Screening and fermentation method of pepper root rot antagonistic bacterium W-5

Abstract

The invention belongs to the technical field of plant cultivation, and particularly relates to a screening and fermentation method of a pepper root rot antagonistic bacterium W-5, which comprises the following steps: 1, preparing experimental materials, namely preparing a test strain, a culture medium, a reagent and an instrument; 2, separating, screening and identifying the antagonistic bacteria; 3, observing the influence of the antagonistic bacteria W-5 on the growth of the mycelia at the front end of the fusarium solani; step 4, detection of pathogenic fungus bacteriostasis spectrum by antagonistic bacteria W-5, the structure is reasonable, the main pathogenic fungus fusarium solani causing the pepper root rot disease is taken as an indicator bacterium, the pepper root rot antagonistic bacteria are obtained through separation by adopting a soil plate dilution method and a plate confrontation method, and the antagonistic bacteria are identified by combining morphology with molecular biology; meanwhile, the broad-spectrum antibacterial activity of the strain W-5 is measured, and the optimal fermentation condition of the strain W-5 is optimized based on a response surface method designed by combining a single-factor test with Box-Behnken, so that ginseng is expected to be provided for development and application of a pepper root rot antagonistic biological agent.

Description

Technical Field

[0001] The invention relates to the technical field of plant planting, and in particular to a method for screening and fermenting antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot. Background Art

[0002] Fusarium solani is the main pathogen that causes root rot of Zanthoxylum bungeanum, which seriously endangers the development of the Zanthoxylum bungeanum industry and brings great difficulties to production.

[0003] Root rot of pepper is a highly harmful soil-borne disease that can cause death of pepper trees and replanting. In previous studies, the pathogens causing root rot in the pepper planting area of ​​Wudu, Longnan, Gansu have been accurately isolated and identified, and Fusarium solani has been determined to be the main pathogen causing the disease. The prevention and control of pepper root rot is also an important and extremely difficult task in pepper production. With the proposal of the concept of reducing pesticide use, the role of biological control has become a hot topic in current research. Therefore, it is very necessary to obtain a sustainable, safe and effective pepper root rot disease prevention and control plan in pepper production. The use of antagonistic bacteria has the advantages of high specificity for pathogens and green environmental protection in the prevention and control of plant diseases. In business, more and more excellent microbial strains have been developed into biocontrol agents, and the biocontrol effect has been well received. Bacillus shows great potential and application prospects in the biological control of plant diseases. Response surface analysis has become a common method for optimizing antagonistic bacteria fermentation culture experiments. The characteristics of this method are that it saves time and reduces the number of experiments, overcoming the shortcomings of single-factor experiments. However, there are currently few antagonistic bacteria resources related to pepper root rot, and excellent strain resources are relatively scarce. The strains that have been reported to have good antagonistic effects on pepper root rot include Paecilomyces lilacinus, Bacillus cereus, Trichoderma viride, Trichoderma harzianum, and Bacillus velez T-1. There are no other related reports.

[0004] Based on the above problems, we proposed a method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum root rot. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to provide a screening and fermentation method for antagonistic bacteria W-5 against root rot of Zanthoxylum bungeanum, taking Fusarium solani, a main pathogenic fungus causing root rot of Zanthoxylum bungeanum, as an indicator bacteria, separating and obtaining antagonistic bacteria against root rot of Zanthoxylum bungeanum by soil plate dilution method and plate confrontation method, identifying the antagonistic bacteria by morphology combined with molecular biology, determining the broad-spectrum antibacterial activity thereof, optimizing the optimal fermentation conditions of strain W-5 by response surface method based on single factor experiment combined with Box-Behnken design, in order to provide reference for the development and application of antagonistic biological agents against root rot of Zanthoxylum bungeanum.

[0008] To solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions:

[0009] A method for screening and fermenting Zanthoxylum bungeanum root rot antagonistic bacteria W-5, comprising the following steps:

[0010] Step 1: Preparation of experimental materials, including test strains, culture media, reagents and instruments;

[0011] Step 2: Isolation, screening and identification of antagonistic bacteria;

[0012] Step 3: Observation of the effect of antagonistic bacteria W-5 on the growth of the front hyphae of Fusarium solani;

[0013] Step 4: Detection of the antibacterial spectrum of antagonistic bacteria W-5 against pathogenic fungi;

[0014] Step 5: In vitro antibacterial test of antagonistic bacteria W-5 on Zanthoxylum bungeanum root slices;

[0015] Step 6: Detection of extracellular enzymes produced by antagonistic bacteria W-5 and siderophore culture medium detection;

[0016] Step 7: Optimization of fermentation conditions of antagonistic bacteria W-5;

[0017] Step 8: Data processing and analysis.

[0018] As a preferred embodiment of the screening and fermentation method of the Zanthoxylum bungeanum root rot antagonistic bacterium W-5 described in the present invention, wherein: in the step 1, the indicator fungus Fusarium solani H1 and the antagonistic bacterium W-5 are respectively isolated from the root tissue and root soil of Zanthoxylum bungeanum suffering from Zanthoxylum bungeanum root rot.

[0019] As a preferred embodiment of the method for screening and fermenting the Zanthoxylum bungeanum root rot antagonistic bacteria W-5 described in the present invention, the culture medium in step 1 comprises:

[0020] PDA medium (g / L): Peel and cut potatoes into pieces and weigh 200.0, glucose 20.0, agar 15.0-20.0, distilled water to 1L;

[0021] LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl. After the solutes were dissolved, the pH was adjusted to 7.0 with 5.0 mol / L NaOH and the volume was adjusted to 1 L with deionized water.

[0022] NYBD medium (g / L): 5.0% yeast extract powder, 8.0% beef extract powder, 10.0% glucose, adjust pH to 7.0-7.5, add distilled water to 1L;

[0023] The above three culture media were sterilized at 103.4 kPa for 21 min.

[0024] As a preferred solution of the screening and fermentation method of the Zanthoxylum bungeanum root rot antagonistic bacteria W-5 described in the present invention, the specific process of step 2 is as follows:

[0025] (1) Isolation of antagonistic bacteria W-5

[0026] Weigh 1.0 g of Zanthoxylum root soil and put it into a 100 mL sterile conical flask. Add 90 mL of sterile water and incubate it on a shaker at 180 r / min for 30 min. After standing for 30-60 min, take the supernatant and dilute it by gradient 10 -1 -10 -8 , take 10 -6 , 10 -7 , 10 -8 200uL of three gradients were spread on LB solid medium and cultured at 32℃ for 24h, then a single colony was picked and numbered as W-5. The single colony was inoculated into LB liquid medium for expansion culture and kept in a 4℃ refrigerator for future use;

[0027] (2) Screening of antagonistic bacteria W-5

[0028] Flat plate standoff method

[13] The antibacterial effect of the isolated antagonistic bacteria W-5 was determined. Fusarium solani was used as the indicator bacteria. The above pathogenic fungi were punched into a 6mm diameter cake with a hole puncher and inoculated in the center of the PDA culture medium. A sterile filter paper was placed in the shape of a finished product 2cm away from the center point. 10uL of the single colony liquid in 1.2.1 was added to the filter paper. The culture was cultured at 30℃ for 5-7d. The diameter of the pathogenic fungal colony was determined and the antibacterial rate was calculated.

[0029] Inhibition rate (%) = (control group colony diameter mm - test group colony diameter mm) / (control group colony diameter mm - hole diameter mm) × 100%

[0030] (3) Molecular biological identification of antagonistic bacteria W-5

[0031] The antagonistic bacteria W-5 was identified by combining morphology with molecular biology methods. The strain was inoculated into LB medium and cultured at 32°C for 24 hours. The morphology was observed under an optical microscope. The single colony was placed under a stereomicroscope (magnification of 150×) for observation and Gram staining was performed. Molecular identification was performed by PCR amplification using bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' / 1492R: 5'-CTACGGCTACCTTGTTAC GA-3', and the PCR product was detected by 1.0% agarose gel electrophoresis.

[0032] As a preferred scheme of the screening and fermentation method of the antagonistic bacteria W-5 against Zanthoxylum root rot described in the present invention, wherein: in the step 3, the antibacterial plate obtained by the plate confrontation method in the above step is placed under a stereo microscope (with a magnification of 150×) to observe the effect of the antagonistic bacteria W-5 on the growth of the front hyphae of the pathogen Fusarium solani.

[0033] As a preferred scheme of the screening and fermentation method of the antagonistic bacteria W-5 for Zanthoxylum root rot described in the present invention, the specific process of step 4 is: using 5 pathogenic fungi (Fusarium graminearum, Fusarium pallidum, Gibberellic acid, Colletotrichum pine needles, and Fusarium lamina) as test indicator bacteria, the plate confrontation method is used to detect the inhibition rate of the antagonistic bacteria W-5 against pathogenic fungi.

[0034] As a preferred embodiment of the method for screening and fermenting the Zanthoxylum bungeanum root rot antagonistic bacteria W-5 described in the present invention, the step 7 specifically comprises:

[0035] (1) Determination of the growth curve of antagonistic bacteria W-5

[0036] In LB liquid medium, the antagonistic bacteria W-5 seed liquid was inoculated at a ratio of 1%, and 3 replicates were set for each detection point. The culture was placed in a shaking incubator at 30°C and 180 r / min, and the OD was measured every 2 h by visible spectrophotometry. 600 Value, a total of 16 points were measured and the growth curve was drawn;

[0037] (2) Single factor experiment to screen the optimal fermentation conditions

[0038] In the previous experiment, it was determined that the best medium for the growth of antagonistic bacteria W-5 was NYBD. Based on this, the effects of different temperatures (26, 28, 30, 32, 34°C), pH (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5), inoculation size (1%, 3%, 5%, 7%, 9%, 11%), and rotation speed (140, 160, 180, 200, 220 r / min) on the growth of antagonistic bacteria W-5 were compared. The OD 600As an indicator, determine the best single factor fermentation conditions;

[0039] (3) Determine the optimal combination using response surface methodology

[0040] Based on the above experiments, four factors (temperature, pH, rotation speed, and inoculum size) were selected as independent variables to antagonize the fermentation OD of strain W-5. 600 A response surface combination test was conducted for the response value, with 1, 0, and -1 representing the high and low levels of the four factors. A response surface analysis experiment with four factors and three levels was carried out to optimize the optimal fermentation conditions of the antagonistic bacteria W-5.

[0041] As a preferred scheme of the screening and fermentation method of the Zanthoxylum bungeanum root rot antagonistic bacteria W-5 described in the present invention, wherein: in the step 8, the obtained data is processed and analyzed using Excel 2007 software; and the response surface results are analyzed using Design-Expert 11 software.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] An antagonistic bacterium with good inhibitory effect on the pathogen of pepper root rot, Fusarium solani, was isolated from the root soil of pepper. It was numbered W-5 and identified as Bacillus subtilis through morphological and molecular biological identification. The inhibition rate of Fusarium solani can reach 82.7%. Under a stereomicroscope, it was observed that W-5 inhibited the growth of the front hyphae of Fusarium solani. Combined with the results of the in vitro test of pepper root slices, it was shown that the antagonistic bacterium W-5 had a good antagonistic effect on the pathogen of pepper root rot. At the same time, the broad-spectrum antibacterial effect was detected. The results showed that it had a good antibacterial effect on the five pathogens tested. The experiment confirmed that W-5 had a wide range of antibacterial activity and could be developed and utilized as an excellent bacterial resource for biocontrol bacteria. The results of the test of the extracellular enzyme and siderophore production ability of the antagonistic bacterium W-5 showed that the bacterium could well secrete protease and cellulase, and had a strong ability to produce siderophore. The biocontrol effect of biocontrol bacteria is achieved by the production of extracellular enzymes by microorganisms to degrade the cell wall of pathogens. The siderophore produced by microorganisms can compete with pathogenic microorganisms for iron nutrition, thereby hindering the growth of pathogens and achieving the effect of biological control. The above test results show that Bacillus subtilis W-5 has good development and application prospects in the prevention and control of pepper root rot.

[0044] The optimization of antagonistic bacteria fermentation conditions by response surface methodology can better explore the antagonistic effect of antagonistic strains. The optimized fermentation system can not only increase the amount of live bacteria of the strains, but also enable the strains to produce more antibacterial metabolites, thereby improving the biocontrol effect.

[25] This study used OD 600The value was used as the consideration index of strain W-5. The fermentation conditions of the strain were optimized by Box-Behnken design combined with response surface analysis. The temperature of 31°C, pH 7.5, inoculation volume of 3.1%, and rotation speed of 181r / min were determined to be the best fermentation parameters for the strain based on comprehensive consideration of the previous test results. Under this condition, OD 600 The value can reach 1.832, which is 2.35 times higher than before optimization, which verifies the feasibility of optimizing fermentation conditions by response surface methodology. The optimized fermentation system not only increases the effective viable count of the bacteria, but also reduces the fermentation cost from the operational aspect. The strain W-5 provides an important theoretical basis for the green prevention and control of pepper root rot as a microbial agent with development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below in combination with the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0046] Figure 1 The morphological characteristics and Gram staining of the antagonistic bacteria W-5 of the present invention (A: colonies of the antagonistic bacteria W-5; B: single colonies under a stereomicroscope (magnification of 150×); C: Gram staining);

[0047] Figure 2 The present invention is to detect the enzyme-producing culture medium and the siderophore culture medium of the antagonistic bacteria W-5 (A: detection of protease; B: detection of cellulase; C: detection of siderophore);

[0048] Figure 3 The antagonistic effect of the strain W-5 of the present invention on Fusarium solani and the influence on the growth of the front hyphae of the pathogen (A: colony of Fusarium solani; B: inhibition of Fusarium solani by the antagonistic bacteria W-5; C: front morphology of normal Fusarium solani hyphae; D: influence of the antagonistic bacteria W-5 on the front hyphae of Fusarium solani);

[0049] Figure 4 The broad-spectrum antibacterial effect of the antagonistic bacteria W-5 of the present invention on pathogenic fungi (A1: Gibberella fujikura; A2: antagonistic effect of antagonistic bacteria W-5 on Gibberella fujikura; B1: Fusarium graminearum; B2: antagonistic effect of antagonistic bacteria W-5 on Fusarium graminearum; C1: Fusarium pallidum; C2: antagonistic effect of antagonistic bacteria W-5 on Fusarium pallidum; D1: Colletotrichum pine needles; D2: antagonistic effect of antagonistic bacteria W-5 on Colletotrichum pine needles; E1: Fusarium laminari; E2: antagonistic effect of antagonistic bacteria W-5 on Fusarium laminari);

[0050] Figure 5 The in vitro antagonistic effect of the antagonistic bacteria W-5 of the present invention on Zanthoxylum bungeanum root slices (A: Zanthoxylum bungeanum root slices with sterile water; B: Zanthoxylum bungeanum root slices with Fusarium solani; C: Zanthoxylum bungeanum root slices with the coexistence of antagonistic bacteria W-5 and Fusarium solani);

[0051] Figure 6 The phylogenetic tree of the antagonistic bacteria W-5 constructed based on the 16S rRNA gene of the present invention;

[0052] Figure 7 Determination of the growth curve of the antagonistic strain W-5 of the present invention;

[0053] Figure 8 The effects of different fermentation factors on the growth of antagonistic strain W-5 of the present invention;

[0054] Fig. 9 This is the response surface of the influence of different factors on the OD value in the W-5 fermentation of the present invention. DETAILED DESCRIPTION

[0055] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0056] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0057] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0058] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0059] The invention provides the following technical scheme: a screening and fermentation method for antagonistic bacteria W-5 against root rot of Zanthoxylum bungeanum, taking Fusarium solani, a main pathogenic fungus causing root rot of Zanthoxylum bungeanum, as an indicator bacterium, separating and obtaining antagonistic bacteria against root rot of Zanthoxylum bungeanum by adopting soil plate dilution method and plate confrontation method, identifying the antagonistic bacteria by combining morphology with molecular biology, and determining the broad-spectrum antibacterial activity thereof, optimizing the optimal fermentation conditions of strain W-5 by combining single factor experiment with response surface method designed by Box-Behnken, so as to provide reference for the development and application of antagonistic biological agents against root rot of Zanthoxylum bungeanum;

[0060] Example 1

[0061] Materials and Methods

[0062] A method for screening and fermenting Zanthoxylum bungeanum root rot antagonistic bacteria W-5, comprising the following steps:

[0063] Step 1: Preparation of experimental materials, including test strains, culture media, reagents and instruments;

[0064] Step 2: Isolation, screening and identification of antagonistic bacteria;

[0065] Step 3: Observation of the effect of antagonistic bacteria W-5 on the growth of the front hyphae of Fusarium solani;

[0066] Step 4: Detection of the antibacterial spectrum of antagonistic bacteria W-5 against pathogenic fungi;

[0067] Step 5: In vitro antibacterial test of antagonistic bacteria W-5 on Zanthoxylum bungeanum root slices;

[0068] Step 6: Detection of extracellular enzymes produced by antagonistic bacteria W-5 and siderophore culture medium detection;

[0069] Step 7: Optimization of fermentation conditions of antagonistic bacteria W-5;

[0070] Step 8: Data processing and analysis.

[0071] Wherein: in the step 1, the indicator fungus Fusarium solani H1 and the antagonistic bacteria W-5 are isolated from the root tissue and root soil of Zanthoxylum bungeanum suffering from Zanthoxylum bungeanum root rot, respectively.

[0072] Wherein: the culture medium in step 1 comprises:

[0073] PDA medium (g / L): Peel and cut potatoes into pieces and weigh 200.0, glucose 20.0, agar 15.0-20.0, distilled water to 1L;

[0074] LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl. After the solutes were dissolved, the pH was adjusted to 7.0 with 5.0 mol / L NaOH and the volume was adjusted to 1 L with deionized water.

[0075] NYBD medium (g / L): 5.0% yeast extract powder, 8.0% beef extract powder, 10.0% glucose, adjust pH to 7.0-7.5, add distilled water to 1L;

[0076] The above three culture media were sterilized at 103.4 kPa for 21 min.

[0077] Wherein: the specific process of step 2 is:

[0078] (1) Isolation of antagonistic bacteria W-5

[0079] Weigh 1.0 g of Zanthoxylum root soil and put it into a 100 mL sterile conical flask. Add 90 mL of sterile water and incubate it on a shaker at 180 r / min for 30 min. After standing for 30-60 min, take the supernatant and dilute it by gradient 10 -1 -10 -8 , take 10 -6 , 10 -7 , 10 -8 200uL of three gradients were spread on LB solid medium and cultured at 32℃ for 24h, then a single colony was picked and numbered as W-5. The single colony was inoculated into LB liquid medium for expansion culture and kept in a 4℃ refrigerator for future use;

[0080] (2) Screening of antagonistic bacteria W-5

[0081] Flat plate standoff method

[13] The antibacterial effect of the isolated antagonistic bacteria W-5 was determined. Fusarium solani was used as the indicator bacteria. The above pathogenic fungi were punched into a 6mm diameter cake with a hole puncher and inoculated in the center of the PDA culture medium. A sterile filter paper was placed in the shape of a finished product 2cm away from the center point. 10uL of the single colony liquid in 1.2.1 was added to the filter paper. The culture was cultured at 30℃ for 5-7d. The diameter of the pathogenic fungal colony was determined and the antibacterial rate was calculated.

[0082] Inhibition rate (%) = (control group colony diameter mm - test group colony diameter mm) / (control group colony diameter mm - hole diameter mm) × 100%

[0083] (3) Molecular biological identification of antagonistic bacteria W-5

[0084] The antagonistic bacteria W-5 was identified by combining morphology with molecular biology methods. The strain was inoculated into LB medium and cultured at 32°C for 24 hours. The morphology was observed under an optical microscope. The single colony was placed under a stereomicroscope (magnification of 150×) for observation and Gram staining was performed. Molecular identification was performed by PCR amplification using bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' / 1492R: 5'-CTACGGCTACCTTGTTAC GA-3', and the PCR product was detected by 1.0% agarose gel electrophoresis.

[0085] Wherein: in the step 3, the antibacterial plate obtained by the plate confrontation method in the above step is placed under a stereo microscope (magnification is 150×) to observe the effect of the antagonistic bacteria W-5 on the growth of the front hyphae of the pathogen Fusarium solani.

[0086] Among them: the specific process of step 4 is: using 5 pathogenic fungi (Fusarium graminearum, Fusarium pallidum, Gibberellic acid, Colletotrichum pine needles, and Fusarium lamina) as test indicator bacteria, the plate confrontation method is used to detect the inhibition rate of the antagonistic bacteria W-5 on pathogenic fungi.

[0087] Wherein: the step 7 specifically includes:

[0088] (1) Determination of the growth curve of antagonistic bacteria W-5

[0089] In LB liquid medium, the antagonistic bacteria W-5 seed liquid was inoculated at a ratio of 1%, and 3 replicates were set for each detection point. The culture was placed in a shaking incubator at 30°C and 180 r / min, and the OD was measured every 2 h by visible spectrophotometry. 600 Value, a total of 16 points were measured and the growth curve was drawn;

[0090] (2) Single factor experiment to screen the optimal fermentation conditions

[0091] In the previous experiment, it was determined that the best medium for the growth of antagonistic bacteria W-5 was NYBD. Based on this, the effects of different temperatures (26, 28, 30, 32, 34°C), pH (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5), inoculation size (1%, 3%, 5%, 7%, 9%, 11%), and rotation speed (140, 160, 180, 200, 220 r / min) on the growth of antagonistic bacteria W-5 were compared. The OD 600 As an indicator, determine the best single factor fermentation conditions;

[0092] (3) Determine the optimal combination using response surface methodology

[0093] Based on the above experiments, four factors (temperature, pH, rotation speed, and inoculum size) were selected as independent variables to antagonize the fermentation OD of strain W-5. 600 A response surface combination test was conducted for the response value, with 1, 0, and -1 representing the high and low levels of the four factors. A response surface analysis experiment with four factors and three levels was carried out to optimize the optimal fermentation conditions of the antagonistic bacteria W-5.

[0094] Wherein: in step 8, Excel 2007 software is used to process and analyze the obtained data; and the response surface results are analyzed using Design-Expert 11 software.

[0095] result

[0096] Morphological characteristics and Gram staining results of antagonistic bacteria W-5

[0097] After the antagonistic bacteria W-5 was cultured in LB solid medium at 32°C for 2 days, the colony morphology was observed under an optical microscope and a stereo microscope. The results showed that the colonies were milky white and opaque, with irregular edges, rough and wrinkled surfaces, and Gram-positive bacteria (see Figure 1 ).

[0098] Plate transparent circle method to detect enzyme production ability of antagonistic bacteria W-5 and siderophore detection

[0099] The results of the three extracellular enzyme tests showed that the antagonistic bacteria W-5 could produce a large degradation zone in the protease and cellulase test medium (see Figure 2 A and 2B), while no degradation zone was produced in the chitinase test medium, and there was an obvious orange-yellow chelation zone of siderophore in the CAS test (see Figure 2 C).

[0100] Antagonistic effect of antagonistic bacteria W-5 on Fusarium solani and its effect on the growth of front hyphae of pathogenic bacteria

[0101] The results of the antagonistic test showed that the diameter of the Fusarium solani pathogen after 5 days of cultivation was 110.0 mm ( Figure 3 A), the diameter of the pathogen in the presence of antagonistic bacteria W-5 is 24.0 mm ( Figure 3 B), the inhibition rate was as high as 82.7%; the effect of antagonistic bacteria W-5 on the front hyphae growth of Fusarium solani under a stereomicroscope showed that the hyphae of the pathogen without antagonistic bacteria were radial, dense, fluffy, and uniform in thickness ( Figure 3 C), in the presence of antagonistic bacteria W-5, the front end of the hyphae of the pathogen was thin, twisted into a deformity, and lost its extensibility ( Figure 3 D) The above test results show that the antagonistic bacteria W-5 does have an inhibitory effect on the pathogen of Zanthoxylum bungeanum root rot and the effect is good.

[0102] Detection results of the antibacterial spectrum of antagonistic bacteria W-5 against pathogenic fungi

[0103] In order to further detect the antibacterial activity of antagonistic bacteria W-5 against other plant pathogens, the antibacterial effect of antagonistic bacteria W-5 on 5 pathogenic fungi was detected by plate confrontation method. The test results showed that antagonistic bacteria W-5 had good antibacterial activity against other plant pathogens, and the antibacterial rate against Fusarium fujikura was 74.3% ( Figure 4 A) The inhibition rate of Fusarium graminearum was 74.6% ( Figure 4 B) The inhibition rate of syphilis Fusarium is 62.5% ( Figure 4 C) The antibacterial rate against Colletotrichum pine needles was 70.3% ( Figure 4 D) The inhibition rate of Fusarium thunbergii was 71.4% ( Figure 4E), indicating that the antagonistic strain W-5 has a wide range of antibacterial activity and has certain development potential in the future.

[0104] In vitro antagonism test of antagonistic bacteria W-5

[0105] The in vitro antagonistic test of Zanthoxylum bungeanum root slices was to verify the antibacterial effect of antagonistic bacteria W-5 on the pathogens of Zanthoxylum bungeanum root rot. The test results showed that the Zanthoxylum bungeanum root slices still retained their original color after being inoculated with sterile water for 7 days, without producing any odor, and the root bark and wood were not separated, and no liquid was produced around the root slices ( Figure 5 A); After 3 days of cultivation, the root slices of Zanthoxylum inoculated with Fusarium solani began to soften and turn black, symptoms gradually appeared, and the hyphae of the pathogen were wrapped around the root slices. The liquid produced by the root slices changed the color of the filter paper, the root bark and the wood were separated, and a foul odor was produced. The above symptoms were the same as those of typical Zanthoxylum root rot in the field ( Figure 5 B); the symptoms of Zanthoxylum bungeanum root slices inoculated with antagonistic bacteria W-5 and pathogens were similar to Figure 5 Compared with B, the symptoms were alleviated, the root slices were lighter in color and did not soften, and the root bark and root slices did not completely separate ( Figure 5 C). The above results are consistent with the conclusions obtained in 2.3. The antagonistic bacteria W-5 still showed good antagonistic effect on the pathogens of Zanthoxylum root rot in vitro.

[0106] Molecular biological identification and phylogenetic tree construction of antagonistic bacteria W-5

[0107] The antagonistic bacteria W-5 was identified by molecular biology using universal primers, and the size of the effective PCR amplification fragment was 1480bp. The sequencing results were compared by BLAST, and 15 strains similar to the antagonistic bacteria W-5 in the database were selected for phylogenetic tree construction. The results showed that strain W-5 was most closely related to HE659512.1 and belonged to the genus Bacillus subtilis. Combined with morphological characteristics, the antagonistic bacteria W-5 was preliminarily determined to be Bacillus subtilis.

[0108] Optimization of Fermentation Conditions of Antagonistic Bacteria W-5 by Response Surface Methodology

[0109] (1) Determination of the growth curve of antagonistic bacteria W-5

[0110] The growth curve of strain W-5 showed (see Figure 7 ): The antagonistic strain W-5 grew faster between 0 and 14 hours after inoculation, the growth of the strain entered a stable period between 16 and 22 hours, and the growth rate of the strain began to gradually decrease after 24 hours. Therefore, the optimal growth period of the antagonistic strain W-5 was determined to be 22 hours.

[0111] (2) Results of single factor test on antagonistic bacteria W-5

[0112] The effects of temperature, pH value, rotation speed and inoculation amount on the growth of antagonistic strain W-5 were determined. The test results showed that the growth of the strain increased first and then decreased when the pH was between 2.5 and 10.5. The antagonistic strain W-5 grew well when the pH was between 5.5 and 9.5. It still had a certain growth ability under weak alkaline conditions, indicating that the strain had a certain alkaline resistance. When the pH was 7.5, the OD 600 The peak value was reached, so the optimal pH for the growth of the strain was determined to be 7.5; the results of the effect of temperature on the growth of the strain showed that the strain could grow between 26-34℃, and 30℃ was the optimal growth temperature of the bacteria; the bacteria grew well when the inoculation amount was between 1% and 11%, and its OD 600 When the speed was between 140-220 r / min, the growth of the bacteria increased and then decreased. When the speed was between 140-220 r / min, the OD 600 The peak value was reached, and the optimal rotation speed of the bacteria was determined to be 180r / min. The data obtained from the above single factor experiment provided theoretical support for the optimization of the subsequent fermentation system.

[0113] (3) Analysis of the results of optimizing fermentation conditions using response surface methodology

[0114] The variance analysis (Table 3) obtained by the response surface method was used to study the effects of various factors on the fermentation culture of antagonistic bacteria W-5. The regression model obtained by the response surface method was:

[0115] Y=1.94-0.0091A+0.0067B+0.0168C+0.0195D+0.0078AB+0.0257AC-0.0021AD+0.0544BC-0.0217BD-0.0516CD-0.2499A2-0.2650B2-0.3046C2

[0116] -0.2700D2. The regression equation analysis and variance analysis results show that the regression model is extremely significant (P < 0.01), indicating that the model fits the response value well. Except for the AD term, which did not reach a significant level, the other linear terms, quadratic terms, and interaction terms (AC, BC, BD, CD) all reached an extremely significant level (P < 0.01).

[0117] OD of item B against antagonistic bacteria W-5 600 There is a significant level (P<0.05), indicating that OD 600The changes are complex, and the experimental factors are not in a simple linear relationship with the response value. The results in Table 3 show that the four factors of temperature, pH, inoculation amount, and rotation speed have a significant impact on the fermentation culture process of antagonistic bacteria W-5. The lack of fit is 0.0601>0.05, indicating that all test points can be described by the model. The determination coefficient of the antagonistic bacteria fermentation culture process model is R 2 =0.9991, indicating that the model has good correlation and can be used to analyze the change of response value and determine the optimal fermentation conditions. 2 adj=0.9983, the two are very close, indicating that the quadratic regression fit of the response surface of antagonistic bacteria fermentation culture is very good. The signal-to-noise ratio Adeq Precision=104.3978>4, indicating that the experimental design of the model is reliable, the credibility is high, and the prediction is relatively accurate. The F value analysis results show that the degree of influence on the fermentation of antagonistic bacteria W-5 is: speed>inoculation amount>pH>temperature.

[0118] Table 2 Response surface methodology design and results

[0119]

[0120]

[0121] Table 3 Analysis of variance of regression equation in response surface experiment

[0122]

[0123]

[0124] Note: The difference was extremely significant (P<0.01) and was represented by **; Significant difference (P<0.05) was represented by *

[0125] The response surface can intuitively reflect the effects of pH value, temperature, inoculation amount, and rotation speed on the fermentation culture of antagonistic bacteria W-5 ( Fig. 9 ), the steepness and gentleness of the slope of the response surface can directly reflect the effect of the four factors on OD 600 The impact of Fig. 9The response surfaces between the four factors are all convex and open downward, indicating that the response values ​​have extremely high values, and the slopes of the surfaces are relatively steep, indicating that the interactions between the factors are complex. The order of the degree of influence of the interactions between the factors on the fermentation culture of the antagonistic bacteria W-5 is from large to small: CD>BC>AC>BD>AB>AD. Except for the AD item, the other interaction factors have a great influence on the antagonistic bacteria W-5OD. 600 The impacts all reached significant levels.

[0126] Based on the above test results, the optimal conditions for the fermentation of antagonistic bacteria W-5 can be determined as follows: temperature 31.3℃, pH 7.5, inoculation volume 3.1%, rotation speed 180.5r / min, and the optimized OD 600 The value reached 1.832. Considering the actual production situation and the convenience of operation, the optimal fermentation conditions of the bacteria were revised to inoculation temperature 31℃, 3.0%, pH 7.5, and rotation speed 181r / min.

[0127] in conclusion

[0128] An antagonistic bacterium with good inhibitory effect on the pathogen of pepper root rot, Fusarium solani, was isolated from the root soil of pepper. It was numbered W-5 and identified as Bacillus subtilis through morphological and molecular biological identification. The inhibition rate of Fusarium solani can reach 82.7%. Under a stereomicroscope, it was observed that W-5 inhibited the growth of the front hyphae of Fusarium solani. Combined with the results of the in vitro test of pepper root slices, it was shown that the antagonistic bacterium W-5 had a good antagonistic effect on the pathogen of pepper root rot. At the same time, the broad-spectrum antibacterial effect was detected. The results showed that it had a good antibacterial effect on the five pathogens tested. The experiment confirmed that W-5 had a wide range of antibacterial activity and could be developed and utilized as an excellent bacterial resource for biocontrol bacteria. The results of the test of the extracellular enzyme and siderophore production ability of the antagonistic bacterium W-5 showed that the bacterium could well secrete protease and cellulase, and had a strong ability to produce siderophore. The biocontrol effect of biocontrol bacteria is achieved by the production of extracellular enzymes by microorganisms to degrade the cell wall of pathogens. The siderophore produced by microorganisms can compete with pathogenic microorganisms for iron nutrition, thereby hindering the growth of pathogens and achieving the effect of biological control. The above test results show that Bacillus subtilis W-5 has good development and application prospects in the prevention and control of pepper root rot.

[0129] The optimization of antagonistic bacteria fermentation conditions by response surface methodology can better explore the antagonistic effect of antagonistic strains. The optimized fermentation system can not only increase the amount of live bacteria of the strains, but also enable the strains to produce more antibacterial metabolites, thereby improving the biocontrol effect.

[25] This study used OD 600The value was used as the consideration index of strain W-5. The fermentation conditions of the strain were optimized by Box-Behnken design combined with response surface analysis. The temperature of 31°C, pH 7.5, inoculation volume of 3.1%, and rotation speed of 181r / min were determined to be the best fermentation parameters for the strain based on comprehensive consideration of the previous test results. Under this condition, OD 600 The value can reach 1.832, which is 2.35 times higher than before optimization, which verifies the feasibility of optimizing fermentation conditions by response surface methodology. The optimized fermentation system not only increases the effective viable count of the bacteria, but also reduces the fermentation cost from the operational aspect. The strain W-5 provides an important theoretical basis for the green prevention and control of pepper root rot as a microbial agent with development potential.

[0130] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for screening and fermenting antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot, characterized in that: The steps include: Step 1: Preparation of experimental materials, including test strains, culture media, reagents and instruments; Step 2: Isolation, screening and identification of antagonistic bacteria; Step 3: Observation of the effect of antagonistic bacteria W-5 on the growth of the front hyphae of Fusarium solani; Step 4: Detection of the antibacterial spectrum of antagonistic bacteria W-5 against pathogenic fungi; Step 5: In vitro antibacterial test of antagonistic bacteria W-5 on Zanthoxylum bungeanum root slices; Step 6: Detection of extracellular enzymes produced by antagonistic bacteria W-5 and siderophore culture medium detection; Step 7: Optimization of fermentation conditions of antagonistic bacteria W-5; Step 8: Data processing and analysis.

2. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: In the step 1, the indicator fungus Fusarium solani H1 and the antagonistic bacteria W-5 are isolated from the root tissue and root soil of Zanthoxylum bungeanum suffering from Zanthoxylum bungeanum root rot, respectively.

3. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: The culture medium in step 1 comprises: PDA medium (g / L): Peel and cut potatoes into pieces and weigh 200.0, glucose 20.0, agar 15.0-20.0, distilled water to 1L; LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl. After the solutes were dissolved, the pH was adjusted to 7.0 with 5.0 mol / L NaOH and the volume was adjusted to 1 L with deionized water. NYBD medium (g / L): 5.0% yeast extract powder, 8.0% beef extract powder, 10.0% glucose, adjust pH to 7.0-7.5, add distilled water to 1L; The above three culture media were sterilized at 103.4 kPa for 21 min.

4. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: The specific process of step 2 is as follows: (1) Isolation of antagonistic bacteria W-5 Weigh 1.0 g of Zanthoxylum root soil and put it into a 100 mL sterile conical flask. Add 90 mL of sterile water and incubate it on a shaker at 180 r / min for 30 min. After standing for 30-60 min, take the supernatant and dilute it by gradient 10 -1 -10 -8 , take 10 -6 , 10 -7 , 10 -8 200uL of three gradients were spread on LB solid medium and cultured at 32℃ for 24h, then a single colony was picked and numbered as W-5. The single colony was inoculated into LB liquid medium for expansion culture and kept in a 4℃ refrigerator for future use; (2) Screening of antagonistic bacteria W-5 Flat plate standoff method [13] The antibacterial effect of the isolated antagonistic bacteria W-5 was determined. Fusarium solani was used as the indicator bacteria. The above pathogenic fungi were punched into a 6mm diameter cake with a hole puncher and inoculated in the center of the PDA culture medium. A sterile filter paper was placed in the shape of a finished product 2cm away from the center point. 10uL of the single colony liquid in 1.2.1 was added to the filter paper. The culture was cultured at 30℃ for 5-7d. The diameter of the pathogenic fungal colony was determined and the antibacterial rate was calculated. Inhibition rate (%) = (control group colony diameter mm - test group colony diameter mm) / (control group colony diameter mm - hole diameter mm) × 100% (3) Molecular biological identification of antagonistic bacteria W-5 The antagonistic bacteria W-5 was identified by combining morphology with molecular biology methods. The strain was inoculated into LB medium and cultured at 32°C for 24 hours. The morphology was observed under an optical microscope. The single colony was placed under a stereomicroscope (magnification of 150×) for observation and Gram staining was performed. Molecular identification was performed by PCR amplification using bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' / 1492R: 5'-CTACGGCTACCTTGTTAC GA-3', and the PCR product was detected by 1.0% agarose gel electrophoresis.

5. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: In the step 3, the antibacterial plate obtained by the plate confrontation method in the above step is placed under a stereo microscope (magnification is 150×) to observe the effect of the antagonistic bacteria W-5 on the growth of the front hyphae of the pathogen Fusarium solani.

6. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: The specific process of step 4 is: using 5 pathogenic fungi (Fusarium graminearum, Fusarium pallidum, Gibberellic acid, Colletotrichum pine needles, and Fusarium lamina) as test indicator bacteria, the plate confrontation method is used to detect the inhibition rate of the antagonistic bacteria W-5 on pathogenic fungi.

7. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: The step 7 specifically includes: (1) Determination of the growth curve of antagonistic bacteria W-5 In LB liquid medium, the antagonistic bacteria W-5 seed liquid was inoculated at a ratio of 1%, and 3 replicates were set for each detection point. The culture was placed in a shaking incubator at 30°C and 180 r / min, and the OD was measured every 2 h by visible spectrophotometry. 600 Value, a total of 16 points were measured and the growth curve was drawn; (2) Single factor experiment to screen the optimal fermentation conditions In the previous experiment, it was determined that the best medium for the growth of antagonistic bacteria W-5 was NYBD. Based on this, the effects of different temperatures (26, 28, 30, 32, 34°C), pH (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5), inoculation size (1%, 3%, 5%, 7%, 9%, 11%), and rotation speed (140, 160, 180, 200, 220 r / min) on the growth of antagonistic bacteria W-5 were compared. The OD 600 As an indicator, determine the best single factor fermentation conditions; (3) Determine the optimal combination using response surface methodology Based on the above experiments, four factors (temperature, pH, rotation speed, and inoculum size) were selected as independent variables to antagonize the fermentation OD of strain W-5. 600 A response surface combination test was conducted for the response value, with 1, 0, and -1 representing the high and low levels of the four factors. A response surface analysis experiment with four factors and three levels was carried out to optimize the optimal fermentation conditions of the antagonistic bacteria W-5.

8. The method for screening and fermenting the antagonistic bacteria W-5 against Zanthoxylum bungeanum root rot according to claim 1, characterized in that: In step 8, Excel 2007 software was used to process and analyze the obtained data; and the response surface results were analyzed using Design-Expert 11 software.

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