CN103173386B - Bio-control strain G1 for preventing and controlling pepper phytophthora blights and applications thereof - Google Patents

Abstract

The invention discloses a biocontrol bacterial strain G1 for preventing and treating pepper blight and application thereof. The present invention provides Paenibacillus polymyxa strain G1, the preservation number of which is CGMCC No.6762. The present invention also provides a method for preparing a G1 microbial agent, comprising the following steps: fermenting the above-mentioned Paenibacillus polymyxa strain G1, and collecting fermentation products to obtain the G1 microbial agent. Experiments of the present invention prove that the present invention is aimed at the current situation of no biocontrol preparations with high control effect on capsicum blight, and provides a single bacterium and preparation thereof for the prevention and treatment of capsicum blight, which is specially used for the prevention and treatment of capsicum blight. An important soil-borne disease has a high control effect, and the control effect of greenhouse pepper blight is up to 71.18%.

Description

Biocontrol strain G1 for preventing and controlling pepper blight and its application

technical field

The invention relates to the field of biotechnology, in particular to a biocontrol strain G1 for preventing and treating pepper blight and application thereof. the

Background technique

Pepper blight is an oomycete disease caused by Phytophthora capsici. Phytophthora belongs to Oomycota, Peronomyces, Pythiumceae, and Phytophthora. Phytophthora survives in soil not in one form, but in different forms, namely mycelium, sporangia, chlamydospores and oospores. The disease can be transmitted through rainwater, irrigation water, and agricultural implements, and is a highly harmful soil-borne disease. With the large-scale planting of peppers in my country, pepper blight also occurs in large areas in open fields, protected areas, and greenhouses. Because the disease has a short onset period and rapid prevalence, it often leads to the death of pepper plants in patches and brings serious economic losses to the pepper industry. Generally, the rate of diseased plants is 20-30%, and in severe cases, it can reach more than 80%. This disease is one of the main factors affecting the economic development of my country's pepper industry. the

Phytophthora capsici is a heterothallic microorganism with two main mating types: A1 and A2. Groves et al. believe that due to the widespread use of fungicides in recent years, Phytophthora capsici has developed resistance, thereby inducing changes in its mating type, which has brought many difficulties to the control of the disease. Special fungicides such as phenylamide fungicides metalaxyl and mefenoxam are widely used to prevent and control the disease, and are currently widely used at home and abroad among the oomycete disease control agents with the highest activity and the largest amount of use. A large class of fungicides, and achieved good control effect. However, since this type of agent is a specific site inhibitor, which has a single site of action on pathogenic bacteria, long-term use can easily lead to drug resistance, resulting in a decline in the control effect. Methoxyacrylate fungicides are another important systemic fungicides for the control of capsicum blight. Due to their novel mechanism of action and no cross-resistance with phenylamide fungicides, they are currently widely used. However, existing studies have shown that some pathogenic bacteria targets have a high frequency of spontaneous mutations that are resistant to such agents, and pathogenic bacteria are prone to form drug-resistant populations under the high selection pressure of agents. the

In summary, the development of new, effective and safe microbial fungicides to control the occurrence of pepper blight is the need to increase the total production of pepper, and it is also the need of the pepper industry economy, pepper food safety and sustainable agricultural development. the

Contents of the invention

An object of the present invention is to provide a Paenibacillus polymyxa bacterial strain G1. the

The Paenibacillus polymyxa strain G1 provided by the invention has a preservation number of CGMCC No.6762. the

Another object of the present invention is to provide a method for preparing the anti-capsicum blight bacterial agent. the

The method provided by the invention comprises the following steps: fermenting the above-mentioned Paenibacillus polymyxa strain G1 to obtain a fermentation product, that is, to obtain a bacterial agent. the

In the above method, after the fermentation product is obtained, the following steps are further included: collecting the bacterial cell precipitate in the fermentation product, and resuspending the bacterial cell precipitate to obtain the microbial agent. the

The suspension used for the above suspension is water. the

In the above method, centrifugation is used to collect the bacterium precipitate in the fermentation product. The specific centrifugation is 11100×g centrifugation for 10 min. the

In the above method, the fermentation conditions are as follows: 26-28°C, 180-200rpm shaking culture for 24-36h; the specific fermentation conditions are as follows: 26°C, 180rpm shaking culture for 24h. the

The bacterial agent prepared by the above method; the total concentration of viable bacteria in the bacterial agent is specifically 1×10 ⁷ -1×10 ¹⁰ cfu/mL, and in the embodiment of the present invention, the total concentration of viable bacteria in the bacterial agent is 1 ×10 ⁷ cfu/mL.

The third object of the present invention is to provide a kind of prevention and treatment of capsicum blight or antibacterial product. the

The product provided by the invention has an active ingredient of the above-mentioned Paenibacillus polymyxa strain G1 or the above-mentioned bacterial agent. the

In the above-mentioned products, the pepper blight is caused by Phytophthora capsici; the above-mentioned Phytophthora capsici is specifically Phytophthora capsici strain SD-33;

The antibacterial is at least one of the following 6 kinds of pathogenic bacteria: poplar rot fungus Valsa sordida, gray mold fungus Botrytis cinerea, chestnut blight fungus Cryphonectria parasitica, melon and fruit Pythium aphanidermatum, Rhizoctonia solani Kühn and Phytophthora capsici. the

The application of the above-mentioned Paenibacillus polymyxa strain G1 or the above-mentioned bacterial agent in the prevention and treatment of pepper blight or antibacterial is also the protection scope of the present invention. the

In the above application, the pepper blight is caused by Phytophthora capsici; the above-mentioned Phytophthora capsici is specifically Phytophthora capsici strain SD-33;

The antibacterial is at least one of the following 6 kinds of pathogenic bacteria: poplar rot fungus Valsa sordida, gray mold fungus Botrytis cinerea, chestnut blight fungus Cryphonectria parasitica, melon and fruit Pythium aphanidermatum, Rhizoctonia solani Kühn and Phytophthora capsici. the

The application methods of the above-mentioned biocontrol fungicides for the prevention and treatment of capsicum blight are:

When raising pepper seedlings in the greenhouse, soak the seeds with an inoculum with a total concentration of 1×10 ⁷ cfu/mL for 1 hour, and then water the inoculum with a total concentration of 1×10 ⁷ live bacteria when sowing; wait until the pepper seedlings grow to 7-8 At the leaf stage, first spray 20mL of G1 bacterial agent (total concentration of viable bacteria is 1×10 ⁷ cfu/mL) evenly on the whole pot of pepper seedlings, and then spray 30mL of G1 bacterial agent (total concentration of viable bacteria is 1× 10 ⁷ cfu/mL) for root irrigation.

Bacteria G1, Paenibacillus polymyxa, was deposited on November 2, 2012 at the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: Datun Road, Chaoyang District, Beijing, Microbiology, Chinese Academy of Sciences Research Institute), the strain preservation number is: CGMCC No.6762. the

The experiment of the present invention proves that the present invention aims at the current situation that there is no biocontrol agent with high control effect on pepper blight, and provides a single bacterium Paenibacillus polymyxa G1 and its bacterial agent for the prevention and treatment of pepper blight, which is specially used for pepper blight The bacterium or its agent has a high control effect on this important soil-borne disease, and the control effect of greenhouse pepper blight is up to 71.18%. Since the bacterial strain G1 of the present invention is a biocontrol bacterial strain specially screened and developed for pepper blight, it has a remarkable control effect in preventing and treating pepper blight compared with other broad-spectrum biological fungicides at present. Because it is a biological agent, there is no series of problems caused by the use of chemical pesticides, which is conducive to the pollution-free production of crops. Farmers can avoid or reduce the amount of other chemical pesticides used to prevent and control pepper blight, which can not only save farmers money. , It is also conducive to the export of chili products. At the same time, the biological control preparation also has the function of increasing production, which can increase economic income for farmers. the

Description of drawings

Figure 1 is the control effect of biocontrol bacteria on pepper blight

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified. the

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. the

Embodiment 1, the separation and identification of biocontrol bacteria G1

1. Isolation and identification of strains

(1) Isolation of strains

The pepper plants collected from the Science and Technology Innovation Park of Shandong Agricultural University were rinsed with tap water, divided into three parts: root, stem, and leaf, and then rinsed twice with sterile water, and the surface moisture was blotted dry with sterile filter paper. Under sterile conditions, soak in 75% ethanol for 1 min, rinse with sterile water for 3 times, transfer to 3% sodium hypochlorite disinfectant for 5 min, rinse with sterile water for 3 times, and dry the surface with sterile filter paper. Place the surface-sterilized roots, stems and leaves in a sterilized mortar, cut them into pieces, add an appropriate amount of sterile water, grind them, and then dilute them into three gradients of 10 ^-1 , 10 ^-2 , and 10 ^-3 . Transfer 200 μL each of the 10 ^-2 , 10 ^-3 dilution gradient grinding solution and the last washing solution into the LA medium plate, and spread evenly. The last washing solution was used as the control CK, and each treatment was repeated 3 times. observe. Select a strain that has no colonies in CK but has colonies growing in the grinding liquid, picks a single colony according to the characteristics of the colony shape, color, etc., and after multiple plate streaks and purifications, number the purified strains and save them on the slope for future use.

LA medium: peptone 10g, yeast powder 5g, NaCl 5g, agar powder 17g, distilled water 1000mL, pH 7.0. the

(2) Screening of antagonistic bacteria

The antagonistic bacteria were screened by the plate confrontation method, and the tested plant pathogenic fungi were Valsa sordida, Botrytis cinerea, Cryphonectria parasitica, Pythium aphanidermatum, Rhizoctonia solani Kühn and Phytophthora capsici strain SD-33 (screening of antagonistic bacteria to Phytophthora capsici, Shandong Agricultural Science, 2012, 44(9):90–92; public available from Shandong Obtained from Agricultural University) were inoculated in the center of the PDA plate respectively, and the bacteria to be tested were spot-connected at a place about 2cm away from the pathogenic bacteria. Three plates were treated for each treatment, and the antibacterial rate was measured after 4 days at 28°C for 4 days (see Table 1 for the results). the

Bacterial inhibition rate = (colony diameter of the control plate - colony diameter of the confrontation plate) / colony diameter of the control plate × 100%. the

An endophytic bacterial strain with strong antagonistic activity against Phytophthora capsici was selected, numbered G1, and stored in 50% glycerol at -80°C. the

Table 1 is the bacteriostatic rate of strain G1 to different pathogenic bacteria

Note: Different letters after the data in the same column indicate significant differences at the 0.05 level. the

2. Morphological and physiological and biochemical characteristics detection of G1

(1) Morphological characteristics of colonies and strains

Strain G1 showed light yellow opaque colonies on LA medium with viscous surface and irregular colony edges. The bacterium is rod-shaped, Gram staining reaction is positive, with perinatal flagella, terminal spores, and oval shape. the

(2) Detection of physiological and biochemical characteristics

Available carbon sources: dextrin, glycogen, L-arabinose, D-fructose, D-galactose, α-D-glucose, α-D-lactose, maltose, D-mannitol, D-succulent milk mushroom Sugar, D-melibiose, β-methyl-D-glucoside, D-psicose, D-raffinose, sucrose, D-trehalose, turanose, glycerol, α-D- Sodium Glycerol Phosphate. the

Carbon sources that cannot be used: α-cyclodextrin, Tween 40, Tween 80, N-acetylglucosamine, D-arabinitol, L-fucose, gentiobiose, m-cellose, L -Rhamnose, D-Sorbitol, Xylitol, Methyl Pyruvate, Monomethyl Succinate, Acetic Acid, D-Galactonolactone, D-Galacturonic Acid, Inosine, Urine Nucleoside , putrescine, α-hydroxybutyric acid, β-hydroxybutyric acid, hydroxybutyric acid, p-hydroxyphenylacetic acid, α-ketoglutaric acid, D,L-lactic acid, propionic acid, succinic acid, succinic acid amide, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-glutamic acid, glycyl-L-glutamic acid, L-pyrrole-glutamic acid, L-Serine, Butylene Glycol, D-Glucose-6-Phosphate. the

3. Analysis of G1's Biolog microbial automatic identification system

Streak inoculation of bacterial strain G1 on LA plate medium, culture at 28°C for 48 hours, after a single colony grows, send to the State Key Laboratory of Microbial Technology of Shandong University for Biolog system identification. The identification steps are as follows: the pure species are expanded and cultivated with Biolog special medium. Prepare a bacterial suspension with a certain turbidity (cell concentration) as required. The bacterial suspension was inoculated onto a microwell identification plate (Microplate). After cultivating for a certain period of time, put the cultured identification plate into the reader for reading, and the software will automatically give the identification result. Start the Biolog Microstation ^TM workstation MicroLog ^TM and enter the MicroLog ^TM 3.4.20 system. Click SET UP to initialize the settings. Enter the sample number (Sample Number), select the identification plate type (Plate Type): GP2, select the strain type (Strain type): GP-Rod SB, and select the incubation time (Incubation Time): 16-24h. Place the incubated identification plate into the tray of the reader and remove the cover. Press the "Read Next" button to start reading. After the reader scans the identification board, it will pop up automatically, and the result will be displayed on the computer screen. Start the Biolog Microstation ^TM workstation MicroLog ^TM and enter the MicroLog ^TM 3.4.20 system. The Biolog Microbial Identification System identification results showed that the Species ID of strain G1 was: Paenibacillus polymyxa, Paenibacillus polymyxa.

According to BIOLOG, strain G1 is a Gram-positive aerobic bacterium, and based on the detection results of complete substrate utilization profiles, strain G1 is Paenibacillus polymyxa (species ID). Among them, the degree of matching with standard strains SIM>0.5; the parameters that can be compared with other identification systems PROB%=100%. the

The system gives identification results, and each result is reflected by three indicators: Probability (PROB), Similarity (SIM) and Distance (DIS). If PROB is close to 100%, and SIM≥0.5, a bacterial species can be identified, while the values of G1 are 100% and 0.765, respectively, indicating that the identification results are reliable. So far, the tentative strain G1 belongs to Paenibacillus polymyxa. the

4. Molecular biological identification of strain G1

Genomic DNA of strain G1 was extracted using TaKaRa MiniBEST Bacterial Genomic DNAKit Ver.2.0 kit. The 16S rDNA was amplified using the bacterial strain G1 genomic DNA as a template by using bacterial universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTAC CTTGTTACGATT-3'). Use 25μl reaction system: 10×PCR Buffer 2.5μL; 25mM Mg ²⁺ 1.5μL; 2.5mM dNTP 2μL; primer 27F 1μL; primer 1492R 1μL; 5U/μL Taq enzyme 0.2μL; genomic template DNA _1μL ; The PCR reaction program was 94°C for 5min, 94°C for 30s, 50°C for 1min, 72°C for 1min, 30 cycles; 72°C for 10min. After the reaction, the amplified products were electrophoresed on 1% agarose gel, and the results were observed. Find the 1500bp target band of 16S rDNA, cut the gel and recover. The target fragment was connected to the vector Peasy-T3 and sent to BGI for sequencing.

The sequencing result shows that the nucleotide sequence of the 16S rDNA of G1 is sequence 1 in the sequence list. the

The BLAST homology sequence comparison analysis was carried out with the known 16S rDNA sequence in the Genbank database, and the results were analyzed with MEG4.0 software. The final identification results showed that the strain G1 was Paenibacillus polymyxa. the

The G1 strain, classified as Paenibacillus polymyxa, was deposited in the General Microbiology Center of the China Committee for Microbial Culture Collection (CGMCC for short) on November 2, 2012, and the address is: Datun Road, Chaoyang District, Beijing, China Academy of Sciences Institute of Microbiology), strain preservation number: CGMCC No.6762. the

Embodiment 2, the application of bacterium G1 in the prevention and treatment of capsicum blight

1. Preparation of G1 bacterial agent

(1) Preparation of seed solution:

The Paenibacillus polymyxa G1CGMCC No.6762 isolated and preserved in Example 1 was inoculated on LA medium containing a concentration of 500 μg/mL streptomycin sulfate (Ster) drug resistance marker. In a 250mL Erlenmeyer flask with 100mL of LB culture medium, shake culture at 28°C and 180rpm for 24h to obtain G1 seed solution. the

LA medium: peptone 10g, yeast powder 5g, NaCl 5g, agar powder 17g, distilled water 1000mL, pH 7.0; LB medium: no agar powder, other ingredients are the same as LA medium. the

(2) Preparation of bacterial agent:

When the G1 seed solution is cultivated to an OD value of 0.5, add the G1 seed solution to the LB culture medium at a volume ratio of 1:100, shake and cultivate at 26°C and 180rpm for 24h; then centrifuge at 11100×g for 10min to remove the supernatant and collect the bacteria precipitation. the

Suspend and dilute the bacterial precipitate with sterile water to obtain the G1 bacterial agent, and the concentration of the bacterial agent is 1×10 ⁷ -1×10 ¹⁰ cfu/mL.

2. The application of bacteria G1 in the prevention and treatment of pepper blight

(1) Preparation of potting soil

Stir evenly with a ratio of 2:1 of substrate and field soil, and sterilize by high-pressure steam before use. the

(2) Pepper pot planting

The seeds of hot pepper (the variety is extra-large eggplant sweet pepper, produced and sold by Zhanxin Seed Industry Co., Ltd., Chifeng City, Inner Mongolia) were germinated at 28°C for 3 days. ⁷ cfu/mL) after soaking for 1 hour, sow the seeds in the nutrient bowl. 50mL of G1 bacterial agent (the total concentration of viable bacteria in G1 bacterial agent is 1×10 ⁷ ) was poured into each bowl, and the tap water treatment was used as the control. There were 3 replicates per treatment, 5 pots per replicate, and 6 seeds per pot.

When the pepper seedlings grow to the 7-8 leaf stage, carry out inoculation treatment with G1 bacterial agent, as follows: First, spray 20mL of G1 bacterial agent (the total concentration of viable bacteria is 1×10 ⁷ cfu/mL) evenly on the whole pot of pepper seedlings After 10 minutes, each pot was irrigated with 30mLG1 bacterial agent (the total concentration of viable bacteria was 1×10 ⁷ cfu/mL). The negative control group was treated with tap water; the positive control group was prepared with the same concentration and the same amount of Paenibacillus polymyxa strain XM (screening of Phytophthora capsici antagonistic bacteria, Shandong Agricultural Science, 2012, 44 (9): 90- 92; publicly available from Shandong Agricultural University).

(3) Phytophthora capsici inoculation

On the second day after inoculation with G1 inoculum, use Phytophthora capsici strain SD-33 (recorded in Phytophthora capsici) pectin methylesterase Pcpme3 gene at a concentration of 1×10 ⁵ cfu/mL Eukaryotic expression and function research, Chinese Agricultural Sciences 2009,42(6):1988-1993; the public can obtain from Shandong Agricultural University) Zoospore suspension root irrigation method inoculation, each plant watered zoospore suspension 5mL. Before inoculation, fill the seedling pot with water in advance, keep it moist for 24 hours after inoculation, place it indoors, and water it at the right time to keep it moist. On the 7th day after inoculation, investigate the incidence (pepper blight) and calculate the control effect, and divide it into 6 grades according to the degree of disease occurrence (Lin Baiqing et al., Preliminary research on the identification method of pepper varieties against blight [J]. Chinese Vegetables, 1994, (4): 21-25):

Level 0: no disease;

Level 1: The base of the seedling stems is slightly blackened, but the plants are healthy and not wilting;

Grade 2: The base of the seedling stem turns black up to 1-2cm, the leaves are irreversibly shriveled, and the lower leaves occasionally fall off;

Grade 3: The base of the stem of the seedlings turns black for more than 2cm, and the leaves are obviously shriveled or fallen off;

Grade 4: The base of the seedling stem turns black and constricted, all leaves except the growth point or the whole plant wilts;

Grade 5: The plants are all dead. the

Disease index = [∑ (number of disease-level plants × representative series)/total number of plants × highest representative series] × 100

Prevention and treatment effect% = (control disease index - treatment disease index) / control disease index × 100

Seedling pot test results are shown in Table 2 and Figure 1. It can be seen that peppers treated with G1 inoculum showed a better control effect, reaching 71.18%. the

Table 2 Greenhouse control effect of biocontrol bacteria on pepper blight

Note: The values in the table are the average of three repetitions. the

Claims (18)

1. Paenibacillus polymyxa paenibacillus polymyxabacterial strain G1, its preserving number is CGMCC No. 6762.

2. prepare a method for control capsicum epidemic disease microbial inoculum, comprise the steps: the Paenibacillus polymyxa according to claim 1 that ferments paenibacillus polymyxabacterial strain G1, obtains tunning, namely obtains microbial inoculum.

3. method according to claim 2, is characterized in that: described obtain tunning after, also comprise the steps: to collect the bacterial sediment in described tunning, bacterial sediment described in settling flux, obtains microbial inoculum.

4. method according to claim 3, is characterized in that: the bacterial sediment in described collection tunning adopts centrifugal mode.

5. method according to claim 2, is characterized in that: the condition of described fermentation is as follows: 26-28 DEG C, 180-200 rpm shaking culture 24-36 h.

6. method according to claim 5, is characterized in that: the condition of described fermentation is as follows: 26 DEG C, 180 rpm shaking culture 24 h.

7. the microbial inoculum prepared by method described in Claims 2 or 3: the viable bacteria total concn in described microbial inoculum is specially 1 × 10 ⁷-1 × 10 ¹⁰cfu/mL.

8. prevent and treat a capsicum epidemic disease product, its activeconstituents is Paenibacillus polymyxa according to claim 1 paenibacillus polymyxabacterial strain G1.

9. prevent and treat a capsicum epidemic disease product, its activeconstituents is microbial inoculum according to claim 7.

10. product according to claim 8 or claim 9, is characterized in that: described capsicum epidemic disease is for by Phytophthora capsici phytophthora capsicicause.

11. 1 kinds of antimicrobial products, its activeconstituents is Paenibacillus polymyxa according to claim 1 paenibacillus polymyxabacterial strain G1.

12. 1 kinds of antimicrobial products, its activeconstituents is microbial inoculum according to claim 7.

13. products according to claim 11 or 12, is characterized in that: described antibacterial be at least one in anti-following 6 kinds of pathogenic bacterias: Valsa sordida bacterium valsa sordida, ash arrhizus bacteria botrytis cinerea, chestnut epidemic disease bacterium cryphonectria parasitica, melon and fruit corruption is mould pythium aphanidermatum, dry thread Pyrenomycetes rhizoctonia solanik ü hn and Phytophthora capsici phytophthora capsici.

14. Paenibacillus polymyxas according to claim 1 paenibacillus polymyxathe application of bacterial strain G1 in control capsicum epidemic disease.

The application of 15. microbial inoculums according to claim 7 in control capsicum epidemic disease.

16. application according to claims 14 or 15, is characterized in that: described capsicum epidemic disease is for by Phytophthora capsici phytophthora capsicicause.

17. Paenibacillus polymyxas according to claim 1 paenibacillus polymyxathe application of bacterial strain G1 in antibacterial, is characterized in that: described antibacterial be at least one in anti-following 6 kinds of pathogenic bacterias: Valsa sordida bacterium valsa sordida, ash arrhizus bacteria botrytis cinerea, chestnut epidemic disease bacterium cryphonectria parasitica, melon and fruit corruption is mould pythium aphanidermatum, dry thread Pyrenomycetes rhizoctonia solanik ü hn and Phytophthora capsici phytophthora capsici.

The application of 18. microbial inoculums according to claim 7 in antibacterial, is characterized in that: described antibacterial be at least one in anti-following 6 kinds of pathogenic bacterias: Valsa sordida bacterium valsa sordida, ash arrhizus bacteria botrytis cinerea, chestnut epidemic disease bacterium cryphonectria parasitica, melon and fruit corruption is mould pythium aphanidermatum, dry thread Pyrenomycetes rhizoctonia solanik ü hn and Phytophthora capsici phytophthora capsici.