / L carrot, chopped and juiced with water for 10 min, filtered with gauze, 15 g / L agar powder, pH = 7.0-7.2) medium plate, place it in an artificial climate box and culture it in the dark at 28°C for 7 days to allow the pathogen to grow all over the plate.

[0049] Preparation of sporangium suspension of litchi downy mildew pathogen: Take 10 mL of sterile water and add it to the plate covered with mycelium, wash the sporangium with a sterile brush, filter the mycelium and impurities into a 500 mL sterile conical flask with a sterile tea strainer, shake evenly, take 1 μL on a slide, observe and count under an inverted biological microscope, add sterile water and shake evenly to adjust the concentration to 5×10 4 sporangium / mL.

[0050] Preparation of zoospore suspension of litchi downy mildew pathogen: Place the prepared sporangium suspension in a 4°C refrigerator for 1 hour, then place it at room temperature for 1 hour, and then place it in a 4°C refrigerator for 1 hour before taking it out. This operation uses temperature difference treatment to stimulate the sporangium to fully release zoospores, and then use a sterilized 0.22μm filter membrane to filter the sporangium and hyphae to obtain a relatively pure zoospore suspension. Shake evenly and take 1μL to count under an inverted biological microscope, add sterile water and shake evenly to adjust its concentration to 35×10 3 Zoospores / mL is sufficient.

[0051] Example 3 Effect of biocontrol agents on mycelial growth of litchi peronophyte

[0052] In this example, the plate confrontation method was used to observe the inhibitory effect of the three components of F32 on the mycelial growth of litchi downy mildew. The specific operation was as follows: the activated litchi downy mildew was evenly punched into a circular bacterial block with a diameter of 5 mm from the outer edge of the colony with a sterilized puncher, and the bacterial block (5 mm) was inoculated into CJA-PDA (CJA: 200 g / L carrots, chopped and juiced for 10 min, filtered with gauze, 15 g / L agar powder, pH = 7.0-7.2 ; PDA: 200g / L potato, chopped and boiled with water for 20min, 20g / L glucose and 15g / L agar powder were added to the filtrate, pH = 7.0-7.2, sterilized separately and mixed in a volume ratio of 1:1) in the middle of the culture medium, 10μL of biocontrol bacteria fermentation liquid (OD600 = 1), bacterial suspension (OD600 = 1), and supernatant (sterilized with 0.22μm filter membrane) were taken and inoculated into four points 27mm around the pathogen. The area of ​​the pathogen was measured by Image J software and the inhibition rate was calculated.

[0053] Inhibition rate = (pathogen area of ​​control group - pathogen area of ​​treatment group) / pathogen area of ​​control group × 100%

[0054] The experimental results showed that there were significant differences among the components of F32 (P < 0.05). The supernatant had no significant inhibitory effect on the mycelial growth of Peronophytum litchii, while the inhibition rates of the bacterial suspension and fermentation broth reached 25% and 32% ( Figure 3 A and B).

[0055] Example 4 Determination of the activity of biocontrol agents on the germination of sporangium of litchi downy mildew pathogen

[0056] This example is intended to explore the inhibitory effect of the three components of Pseudomonas oryzae F32 on the spore germination of Peronophytum litchii, and the specific operation is as follows:

[0057] Experimental group: Use a pipette to take 4.5 mL of 5×10 4 Sporangia / mL sporangium suspension was mixed with 0.5mL of biocontrol bacteria fermentation liquid (OD600=1), bacterial suspension (OD600=1), and supernatant (sterilized with 0.22μm filter) in a test tube. Control group: 4.5mL of sporangium suspension was mixed with 0.5mL of sterile water in a test tube with a pipette, and cultured in a constant temperature incubator at 30℃ and 180r / min. Each treatment was repeated 6 times. 1μL was taken out of every 3 repetitions at 6h and 9h to count the number of germinated sporangia and the number of ungerminated sporangia under a microscope ( Figure 4 C), every 200 sporangia were counted as one replicate, the data were recorded, and the sporangium germination rate and inhibition rate were calculated.

[0058] Germination rate = germination number / total number,

[0059] Inhibition rate = (germination rate of control group - germination rate of treatment group) / germination rate of control group.

[0060] The experimental results show that ( Figure 4 A and B), when co-cultured for 6 hours, the sporangium germination rates of the bacterial suspension and fermentation broth were only 41% and 11%, while the germination rates of the supernatant and the control group were 85% and 80%, and the inhibition rates were 48.5%, 83.4%, -6.7%, and 0%, respectively; when co-cultured for 9 hours, the sporangium germination rates of the bacterial suspension and fermentation broth were 31% and 30%, respectively, while the germination rates of the supernatant and the control group were 52% and 81%, and the inhibition rates were 61.4%, 62.9%, 35.3%, and 0%, respectively. Through statistical analysis, it can be seen that there are significant differences between the various components of F32 and the control group (P < 0.05). It can be seen that the fermentation broth and bacterial suspension of Pseudomonas F32 have an inhibitory effect on the sporangium germination of litchi downy mildew, and the inhibition rate can reach 47% to 81%, among which the inhibitory effect of the bacterial suspension does not weaken with the change of time, and has long-term inhibition potential.

[0061] Example 5 Determination of the activity of biocontrol agents on zoospore germination of litchi downy mildew pathogen

[0062] This example is intended to explore the inhibitory effect of the three components of Pseudomonas F32 on the germination of zoospores of litchi downy blight, and the specific operation is as follows:

[0063] Experimental group: Use a pipette to take 4.5 mL of 5×10 4 Zoospores / mL zoospore suspension was mixed with 0.5mL of biocontrol bacteria fermentation liquid (OD600=1), bacterial suspension (OD600=1), and supernatant (sterilized with 0.22μm filter) in a test tube. Control group: 4.5mL of zoospore suspension was mixed with 0.5mL of sterile water in a test tube with a pipette, and cultured in a constant temperature incubator at 30℃ and 180r / min. Each treatment was repeated 6 times. 1μL was taken out of every 3 repetitions at 3h and 5h to count the number of germinated zoospores and the number of ungerminated zoospores under a microscope ( Figure 5 C), every 120 zoospores were counted as one replicate, the data were recorded, and the sporangium germination rate and inhibition rate were calculated.

[0064] Germination rate = germination number / total number,

[0065] Inhibition rate = (germination rate of control group - germination rate of treatment group) / germination rate of control group

[0066] The experimental results show that ( Figure 5A and B), at 3h of culture, the zoospore germination rates of the bacterial suspension and fermentation broth were 61% and 37%, respectively, while the germination rates of the supernatant and the control group were 80% and 84%, and the inhibition rates were 27.8%, 55.9%, 5.2% and 0%, respectively. At 5h of culture, the sporangium germination rates of the bacterial suspension and fermentation broth were only 33% and 30%, while the germination rates of the supernatant and the control group were 78% and 84%, and the inhibition rates were 59.7%, 63.3%, 6.6% and 0%, respectively. Through statistical analysis, it can be seen that there is no significant difference between the supernatant of F32 and the control group, while there are significant differences between the bacterial suspension and the fermentation broth and the control group (P < 0.05). It can be seen that the fermentation broth and bacterial suspension of Pseudomonas F32 have an inhibitory effect on the germination of sporangium of litchi downy mildew, and the inhibition rate can reach 28% to 63%, which has a good inhibitory effect. By comparing the culture time, it can be seen that the inhibition rate of the fermentation broth increased from 55% to 63%, while the inhibition rate of the bacterial suspension increased from 28% to 57%. This shows that the bacterial suspension has better long-term inhibitory ability on the germination of zoospores of litchi downy mildew pathogen than the fermentation broth.

[0067] Example 6 Determination of the efficacy of biocontrol agents on downy blight of litchi fruit in vitro

[0068] This example combines the experimental results of Examples 3, 4 and 5, selects F32 bacterial suspension to simulate field control efficacy determination, and explores the biological control effect of F32 bacterial suspension on litchi downy mildew. The specific operation is as follows:

[0069] The leaves or fruits of healthy litchi 'Fei Zi Xiao' picked from the field were treated as follows: A. Experimental group: the suspension of biocontrol bacteria was diluted 50 times and sprayed on the leaves or fruits; B. Control group: the sterilized water culture solution with the same dilution multiple. Three replicates were set for each treatment, and 30 fruits or several leaves were placed in a fresh-keeping box for each replicate. A total of 300 mL was sprayed for each treatment (3 replicates) in the treatment group and the control group. After 24 hours, the suspension of pathogenic sporangia (5×10 4 The disease index was investigated at 72 and 84 hpi (hours post inoculation) and the biocontrol efficacy was calculated.

[0070] According to the disease severity survey method ( Figure 6 A and B): Level 1: Slight lesions at the inoculation point, with a necrotic area of ​​less than 5%; Level 3: Necrotic area 5%-10%; Level 5: Necrotic area 10%-25%; Level 7: Necrotic area 25%-50%; Level 9: Necrotic area exceeds 50% or all browning. Statistical calculation method of disease index and biocontrol effect: Disease index (%) = [∑ (number of disease levels × number of fruits at the disease level) / (highest disease level × total number of fruits)] × 100; Biocontrol effect (%) = [(control disease index - treatment group disease index) / control disease index] × 100.

[0071] The experimental results show that ( Figure 7 and Figure 8 )F32 bacterial suspension has a good control effect on litchi downy mildew. Among them, the disease index of 72hip and 84hip in the in vitro fruit control effect is 4.28% and 42.59% respectively, which is lower than the severity of the control group. The biological control effect reaches 91.7% and 51.18% respectively, indicating that F32 bacterial suspension has an inhibitory effect on the occurrence of litchi downy mildew on in vitro fruits. In the in vitro leaf control effect, the disease index in 84hip is 62.73%, which is lower than the severity of the control group. The biological control effect reaches 31.45%, indicating that F32 bacterial suspension has an inhibitory effect on the occurrence of litchi downy mildew on in vitro leaves. In summary, F32 bacterial suspension has a good application prospect in the field of biological control of litchi downy mildew.

Claims

1. A strain of Pseudomonas oryzihabitans F32 for controlling litchi downy mildew, classified as Pseudomonas oryzihabitans, with a strain collection number of GDMCC No.65232, and a preservation unit named Guangdong Microbiological Culture Collection Center, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation date of October 8, 2024.

2. A bacterial agent prepared by the Pseudomonas oryzihabitans F32 according to claim 1.

3. The bacterial agent according to claim 1, characterized in that The seed liquid of Pseudomonas oryzihabitans F32 was inoculated into the liquid LB culture medium, and the culture was placed in a constant temperature oscillator at 28° C. and 200 r / min for 8-12 hours to obtain the fermentation liquid as the bacterial agent.

4. The bacterial agent according to claim 1, characterized in that The seed liquid of Pseudomonas oryzihabitans F32 was inoculated into liquid LB culture medium, and the culture was placed in a constant temperature oscillator at 28°C and 200 r / min for 8-12 hours to obtain a fermentation liquid. The fermentation liquid was filtered, and the bacterial bodies were resuspended with sterile water to obtain a bacterial suspension as a bacterial agent.

5. Use of Pseudomonas oryzihabitans F32 according to claim 1 in preventing and controlling litchi downy mildew.

6. Use of the Pseudomonas oryzihabitans F32 according to claim 1 in preparing a product for preventing and controlling litchi downy mildew.

7. Use of the bacterial agent according to any one of claims 2 to 4 in preventing and controlling litchi downy mildew.

8. The use according to claim 7, characterized in that: Use of the bacterial agent according to claim 3 in inhibiting the mycelial growth of litchi peronophyte, inhibiting the germination of sporangia of litchi peronophyte and / or inhibiting the germination of zoospores of litchi peronophyte.

9. The use according to claim 7, characterized in that: The use of the bacterial agent described in claim 4 in inhibiting the growth of litchi downy mildew hyphae, inhibiting the germination of sporangium of litchi downy mildew and / or inhibiting the germination of zoospores of litchi downy mildew, and improving the prevention effect of litchi downy mildew on in vitro litchi fruits and leaves.

10. Use of the bacterial agent according to any one of claims 2 to 4 in the preparation of an in vitro litchi preservative or a product for preventing and treating in vitro litchi downy mildew.

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