Biocontrol bacterium F14 for preventing and treating peronophythora litchii and application of biocontrol bacterium F14

Abstract

The invention discloses a biocontrol bacterium for preventing and treating peronophythora litchii and application of the biocontrol bacterium. The biocontrol bacterium is sphingomonas, and the preservation number of the biocontrol bacterium is GDMCC No: 65231. The invention also discloses a microbial inoculum prepared from the biocontrol bacterium. The biocontrol bacterium and the fungicide are applied to prevention and treatment of litchi downy mildew. 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 present invention relates to the field of agricultural microorganisms, and specifically relates to a biocontrol agent for preventing and controlling litchi downy blight and its application. Background Art

[0002] Litchi (Litchi chinensis Sonn.) is one of the most beloved fruits in summer and belongs to the genus Litchi of the Sapindaceae family. Litchi downy blight is currently the most serious disease during the production, storage and transportation of litchi. This disease mainly harms mature litchi fruits, and young fruits and young leaves are also occasionally affected. When the fruit is diseased, brown lesions are formed. The lesions develop rapidly and a white mold layer is formed on the surface. In the later stage, the whole fruit turns brown and rots, and then a large number of fruits drop, ultimately harming the quality and yield of litchi fruits.

[0003] At present, the agricultural control of this disease mainly focuses on strengthening cultivation management and paying attention to orchard cleaning. In China, there is a rich variety of fungicides for preventing and controlling litchi downy blight. The main protective fungicides include mancozeb, chlorothalonil, copper hydroxide, etc.; fungicides with both protective and therapeutic effects such as metalaxyl-mancozeb, propamocarb, azoxystrobin, etc. However, fruit farmers have problems such as overuse of pesticides, chaotic use of pesticides, missing the best period for applying the fungicide, and lack of awareness of rotational use of pesticides during the pesticide application process, making it impossible to effectively control this disease or easily resulting in drug resistance.

[0004] Due to the serious environmental pollution and pesticide residue problems caused by the long-term irregular use of chemical agents, biological control has attracted more and more attention. In 2010, an article reported that after treatment with Bacillus subtilis, the active oxygen metabolism and the activity of pathogenesis-related proteins in litchi fruits can be significantly up-regulated, which has a good effect on preventing and controlling litchi downy blight of postharvest fruits. In 2018, it was also confirmed that the volatile substances of a Streptomyces fimicarius can damage the cell wall and inner membrane system of litchi downy blight, thus playing a role in inhibiting its growth. Summary of the Invention

[0005] The purpose of the present invention is to provide a biocontrol agent prepared from Sphingomonas sp. aiming at the deficiencies of the biological control technology for litchi downy blight. This biocontrol agent has a strong antagonistic effect against litchi downy blight and can prevent and control litchi downy blight in agricultural production.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] A Sphingomonas sp. F14 for controlling litchi downy blight, with the taxonomic name Sphingomonas sp., the preservation unit name is Guangdong Provincial Microbial Culture Collection Center, the address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, the preservation time is October 8, 2024, and the culture collection number is GDMCC No: 65231.

[0008] A bacterial agent prepared from the Sphingomonas sp. F14 described above.

[0009] As a preference of the present invention, the fermentation broth obtained by inoculating the F14 seed liquid into a liquid LB medium and culturing it in a constant temperature oscillator at 28°C and 200 r / min for 8 - 12 h is used as the bacterial agent.

[0010] As a preference of the present invention, the fermentation broth obtained by inoculating the F14 seed liquid into a liquid LB medium and culturing it in a constant temperature oscillator at 28°C and 200 r / min for 8 - 12 h, and the fermentation broth is filtered, and the bacterial suspension obtained after resuspending the thalli with sterile water is used as the bacterial agent.

[0011] The application of the Sphingomonas sp. F14 described above in controlling litchi downy mildew.

[0012] The application of the Sphingomonas sp. F14 described above in preparing a product for controlling litchi downy mildew.

[0013] The application of the bacterial agent described above in controlling litchi downy mildew.

[0014] As a preference of the present invention, the application of the bacterial agent prepared from the F14 fermentation broth in inhibiting the germination of sporangia of Peronophythora litchi and / or inhibiting the germination of zoospores of Peronophythora litchi.

[0015] As a preference of the present invention, the application of the bacterial agent prepared from the F14 bacterial suspension in inhibiting the growth of mycelia of Peronophythora litchi and inhibiting the germination of sporangia of Peronophythora litchi.

[0016] The application of the bacterial agent described above in preparing an in vitro litchi preservative or a product for controlling in vitro litchi downy mildew.

[0017] The present invention has the following two advantages:

[0018] (1) The bacterial agent provided by the present invention is composed of Sphingomonas sp., which is isolated from litchi pericarp, avoiding the situation of alien species invasion and the restriction of biocontrol bacteria by the environment in biological control, and at the same time preventing the environmental pollution problem and drug resistance problem caused by chemical drugs.

[0019] (2) The biocontrol strain used in the biocontrol agent provided by the present invention has high control efficacy, simple culture conditions and low price. It has been industrialized and can be widely promoted and applied on a large scale, showing good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Single colony map of biocontrol bacterium F14

[0021] As can be seen from the figure, the F14 colony color is light yellow, the shape is round, the edge is smooth, the texture is smooth in the early stage, and a biofilm is formed in the later stage.

[0022] Figure 2 Effects of three different components (supernatant, fermentation broth, bacterial suspension) of biocontrol bacterium F14 on hyphae

[0023] Figure 3 Effects of three components of biocontrol bacterium F14 on the germination of sporangia of Peronophythora litchi

[0024] Figure 4 Effects of three components of F14 on the germination of zoospores of Peronophythora litchi

[0025] Figure 5 Schematic diagram of disease grading for the infection of litchi fruits and leaves by Peronophythora litchi

[0026] Figure 6 Disease index and biocontrol effect of F14 on detached litchi fruits

[0027] Figure 7 Disease index and biocontrol effect of F14 on detached litchi leaves

[0028] Biological sample preservation information

[0029] Sphingomonas sp. F14, with the taxonomic name of Sphingomonas sp., the preservation unit is the Guangdong Microbial Culture Collection Center, the address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, the preservation time is October 8, 2024, and the culture collection number is GDMCC No: 65231. DETAILED DESCRIPTION OF THE INVENTION

[0030] Example 1 Isolation and purification of F14

[0031] Culture medium preparation: The formula of LB solid medium (10 g of sodium chloride, 10 g of tryptone, 5 g of yeast powder, 15 g of agar powder, made up to 1 L with distilled water, adjust the pH to approximately 7.2) was sterilized at 121 °C for 20 min; the formula of R2A solid medium (0.5 g / L of yeast powder, 0.25 g / L of casein peptone, 0.25 g / L of meat peptone, 0.5 g / L of hydrolyzed casein, 0.5 g / L of glucose, 0.5 g / L of starch, 0.3 g of pyruvic acid, pH = 7.2) was sterilized at 121 °C for 20 min.

[0032] Strain isolation: Weigh 3 g of the pericarp of litchi 'Feizixiao', place it in a sterilized mortar, add 27 mL of sterilized 0.85% sodium chloride and grind it (including sterilized glass beads), shake it on a shaker at 180 rmp for 30 min, and let it stand for 5 min. Filter it through sterile cotton cloth to obtain a -1 bacterial suspension.

[0033] Dilution and spreading: Pipette 0.1 mL of the supernatant, and successively add it to 900 μL of sterile 0.85% NaCl and mix well to obtain a -2 sample dilution. And so on, finally obtaining dilutions of 10 -3 、10 -4 、10-5、10 -6 respectively. Take 10 -4 、10 -5 、10 -6 of the three gradient dilutions, 100 μL each, and add them to the 2 R2A (0.5 g / L of yeast powder, 0.25 g / L of casein peptone, 0.25 g / L of meat peptone, 0.5 g / L of hydrolyzed casein, 0.5 g / L of glucose, 0.5 g / L of starch, 0.3 g of pyruvic acid, pH = 7.2) solid medium plates. Spread them evenly with a spreader until completely absorbed. There are 3 replicates for each gradient, and incubate them in an inverted position at 28 °C for 48 - 72 h, observe the results, and isolate and purify the strains from them.

[0034] Identification sequencing: Pick a single colony of the purified and preserved bacteria and inoculate it into LB liquid medium to prepare a fermentation broth of beneficial bacteria. Take 1.5 mL for standby. Collect bacterial cells by centrifugation and extract according to the AxyPrep Bacterial Genomic DNA Mini Kit. Take 5 μL of the extracted DNA and perform electrophoresis detection using 0.8% agarose gel. Select a Marker as a reference according to the predicted size of the target fragment. After completion, move the gel to an ultraviolet transilluminator for observation. If the band is bright, clear and conforms to the predicted size, it can be stored at -20 °C for standby. Using the extracted bacterial DNA as a template, amplify the 16S rRNA gene with universal primers. The primer sequences are: U8-27(F): 5’-AGAGTTTGATC(AC)TGGCTCAG-3’; L1494-1514(R): 5’-CTACGG(AG)TACCTTGTTACGAC-3’. The reaction system is: 25 μL Mix, 2 μL primer F, 2 μL primer R, 2 μL template DNA and 19 μL ultrapure water. The reaction procedure is: pre-denature at 95 °C for 3 min first; then denature at 95 °C for 10 s, anneal at 56 °C for 40 s, extend at 72 °C for 1 min, and cycle 28 times; finally, perform post-extension at 72 °C for 5 min. Take 5 μL of the product, add 1 μL of loading buffer and mix well, perform 1.0% agarose gel electrophoresis detection at a voltage of 120 v for 25 min, soak in EB for 15 min, and then observe the target band using a BIO-RAD gel system imaging system; observe the band, and send the amplified product that is bright, clear and consistent with the expected size to Shanghai Sangon Biotech Co., Ltd. for sequencing. BLAST align the original sequence on NCBI, select the strain with the highest homology, and finally identify F14 as Sphingomonas sp.

[0035] Example 2 Preparation of biocontrol agent, pathogen mycelium, sporangium, and zoospore

[0036] Preparation of biocontrol bacteria fermentation broth: Use a toothpick to pick an activated single colony into 2 mL of liquid LB medium, place it in a constant temperature oscillator at 30 °C and 180 r / min for 12 h to obtain a seed solution. Then take 1 mL of the seed solution and add it to 19 mL of liquid LB medium, and place it in a constant temperature oscillator at 30 °C and 200 r / min for 8 h to obtain a fermentation broth.

[0037] Preparation of biocontrol bacteria suspension: Dispense and balance the prepared fermentation broth, centrifuge at 4 °C, 8000 r / min for 10 min, pour off the supernatant, resuspend the wet bacterial cells with sterile water, and adjust the concentration to OD600 = 1 measured by an enzyme-linked immunosorbent assay instrument.

[0038] Preparation of supernatant of biocontrol bacteria: The prepared fermentation broth was dispensed and balanced, placed at 4°C, centrifuged at 8000 r / min for 10 min, and the supernatant was taken and filtered through a sterilized 0.22 μm filter membrane.

[0039] Activation of pathogenic bacteria: Take the plate of Peronophythora litchii strain cultured in the dark at 26°C. Use the sterilized yellow pipette tip to pick the fresh bacterial cake on the outermost layer of the plate, and inoculate it at the center position of the CJA (200 g / L of carrots, chopped and juiced with water for 10 min, filtered through gauze, 15 g / L of agar powder, pH = 7.0 - 7.2) medium plate, and place it in an artificial climate chamber for dark culture at 28°C for 7 d to make the pathogenic bacteria cover the entire plate.

[0040] Preparation of sporangium suspension of Peronophythora litchii: Add 10 mL of sterile water to the plate covered with mycelium, use a sterilized writing brush to wash off the sporangia, filter the mycelium and impurities through a sterilized tea strainer into a 500 mL sterilized conical flask, shake well, take 1 μL on a glass slide, observe and count under an inverted biological microscope, add sterile water and shake well to adjust its concentration to 5×10 4 sporangia / mL.

[0041] Preparation of zoospore suspension of Peronophythora litchii: Put the prepared sporangium suspension into a 4°C refrigerator for 1 h, then place it at room temperature for 1 h, and then put it into a 4°C refrigerator for 1 h and take it out. This operation uses temperature difference treatment to stimulate the sporangia to fully release zoospores, and then uses a sterilized 0.22 μm filter membrane to filter the sporangia and mycelium to obtain a relatively pure zoospore suspension. Shake well and take 1 μL to count under an inverted biological microscope, add sterile water and shake well to adjust its concentration to 35×10 3 zoospores / mL.

[0042] Example 3 Effect of F14 bacterial agent on the mycelial growth of Peronophythora litchii

[0043] In this example, the plate confrontation method was used to observe the inhibitory effects of the supernatant, fermentation broth, and bacterial suspension of Sphingomonas F14 on the mycelial growth of Peronophythora litchii. The specific operation is as follows:

[0044] The well-activated Phytophthora litchii was evenly punched into circular blocks with a diameter of 5 mm from the outer edge of the colony using a sterilized borer. The blocks (5 mm) were inoculated into the middle of the CJA-PDA medium (CJA: 200 g / L of carrots, chopped and juiced with water for 10 min, filtered through gauze, 15 g / L of agar powder, pH = 7.0 - 7.2; PDA: 200 g / L of potatoes, chopped and boiled with water for 20 min, 20 g / L of glucose added to the filtrate, 15 g / L of agar powder, pH = 7.0 - 7.2, sterilized separately and then mixed together at a volume ratio of 1:1). 10 μL of the fermentation broth of the biocontrol bacterium (OD600 = 1), bacterial suspension (OD600 = 1), and supernatant (filtered through a 0.22 μm sterilized filter membrane) were respectively pipetted and inoculated at four points 27 mm away from the pathogen, as follows Figure 2 . The inhibition rate was calculated by measuring the area of the pathogen

[0045] Inhibition rate = (area of the pathogen in the control group - area of the pathogen in the treatment group) / area of the pathogen in the control group × 100%

[0046] The experimental results are as follows Figure 3 , there were significant differences among the components of F14 (P < 0.05). The supernatant and fermentation broth had no obvious inhibitory effect on the mycelial growth of Phytophthora litchii, while the bacterial suspension reached 78.33%.

[0047] Example 4 Determination of the sporangium germination activity of the F14 bacterial agent against Phytophthora litchii

[0048] In this example, to explore the inhibitory effects of the three components of F14 on the sporangium germination of Phytophthora litchii, the specific operation was as follows: Experimental group: 4.5 mL of a 5×10 4 sporangia / mL sporangium suspension was taken with a pipette and mixed with 0.5 mL of the fermentation broth of the biocontrol bacterium (OD600 = 1), bacterial suspension (OD600 = 1), and supernatant (filtered through a 0.22 μm sterilized filter membrane) in a test tube. Control group: 4.5 mL of sporangium suspension was taken with a pipette and mixed with 0.5 mL of sterile water in a test tube. They were placed in a constant temperature incubator at 30 °C and 180 r / min for cultivation. Each treatment had 6 replicates. For every 3 replicates, at 6 h and 9 h respectively, 1 μL was taken and observed and counted for the number of germinated sporangia and ungerminated sporangia under a microscope( Figure 4 C). Every 200 sporangia were regarded as one replicate, and the data were recorded to calculate the sporangium germination rate and inhibition rate

[0049] Germination rate = number of germinated / total number

[0050] Inhibition rate = (germination rate in the control group - germination rate in the treatment group) / germination rate in the control group

[0051] The experimental results showed that when co-cultured for 6 h, the sporangium germination rates of the bacterial suspension and the fermentation broth were only 36.91% and 11.33% respectively, while those of the supernatant and the control group were 60.87% and 80.4%; when co-cultured for 9 h, the sporangium germination rates of the bacterial suspension and the fermentation broth were 18.65% and 52.45% respectively, while those of the supernatant and the control group were 50.3% and 85.23%. Through statistical analysis, it was found that there were significant differences between each component of F14 and the control group (P < 0.05). Thus, it can be seen that the bacterial suspension of Sphingomonas sp. F14 has an inhibitory effect on the sporangium germination of Peronophythora litchii, and the inhibition rate can reach 68.9%.

[0052] Example 5 Determination of the Germination Activity of Zoospores of F14 Bacterial Agent against Peronophythora litchii

[0053] This case explored the inhibitory effects of three components of F14 on the germination of zoospores of Peronophythora litchii. The specific operations were as follows: Experimental group: Use a pipette to take 4.5 mL of a 5×10 4 zoospores / mL zoospore suspension and mix it with 0.5 mL of the fermentation broth of the biocontrol bacterium (OD600 = 1), the bacterial suspension (OD600 = 1), and the supernatant (filtered through a 0.22 μm sterilized filter membrane) in a test tube. Control group: Use a pipette to take 4.5 mL of the zoospore suspension and mix it with 0.5 mL of sterile water in a test tube, and place it in a constant temperature incubator at 30 °C and 180 r / min for cultivation. Each treatment had 6 replicates. At 3 h and 5 h respectively for every 3 replicates, pipette 1 μL and observe and count the number of germinated zoospores and ungerminated zoospores under a microscope (Figure), and record the data. Consider every 120 zoospores as one replicate, and calculate the sporangium germination rate and the inhibition rate.

[0054] Germination rate = number of germinated spores / total number

[0055] Inhibition rate = (germination rate of the control group - germination rate of the treatment group) / germination rate of the control group

[0056] The experimental results showed that at 3h of culture, the zoospore germination rates of the bacterial suspension and fermentation broth were 69.82% and 37.78%, respectively, while the germination rates of the supernatant and the control group were 78.84% and 82.55%. At 5h of culture, the sporangium germination rates of the bacterial suspension and fermentation broth were only 69.75% and 29.72%, while the germination rates of the supernatant and the control group were 77.86% and 79.66%. Through statistical analysis, it can be seen that there is no significant difference between the supernatant of F14 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 of F14 has an inhibitory effect on the germination of sporangium of litchi downy mildew, and its inhibition rate can reach 64.7%, which has a good inhibitory effect. Comparing the culture time, it can be seen that the inhibition rate of the fermentation broth increased from 55.3% to 64.7%, which shows that the fermentation broth has better long-term inhibitory ability on the germination of zoospores of litchi downy mildew pathogen than the bacterial suspension.

[0057] Example 6: Effect of F14 on Downy Blight of Litchi Fruits and Leaves in Vitro

[0058] In this example, the above test results are summarized, and the F14 suspension is selected to simulate the field control effect determination to explore the biological control effect of the F14 bacterial suspension on litchi downy mildew. The specific operation is as follows:

[0059] Freshly picked litchi 'Fei Zi Xiao' fruits and leaves were treated as follows: A. Experimental group: F14 bacterial suspension diluted 50 times and sprayed on leaves or fruits; B. Control group: The culture solution was diluted 50 times and then sprayed. Three replicates were set for each treatment, and 30 fruits or leaves were placed in a fresh-keeping box for each replicate. A total of 300 mL was sprayed for each treatment and control group. After 24 hours, the pathogen sporangium suspension (5×10 4 The disease index was investigated after 72 and 84 hpi hours postinoculation and the biocontrol effect was calculated.

[0060] According to the disease severity survey method ( Figure 6 A and B): Level 1: slight lesions at the inoculation site, 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.

[0061] Statistical calculation method for disease index and biocontrol effect:

[0062] Disease index (%) = [∑ (disease level × number of results for that disease level) / (highest disease level × total number of results)] × 100;

[0063] Biocontrol effect (%) = [(control disease index - treatment group disease index) / control disease index] × 100.

[0064] The experimental results showed that the F14 bacterial suspension had a good control effect on preventing and controlling postharvest litchi downy blight. Among them, the control effects on fruits were 13.19% and 71.53% at 72 hpi and 84 hpi of disease index respectively, and the disease severity was lower than that of the control group. The biocontrol effect was the highest at 74.44% at 72 hpi, but the control effect decreased significantly at 84 hpi, indicating that the F14 bacterial suspension had an obvious inhibitory effect on the incidence of litchi downy blight on fruits at 72 hpi and the effect was the best.

[0065] In the control effect on leaves, the disease index was 51.75% at 84 hpi. Compared with the disease index of 94.17% in the control group, the disease severity was lower, and the biocontrol effect reached 42.42%, indicating that the F14 bacterial suspension had an inhibitory effect on the incidence of litchi downy blight on leaves. In summary, the F14 bacterial suspension has good application prospects in the field of biological control of litchi downy blight.

Claims

1. A Sphingomonas sp. F14 for preventing and controlling litchi downy blight, classified as Sphingomonas sp., the name of the preservation unit is Guangdong Microbiological Culture Collection Center, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation time is October 8, 2024, and the culture collection number is GDMCC No: 65231.

2. A bacterial agent prepared by the Sphingomonas sp. F14 according to claim 1.

3. The bacterial agent according to claim 1, characterized in that The F14 seed liquid was inoculated into liquid LB culture medium, and cultured 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 F14 seed liquid 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 cells were resuspended with sterile water to obtain a bacterial suspension as a bacterial agent.

5. Use of Sphingomonas sp. F14 according to claim 1 in preventing and controlling litchi downy mildew.

6. Use of the Sphingomonas sp. F14 according to claim 1 in preparing a product for preventing and treating 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 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: Use of the bacterial agent described in claim 4 in inhibiting the growth of mycelium of litchi downy mildew and inhibiting the germination of sporangia of litchi downy mildew.

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

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