Serratia pumila MM, fungicide and application of serratia pumila MM in prevention and treatment of pepper phytophthora blight

Abstract

The invention relates to the technical field of biology, in particular to application of serratia pumila MM fermentation liquor and a microbial agent in inhibiting growth of phytophthora capsici and preventing and treating phytophthora blight of capsici, serratia pumila MM is preserved in China General Microbiological Culture Collection Center on June 2, 2022, the preservation number is CGMCC No.24997, and the preservation number is CGMCC No.24997. The serratia pumila MM is preserved in China General Microbiological Culture Collection Center on June 2, 2022. The compound has a good inhibition effect on phytophthora capsici, and the bacteriostasis rate reaches 79.05%; the growth of pathogenic bacteria hyphae can be effectively inhibited, the hyphae are damaged, and the hyphae are broken, shrunk, deformed and the like. In a pot experiment, the prevention and treatment effects of a prevention group and a treatment group on the pepper phytophthora blight are 88.33% and 58.30% respectively, and the morbidity of the pot prevention group is only 14.6%. In a potted plant growth promoting test, the plant height, root length, fresh weight and stem diameter of pepper seedlings are obviously improved and are respectively increased by 29.56%, 51.41%, 25.90% and 20.00% compared with a contrast, and the growth promoting effect is obvious. In a field experiment, a Serratia uchenensis MM agent treatment group is excellent in performance, the control effect on the pepper phytophthora blight is 82.2%, plants grow robustly, the disease incidence rate is low, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of Serratia plymuthica MM and its bacterial agent in preventing and controlling Phytophthora capsici of peppers and promoting the growth of pepper plants. Background Art

[0002] Pepper (Capsicum annuum L.) is a solanaceous plant, which is a widely used condiment and vegetable and has rich nutritional value. As an important crop, pepper is widely planted globally, and its planting area and yield occupy a place among global vegetables. However, with the expansion of pepper planting area and continuous cropping, the disease problems faced by peppers are increasing. Among them, Phytophthora blight of pepper is a relatively serious disease in pepper production, posing a major threat to the growth and yield of peppers, and can occur throughout the entire growth period of peppers. Its pathogen, Phytophthora capsici, belongs to the facultative parasite of the genus Phytophthora in the phylum Oomycota, and can infect and seriously damage multiple types of plants such as solanaceous, leguminous, and cucurbitaceous plants, and can survive in the soil for a long time, so it is difficult to cure. When Phytophthora blight of pepper occurs at the base of the pepper plant stem, water-soaked dark green lesions will appear and quickly expand around the stem, resulting in the wilting and death of the plant, bringing huge economic losses to agricultural production.

[0003] Chemical control methods are currently the main control measures for Phytophthora blight of peppers, but it is difficult to eradicate. Long-term use will cause the pathogen to develop drug resistance, and at the same time will cause environmental pollution and affect human health. With the in-depth research on biological control, the discovery of high-quality biocontrol strains and the development and application of efficient biocontrol agents have also become more extensive. Serratia plymuthica, as a biocontrol bacterium, has a wide range of applications in the field of agricultural biological control. This bacterium can produce a variety of antifungal factors, including cell wall degrading enzymes such as chitinase and β-1,3-glucanase, as well as pyrrolnitrin (PRN), etc. These metabolites have a broad-spectrum inhibitory effect on plant pathogenic fungi, bacteria, and nematodes, providing an environmentally friendly and sustainable solution for crop protection.

[0004] Chinese Patent Publication No.: CN 101189942A provides a method for preventing and controlling Phytophthora blight of peppers by combining a biocontrol bacterium with a solid water retainer to treat the seedling substrate. However, the incidence rate of the single biocontrol bacterium treatment group is as high as 32.6%, while the incidence rate of the preventive treatment group of Serratia plymuthica MM in the present invention is only 14.6%, which is 18.0% lower than that of it, and the control effect is more excellent. Summary of the Invention

[0005] In view of the above technical problems, the first object of the present invention is to provide the application of the fermentation broth of Serratia plymuthica MM in preventing and controlling Phytophthora blight of peppers.

[0006] Preferably, the Serratia plymuthica MM was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 2, 2022, with the deposit number CGMCC No. 24997, the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the contact phone number being: 010 - 64807355.

[0007] Preferably, the pathogen of the Phytophthora blight of peppers is Phytophthora capsici.

[0008] The second object of the present invention is to provide the application of Serratia plymuthica MM in promoting the growth of pepper plants.

[0009] The third object of the present invention is to provide a preparation method of the biocontrol agent, and its components and weight percentages are as follows: the active ingredient Serratia plymuthica 18%, the carrier diatomaceous earth 64%, light calcium carbonate 12%, sodium chloride 3%, Tween - 80 2%, antioxidant 1%.

[0010] The fourth object of the present invention is to provide the application of the Serratia plymuthica MM agent in field prevention and control of Phytophthora blight of peppers.

[0011] The beneficial effects of the present invention are as follows: The present invention provides an application of Serratia plymuthica MM in controlling Phytophthora capsici. The Serratia plymuthica MM was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 2, 2022, with the deposit number CGMCC No. 24997. It has a good inhibitory effect on Phytophthora capsici, with an inhibition rate of 79.05%. It can effectively inhibit the mycelial growth of the pathogen, damage the mycelia, causing the mycelia to break, constrict, and deform. It can be applied to the control of Phytophthora capsici. In the pot experiment, the control effects of the prevention group and the treatment group on Phytophthora capsici were 88.33% and 58.30% respectively, and the incidence rate of the pot prevention group was only 14.6%. In the pot growth promotion experiment, the plant height, root length, fresh weight, and stem diameter of pepper seedlings were significantly increased, increasing by 29.56%, 51.41%, 25.90%, and 20.00% respectively compared with the control, showing an obvious growth promotion effect. In the field experiment, the treatment group with the Serratia plymuthica MM agent showed excellent performance, with a control effect on Phytophthora capsici of 82.2%, the plants grew robustly, and the disease incidence rate was low. The Serratia plymuthica MM described in the present invention has a good biocontrol effect on Phytophthora capsici, and the prevention effect is significantly better than the treatment effect, with broad application prospects. Description of the Drawings

[0012] Figure 1 Colony map of Serratia plymuthica MM

[0013] Figure 2 Confrontation test of laboratory biocontrol bacteria and Serratia plymuthica MM with Phytophthora capsici

[0014] Figure 3 Electron microscope observation diagram of the inhibitory effect of the sterile filtrate of Serratia plymuthica MM on the mycelia of Phytophthora capsici

[0015] Note: A: CK; B, C, D: Treatment groups with the sterile filtrate of Serratia plymuthica MM

[0016] Figure 4 Pot control effect of the fermentation broth of Serratia plymuthica MM on Phytophthora capsici

[0017] Figure 5 Field control effect of the Serratia plymuthica MM agent on Phytophthora capsici Detailed Description of the Invention

[0018] The protection scope of the present invention will be elaborated in detail below in combination with specific embodiments. It should be noted that the protection scope of the present invention is not limited by the following embodiments.

[0019] In the following embodiments, unless otherwise specified, the methods are all conventional methods, and the reagents / consumables can all be obtained through commercial channels.

[0020] In the following examples, the pathogenic bacterium Phytophthora capsici was donated by the Gansu Academy of Agricultural Sciences.

[0021] Example 1: Screening of antagonistic strains against the pathogenic bacterium of Phytophthora blight of pepper

[0022] Select the existing biocontrol bacteria in the laboratory, Bacillus mucilaginosus, Paenibacillus polymyxa, Bacillus amyloliquefaciens, Bacillus velezensis, Serratia plymuthica MM, etc. Through the plate confrontation method, the biocontrol bacteria with antagonistic effects against Phytophthora capsici were screened out, and the inhibition rate of their mycelial growth against Phytophthora capsici was calculated:

[0023]

[0024] The test results are as Figure 2 shown in Table 1. It can be seen that each biocontrol bacterium in the laboratory has the effect of inhibiting Phytophthora capsici. Among them, Serratia plymuthica MM described in the present invention has the highest inhibition rate against Phytophthora capsici, reaching 79.05%, which is significantly higher than other biocontrol bacteria. Serratia plymuthica MM is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 24997. The colony streaking map of it on the solid LB medium is as Figure 1 .

[0025] Table 1 Determination of the antibacterial rate of biocontrol bacteria

[0026] Biocontrol bacteria Colony radius (mm) Inhibition rate / % Bacillus mucilaginosus <![CDATA[20.65±1.30 a > <![CDATA[39.33±0.58 e > Paenibacillus polymyxa <![CDATA[14.21±1.48 d > <![CDATA[64.00±1.73 b > Bacillus amyloliquefaciens <![CDATA[19.22±1.00 b > <![CDATA[47.00±3.00 d > Bacillus velezensis <![CDATA[16.98±1.94 c > <![CDATA[56.33±1.53 c > Serratia plymuthica MM <![CDATA[9.70±0.64 e > <![CDATA[79.05±0.58 a >

[0027] Note: Different letters represent the standard deviation of repeated values and there are significant differences (p < 0.05)

[0028] Example 2: Inhibitory effect of the sterile fermentation broth of Serratia plymuthica MM on the mycelia of Phytophthora capsici

[0029] Inoculate Serratia plymuthica MM from Example 1 onto the sterilized LB medium plate. The culture conditions are as follows: the temperature is 30 °C, and the culture time is 48 h; pick a single colony and inoculate it into the LB liquid medium, and culture it on a shaker at 30 °C and 180 r / min for 24 h to prepare the seed solution.

[0030] Inoculate the fresh seed liquid into LB medium and culture it on a shaker for 48 h. Centrifuge at 5000 r / min for 10 min at room temperature, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the sterile fermentation filtrate for standby.

[0031] Use a scanning electron microscope to observe the mycelia of Phytophthora capsici before and after treatment with the sterile fermentation filtrate of Serratia plymuthica MM. The results are as Figure 4 shown. The mycelia treated with the sterile fermentation filtrate showed phenomena such as breakage, constriction, and deformation, indicating that the sterile fermentation filtrate of Serratia plymuthica MM damaged the mycelia and could effectively inhibit the growth of Phytophthora capsici mycelia.

[0032] Example 3. Pot control effect of the fermentation broth of Serratia plymuthica MM on Phytophthora blight

[0033] Transplant the pepper seedlings with uniform growth into pots. Three groups are designed in the experiment, with 10 pots for each treatment, which are: the control group is inoculated with a spore suspension of Phytophthora capsici (10 7 CFU / mL); the prevention group is inoculated with the fermentation broth of Serratia plymuthica MM, and then inoculated with a spore suspension of Phytophthora capsici (10 8 CFU / mL) after 24 h; the treatment group is inoculated with a spore suspension of Phytophthora capsici, and then inoculated with the fermentation broth of Serratia plymuthica MM after 24 h.

[0034] After 14 d, statistically analyze the growth of the plants in each treatment, investigate the incidence and disease index of Phytophthora blight, and calculate the control effect of the biocontrol bacteria. The grading standard for Fusarium wilt at the pepper seedling stage: grade 0 means that the vascular bundle in the stem is normal and there are no external symptoms; grade 1 means that the vascular bundle at the root and stem of the seedling is slightly blackened and the leaves do not wilt or have recoverable wilting; grade 2 means that the vascular bundle at the root and stem of the seedling is blackened by 1 cm - 2 cm, the leaves have non-recoverable wilting, and the lower leaves occasionally fall off; grade 3 means that the vascular bundle at the root and stem of the seedling is blackened by more than 2 cm, the leaves are significantly wilted or the leaves fall off significantly; grade 4 means that the root and stem of the seedling are blackened and constricted, and all leaves except the growth point fall off or the plant wilts; grade 5 means that the whole plant dies.

[0035]

[0036]

[0037] Table 2 Control effect of the fermentation broth of Serratia plymuthica MM on Phytophthora blight in pots

[0038] Group Disease index Incidence rate (%) Relative control efficacy (%) Control group <![CDATA[4.8 ±0.48 a > <![CDATA[100% a > - Prevention group <![CDATA[0.53 ±0.12 c > <![CDATA[14.6 c > <![CDATA[88.89 ±0.06 a <!-- 3 -->]]> Treatment group <![CDATA[1.34 ±0.36 b > <![CDATA[32.5 b > <![CDATA[72.22 ±0.13 b >

[0039] Note: Different letters indicate significant differences (p < 0.05)

[0040] The growth of the plants is as Figure 4, it can be seen that the stems of the pepper plants in the control group turned black and constricted, all the leaves fell severely, or the plants wilted and died; the preventive group had no symptoms or the rhizomes of the pepper plant seedlings turned slightly brown; the rhizomes of the pepper plant seedlings in the treatment group turned slightly black, and the leaves did not wilt or showed recoverable wilting. This indicates that Serratia plymuthica MM has a strong control effect on preventing and controlling Phytophthora blight of pepper.

[0041] The statistical results of the incidence of Phytophthora blight of pepper in pots treated with Serratia plymuthica MM are shown in Table 2. It can be seen that the incidence rate of the treatment group was 32.5% and the control effect was 72.22%, the incidence rate of the preventive group was 14.6%, and the control effect was 88.89%. Both were significantly lower than the incidence rate of the control group (100%), and the control effect was excellent. This shows that Serratia plymuthica MM has both therapeutic and preventive effects on Phytophthora blight of pepper, and the preventive effect is significantly better than the therapeutic effect.

[0042] Example 4. Promoting growth effect of the fermentation broth of Serratia plymuthica MM on pepper plants in pots

[0043] Prepare the fermentation broth of strain MM, and adjust the concentration of the bacterial suspension to 10 8 CFU / mL. Select healthy and plump pepper seeds, surface disinfect them and sow them in sterile soil. When the seedlings grow to 2 - 3 true leaves, add the fermentation broth of Serratia plymuthica MM to the potting substrate of the treatment group, while the control group adds an equal amount of sterile medium. After culturing for 14 days in an environment of 25°C with 12 h light-dark alternation, measure the growth indexes of each group. Each treatment includes 10 pots, and this experiment is independently repeated three times. Compared with the control group, after adding the fermentation broth of strain Serratia plymuthica MM, the pepper seedlings were significantly improved in plant height, root length, fresh weight and stem diameter, increasing by 29.56%, 51.41%, 25.90% and 20.00% respectively (Table 3).

[0044] Plant height (cm) Root length (cm) Fresh weight (g) Stem diameter (mm) Control group <![CDATA[16.04±2.83 b > <![CDATA[8.52±1.34 b > <![CDATA[9.77±0.85 b > <![CDATA[3.10±0.51 b > Treatment group with strain MM <![CDATA[22.73±1.68 a > <![CDATA[12.9±0.75 a > <![CDATA[12.3±1.42 a > <![CDATA[3.72±0.24 a > Growth promotion effect (%) 29.56% 51.41% 25.90% 20.00%

[0045] Table 3 Evaluation of the growth-promoting effect of the fermentation broth of Serratia plymuthica MM on pepper plants

[0046] Note: Different letters represent significant differences (p<0.05)

[0047] Example 5. Field control effect of Serratia plymuthica MM microbial agent on Phytophthora blight of pepper

[0048] According to the results of the above pot experiment, a field experiment was carried out in 2022 in Changcheng Village, Jiayuguan City. Set 10 10Three treatments were set up, namely the wettable powder of Serratia plymuthica MM diluted 100 times, the wettable powder of mancozeb 70% diluted 600 times, and the water control. Each treatment was replicated 3 times. When transplanting the pepper plants, the plants were dipped in the bacterial agent and the wettable powder of mancozeb 70% at the roots. At 40 days after transplantation, each treatment was drip-irrigated again at the above concentrations. At the peak of the disease incidence, three points were randomly selected for investigation, with 50 plants at each point, and the disease index and control effect were calculated.

[0049] Disease grading standard: Grade 0: No disease; Grade 1: No lesions on the root and stem, and individual branches or fruits are diseased; Grade 3: Slight disease on the root and stem, or less than half of the branches or fruits are diseased; Grade 5: Moderate disease on the root and stem, or more than half of the branches or fruits are diseased; Grade 7: Severe disease on the root and stem, and most of the plants are withered; Grade 9: All plants are withered and dead. According to the investigation results, and in accordance with the following formula:

[0050]

[0051]

[0052] Table 4 Evaluation of the field control effect of Serratia plymuthica MM agent on Phytophthora blight of pepper

[0053] Treatment Disease index Control efficacy (%) Serratia plymuthica MM <![CDATA[6.33±1.7 c > 82.2 Mancozeb <![CDATA[19.67±2.7 b > 66.5 Control <![CDATA[58.66±2.5 a > -

[0054] Note: Different letters represent the standard deviation of repeated values and are significantly different (p < 0.05)

[0055] In the field experiment, the control effects of the wettable powder of Serratia plymuthica MM treatment and the wettable powder of mancozeb on Phytophthora blight of pepper were 82.2% and 66.5% respectively (Table 4). The control effect of the wettable powder of Serratia plymuthica MM treatment on Phytophthora blight of pepper was 15.7 percentage points higher than that of the wettable powder of mancozeb treatment group. Moreover, the wettable powder of Serratia plymuthica MM treatment group showed excellent performance, and the plants grew vigorously ( Figure 5 ) and could effectively control the occurrence of Phytophthora blight of pepper, having good popularization and application value.

Claims

1. Application of Serratia plymuthica ( Serratia plymuthica ) MM fermentation broth and bacterial agent in preventing and controlling Phytophthora capsici.

2. The application according to claim 1, wherein The described Serratia plymuthica MM was deposited at the General Microbiology Center of the China Center for Type Culture Collection on June 2, 2022, with the deposit number CGMCC No. 24997.

3. The application according to claim 1, characterized in that The pathogen of the described Phytophthora blight of pepper is Phytophthora capsici ( Phytophthora capsici ).

4. Application of Serratia plymuthica ( Serratia plymuthica ) MM fermentation broth and microbial agent in promoting the growth of pepper plants.

5. A wettable powder for preventing and controlling Phytophthora capsici, characterized in that, The described biocontrol agent includes Serratia plymuthica MM.

6. A wettable powder according to claim 5, wherein, The components and their weight percentages are as follows: 18% of the active ingredient Serratia plymuthica, 64% of the carrier diatomaceous earth, 12% of light calcium carbonate, 3% of sodium chloride, 2% of Tween-80, and 1% of antioxidant.

7. Use of the biocontrol agent according to claim 6 in the field control of Phytophthora blight of peppers.

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