A bacterial wilt antagonistic strain F24 and its screening method and application

Abstract

The present invention relates to a bacterial wilt antagonistic strain F24 and a screening method and application thereof. The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, with a deposit date of June 16, 2022, a deposit number of CGMCC No.25103, and a classification name of dispersed pantoea. The screening method includes randomly collecting 3 healthy tomato seedlings that have been treated, collecting rhizosphere soil; preliminarily screening the obtained bacterial wilt antagonistic bacteria by a plate confrontation method; continuing to drip the initial screening bacterial solution on the pathogen plate to obtain the re-screened antagonistic bacteria; purifying the re-screened antagonistic bacteria in a solid culture medium to obtain the antagonistic strain F24. The present invention has a significant inhibitory effect on Ralstonia solanacearum, and its plate inhibition ability is stronger than that of other Bacillus, and the anti-wilt effect of the biological organic fertilizer produced by combining with organic fertilizer is more significant; and the screening of the bacterial wilt antagonistic strain F24 is screened from the rhizosphere soil of healthy plants treated with organic fertilizers with good disease prevention effects, and the antagonistic strain has a stronger adaptability.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a bacterial wilt antagonistic strain F24 and a screening method and application thereof. Background Art

[0002] In the Yangtze River Basin and areas south of it, the area affected by tomato bacterial wilt in general years accounts for about 10% of the planted area, causing a yield loss of 10% to 15%; in years of repeated outbreaks, the yield loss can reach more than 50%, and some plots even have no harvest. The wide outbreak area, serious damage, and huge losses have become a major bottleneck and problem in tomato production and cultivation. In addition, with the changes in my country's climate and planting patterns in recent years, the scope of tomato bacterial wilt has gradually moved northward, and tomato bacterial wilt has become one of the main obstacles to sustainable agricultural development.

[0003] Compared with other agricultural and chemical control measures, biological control is an environmentally friendly control measure that can effectively reduce the use of pesticides and inhibit the development of diseases. Screening antagonistic bacteria has become a research hotspot for the prevention and control of pests and diseases. In the process of tomato bacterial wilt control, biological control measures play an important role in ensuring green tomato planting and protecting the ecological environment, and are also conducive to restoring soil health and improving food safety.

[0004] Problems with the existing technology: Using organic fertilizer products to control soil-borne diseases may have the negative effect of promoting diseases, which to a certain extent limits the large-scale use of organic fertilizers in plant disease prevention and control. Due to the variety of soil-borne pathogens and the different crop varieties, the biological organic fertilizers produced on the market have broad-spectrum disease inhibition characteristics, but there are also cases where they only have an inhibitory effect on a certain type of soil-borne disease of a specific crop, and have no inhibitory effect on other diseases of other crop varieties or even aggravate the disease. The disease inhibition effect is unstable and there is great uncertainty. In view of this, there is an urgent need for a biological organic fertilizer with strong specificity and good disease inhibition effect to inhibit tomato bacterial wilt. At present, the antagonistic bacteria reported for the prevention and control of tomato bacterial wilt mainly include Bacillus, Pseudomonas and Streptomyces, and there is little research on the application of dispersed Pantoea in the prevention and control of tomato bacterial wilt. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide a bacterial wilt antagonistic strain F24 and its screening method and application to solve the above-mentioned problems mentioned in the background technology.

[0006] In order to achieve the above objectives, this application provides the following technical solutions.

[0007] A bacterial wilt antagonistic strain F24, the strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation date is June 16, 2022, the preservation number is CGMCC No.25103, and the classification name is dispersed Pantoea.

[0008] Preferably, the strain has the following morphological characteristics: light yellow, round, convex, opaque, and neatly marginated.

[0009] The screening method of the bacterial wilt antagonistic strain F24 comprises the following steps:

[0010] 101. Randomly select three healthy tomato seedlings from the tomato seedlings treated with organic fertilizer that has the effect of inhibiting tomato bacterial wilt, and collect the rhizosphere soil;

[0011] 102. Using the plate confrontation method, the gradient diluted soil suspension is spread on the plate containing the bacterial wilt pathogen. The bacterial wilt antagonists obtained by preliminary screening have transparent inhibition zones around the pathogens.

[0012] 103. Continue to drip the primary screening bacterial solution onto the plate containing the bacterial wilt pathogen, and select a certain standard strain as the secondary screening antagonistic bacteria;

[0013] 104. The rescreened antagonistic bacteria were purified in LB solid culture medium to finally obtain dispersed Pantoea F24.

[0014] Preferably, the criteria for selecting the rescreened antagonistic bacteria in step 103 are obvious inhibition of the transparent zone and D / d greater than 6.5, wherein D is the diameter of the transparent zone and d is the diameter of the colony.

[0015] Preferably, the LB solid culture medium in step 104 comprises the following components and weight portions: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, 20 g of agar powder, the pH is adjusted to 7.0, and sterilized at 121° C. for 20 min.

[0016] The application of the above-mentioned bacterial wilt antagonistic strain F24, and the application of the antagonistic strain to prevent and control tomato bacterial wilt.

[0017] A bio-organic fertilizer, prepared by inoculating compost with the above-mentioned bacterial wilt antagonistic strain F24, wherein the concentration of the antagonistic strain is 10 9 CFU / mL and above.

[0018] The preparation method of the above-mentioned bio-organic fertilizer comprises the following steps:

[0019] 201. Evenly mix the organic fertilizer raw materials according to the mass ratio to obtain a mixed material, and load the mixed material into a composting reactor;

[0020] 202. Set the aeration volume of the composting reactor, aerate for a certain period of time at a certain interval, and keep circulating; turn the compost once at a certain interval to perform aerobic composting;

[0021] 203. During the composting stage, antagonistic bacteria liquid is inoculated to produce biological organic fertilizer.

[0022] Preferably, step 201 also includes adjusting the initial moisture content of the mixed material to 55-65% and the C / N ratio to 20-40:1.

[0023] Preferably, the aeration rate of the composting reactor in step 202 is set to 20-25 L·L -1 ·min -1 ; Aerate for 15 to 25 minutes every 25 to 35 minutes, and keep circulating; Turn the pile every 2 to 5 days, and measure the temperature every day with a temperature probe; Carry out aerobic composting for 35 to 45 days.

[0024] Beneficial technical effects obtained by the present invention:

[0025] 1) The solanacearum antagonistic strain F24 in the present invention has a significant inhibitory effect on Ralstonia solanacearum, and its plate inhibition ability is stronger than that of other Bacillus, and the anti-wilt effect of the biological organic fertilizer produced by combining with organic fertilizer is more significant; and the solanacearum antagonistic strain F24 is screened from the rhizosphere soil of healthy plants treated with organic fertilizer with good disease prevention effect. Based on the principle of "where you come from, where you go", the screened antagonistic strain has stronger adaptability.

[0026] 2) The organic biofertilizer prepared by the antagonistic strain F24 in the present invention has a strong effect of preventing and treating tomato bacterial wilt, and has low cost, considerable economic benefits, strong feasibility in production, and easy to promote and implement.

[0027] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a preferred embodiment of the present application and is described in detail with the accompanying drawings as follows.

[0028] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0030] Figure 1 is an antagonistic bacteria inhibition circle diagram in one embodiment of the present disclosure;

[0031] Figure 2 This is a plate streak diagram of the antagonistic strain F24 in one embodiment of the present disclosure;

[0032] Figure 3 is a phylogenetic tree of antagonistic strains in one embodiment of the present disclosure;

[0033] Figure 4 This is a comparison chart of the diameters of the inhibition zones of the antagonistic strains in the plate antibacterial experiment;

[0034] Figure 5 This is a comparison chart of the disease index of tomato bacterial wilt under different treatments;

[0035] Figure 6 This is a comparison chart of the disease prevention effects under different treatments. DETAILED DESCRIPTION

[0036] To make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all of the embodiments. In the following description, specific details such as specific configuration and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures is omitted in the embodiment.

[0037] It should be understood that the references to "one embodiment" or "this embodiment" throughout the specification mean that the specific features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present application. Therefore, the references to "one embodiment" or "this embodiment" appearing throughout the specification do not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0038] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity, and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0039] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the previous and next associated objects are in an "or" relationship.

[0040] The term "at least one" in this article is merely a description of the association relationship of associated objects, indicating that there may be three relationships. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0041] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusions.

[0042] Example 1

[0043] A bacterial wilt antagonistic strain F24, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of June 16, 2022, a deposit number of CGMCC No.25103, and a classification name of Pantoea dispersa.

[0044] The screening method of the bacterial wilt antagonistic strain F24 comprises the following steps:

[0045] 101. Randomly select three healthy tomato seedlings from the tomato seedlings treated with organic fertilizer that has the effect of inhibiting tomato bacterial wilt, and collect the rhizosphere soil.

[0046] Specifically, the whole plant was pulled out of the soil, large pieces of soil were shaken off, and the soil attached to the plant roots was removed as much as possible. Then the tomato roots were cut and placed in a 50 mL centrifuge tube containing 10 mL of 0.85% sodium chloride solution. The roots were shaken vigorously for washing. The root tissue was taken out and shaken at 30 ° C and 150 r / min for 30 min. After standing for 20 to 30 s, the supernatant was taken for 10-fold serial dilution. 3 ~10 5 Serial dilutions.

[0047] 102. Using the plate confrontation method, the gradient diluted soil suspension is spread on the plate containing the bacterial wilt pathogen. The antagonistic bacteria against bacterial wilt obtained by preliminary screening have transparent inhibition zones around the pathogens.

[0048] Specifically, 100 μL of the dilution in step 101 was applied to a plate containing pathogens. The plate culture medium was 5 g of yeast extract powder, 5 g of peptone, 10 g of glucose, 18 g of agar powder, and 1000 mL of water. The pH was adjusted to 7.0. After heating and dissolving, the solution was dispensed into shake bottles, with 100 mL of liquid per bottle. After sterilization, the solution was cooled to about 50°C. In a sterile environment, 2 mL of 1.0×10 7 CFU / mL Ralstonia solanacearum, use a magnetic stirrer to shake thoroughly, pour the plate and blow dry the water vapor on the plate, repeat each gradient three times, culture at 30℃, the antagonistic strains with transparent inhibition circles around the pathogens, select single colonies with transparent circles for purification and culture to obtain the primary screening antagonistic bacteria,

[0049] 103. Continue to drop the primary screening bacterial solution on the plate containing the bacterial wilt pathogen, and select the strains with obvious inhibition transparent zone and D / d greater than 6.5 as the secondary screening antagonistic bacteria, where D is the diameter of the transparent zone and d is the colony diameter.

[0050] 104. The antagonistic bacteria screened were purified in LB solid medium to obtain dispersed Pantoea F24. Figure 1 and 2 shown.

[0051] LB solid medium includes the following components and weight portions: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, 20 g of agar powder, adjusted to pH 7.0, and sterilized at 121° C. for 20 min.

[0052] The dispersed Pantoea F24 obtained above was stored at 4° C. and used for the subsequent preparation of compost antagonistic bacteria seed solution.

[0053] The morphological characteristics of the above strains are: light yellow, round, convex, opaque, and have neat edges.

[0054] Physiological and biochemical tests of the above strains are shown in Table 1.

[0055] Table 1

[0056]

[0057] The above strains are motile; the contact enzyme test is positive; they can decompose glucose to produce acid, but cannot decompose sucrose and lactose to produce acid; the VP test and MR test are negative, indicating that the pyruvate produced by the strain decomposing glucose cannot be decarboxylated by carboxylase, nor can it be decomposed to produce organic acids such as formic acid and acetic acid; they can use citrate as a carbon source for growth, and do not produce phenylalanine deacidase.

[0058] DNA of strain F24 was extracted using a bacterial DNA rapid extraction kit. The stock DNA solution was diluted 1:10 with ddH2O, and primers 27F and 1492R were used for 16S rRNA amplification and gene sequencing. The base sequence was analyzed for homology using BLAST. The results showed that strain F24 belonged to the genus Pantoea dispersa. Figure 3 shown.

[0059] An application of a bacterial wilt antagonistic strain F24, and application of the antagonistic bacteria to the prevention and treatment of tomato bacterial wilt.

[0060] A biological organic fertilizer, prepared by inoculating compost with the above-mentioned bacterial wilt antagonistic strain F24, wherein the concentration of the antagonistic strain is 10 9 CFU / mL and above.

[0061] The preparation method of the above-mentioned bio-organic fertilizer comprises the following steps:

[0062] 201. The organic fertilizer raw materials are uniformly mixed according to the mass ratio to obtain a mixed material, the initial moisture content of the mixed material is adjusted to 55-65%, the C / N ratio is adjusted to 20-40:1, and the mixed material is loaded into a 50kg composting reactor.

[0063] 202. Set the aeration volume of the composting reactor to 20-25 L·L -1 ·min -1 ; Aerate for 15 to 25 minutes every 25 to 35 minutes, and keep circulating; Turn the pile every 2 to 5 days, and measure the temperature every day with a temperature probe; Carry out aerobic composting for 35 to 45 days.

[0064] 203. During the compost maturity stage (temperature drops to room temperature, moisture content ≤ 30%), inoculate antagonistic bacteria seed solution to produce biological organic fertilizer.

[0065] In one embodiment, the organic fertilizer raw materials in step 201 include mushroom residues and crop straws.

[0066] In one embodiment, in step 202, the aeration rate of the reactor is set to 21.3 L·L-1·min-1; aeration is performed for 20 minutes every 30 minutes, and the cycle is continued; the compost is turned every 3 days, and the temperature is measured every day using a temperature probe; and aerobic composting is performed for 40 days.

[0067] Preparation of the above-mentioned antagonistic bacteria seed solution: inoculate one loop of the antagonistic bacteria into 10 mL of LB liquid culture medium, and culture it at 37°C and 200 rpm for 1 day; take 1 mL of the culture solution and add it to a new 100 mL of LB liquid culture medium, and gradually expand the antagonistic bacteria seed solution in this way; ensure that the number of viable bacteria in the antagonistic bacteria seed solution is 10 9 CFU / mL and above.

[0068] Furthermore, the LB liquid culture medium includes the following components and weight portions: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1000 mL of distilled water, and the pH is adjusted to 7.0.

[0069] Greenhouse protection effectiveness verification test

[0070] In order to verify the control effect of the above-mentioned biological organic fertilizer on tomato bacterial wilt, a potted experiment was carried out in the artificial climate greenhouse of the Institute of Organic Resource Recycling of China Agricultural University (Suzhou). The experiment is as follows:

[0071] (1) Tomato seeds were evenly spread on filter paper moistened with distilled water and cultured in a dark greenhouse for 3 days; then the germinated seeds were placed in a plug tray using tweezers and cultured in a greenhouse. The greenhouse culture conditions were a cycle of 16 h / 8 h, 22° C., and 70% relative humidity.

[0072] (2) When tomatoes reached the 3-leaf stage, tomato seedlings with uniform growth were selected and transplanted into 350 mL plastic cups. Each cup contained approximately 200 g of substrate soil (Northeast black soil, vermiculite, and perlite were mixed in a ratio of 3:1:1). There was one seedling per cup, and 24 replicates were set up for each treatment.

[0073] (3) Four treatments were set up in the experiment, namely, matrix soil as the control group, treatment 1 (CK); matrix soil + mushroom residue organic fertilizer (6 g), treatment 2 (OF); matrix soil + commercially available biological organic fertilizer (6 g), treatment 3 (BOF); matrix soil + biological organic fertilizer (6 g), treatment 4 (MGBOF).

[0074] (4) On the second day after transplanting, 20 mL of 5.0 × 10 7CFU / mL (OD600 = 0.5) HN4 Ralstonia solanacearum. The treated tomato seedlings were placed in a greenhouse at a temperature of 28 ° C, a relative humidity of 90%, and a photoperiod of 16h / 8h for cultivation. Referring to the bacterial wilt disease grade standard proposed by Kempe, the occurrence of the disease in each group was observed and counted: Grade 0: The whole plant showed no symptoms of bacterial wilt; Grade 1: Less than 25% (including 25%) of the leaves of the whole plant showed wilt symptoms; Grade 2: Less than 50% (including 50%) of the leaves of the whole plant showed wilt symptoms; Grade 3: Less than 75% (including 75%) of the leaves of the whole plant showed wilt symptoms; Grade 4: Less than 100% (including 100%) of the leaves of the whole plant showed wilt symptoms. Statistics were stopped when the incidence rate of the control group was above 90%.

[0075]

[0076]

[0077] Results and Analysis:

[0078] As attached Figure 4 As shown, in the plate antibacterial experiment, the strain F24 among the screened antagonistic bacteria had the largest inhibition zone and the strongest antibacterial effect.

[0079] As attached Figure 5 and 6 As shown, the above-mentioned biological organic fertilizer can significantly reduce the disease index of tomato bacterial wilt, and the prevention efficiency of tomato bacterial wilt is as high as 79.31%, which is 60.92% and 37.93% higher than ordinary organic fertilizer and biological organic fertilizer purchased on the market.

[0080] The above results show that the present invention has discovered a strain of Pantoea dispersa F24 with strong antagonistic ability to bacterial wilt, which has stronger plate inhibition ability than other Bacillus sp., and the biological organic fertilizer prepared by using the strain has a strong effect of preventing and controlling tomato bacterial wilt.

[0081] The above description is only the preferred embodiment of the present invention, which does not limit the protection scope of the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. Within the spirit and principle of the present invention, any change, modification, replacement, integration and parameter change of these embodiments by conventional substitution or capable of achieving the same function without departing from the principle and spirit of the present invention shall fall within the protection scope of the present invention.

Claims

1. A bacterial wilt antagonistic strain F24, characterized in that The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 16, 2022, with a deposit number of CGMCC No.25103 and a classification name of Pantoea dispersa ( Pantoea dispersa ).

2. The use of the bacterial wilt antagonistic strain F24 according to claim 1, characterized in that: The antagonistic strain is used to prevent and treat tomato bacterial wilt.

3. A biological organic fertilizer, characterized in that: The bio-organic fertilizer is prepared by inoculating compost with the bacterial wilt antagonistic strain F24 according to claim 1, wherein the concentration of the antagonistic strain is 10 9 CFU / mL and above.

4. The preparation method of bio-organic fertilizer according to claim 3, characterized in that, The following steps are involved:

201. Evenly mix the organic fertilizer raw materials according to the mass ratio to obtain a mixed material, and load the mixed material into a composting reactor; 202. Set the aeration volume of the composting reactor, aerate for a certain period of time at a certain interval, and keep circulating; turn the compost once at a certain interval to perform aerobic composting; 203. During the composting stage, antagonistic bacteria liquid is inoculated to produce biological organic fertilizer.

5. The preparation method of bio-organic fertilizer according to claim 4, characterized in that, The step 201 also includes adjusting the initial moisture content of the mixed material to 55-65% and the C / N ratio to 20-40:

1.

6. The preparation method of bio-organic fertilizer according to claim 4, characterized in that, In step 202, the aeration rate of the composting reactor is set to 20-25 L·L -1 ·min -1 ; Aerate for 15-25 minutes every 25-35 minutes, and keep circulating; Turn the pile every 2-5 days, and measure the temperature every day with a temperature probe; Carry out aerobic composting for 35-45 days.

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