CN114875016A - Preparation carrier suitable for pseudomonas fluorescens and microbial inoculum thereof - Google Patents

Abstract

The present invention further researches and develops a formulation carrier suitable for the Pseudomonas fluorescens LSW-4 on the basis of isolating and obtaining the Pseudomonas fluorescens resistant to R. solanacearum, wherein the carrier is grain bran, straw powder or diatomite. Using the inoculum medium obtained by compounding the carrier, Pseudomonas fluorescens LSW-4 can be made into a solid microbial inoculum with a high number of viable cells and a long storage time, which solves the problem of transportation and storage of Pseudomonas fluorescens LSW-4. The method for preparing the solid microbial inoculum is simple, and it is convenient to further industrialize the isolated Pseudomonas fluorescens LSW-4 with biocontrol effect and apply it to the field to prevent and control tobacco bacterial wilt, and give full play to its actual role and effect. .

Description

A formulation carrier suitable for Pseudomonas fluorescens and its bacterial agent

technical field

The invention belongs to the technical field of agricultural microorganism control, and relates to a formulation carrier suitable for Pseudomonas fluorescens and a bacterial agent thereof.

Background technique

Tobacco bacterial wilt is a devastating soil-borne disease caused by Ralstonia solanacearum. Serious, resulting in higher economic losses. For the prevention and control technology of tobacco bacterial wilt, domestic and foreign scholars have studied the prevention and control from chemical, biological, agricultural measures, etc. Although the occurrence of tobacco bacterial wilt can be alleviated to a certain extent, there are still certain limitations. In recent years, the use of biocontrol bacteria to control tobacco bacterial wilt is an effective control measure, which has the advantages of environmental friendliness and long-term effects, and has broad application prospects. The biocontrol bacteria that have been isolated from soil and can be used to control tobacco bacterial wilt mainly include Bacillus subtilis, arbuscular mycorrhizal fungi, Streptomyces, Pseudomonas and so on. All of these isolated strains can be further exploited for their potential as microbial pesticides.

However, because Pseudomonas fluorescens lives in an obligate aerobic life, it can grow in an environment of pH 5-8 and 7% NaCl, and the optimum growth temperature is about 25-30 °C. It grows under low temperature conditions, is not resistant to high temperature, does not grow above 41 °C, and cannot produce dormant bodies such as spores. Its application has certain limitations. In order to be further applied to microbial pesticides, the research and development of inoculants and the selection of carriers for inoculants are particularly important. Carriers and other auxiliary components in inoculants are the key factors affecting the number of viable bacteria in microbial inoculants. The preparation process of liquid bacterial preparation is simple, and the bacterial body is in a suspended and homogeneous state under liquid conditions, which is conducive to the diffusion of bacterial species and products, and facilitates the control and detection of fermentation bacterial liquid, but general liquid fermentation preparations are not easy to transport. , the storage conditions are harsh, and the number of viable bacteria decreases rapidly. The advantages of solid microbial preparations are low packaging cost, convenient transportation, high bacterial content, storage at room temperature and not easy to inactivate, can contain many types of bacteria, and can be combined with compound enzyme preparations for better effect and easier use. It is convenient, can be granulated at high temperature, and is convenient for the large-scale use of automatic feeding systems.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a formulation carrier suitable for Pseudomonas fluorescens, and an inoculum medium prepared by using the carrier. The present invention also aims to provide a bacterial agent of Pseudomonas fluorescens LSW-4.

To achieve the above object, the present invention provides the following technical solutions:

1. A formulation carrier suitable for Pseudomonas fluorescens, the carrier being grain bran, straw powder or diatomaceous earth.

Further, the preferred formulation carrier grain bran suitable for Pseudomonas fluorescens.

Further, in the formulation carrier suitable for Pseudomonas fluorescens, the Pseudomonas fluorescens is Pseudomonas fluorescens LSW-4, which is preserved in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: April 11, 2022, preservation number: CGMCC24668.

2. The present invention also provides a microbial inoculant for preventing and treating tobacco bacterial wilt, comprising Pseudomonas fluorescens LSW-4 and a carrier, and the preservation number of Pseudomonas fluorescens LSW-4 is: CGMCC 24668.

Further, the microbial inoculum for preventing and treating tobacco bacterial wilt disease also includes inoculum medium: tryptone 1%, yeast extract 0.5%, sodium chloride 1%, sodium carboxymethyl cellulose 0.01%-0.02%, dextrin 0.5%-1.5%, sodium caseinate 2%-3%.

Further, in the microbial inoculum for preventing and treating bacterial wilt disease of tobacco, the ratio of the bacterial liquid of Pseudomonas fluorescens LSW-4 to the carrier is 0.5-3ml:1g.

Preferably, in the microbial inoculum for preventing and treating bacterial wilt disease of tobacco, the ratio of Pseudomonas fluorescens LSW-4 bacterial liquid to the carrier is 0.5-1.5ml:1g.

3. The present invention also provides a method for preparing a microbial inoculant for preventing and treating tobacco bacterial wilt, the specific steps comprising:

(1) Inoculate Pseudomonas fluorescens LSW-4 into LB liquid medium, place it on a constant temperature shaker at 28°C-30°C for 24h-48h, and centrifuge the cultured bacterial fermentation broth. Suspend the inoculum medium, gently shake to mix;

(2), add the suspended bacterial liquid to the carrier, cultivate at 28°C-30°C constant temperature shaker for 30-40min, and finally dry to constant weight.

Further, in the preparation method of microbial inoculum, the carrier is grain bran, straw powder or diatomaceous earth; the inoculum medium is 1% tryptone, 0.5% yeast extract, 1% sodium chloride, carboxymethyl Cellulose sodium 0.01%-0.02%, dextrin 0.5%-1.5%, sodium caseinate 2%-3%.

Further, in the preparation method of the microbial inoculum, the ratio of the bacterial liquid to the carrier in step (2) is 0.5-3ml:1g.

Preferably, in the preparation method of the microbial inoculum, the ratio of the bacterial liquid to the carrier in step (2) is 1-1.5 ml:1 g.

Further, in the preparation method of the microbial inoculum, the concentration of Pseudomonas fluorescens LSW-4 in the bacterial solution is 1×10 ¹⁰ cfu/ml to 1×10 ¹² cfu/ml.

4. The application of any one of the above microbial inoculants in the prevention and control of tobacco bacterial wilt is also within the scope of protection of the present invention.

The beneficial effects of the present invention are: the Pseudomonas fluorescens LSW-4 used in the present invention has better inhibitory and antagonistic effects on Ralstonia solanacearum, and the inhibitory effects on Ralstonia solanacearum under different concentrations The bacterial circle can reach 12.30mm-33.76mm. The pot experiment showed that the Pseudomonas fluorescens LSW-4 had a good direct disease control effect on tobacco bacterial wilt. In further field experiments, the Pseudomonas fluorescens LSW-4 4 also showed excellent resistance to tobacco bacterial wilt, the incidence rate was only 10.00%, the disease index was 5.78, and the control effect was 79.97%, which fully proved that Pseudomonas fluorescens LSW-4 in the field can be effective against tobacco plants. Bacterial wilt is effectively controlled and has a certain growth-promoting effect. It can be fully applied to actual tobacco planting and production. In order to further solve the problem of transportation and storage of Pseudomonas fluorescens LSW-4, talcum powder, diatomite, oyster shell powder, grain bran, straw powder, etc. were screened from different carriers. Five kinds of vectors were suitable for the growth of Pseudomonas fluorescens LSW-4, and the most suitable vector for Pseudomonas fluorescens was selected. Further using the inoculum medium obtained by compounding the carrier, Pseudomonas fluorescens LSW-4 can be made into a solid microbial inoculum, which is suitable for long-term storage. It has been tested by the number of viable bacteria in the inoculum and the pot experiment test, showing a relative control effect. Up to 54.65%, the method for preparing the solid microbial inoculum is simple, and the activity of the strain is high, which is suitable for industrial production, and is beneficial to the preservation and transportation of the strain. The isolated Pseudomonas fluorescens LSW-4 with biocontrol effect can be further industrialized and applied to field control of tobacco bacterial wilt.

biological material preservation

In the present invention, Pseudomonas fluorescens LSW-4 is preserved in the General Microorganism Center of the China Microorganism Culture Collection and Administration Commission, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: April 2022 December 11, deposit number: CGMCC 24668.

Description of drawings

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

Figure 1 is a partial plate diagram of the bacteriostatic effect of rescreened antagonistic bacteria on R. solanacearum.

Figure 2 shows the partial results of pot pre-test screening.

Figure 3 shows the effect of rhizosphere regulation of different microbial agents on the incidence of tobacco bacterial wilt resistance in tobacco plants.

Figure 4 shows the effect of rhizosphere regulation of different microbial agents on the disease index of tobacco bacterial wilt resistance of tobacco plants.

Figure 5 is a morphological diagram of a single colony of LSW-4.

Fig. 6 is a view of bacterial morphology observed by LSW-4 transmission electron microscope.

Figure 7 is a phylogenetic tree of LSW-4 based on 16S rDNA.

Figure 8 is a growth curve of Pseudomonas fluorescens LSW-4.

Figures 9 to 13 are test charts of the number of viable bacteria sampled at different time points for inoculum prepared by different carriers; the carrier in Figure 9 is diatomaceous earth, the carrier in Figure 10 is straw powder, the carrier in Figure 11 is grain bran, and the carrier in Figure 12 The carrier of Fig. 13 is talc powder, and the carrier of Fig. 13 is oyster shell powder.

Figures 14 to 16 are respectively the culture diagrams of the number of viable bacteria sampled at different time points of the inoculum obtained from each inoculum culture medium of Example 7.

Figure 17 is the pot test results of different Pseudomonas fluorescens LSW-4 solid inoculants.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental methods for which specific conditions are not indicated in the examples are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Example 1

1. Soil sample collection: Soil samples were collected on August 29, 2019. The soil sample collection information is shown in Table 1. Poke the surface soil of the healthy tobacco plants, collect the soil layer 5-25cm below the surface, dig out the tobacco roots, and mix the rhizosphere soil of the three healthy tobacco plants in the collection bag with the soil from the roots of the tobacco. Repeat, seal and mark.

Table 1 Soil sample collection information table

2. Activation of the tested pathogenic bacteria: The pathogenic bacteria Ralstonia solanacearum CQPS-1 was isolated from the tobacco strain of tobacco bacterial wilt disease in Pengshui, Chongqing, and the identification and characteristics of this pathogenic bacteria have been completed. Tobacco R. solanacearum CQPS-1 was activated first, and propagated in liquid medium B. The propagation conditions were constant temperature shaker at 30°C, 180rpm/min for about 12h, and the OD value was detected by spectrophotometer=0.8-1.0 , the concentration of R. solanacearum was about 1×10 ⁹ cfu/mL. The culture medium used in the present embodiment and its specific formula are as follows:

NA medium: 5.0 g of bacterial peptone, 1.0 g of yeast powder, 10.0 g of glucose, 3.0 g of beef powder, 20.0 g of agar powder, 1000 mL of distilled water, pH 6.8-7.2; sterilized at 121°C for 20 min.

LB medium: yeast extract 5.0 g, sodium chloride 10.0 g, tryptone 10.0 g, distilled water 1000 mL; sterilized at 121° C. for 20 min.

B liquid medium: 1.0 g of casein amino acid, 5.0 g of glucose, 10.0 g of bacterial peptone, 1.0 g of yeast extract, 1000 mL of distilled water; sterilized at 121° C. for 20 min.

3. Isolation and purification of strains

The test adopts the dilution plate coating method to separate and purify the bacteria in the soil samples. The specific operation is as follows: Weigh 10 g of soil samples and add 90 mL of sterile water, shake them slightly, and shake them on a constant temperature shaker at 220 r/min for 30 min after fully mixing. Let stand for 10min; take the supernatant of the soil bacteria suspension after standing, adopt the method of gradient dilution, configure ^10-2 , ^10-3 , ^10-4 and ^10-5 dilutions; take the dilution of each concentration gradient Add 100 μL of the solution to the NA plate with coated beads, shake it back and forth evenly to make the bacterial suspension evenly spread, and repeat 3 times for each concentration gradient; invert the coated NA plate in a constant temperature incubator at 30 °C. After 2 days, take out the NA plate and observe the growth of the colonies in the ultra-clean workbench. According to the colony shape and color as the judgment basis, pick out different single colonies in the LB liquid medium, and place them at 30°C, 170r/min for 12h. After that, adopt the method of streaking on the plate, place it at 30°C for 48 hours, and repeat 3 replicates for each streak plate to purify the isolated bacteria, and repeat the step of picking colonies on the NA plate three times until all the colonies on the NA plate are repeated. Consistent, this is the completion of the purification.

After separating and purifying the bacteria in the collected soil samples, the difference in the shape and color of the bacteria was visually judged, and a total of 184 bacteria were isolated. The 184 bacteria isolated by the plate confrontation method were further used to evaluate their effect on tobacco bacterial wilt. The antagonism of the bacteria was initially screened, and the specific operation method was as follows: take 100 μL of the activated bacterial suspension (10 ⁶ cfu/mL) of tobacco R. A 6mm filter paper sheet was placed in the center of the NA plate coated with R. solanacearum; 5 μL of each purified bacteria to be tested was placed on the filter paper sheet, and each treatment was repeated 3 times. After 48 hours of inverted culture in a constant temperature incubator at 30°C, the formation of a bacteriostatic zone was observed.

A total of 49 strains of bacteria with antagonistic effects against R. solanacearum were isolated from the primary screening, and there were obvious inhibition zones, but the bacteriostatic effects were different, and the diameters of the inhibition zones were different. The results are shown in Table 2.

Table 2 The diameter of the inhibition zone in the primary screening

The antagonistic bacteria that were initially screened were further screened again, and the specific operation method was as follows: take 100 μL of the bacterial suspension (10 ⁶ cfu/mL) of the activated R. solanacearum in advance, add it to the NA plate with sterilized coated beads, and spread it evenly. The 6mm filter paper sheet is placed in the center of the NA plate that has been coated with R. nicotianae; In the preliminary screening results, the bacterium that has antagonistic effect on R. nicotianae is transferred to LB liquid medium and cultivated to 10 ⁹ cfu/mL (OD = 0.8-1), to ensure that the concentration of the bacteria to be tested is consistent, take 5 μL of the bacteria to be tested and connect it to the filter paper, and each treatment is repeated 3 times. After being placed in a 30°C constant temperature incubator for 48 hours of inverted culture, in addition to observing the formation of the inhibition zone, the size of each inhibition zone was measured by the cross method. From the 49 strains of antagonistic bacteria screened in the initial screening, 24 strains of bacteria that still have antagonistic effects on R. solanacearum were rescreened. Bacterial circle diameter table.

Table 3 Rescreening inhibition zone diameter table

Example 2

In order to effectively prevent and control tobacco bacterial wilt, the screening of antagonistic bacteria against tobacco bacterial wilt is not limited to evaluating the direct inhibitory effect of antagonistic bacteria on tobacco R. Direct disease control effect.

Select the same size and healthy tobacco seedlings with four leaves and one heart for pot experiment. After the re-screened antagonistic bacteria are activated, they are transferred to LB liquid medium for cultivation, placed at 30 ° C, 170 r/min for 12 h, and detected with a spectrophotometer. To OD=0.8-1, the method of root irrigation was adopted, each tobacco seedling was connected with 10 mL of bacterial liquid, 10 tobacco seedlings were treated with each treatment, and the blank control was connected with 10 mL of sterile water. The cultivation conditions in the greenhouse were set as follows: light for 12 hours, temperature of 28°C, and humidity of 85%. During the growth period of tobacco seedlings, irrigate with sterile water regularly to ensure the normal growth of tobacco seedlings. From the time when tobacco bacterial wilt disease begins to occur, according to the national standard for bacterial wilt disease GB/T2322-2008 and indoor grading standards for tobacco bacterial wilt disease The incidence rate was investigated. Figure 2 shows the results of pot pre-test screening. Table 4 shows the effect of potted pre-test screening of antagonistic bacteria on tobacco bacterial wilt.

Table 4

Example 3

In order to clarify the effect of LSW-4 concentration and inoculation time on tobacco bacterial wilt disease, the four-leaf and one-xin Yunyan 87 tobacco seedlings were selected for the laboratory test using common seedling trays, and the concentrations were 1×10 ⁸ cfu/mL , 1 × 10 ⁷ cfu/mL, 1 × 10 ⁶ cfu/mL antagonistic bacteria concentration for root irrigation.

Each tobacco strain was inoculated with antagonistic bacteria LSW-4 in 10 mL of different treatment concentrations. After 3 days of inoculation with antagonistic bacteria LSW-4, each tobacco strain was inoculated with R. solanacearum CQPS-1 (1×10 ⁷ cfu/mL 10 mL). Each treatment 10 tobacco seedlings, repeated 3 times, the greenhouse culture conditions are light for 12 hours, temperature 28 ℃, humidity 85%, starting from the occurrence of tobacco bacterial wilt, according to the national standard GB/T2322-2008 and indoor according to the occurrence degree of bacterial wilt The grading standard was investigated, and the occurrence of bacterial wilt was recorded every 24 hours. It was found that the antagonistic bacteria LSW-4 had a concentration effect on the prevention and control of tobacco bacterial wilt, that is, the higher the concentration of antagonistic bacteria LSW-4, the better the control effect on tobacco bacterial wilt. When the incidence rate of blank control was 66.66%, the incidence rates of 1×10 ⁸ cfu/mL, 1×10 ⁷ cfu/mL and 1×10 ⁶ cfu/mL were 11.11%, 25.00% and 44.45%, respectively. were 11.11, 23.61, and 44.45, and the relative control effects of different concentrations were divided into 83.33%, 64.58%, and 33.32%.

Further through the potted plant pre-test of Example 2, several antagonistic bacteria LSW-4, LSW-32 and PSG-26 that did not develop disease were selected in the field test (1143m above sea level) in Cangba Village, Cangling Town, Youyang, Chongqing in 2019. Cangba Village, Cangling Town, Youyang County, whose longitude and latitude are E108°34′22.99″ east longitude and N29°0′42.83″ north latitude, with an average of about 1,200 transplanted tobacco seedlings per acre), the test variety is Yunyan 87, which supplies Test materials: (1) Self-extracting antagonistic bacteria (LSW-4, PSG-26, LSW-32); (2) Trichoderma harzianum; (3) Bacillus subtilis; (4) Strong seedlings; (5) Polymyxa Bacillus. (2)-(5) were purchased from the market, and each inoculum was irrigated and applied to the roots. Treatment 1: Paenibacillus polymyxa; Treatment 2: Trichoderma harzianum agent; Treatment 3: Seed hardy complex bacterial agent; Treatment 4: Bacillus subtilis agent; Treatment 5: LSW-4; Treatment 6: PSG-26; Treatment 7 : LSW-32; Treatment 8 (CK): clear water control, the concentration of each bacterial agent was 1×10 ⁸ cfu/mL. After activating the re-screened antagonistic bacteria, they were transferred to LB liquid medium for cultivation, placed at 30°C, 170 r/min for 12 h, detected with a spectrophotometer to OD=0.8-1, and the method of root irrigation was adopted.

Cultivation situation: In the early stage, floating seedlings were used for the cultivation of tobacco seedlings, and the management of tobacco plant growth was carried out in accordance with relevant technical standards. , topped on July 7 and harvested on July 18. The effects of rhizosphere regulation of different microbial agents on agronomic traits of tobacco plants are shown in Table 5. It can be seen from Table 5 that the antagonistic bacteria LSW-4, LSW-32 and PSG-26 isolated by the present invention have good effects on agronomic traits (according to YC/T142-1998 "Method for Investigation of Tobacco Agronomic Traits") at the clustering stage of tobacco plants. influences.

table 5

The plots selected in this experiment are high incidence areas of perennial bacterial wilt. The effects of rhizosphere regulation of different microbial agents on tobacco bacterial wilt resistance of tobacco plants are shown in Figure 3 (incidence rate) and Figure 4 (disease index). The incidence rates of different microbial inoculants rhizosphere regulation treatments were lower than those of the control treatments, and there was a significant difference with the clean water control, indicating that rhizosphere regulation could affect the resistance of tobacco plants to tobacco bacterial wilt to a certain extent. . The results showed that the antagonistic bacteria LSW-4 had the best disease control effect, with a late incidence rate of only 10.00%, followed by Bacillus subtilis with an incidence rate of 15.00%, Paenibacillus polymyxa, Miaoqiang, PSG-26, LSW The incidence rates of -32 were 43.33%, 38.33%, 38.33% and 41.67% respectively, while the incidence rate of the blank control was 61.67%. The trend of disease index and incidence rate is consistent, LSW-4 is 5.78, Bacillus subtilis disease index is 7.22, Paenibacillus polymyxa, Miao strong, PSG-26, LSW-32 disease index are 16.13, 18.44, 20.46 respectively , 17.96, the disease index of the blank control was 28.85, and the control effect of LSW-4 was 79.97%, which fully proved that LSW-4 can effectively control the bacterial wilt of tobacco plants in the field, and also has a certain growth-promoting effect. The tobacco plants irrigated with LSW-4 entered the cluster stage 2 days earlier than the control, and their plant height, effective leaf number, leaf length, leaf width and leaf area were all superior to those of the control.

Example 4

The identification of antagonistic bacteria LSW-4, Figure 5 is the morphological diagram of LSW-4 single colony, Figure 6 is the morphological observation diagram of LSW-4 transmission electron microscope. After culturing LSW-4 in TSA medium for 48 hours, the morphology of single colony was observed and gram stained. It was observed that LSW-4 was a Gram-negative bacteria, and the morphology of single colony was round colony and the color was milky white. , the surface is smooth, the edges are neat, and the middle is slightly raised. The results of transmission electron microscopy showed that the cells were rod-shaped and had terminal flagella.

Bacterial DNA was extracted (DNA extraction kit from Beijing Sobaole Technology Co., Ltd.) and PCR amplification was performed on the 27F\1492R range of bacterial DNA according to the amplification primers (27F and 1492R). 27F: AGAGTTTGATCCTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT. The PCR amplification products were sent to BGI for sequencing, and the sequencing results were compared in GenBank by BLAST. The 16S rDNA sequence with higher homology was selected as the reference object, and the Neighbour-joining method was used to construct a phylogenetic tree. Figure 7 is based on 16S rDNA to establish LSW-4 phylogenetic tree. The strain LSW-4 was identified as Pseudomonas fluorescens. For the antagonistic bacteria LSW-4, its OD value was measured by spectrophotometer to clarify its growth status and understand its growth law. After a total of 72 hours of measurement, it was observed that its OD value had a significant downward trend. Between 10h and 12h, LSW -4 reached the logarithmic phase of growth. Figure 8 is a growth curve of Pseudomonas fluorescens LSW-4.

The strain LSW-4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, preservation date: April 11, 2022, preservation number: CGMCC 24668.

Example 5

The soil after the application of antagonistic bacteria LSW-4 was collected in two times. The collection period was June 23 (prosperous period), and July 18 (the late topping period) with two data before and after comparison, three replicates, and the analysis was in Changes in soil microbial communities following the use of antagonistic bacteria LSW-4.

By using high-throughput sequencing from the DNA samples of soil treated with LSW-4 in different periods, and analyzing with the help of the Meiji cloud platform, the following conclusions were obtained:

(1) By analyzing the soil samples collected on June 23 and July 18, the level community composition of bacteria and fungi was compared, and the soil samples collected on June 23 and July 18 were compared. After the antagonistic bacteria LSW After -4 treatment, the proportion of Chloroflexi in the bacterial community showed an increasing trend, the proportion of Ascomycota in the fungal community decreased by 11.41%, and the proportion of Mortierellomycota increased by 3.54%. The proportion of Basidiomycota increased by 3.76%.

(2) Statistical analysis of β-diversity showed that at the level of OTU, in the soil on June 23, whether it was bacteria or fungi, the difference between the LSW-4 treatment and the blank control was not very obvious. In the soil on July 18, after LSW-4 treatment, the bacterial community was greatly affected, and there were obvious differences.

(3) Through the discriminant analysis of LEfSe of the soil samples treated with LSW-4, the soil samples collected on June 23 showed that at the level of bacterial community genus, the bacteria that were significantly enriched after LSW-4 treatment was Bacillus tau Genus (Rhodanobacter); Pseudeurotium, Setophoma, Rickenella, Cotylidia were significantly enriched for fungi after LSW-4 treatment. In the soil sample on July 18, Obscuribacterales was significantly enriched after LSW-4 treatment in the bacterial community, while at the level of fungi, Codinaea and Scytalidium were significantly enriched after LSW-4 treatment. .

As a biocontrol strain, LSW-4 can not only directly inhibit the proliferation of pathogenic bacteria and induce disease resistance in plants, but also play a role in regulating the soil micro-ecological structure in the soil. Soil is the environment for plants to grow directly, and it is also an important medium for pathogen-plant interactions. The health of the soil directly affects the health of the plant. The increasingly serious soil-borne diseases are often caused by the destruction of soil micro-ecological balance caused by continuous cropping all year round. Soil microorganisms are considered to be an important part of the soil ecosystem and an important indicator of whether the soil is healthy or not. Changes in soil microbial composition and the occurrence of soil-borne diseases are closely related to plant health. Tobacco rhizosphere soil microbial community structure was affected after application of the antagonistic bacterium LSW-4, Pseudomonas fluorescens, which could regulate the abundance of some specific microbial populations in the microbial community composition , reduce the occurrence of soil-borne disease bacterial wilt. The soil samples collected on June 23 showed that at the level of bacterial community, Rhodanobacter was significantly enriched after LSW-4 treatment; for fungi, it was significantly enriched after LSW-4 treatment. The ones are Pseudeurotium, Setophoma, Rickenella, Cotylidia. In the soil sample on July 18, Obscuribacterales was significantly enriched after LSW-4 treatment in the bacterial community, while at the level of fungi, Codinaea and Scytalidium were significantly enriched after LSW-4 treatment. .

Example 6

In order to further apply the strain LSW-4 to commercial products, on the basis of the previous research, continue to investigate the formulation carrier and solid microbial inoculum suitable for Pseudomonas fluorescens to solve the transportation and storage problems of Pseudomonas fluorescens , it is convenient to be used in the field to control tobacco bacterial wilt.

1. Preparation of liquid culture medium

LB liquid medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, constant volume 1000ml, natural pH;

LB solid medium: LB liquid medium plus agar 15%-20% (w/v).

2. Carrier

Carrier materials: oyster shell powder (Haozhou Chujian Huakai Electronic Commerce Co., Ltd. 500g/bag), diatomite (Dengfeng Fine Chemical AR500g/bag), talc (Fuchen Fine Chemicals LR500g/bottle), corn straw Powder (Lianfeng Agricultural Products Deep Processing 250g/bag of 20 meshes), Grain Bran Powder (Lianfeng Agricultural Products Deep Processing 250g/bag of 20 meshes).

Oyster shell powder, diatomaceous earth, talc powder, straw powder, and grain bran powder were taken respectively, sterilized by high pressure steam at 121°C for 30 minutes, and then dried in a constant temperature oven at 60°C to a constant state. Table 6 shows the pH values of different carriers.

Table 6 pH values of different carriers

3. Bacterial culture

Pseudomonas fluorescens LSW-4 was inoculated into LB liquid medium according to the inoculum amount of 1%-1.5%, placed in a constant temperature shaker at 30°C at 180 r/min, and cultured for 24h-48h.

4. Centrifugation

The cultured bacterial fermentation broth was centrifuged at 8000r for 5min with a 50ml centrifuge tube sterilized in advance.

5. Suspended

The centrifuged bacteria were added to the LB liquid medium, which was about one-tenth of the volume of the original bacteria solution, and the suspension was gently shaken and mixed. The concentration of the mixed bacterial solution was about 1×10 ¹² cfu/ml.

6. Mix

Add the suspended bacterial liquid to the aseptically treated oyster shell powder, diatomite, talcum powder, straw powder, grain bran and other carriers according to the volume-to-mass ratio (ml:g), and shake at a constant temperature of 30 °C for 180 r. Mix for 30-40min under the condition of /min, and finally ventilate and dry to constant weight in a sterile workbench, then put them into sterilized glass bottles respectively, and stir evenly with a sterile glass rod.

7. Save

Sealed with parafilm and stored at room temperature, samples were taken at 10 d, 30 d and 90 d respectively, the number of viable bacteria was measured, and the carrier with the largest number of viable bacteria was screened. Sampling was carried out for gradient dilution coating, and the number of viable bacteria was counted (LB solid medium, 3 samples were taken each time, weighed 1 g, each sample had 3 concentrations, and each concentration had 3 plates).

The ratio of bacterial liquid and carrier was tested according to 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1 (ml:g), oyster shell powder, talcum powder, straw powder, grain bran When the ratio of the inoculum to the carrier is greater than 1.5:1, the number of viable bacteria of the solid inoculum prepared by the same carrier does not change much during the test on the 10th day. When the ratio of the solid inoculum prepared by diatomite is greater than 2.5:1, The number of viable bacteria changed little when tested on day 10. Through experiments, it can be seen that the suitable carriers for the formulation of Pseudomonas fluorescens LSW-4 are grain bran, straw powder and diatomaceous earth, preferably grain bran. And grain bran is a convenient and cheap source of raw materials, and the quantity is large, so choose grain bran as the best carrier for further testing and research.

Figures 9-13 show the number of viable bacteria at different time points when the ratio of bacterial liquid to carrier is 1:1, wherein the carrier in Figure 9 is diatomaceous earth, the carrier in Figure 10 is straw powder, and the carrier in Figure 11 is grain bran , the carrier in Figure 12 is talc, and the carrier in Figure 13 is oyster shell powder. Table 7 shows the number of viable bacteria measured by sampling at different time points when the ratio of bacterial liquid to carrier is 1:1.

Table 7 is the number of viable bacteria (cfu/g) measured by sampling at different time points of each carrier

carrier 10d 30d 90d bran 2.1×10¹¹ 5.7×10¹⁰ 1.3×10⁹ straw powder 1.3×10⁹ 9.0×10⁷ 1.2×10⁸ talcum powder 2.5×10¹⁰ 4.7×10⁷ \ diatomite 7.7×10¹¹ 5.2×10⁷ 9.6×10⁶ Oyster Shell Powder 5.1×10¹⁰ 2.1×10⁸ \

Example 7

Inoculate Pseudomonas fluorescens LSW-4 into LB liquid medium according to the inoculum amount of 1% to 1.5%, place it on a constant temperature shaker at 30°C for 180 r/min, cultivate for 24h-48h, and sterilize the cultured bacterial fermentation broth with Centrifuge the bacteria centrifuge tube at 8000 r for 5 min; after the centrifugation, the bacteria are suspended in the inoculum medium with 1/8 to 1/12 of the original bacteria liquid volume, and gently shake to mix.

Inoculum medium 1: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, dextrin 5-15g, sodium caseinate 20-30g, constant volume 1000ml , pH natural.

Inoculum medium 2: carrier is grain bran, inoculum medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, sodium caseinate 20-30g , constant volume 1000ml, pH natural.

Inoculum medium 3: the carrier is grain bran, the inoculum medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, constant volume 1000ml, natural pH.

Using grain bran as a carrier, add the bacterial liquid suspended with different bacterial agent medium into the aseptically treated carrier in a volume-to-mass ratio of 0.5 to 1.5:1 (ml:g), and place it on a constant temperature shaker at 30°C for 180 r. Mix for 30-40 min under the condition of /min, and finally ventilate and dry to constant weight in a sterile workbench. In order to speed up the drying speed, it is also possible to perform aseptic filtration after mixing, and then ventilate and dry to constant weight under aseptic conditions to obtain a solid inoculum of Pseudomonas fluorescens LSW-4. It can also be carried out by freeze-drying or spray-drying.

The inoculum culture medium of the present invention has been tested with various component combinations and different concentrations, using grain bran as a carrier, and investigating the effective viable bacteria for 10 days, 30 days and 90 days under the condition that the ratio of bacterial liquid to carrier is 1:1 Quantity, confirm the optimal inoculum medium, Table 8 is the statistics of the number of viable bacteria obtained by sampling at different time points of the inoculum obtained by each inoculum medium, Figure 14-Figure 16 are respectively the bacteria obtained by each inoculum medium in turn. The number of viable bacteria sampled at different time points was cultured. Finally, using grain bran as a carrier, the LB liquid medium was screened under the condition of 1:1 bacteria liquid and carrier, adding 0.01-0.02% of sodium carboxymethylcellulose, 0.5-1.5% of dextrin, and 2-3% of sodium caseinate. % (w/v) of the inoculum medium can improve the loading rate and the number of effective viable bacteria of Pseudomonas fluorescens LSW-4 on the grain bran carrier. content and data.

Table 8 Statistics of viable count (cfu/g)

Carrier + inoculum medium 10d 30d 90d Grain bran + bacterial agent 1 4.8×10¹¹ 1.9×10¹¹ 5.7×10¹⁰ Grain bran + bacterial agent 2 9.7×10¹⁰ 1.3×10¹⁰ 7.3×10⁹ Grain bran + bacterial agent 3 7.5×10⁹ 3.6×10⁸ 8.3×10⁷

Example 8

Through the screening of the carrier and the inoculum medium, the Pseudomonas fluorescens LSW-4 solid inoculum prepared by different methods was subjected to an indoor pot experiment for further comparison and confirmation. The Yunyan 87 tobacco seedlings with four leaves and one heart were selected to be tested in the laboratory by the way of raising seedlings in common seedling trays. Three replicates were set for each treatment, with 12 plants in each replicate. When transplanting, each tobacco plant is about 60g of substrate, and 2 grams of bacterial agent is applied in the nest. After transplanting, the tobacco plants are cultivated at 30 ° C, light for 16 hours and night for 8 hours, and the air humidity is 85% in a constant temperature greenhouse, and watered in time. After slowing the seedlings for 3 days, the roots were irrigated with R. solanacearum with a bacterial concentration of 1×10 ⁷ cfu/ml, and 10 ml of each tobacco was irrigated. Disease surveys were carried out from the initial stage of disease onset, data were recorded and analyzed.

The preparation methods of the following different Pseudomonas fluorescens LSW-4 solid inoculants are the same as in Example 2, the carrier and the inoculum medium are as follows, and the ratio of the bacterial liquid to the carrier is 1:1 for investigation under the same conditions.

Bacterial agent 1: the carrier is straw powder, the inoculum agent medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, constant volume 1000ml, natural pH.

Bacterial agent 2: the carrier is grain bran powder, the inoculum agent medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, constant volume 1000ml, natural pH.

Bacterial agent 3: the carrier is straw powder, the inoculum medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, dextrin 5-15g, casein acid Sodium 20～30g, constant volume 1000ml, natural pH.

Bacterial agent 4: the carrier is straw powder, the inoculum agent medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, constant volume 1000ml, pH natural.

Bacterial agent 5: the carrier is grain bran powder, the inoculum medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, constant volume 1000ml, pH natural.

Bacterial agent 6: the carrier is grain bran powder, the inoculum agent medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, sodium caseinate 20-30g, Constant volume 1000ml, pH natural.

Inoculum 7: The carrier is grain bran powder, and the inoculum medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, sodium carboxymethyl cellulose 0.1-0.2g, dextrin 5-15g, casein Sodium 20～30g, constant volume 1000ml, pH natural.

CK: clear water control.

The results are shown in FIG. 17 , A1-A7 correspond to the bacterial agents with the same numbers, respectively.

A1: The incidence rate is 66.67%, the disease index is 52.08, and the relative control effect is 12.79%;

A2: The incidence rate is 58.33%, the disease index is 49.31, and the relative control effect is 17.44%;

A3: The incidence rate is 52.78%, the disease index is 42.36, and the relative control effect is 29.07%;

A4: The incidence rate is 66.67%, the disease index is 51.04, and the relative control effect is 14.53%;

A5: The incidence rate is 58.33%, the disease index is 50.00, and the relative control effect is 16.28%;

A6: The incidence rate is 44.44%, the disease index is 38.89, and the relative prevention effect is 34.88%;

A7: The incidence rate is 29.17%, the disease index is 27.08, and the relative prevention effect is 54.65%;

CK: incidence 69.44% disease index 59.72.

The number of viable bacteria and indoor pot experiment data show that the effect of using grain bran as the carrier of strain LSW-4 is better than other carriers. Pseudomonas fluorescens LSW-4 solid inoculum prepared with 1.5%, 2-3% (w/v) sodium caseinate inoculum medium has the highest strain activity, which can meet the long-term normal temperature storage conditions, which is beneficial to the strain It has the best relative control effect on tobacco bacterial wilt, and is suitable for industrial production. The isolated strain can be fully applied to the field to prevent and control tobacco bacterial wilt, and its practical role and effect can be fully exerted.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should Various changes may be made in details without departing from the scope of the invention as defined by the claims.

Claims (10)

1. A formulation carrier suitable for Pseudomonas fluorescens, characterized in that the carrier is grain bran, straw powder or diatomaceous earth. 2. preparation carrier according to claim 1, is characterized in that, described Pseudomonas fluorescens is Pseudomonas fluorescens (Pseudomonas fluorescens) LSW-4, is preserved in China Microorganism Culture Collection General Microorganism Center , Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: April 11, 2022, preservation number: CGMCC24668. 3. A microbial inoculant for preventing and treating tobacco bacterial wilt disease, characterized in that it comprises Pseudomonas fluorescens LSW-4 and a carrier, and the preservation number of Pseudomonas fluorescens LSW-4 is: CGMCC 24668. 4. The microbial inoculum for preventing and treating tobacco bacterial wilt according to claim 3, further comprising inoculum medium: tryptone 1%, yeast extract 0.5%, sodium chloride 1%, carboxymethyl cellulose Sodium 0.01%-0.02%, dextrin 0.5%-1.5%, sodium caseinate 2%-3%. 5 . The microbial inoculant for preventing and controlling tobacco bacterial wilt according to claim 3 , wherein the ratio of Pseudomonas fluorescens LSW-4 bacterial liquid to the carrier is 0.5-3 ml:1 g. 6 . 6. the preparation method of the microbial inoculum of any one of claim 3-5 preventing and controlling tobacco bacterial wilt disease, is characterized in that, concrete steps comprise: (1) Inoculate Pseudomonas fluorescens LSW-4 into LB liquid medium, place it on a constant temperature shaker at 28-30°C for 24h-48h, centrifuge the cultured bacterial fermentation broth, and use bacteria for the centrifuged cells Suspend the agent medium, gently shake to mix; (2), add the suspended bacterial liquid to the carrier, cultivate at 28-30°C constant temperature shaker for 30-40min, and finally dry to constant weight. 7. The preparation method of microbial inoculum according to claim 6, wherein the carrier is grain bran, straw powder or diatomaceous earth; the inoculum medium is 1% tryptone and 0.5% yeast extract , 1% of sodium chloride, 0.01% to 0.02% of sodium carboxymethyl cellulose, 0.5% to 1.5% of dextrin, and 2% to 3% of sodium caseinate. 8 . The method for preparing a microbial inoculum according to claim 6 , wherein the ratio of the bacterial liquid to the carrier in step (2) is 0.5-3 ml:1 g. 9 . 9 . The method for preparing a microbial inoculum according to claim 6 , wherein the concentration of Pseudomonas fluorescens LSW-4 in the bacterial solution is 1×10 ¹⁰ cfu/ml to 1×10 ¹² cfu/ml. 10 . 10. The application of any one of claims 3-5 microbial inoculants in the control of tobacco bacterial wilt.

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