CN114875016A

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

Info

Publication number: CN114875016A
Application number: CN202210491642.0A
Authority: CN
Inventors: 丁伟; 王珍珍; 厉阗; 冉渝澳; 吴晓敏
Original assignee: Chongqing Xinong Plant Protection Technology Development Co ltd; Southwest University
Current assignee: Chongqing Xinong Plant Protection Technology Development Co ltd; Southwest University
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2022-08-09
Anticipated expiration: 2042-04-29
Also published as: CN114875016B

Abstract

On the basis of separating and obtaining pseudomonas fluorescens LSW-4 with resistance to ralstonia solanacearum, the invention further researches and develops a preparation carrier suitable for the pseudomonas fluorescens, wherein the carrier is bran coat, straw powder or diatomite. The solid microbial agent with high viable count and long storage time can be prepared from the pseudomonas fluorescens LSW-4 by using the microbial agent culture medium compounded by the carrier, so that the problems of transportation and storage of the pseudomonas fluorescens LSW-4 are solved.

Description

Preparation carrier suitable for pseudomonas fluorescens and microbial inoculum thereof

Technical Field

The invention belongs to the technical field of agricultural microbial control, and relates to a preparation carrier suitable for pseudomonas fluorescens and a microbial inoculum thereof.

Background

Tobacco bacterial wilt is a devastating soil-borne disease caused by Ralstonia solanacearum. In severe cases, higher economic losses are caused. Aiming at the control technology of tobacco bacterial wilt, scholars at home and abroad research the control in aspects of chemical, biological and agricultural measures and the like, and although the occurrence of the tobacco bacterial wilt can be reduced to a certain extent, the control technology still has certain limitations. In recent years, the biocontrol bacteria is used for preventing and treating the tobacco bacterial wilt, is an effective prevention and treatment measure, has the advantages of environmental friendliness, long acting and the like, and has wide application prospect. The biocontrol bacteria which are separated from soil at present and can be used for preventing and controlling tobacco bacterial wilt mainly comprise bacillus subtilis, arbuscular mycorrhizal fungi, streptomyces, pseudomonas and the like. These isolated strains can be further developed into microbial pesticides.

However, the pseudomonas fluorescens is an obligate aerobic life, can grow in the environment with the pH value of 5-8 and 7% NaCl, has the optimal growth temperature of about 25-30 ℃, can grow under the low temperature condition of 4 ℃ or below 4 ℃, can not resist high temperature, can not grow at the temperature of more than 41 ℃, can not generate dormancy bodies such as spores and the like, and has certain limitation in application. For further application to microbial pesticides, the research and development of microbial agents and the selection of carriers of the microbial agents are particularly important, and carriers and other auxiliary components in the microbial agents are key factors influencing the number of live bacteria in the microbial agents. The preparation process of the liquid bacterium preparation is simple, the bacterium is in a suspended and homogeneous state under the liquid condition, the diffusion of the bacterium and the product is facilitated, and the control and the detection of the zymocyte liquid are convenient. The solid microbial preparation has the advantages of low packaging cost, convenient transportation, high bacteria content, preservation at normal temperature, difficult inactivation, various strains, better combination effect with the complex enzyme preparation, more convenient use, high-temperature granulation, convenient large-scale application of an automatic feeding system and the like; the disadvantage is that the activity is low, and the use needs a certain time to activate to produce the effect.

Disclosure of Invention

In view of the above, the present invention aims to provide a preparation carrier suitable for pseudomonas fluorescens, and a microbial inoculum culture medium prepared by compounding the carrier. The invention also aims to provide a pseudomonas fluorescens LSW-4 microbial inoculum.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a preparation carrier suitable for pseudomonas fluorescens is prepared from bran coats, straw powder or diatomite.

Further, a carrier bran suitable for formulation of Pseudomonas fluorescens is preferable.

Further, in the formulated vector suitable for Pseudomonas fluorescens, the Pseudomonas fluorescens is Pseudomonas fluorescens (LSW-4) which is preserved in the general microbiological center of the china committee for culture collection of microorganisms at the address: west road No. 1 hospital No. 3, north jing, chaoyang district, preservation date: 11/4/2022, accession number: CGMCC 24668.

2. The invention also provides a microbial agent for preventing and treating tobacco bacterial wilt, which comprises pseudomonas fluorescens LSW-4 and a carrier, wherein the preservation number of the pseudomonas fluorescens LSW-4 is as follows: CGMCC 24668.

Further, the microbial agent for preventing and treating the tobacco bacterial wilt further comprises a microbial agent culture medium: 1% of tryptone, 0.5% of yeast extract, 1% of sodium chloride, 0.01% -0.02% of sodium carboxymethyl cellulose, 0.5% -1.5% of dextrin and 2% -3% of sodium caseinate.

Further, in the microbial agent for preventing and treating tobacco bacterial wilt, the ratio of pseudomonas fluorescens LSW-4 bacterial liquid to a carrier is 0.5-3 ml: 1g of the total weight of the composition.

Preferably, in the microbial agent for preventing and treating tobacco bacterial wilt, the ratio of pseudomonas fluorescens LSW-4 bacterial liquid to the carrier is 0.5-1.5 ml: 1g of the total weight of the composition.

3. The invention also provides a preparation method of the microbial agent for preventing and treating tobacco bacterial wilt, which comprises the following specific steps:

(1) inoculating pseudomonas fluorescens LSW-4 into an LB liquid culture medium, placing the liquid culture medium into a constant temperature shaking table at the temperature of 28-30 ℃ for culturing for 24-48 h, centrifuging the cultured bacterial fermentation liquor, suspending the centrifuged thallus by using a microbial inoculum culture medium, and slightly oscillating and uniformly mixing the suspended thallus;

(2) adding the suspended bacterial liquid into a carrier, carrying out shake cultivation for 30-40min at a constant temperature of 28-30 ℃, and finally drying to constant weight.

Further, in the preparation method of the microbial agent, the carrier is rice bran, straw powder or diatomite; the microbial inoculum culture medium is 1 percent of tryptone, 0.5 percent of yeast extract, 1 percent of sodium chloride, 0.01 to 0.02 percent of sodium carboxymethylcellulose, 0.5 to 1.5 percent of dextrin and 2 to 3 percent of sodium caseinate.

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

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

Further, in the preparation method of the microbial agent, the concentration of the pseudomonas fluorescens LSW-4 in the bacterial liquid is 1 x 10 ¹⁰ cfu/ml～1×10 ¹² cfu/ml。

4. The application of any one of the microbial agents in the prevention and treatment of tobacco bacterial wilt is also within the protection scope of the invention.

The invention has the beneficial effects that: the pseudomonas fluorescens LSW-4 used in the invention has better inhibition and antagonism on the ralstonia solanacearum, the inhibition zone on the ralstonia solanacearum under different concentrations can reach 12.30mm-33.76mm, pot preliminary experiments show that the pseudomonas fluorescens LSW-4 has good direct disease control effect on the tobacco bacterial wilt, in further field experiments, the pseudomonas fluorescens LSW-4 also shows excellent resistance to the tobacco bacterial wilt, the disease incidence is only 10.00%, the disease index is 5.78, the control effect is 79.97%, and the pseudomonas fluorescens LSW-4 is fully proved to be capable of effectively controlling the tobacco bacterial wilt in a field and has a certain growth promotion effect. The carrier can be fully applied to the actual tobacco planting production, in order to further solve the problem of transportation and storage of the pseudomonas fluorescens LSW-4, 5 carriers suitable for growth of the pseudomonas fluorescens LSW-4, such as talcum powder, diatomite, oyster shell powder, rice bran, straw powder and the like, are screened from different carriers, and the carrier which is most suitable for the load of the pseudomonas fluorescens is selected from the carriers. The solid microbial inoculum prepared by compounding the carrier can be used for preparing the pseudomonas fluorescens LSW-4 into the solid microbial inoculum which is suitable for long-time storage, and the relative prevention effect can reach 54.65 percent through the viable count test and the potted plant experiment test of the microbial inoculum. The separated pseudomonas fluorescens LSW-4 with the biocontrol effect can be further applied to the field control of tobacco bacterial wilt.

Biological material preservation

The Pseudomonas fluorescens (Pseudomonas fluorescens) LSW-4 is preserved in China general microbiological culture Collection center, and the address is as follows: west road No. 1 hospital No. 3, north jing, chaoyang district, preservation date: 11/4/2022, accession number: CGMCC 24668.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a partial plan view of the bacteriostatic effect of the rescreened antagonistic bacteria on Ralstonia solanacearum.

FIG. 2 shows the results of the screening part of the pot pre-test.

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

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

FIG. 5 is a morphological diagram of LSW-4 single colonies.

FIG. 6 is the LSW-4 transmission electron microscope bacterial morphology observation picture.

FIG. 7 is the creation of LSW-4 phylogenetic trees based on 16S rDNA.

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

FIGS. 9-13 are graphs showing the number of viable bacteria sampled at different time points for microbial agents prepared by different carriers; wherein the carrier in figure 9 is diatomite, the carrier in figure 10 is straw powder, the carrier in figure 11 is rice bran, the carrier in figure 12 is talcum powder, and the carrier in figure 13 is oyster shell powder.

FIGS. 14 to 16 are graphs sequentially showing viable cell count of samples of the microbial inoculum obtained from the medium of each microbial inoculum in example 7 at different time points.

FIG. 17 shows the results of potting experiments with different Pseudomonas fluorescens LSW-4 solid inoculum.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

Example 1

1. Collecting a soil sample: soil samples were collected at 29/8/2019, and the soil sample collection information is shown in table 1. Pulling out the soil on the surface of the healthy tobacco plants, collecting soil layers 5-25cm below the ground surface, digging out tobacco roots, uniformly mixing the soil on the tobacco roots with rhizosphere soil of 3 healthy tobacco plants in the collection bag, repeating the steps for 3 times, and sealing and marking.

TABLE 1 soil sample Collection information sheet

2. Test pathogen activation: pathogenic bacteria Ralstonia solanacearum CQPS-1 separated from tobacco strains with bacterial wilt of Pengpeng hydropyronie Chongqing is used for researching the identification and the characteristics of the pathogenic bacteria. Activating tobacco ralstonia solanacearum CQPS-1, performing propagation in liquid culture medium B under conditions of constant temperature of 30 deg.C and 180rpm/min for 12 hr, and detecting OD value of 0.8-1.0 with spectrophotometer to obtain tobacco ralstonia solanacearum concentration of 1 × 10 ⁹ cfu/mL. The culture medium used in this example and its specific formulation were as follows:

NA medium: 5.0g of bacterial peptone, 1.0g of yeast powder, 10.0g of glucose, 3.0g of beef powder, 20.0g of agar powder and 1000mL of distilled water, wherein the pH value is 6.8-7.2; sterilizing at 121 deg.C for 20 min.

LB culture medium: 5.0g of yeast extract, 10.0g of sodium chloride, 10.0g of tryptone and 1000mL of distilled water; sterilizing at 121 deg.C for 20 min.

B, liquid culture medium: 1.0g of casamino acid, 5.0g of glucose, 10.0g of bactopeptone, 1.0g of yeast extract and 1000mL of distilled water; sterilizing at 121 deg.C for 20 min.

3. Isolation and purification of the strains

In the test, a dilution plate coating method is adopted to separate and purify bacteria in a soil sample, and the specific operation is as follows: weighing 10g of soil sample, adding 90mL of sterile water, slightly shaking, fully and uniformly mixing, then shaking for 30min on a constant temperature shaking table at 220r/min, and standing for 10 min; taking supernatant of the soil bacterium suspension after standing, adopting a gradient dilution method to prepare 10 ^-2 、10 ^-3 、10 ^-4 And 10 ^-5 The diluent of (4); taking 100 mu L of diluent of each concentration gradient, adding the diluent to a NA flat plate with coated beads, and uniformly oscillating back and forth to uniformly coat the bacterial suspension, wherein each concentration gradient is repeated for 3 times; inversely culturing the coated NA plate in a constant-temperature incubator at 30 ℃; 2d, taking out the NA plate, observing the growth condition of the bacterial colony in an ultra-clean workbench, selecting different single bacterial colonies in an LB liquid culture medium according to the shape and color of the bacterial colony as a judgment basis, culturing at 30 ℃ at 170r/min for 12h, and then culturing at 30 ℃ by adopting a plate streaking methodAnd culturing for 48h, repeating 3 times on each scribing plate, purifying the separated bacteria, repeating the step of picking a single colony on the NA plate three times back and forth until all colonies on the NA plate are consistent, and finishing the purification.

After bacteria in the collected soil sample are separated and purified, visually judging the difference of the shapes and colors of the bacteria, separating 184 strains of bacteria together, and further adopting a plate confrontation method to evaluate the antagonistic action of the 184 strains of bacteria on the ralstonia solanacearum to carry out primary screening work, wherein the specific operation method comprises the following steps: taking bacterial suspension (10) of activated tobacco ralstonia solanacearum ⁶ cfu/mL) of 100 mu L, adding the mixture into an NA flat plate of sterilized coating beads, and uniformly coating; placing a 6mm filter paper sheet in the center of an NA plate coated with tobacco ralstonia solanacearum; 5 μ L of each purified test bacterium was attached to a filter paper sheet, and each treatment was repeated 3 times. And (5) placing the mixture in a constant temperature incubator at 30 ℃ for inverted culture for 48 hours, and observing whether a bacteriostatic zone is formed.

49 bacteria with antagonistic action on tobacco ralstonia solanacearum are separated out through primary screening, and an obvious inhibition zone is formed, but the inhibition action is different, the diameters of the inhibition zones are different, and the results are shown in table 2.

TABLE 2 diameter table for primary screening of bacteriostatic circle

Further re-screening the preliminarily screened antagonistic bacteria, wherein the specific operation method comprises the following steps: collecting bacterial suspension (10) of previously activated tobacco ralstonia solanacearum ⁶ cfu/mL) of 100 mu L, adding the mixture into an NA flat plate of sterilized coating beads, and uniformly coating; placing a 6mm filter paper sheet in the center of an NA plate coated with tobacco ralstonia solanacearum; transferring the bacteria with antagonistic effect on tobacco ralstonia solanacearum in the primary screening result to LB liquid culture medium to 10 ⁹ cfu/mL (OD is 0.8-1), the concentration of the bacteria to be tested is ensured to be consistent, 5 mu L of the bacteria to be tested is connected onto a filter paper sheet, and each treatment is repeated for 3 times. And (3) placing the mixture in a constant-temperature incubator at 30 ℃ for inverted culture for 48h, and measuring the size of each inhibition zone by adopting a cross method except observing whether the inhibition zones are formed. Re-screening 24 strains of ralstonia solanacearum from the 49 preliminarily screened antagonistic bacteriaThe figure 1 is a partial flat-sheet diagram of the bacteriostatic effect of the rescreened antagonistic bacteria on ralstonia solanacearum, and the table 3 is a diameter table of the rescreened bacteriostatic circle.

TABLE 3 diameter meter for double-sifting bacteriostatic circle

Example 2

In order to effectively prevent and control the tobacco bacterial wilt, the screening of the tobacco bacterial wilt antagonistic bacteria is not only limited to the evaluation of the direct inhibition effect of the antagonistic bacteria on the tobacco bacterial wilt, but also needs to confirm the direct disease control effect of the antagonistic bacteria on the tobacco bacterial wilt through pot preliminary tests.

Selecting healthy four-leaf one-heart tobacco seedlings with consistent sizes for pot experiment, activating the rescreened antagonistic bacteria, transferring the activated antagonistic bacteria to an LB liquid culture medium for culture, culturing at 30 ℃ at 170r/min for 12h, detecting until OD is 0.8-1 by using a spectro-gradiometer, inoculating 10mL of bacterial liquid to each tobacco seedling by adopting a root irrigation mode, treating 10 tobacco seedlings each, and inoculating 10mL of sterile water to a blank control. The cultivation conditions of the greenhouse are as follows: the light is irradiated for 12 hours, the temperature is 28 ℃, and the humidity is 85 percent. And (3) periodically irrigating with sterile water during the growth period of the tobacco seedlings to ensure the normal growth of the tobacco seedlings, and when the tobacco bacterial wilt starts to occur, investigating the incidence rate of the tobacco bacterial wilt according to the national bacterial wilt standard GB/T2322-2008 and an indoor grading standard, wherein a part of screening results of a potting preliminary test are shown in a figure 2, and a table 4 shows the effect of antagonistic bacteria on the tobacco bacterial wilt in the potting preliminary test screening.

TABLE 4

Example 3

In order to determine the influence of LSW-4 concentration and inoculation time on tobacco bacterial wilt, 87 tobacco seedlings of Yunyan tobacco with four leaves and one core are selected to be tested in a laboratory by adopting a common seedling tray seedling raising mode, and the concentration is 1 multiplied by 10 respectively ⁸ cfu/mL、1×10 ⁷ cfu/mL、1×10 ⁶ cfu/mL antagonismAnd (5) irrigating roots according to the bacterial concentration.

Inoculating antagonistic bacteria LSW-4 into each tobacco strain at different treatment concentrations of 10mL, irrigating roots and inoculating ralstonia solanacearum CQPS-1(1 × 10) ⁷ cfu/mL 10 mL). And (3) repeating the steps for each 10 tobacco seedlings, wherein the greenhouse culture conditions comprise that the illumination is 12 hours, the temperature is 28 ℃, the humidity is 85 percent, the investigation is carried out from the occurrence of the tobacco bacterial wilt according to the national standard GB/T2322-2008 and the indoor classification standard according to the occurrence degree of the bacterial wilt, the observation is carried out once every 24 hours, and the occurrence condition of the bacterial wilt is recorded. The antagonistic bacterium LSW-4 is found to have a concentration effect on the prevention and control of the tobacco bacterial wilt, namely the higher the concentration of the antagonistic bacterium LSW-4 is, the better the prevention and control effect on the tobacco bacterial wilt is. When the incidence rate of blank control is 66.66%, 1X 10 ⁸ cfu/mL、1×10 ⁷ cfu/mL、1×10 ⁶ The morbidity of cfu/mL is 11.11%, 25.00% and 44.45% respectively, the disease index is 11.11, 23.61 and 44.45 respectively, and the relative prevention effect of different concentrations is 83.33%, 64.58% and 33.32%.

Further, by the pot pre-test of example 2, several antagonistic bacteria LSW-4, LSW-32, PSG-26 which did not develop disease were selected in the field test conducted in 2019 in the city of junqing jungle town cang dam village (the city of jungle county cang town cang dam village with altitude 1143m, longitude and latitude of east longitude E108 ° 34 '22.99 ″, north latitude N29 ° 0' 42.83 ″, average number of tobacco seedlings transplanted per mu was about 1200), the variety to be tested was yunyan 87, and the test material: (1) self-extracting antagonistic bacteria (LSW-4, PSG-26, LSW-32); (2) trichoderma harzianum; (3) b, bacillus subtilis; (4) strengthening seedlings; (5) paenibacillus polymyxa. (2) - (5) the microbial inoculum is applied by root irrigation for market purchase. Treatment 1: paenibacillus polymyxa; and (3) treatment 2: trichoderma harzianum; and (3) treatment: strengthening the seedling with the compound bacterial agent; and (4) treatment: a bacillus subtilis agent; and (4) treatment 5: LSW-4; and (6) treatment: PSG-26; and (7) treatment: LSW-32; process 8 (CK): the concentration of each microbial inoculum is 1 multiplied by 10 by clear water contrast ⁸ cfu/mL. Activating the rescreened antagonistic bacteria, transferring to an LB liquid culture medium for culture, placing at 30 ℃, culturing at 170r/min for 12h, detecting by a spectro-gradiometer until OD is 0.8-1, and adopting a root irrigation mode.

The cultivation condition is as follows: floating seedling culture is adopted for tobacco seedling culture in the early stage, tobacco plant growth management is unified field management according to related technical standards, transplanting time is 4 months and 30 days, axillary buds are controlled by 12.5% flumetralin EC when a central flower is bloomed, topping is carried out for 7 months and 7 days, and harvesting is carried out for 18 days in 7 months. The effect of rhizosphere regulation of different microbial agents on the agronomic traits of tobacco plants is shown in table 5. As can be seen from Table 5, the antagonistic bacteria LSW-4, LSW-32 and PSG-26 isolated by the present invention all have good influence on the agronomic traits of tobacco plants in the mass stage (according to YC/T142-1998 tobacco agronomic traits research method).

TABLE 5

The plots selected in the experiment are perennial bacterial wilt high-incidence plots, the results of the effects of rhizosphere regulation and control of different microbial agents on the tobacco bacterial wilt resistance of the tobacco plants are shown in fig. 3 (incidence) and fig. 4 (disease index), the incidence of rhizosphere regulation and control treatment of different microbial agents is lower than that of control treatment, and the significant difference exists in comparison with clear water, which indicates that the resistance of the tobacco plants to the tobacco bacterial wilt can be influenced to a certain extent by the mode of rhizosphere regulation and control. The results show that: the disease control effect is best to antagonize the bacteria LSW-4, the later-period disease incidence rate is only 10.00 percent, the subsequent disease incidence rate is 15.00 percent, the disease incidence rates of the paenibacillus polymyxa, the seedling strengthening, the PSG-26 and the LSW-32 are respectively 43.33 percent, 38.33 percent and 41.67 percent, and the disease incidence rate of the blank control is 61.67 percent. The disease index is consistent with the disease incidence trend, the LSW-4 index is 5.78, the disease index of the bacillus subtilis is 7.22, the disease indexes of the paenibacillus polymyxa, the seedling is strong, the PSG-26 index and the LSW-32 index are 16.13, 18.44, 20.46 and 17.96 respectively, the disease index of a blank control is 28.85, and the control effect of the LSW-4 is 79.97%, so that the LSW-4 can effectively control bacterial wilt of tobacco plants in a field and has a certain growth promoting effect, tobacco plants irrigated by the LSW-4 enter a bulk stage 2d ahead of the control, and the plant height, the effective leaf number, the leaf length, the leaf width and the leaf area of the tobacco plants are all superior to the control.

Example 4

Identification of antagonistic bacteria LSW-4, wherein FIG. 5 is a LSW-4 single colony morphology, and FIG. 6 is a LSW-4 transmission electron microscope bacteria morphology observation picture. After LSW-4 is cultured in TSA culture medium for 48h, the morphology of a single colony of the LSW-4 is observed and gram-stained, and the LSW-4 is observed to be gram-negative bacteria, the morphology of the single colony is circular, the color is milky white, the surface is smooth, the edge is neat, and the middle is slightly raised. The results of transmission electron microscopy showed that the cells were rod-shaped with terminal flagella.

Bacterial DNA (DNA extraction kit of Beijing Soxholey science and technology Co., Ltd.) was extracted, and PCR amplification was performed on the 27F \1492R region of the bacterial DNA according to the amplification primers (27F and 1492R). 27F: AGAGTTTGATCCTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT. The PCR amplification product is sent to Huada Gene company for sequencing, BLAST comparison is carried out on the sequencing result in GenBank, a 16S rDNA sequence with higher homology is selected as a reference object, a Neighbour-join method is adopted to construct a phylogenetic tree, and FIG. 7 shows that an LSW-4 phylogenetic tree is established based on the 16S rDNA. The strain LSW-4 is identified as Pseudomonas fluorescens (Pseudomonas fluorescens). The growth condition of antagonistic bacteria LSW-4 is determined by measuring the OD value of the antagonistic bacteria LSW-4 by a spectrophotometer, the growth rule is known, the OD value is observed to have a trend of obviously decreasing after the growth rule is measured for 72 hours, and the LSW-4 reaches the logarithmic growth phase between 10 hours and 12 hours. FIG. 8 is a growth curve of Pseudomonas fluorescens LSW-4.

The strain LSW-4 is preserved in China general microbiological culture Collection center, addresses: west road No. 1 hospital No. 3, north jing, chaoyang district, preservation date: 11/4/2022, accession number: CGMCC 24668.

Example 5

The soil after the application of the antagonistic bacterium LSW-4 was collected in two times, with a collection period of 6 months and 23 days (vigorous growth period), 7 months and 18 days (late topping period) and compared before and after the two data, 3 replicates, and the changes in the soil microbial community after the application of the antagonistic bacterium LSW-4 were analyzed.

The following conclusions were obtained by high throughput sequencing from DNA samples of soil treated with LSW-4 at different times and analysis with the aid of the magenton platform:

(1) by analyzing the horizontal community composition of bacteria and fungi in the soil samples collected at 23 th and 18 th days, and comparing the soil samples collected at 23 th and 18 th days, after the soil samples collected at 23 th and 18 th days are treated by antagonistic bacteria LSW-4, the ratio of closteron (Chloroflexi) in the bacterial community shows an increasing trend, the ratio of Ascomycota (Ascomycota) in the fungal community is reduced by 11.41%, the ratio of Mortierella (Mortierella) is increased by 3.54%, and the ratio of Basidiomycota (Basidiomycota) is increased by 3.76%.

(2) Statistical analysis of beta diversity found that at the OTU level, in soil at 23 days 6 months, whether bacterial or fungal, the difference from the blank after LSW-4 treatment was not very significant. In the soil of 7 months and 18 days, after the LSW-4 treatment, the influence on the bacterial flora is large, and a remarkable difference appears.

(3) Through LEfSe discriminant analysis of the soil sample treated by LSW-4, the soil sample collected at 23 days after 6 months shows that the soil sample treated by LSW-4 is significantly enriched in the genus of Roehelia (Rhodanobacter) on the level of the genus of bacteria; the fungi which are significantly enriched after being treated by LSW-4 are Pseudoaerobium beckei (Pseudoeurotium), Setophoma, Rickenella and Cotylidia. The soil sample of day 18 in 7 months is obviously enriched with obscuribacteria after being treated by LSW-4 in bacterial flora, and is obviously enriched with Codinaea and Scytalidium after being treated by LSW-4 in the level of fungi.

The LSW-4 serving as a biocontrol strain can directly inhibit the proliferation of pathogenic bacteria and induce plants to generate disease-resistant characteristics, and can play a role in regulating and controlling the soil microecological structure in soil. The soil is the environment for the direct growth of plants and is also an important medium for the interaction of pathogenic plants. The health of the soil directly affects the health of the plants. The growing severity of soil-borne diseases is often caused by the disruption of soil microecological balance due to continuous cropping throughout the year. Soil microorganisms are considered to be an important component of the soil ecosystem and also 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. After antagonistic bacterium LSW-4 Pseudomonas fluorescens (Pseudomonas fluorescens) is applied, the microbial community structure of the tobacco rhizosphere soil is affected, and the Pseudomonas fluorescens LSW-4 can regulate and control the abundance of certain specific microbial populations in the microbial community composition and reduce the occurrence of soil-borne disease bacterial wilt. The soil samples collected at 23 days 6 months showed significant enrichment of the bacteria in the genus Roehenia (Rhodanobacter) after LSW-4 treatment at the level of the bacterial flora; the fungi which are significantly enriched after being treated by LSW-4 are Pseudoaerobium beckei (Pseudoeurotium), Setophoma, Rickenella and Cotylidia. The soil sample of day 18 in 7 months is obviously enriched with obscuribacteria after being treated by LSW-4 in bacterial flora, and is obviously enriched with Codinaea and Scytalidium after being treated by LSW-4 in the level of fungi.

Example 6

In order to further apply the strain LSW-4 to commercial products, a preparation carrier and a solid microbial agent suitable for the pseudomonas fluorescens are continuously investigated on the basis of the previous research so as to solve the problems of transportation and storage of the pseudomonas fluorescens and be conveniently applied to the field for preventing and controlling the tobacco bacterial wilt.

1. Preparation of liquid Medium

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

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

2. Carrier

Carrier material: oyster shell powder (500 g/bag of Mezhou city first-time blossoming electronic commerce Co., Ltd.), diatomite (Dengfeng fine chemical AR500 g/bag), talcum powder (Fucheng fine chemical LR500 g/bottle), corn straw powder (deep processing 250 g/bag of Lifeng agricultural products with 20 meshes) and rice bran powder (deep processing 250 g/bag of Lifeng agricultural products with 20 meshes).

Sterilizing oyster shell powder, diatomite, talcum powder, straw powder and rice bran powder by high-pressure steam at 121 ℃ for 30 minutes, and then placing the sterilized materials in a constant-temperature oven at 60 ℃ to dry the sterilized materials to a constant state. Table 6 shows the different carrier pH values.

TABLE 6 different carrier pH values

3. Bacterial culture

Inoculating pseudomonas fluorescens LSW-4 into LB liquid culture medium according to the inoculation amount of 1-1.5%, placing the liquid culture medium in a constant temperature shaking table at 30 ℃ for 180r/min, and culturing for 24-48 h.

4. Centrifugation

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

5. Suspended in water

Adding the centrifuged bacteria into LB liquid culture medium which is about one tenth of the volume of the original bacteria liquid, suspending, gently shaking and uniformly mixing. The concentration of the mixed bacterial liquid is about 1 multiplied by 10 ¹² cfu/ml。

6. Mixing

Adding the suspended bacteria liquid into carriers such as oyster shell powder, diatomite, talcum powder, straw powder, rice bran and the like which are subjected to sterile treatment according to the volume mass ratio (ml: g), uniformly mixing for 30-40min under the condition of a constant temperature shaking table at 30 ℃ for 180r/min, finally ventilating and drying in an aseptic workbench to constant weight, then respectively filling into sterilized glass bottles, and uniformly stirring by using an aseptic glass rod.

7. Preservation of

Sealing with sealing film, storing at room temperature, sampling at 10d, 30d and 90d, respectively, measuring viable count, and screening to obtain carrier with maximum viable count. Samples were taken for gradient dilution plating and counted for viable count (LB solid medium, 3 samples taken each time, weighing 1g, 3 concentrations per sample, 3 plates per concentration).

The ratio of the bacteria liquid to the carrier is respectively tested according to 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1 and 3:1 (ml: g), when the ratio of the bacteria liquid to the carrier is more than 1.5:1, the number of live bacteria of the solid bacteria prepared by the same carrier is not greatly changed on the 10 th day, and when the ratio of the solid bacteria prepared by the diatomite is more than 2.5:1, the number of live bacteria is not greatly changed on the 10 th day. Experiments show that the carriers suitable for preparing the pseudomonas fluorescens LSW-4 are rice bran, straw powder and diatomite, and the rice bran is preferred. And the bran coat is convenient and cheap as a raw material source, and has a large quantity, so the bran coat is selected as an optimal carrier for further testing and research.

Fig. 9-13 show viable count at different time points when ratio of bacteria liquid to carrier is 1:1, wherein carrier in fig. 9 is diatomite, carrier in fig. 10 is straw powder, carrier in fig. 11 is rice bran, carrier in fig. 12 is talcum powder, and carrier in fig. 13 is oyster shell powder. Table 7 shows the viable count of bacteria sampled at different time points when the ratio of bacteria liquid to carrier is 1: 1.

TABLE 7 number of viable bacteria (cfu/g) measured for each carrier at different time points

Carrier	10d	30d	90d
				Bran coat	2.1×10 ¹¹	5.7×10 ¹⁰	1.3×10 ⁹
Straw powder	1.3×10 ⁹	9.0×10 ⁷	1.2×10 ⁸
				Talcum powder	2.5×10 ¹⁰	4.7×10 ⁷	\
Diatomite	7.7×10 ¹¹	5.2×10 ⁷	9.6×10 ⁶
				Oyster shell powder	5.1×10 ¹⁰	2.1×10 ⁸	\

Example 7

Inoculating pseudomonas fluorescens LSW-4 into an LB liquid culture medium according to the inoculation amount of 1-1.5%, placing the liquid culture medium in a constant temperature shaking table at 30 ℃ for 180r/min, culturing for 24-48 h, and centrifuging the cultured bacterial fermentation liquid for 8000r and 5min by using a sterilized centrifugal tube; and suspending the centrifuged bacteria in a bacterial agent culture medium with the volume of the original bacterial liquid of 1/8-1/12, and lightly shaking and uniformly mixing.

Microbial inoculum culture medium 1: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethylcellulose, 5-15g of dextrin and 20-30 g of sodium caseinate, wherein the volume is fixed to 1000ml, and the pH is natural.

And (3) microbial inoculum culture medium 2: the carrier is bran coat, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethylcellulose, 20-30 g of sodium caseinate, 1000ml of constant volume and natural pH.

And (3) microbial inoculum culture medium: the carrier is bran coat, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethyl cellulose, 1000ml of constant volume and natural pH.

Taking bran as a carrier, adding bacterial liquid suspended by different microbial inoculum culture media into the carriers after aseptic treatment according to the volume mass ratio of 0.5-1.5: 1 (ml: g), mixing uniformly for 30-40min under the condition of a constant temperature shaking table at 30 ℃ for 180r/min, and finally airing in an aseptic workbench until the weight is constant. In order to accelerate the drying speed, the pseudomonas fluorescens LSW-4 solid microbial inoculum can also be obtained by uniformly mixing, then carrying out sterile filtration and then carrying out ventilation drying under the sterile condition to constant weight. It can also be freeze-dried or spray-dried.

The microbial inoculum culture medium is tested by combining various components and different concentrations, the effective viable count of 10 days, 30 days and 90 days is inspected by taking bran as a carrier under the condition that the ratio of a bacterial liquid to the carrier is 1:1, the optimal microbial inoculum culture medium is determined, the viable count statistics is obtained by sampling the microbial inoculum obtained by each microbial inoculum culture medium at different time points in table 8, and fig. 14-16 are culture graphs of the sampled viable count of the microbial inoculum obtained by each microbial inoculum culture medium at different time points in sequence. Finally, taking the bran as a carrier, and adding a microbial inoculum culture medium of 0.01-0.02% of sodium carboxymethylcellulose, 0.5-1.5% of dextrin and 2-3% (w/v) of sodium caseinate into an LB liquid culture medium screened under the condition that the ratio of bacteria liquid to carrier is 1:1, so that the loading rate and the number of effective viable bacteria of pseudomonas fluorescens LSW-4 on the bran carrier can be improved, and the rest data are more, and only the contents and data related to the invention are shown.

TABLE 8 viable count statistics (cfu/g)

Carrier and microbial inoculum culture medium	10d	30d	90d
				Bran coat and microbial inoculum 1	4.8×10 ¹¹	1.9×10 ¹¹	5.7×10 ¹⁰
Bran coat and microbial inoculum 2	9.7×10 ¹⁰	1.3×10 ¹⁰	7.3×10 ⁹
				Bran coat and microbial inoculum 3	7.5×10 ⁹	3.6×10 ⁸	8.3×10 ⁷

Example 8

Screening a carrier and a microbial inoculum culture medium, and then carrying out an indoor pot experiment on the pseudomonas fluorescens LSW-4 solid microbial inoculum prepared by different methods for further comparison and confirmation. Four-leaf and one-core Yunyan 87 tobacco seedlings are selected to be tested in a laboratory by adopting a common seedling tray seedling raising mode, three times of treatment are set, and 12 plants are repeated. During transplanting, about 60g of tobacco matrix is applied to each tobacco plant, 2g of microbial inoculum is applied to each tobacco plant, the transplanted tobacco plants are cultured under the conditions of 30 ℃, 16h of illumination for 8 hours at night and 85% of air humidity, and watering is carried out in time. 3 days after the seedling is revived, the concentration of the adopted bacteria is 1 multiplied by 10 ⁷ And (5) irrigating roots with 10ml of cfu/ml of ralstonia solanacearum, wherein the roots are irrigated with 10ml of each strain of tobacco. Disease investigation was conducted from the initial stage of disease onset, and data was recorded and analyzed.

The following preparation methods of different Pseudomonas fluorescens LSW-4 solid microbial agents were the same as example 2, and the carriers and microbial agent culture media were as follows, and the ratio of the bacterial liquid to the carriers was 1:1, and the investigation was performed under the same conditions.

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

And (2) microbial inoculum: the carrier is rice bran powder, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 1000ml of constant volume and natural pH.

And (3) microbial inoculum: the carrier is straw powder, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethylcellulose, 5-15g of dextrin and 20-30 g of sodium caseinate, wherein the volume is fixed to 1000ml, and the pH is natural.

And (4) microbial inoculum: the carrier is straw powder, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethyl cellulose, 1000ml of constant volume and natural pH.

And (5) microbial inoculum: the carrier is rice bran powder, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethyl cellulose, 1000ml of constant volume and natural pH.

And (6) microbial inoculum: the carrier is rice bran powder, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethylcellulose, 20-30 g of sodium caseinate, 1000ml of constant volume and natural pH.

And (7) microbial inoculum: the carrier is rice bran powder, and the microbial inoculum culture medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 0.1-0.2 g of sodium carboxymethylcellulose, 5-15g of dextrin and 20-30 g of sodium caseinate, wherein the volume is fixed to 1000ml, and the pH is natural.

CK: and (5) clear water control.

As shown in FIG. 17, A1-A7 correspond to the same numbers of microbial agents, respectively.

A1: the morbidity is 66.67%, the disease index is 52.08, and the relative prevention effect is 12.79%;

a2: the disease index of the disease is 49.31 with the incidence rate of 58.33 percent and the relative prevention effect is 17.44 percent;

a3: the morbidity is 52.78%, the disease index is 42.36, and the relative control effect is 29.07%;

a4: the morbidity is 66.67%, the disease index 51.04 is 14.53% relative to the control effect;

a5: the disease index of the disease is 50.00 and the relative prevention effect is 16.28 percent, wherein the disease rate is 58.33 percent;

a6: the morbidity is 44.44%, the disease index is 38.89%, and the relative prevention effect is 34.88%;

a7: the disease index of 29.17 percent of the disease rate is 27.08, and the relative prevention effect is 54.65 percent;

CK: the incidence rate is 69.44%, and the disease index is 59.72.

The number of live bacteria and indoor pot culture test data show that the effect of the carrier taking the bran as the strain LSW-4 is better than that of other carriers, the activity of the strain in the pseudomonas fluorescens LSW-4 solid microbial inoculum prepared by the microbial inoculum culture medium adding 0.01-0.02% of sodium carboxymethylcellulose, 0.5-1.5% of dextrin and 2-3% (w/v) of sodium caseinate is highest, the long-time normal-temperature preservation condition can be met, the preservation and the transportation of the strain are facilitated, the relative prevention effect on the tobacco bacterial wilt is the best, the method is suitable for industrial production, the separated strain can be fully applied to field tobacco bacterial wilt prevention and control, and the actual action and the effect are fully exerted.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A preparation carrier suitable for pseudomonas fluorescens is characterized in that the carrier is rice bran, straw powder or diatomite.

2. The formulation vehicle according to claim 1, wherein the Pseudomonas fluorescens is Pseudomonas fluorescens (LSW-4) deposited at the china general microbiological culture collection center, address: west road No. 1 hospital No. 3, north jing, chaoyang district, preservation date: 11/4/2022, accession number: CGMCC 24668.

3. The microbial agent for preventing and treating the tobacco bacterial wilt is characterized by comprising pseudomonas fluorescens LSW-4 and a carrier, wherein the preservation number of the pseudomonas fluorescens LSW-4 is as follows: CGMCC 24668.

4. The microbial agent for preventing and treating tobacco bacterial wilt according to claim 3, further comprising a microbial agent culture medium: 1% of tryptone, 0.5% of yeast extract, 1% of sodium chloride, 0.01% -0.02% of sodium carboxymethyl cellulose, 0.5% -1.5% of dextrin and 2% -3% of sodium caseinate.

5. The microbial agent for preventing and treating tobacco bacterial wilt according to claim 3, wherein the ratio of pseudomonas fluorescens LSW-4 bacterial liquid to carrier is 0.5-3 ml: 1g of the total weight of the composition.

6. The preparation method of the microbial agent for preventing and treating tobacco bacterial wilt as claimed in any one of claims 3 to 5, characterized by comprising the following specific steps:

(1) inoculating pseudomonas fluorescens LSW-4 into an LB liquid culture medium, placing the liquid culture medium into a constant temperature shaking table at 28-30 ℃ for culturing for 24-48 h, centrifuging the cultured bacterial fermentation liquor, suspending the centrifuged thallus by using a microbial inoculum culture medium, and slightly oscillating and uniformly mixing the suspended thallus;

(2) adding the suspended bacterial liquid into a carrier, carrying out shake cultivation at a constant temperature of 28-30 ℃ for 30-40min, and finally drying to constant weight.

7. The method for preparing a microbial agent according to claim 6, wherein the carrier is rice bran, straw powder or diatomaceous earth; the microbial inoculum culture medium is 1 percent of tryptone, 0.5 percent of yeast extract, 1 percent of sodium chloride, 0.01 to 0.02 percent of sodium carboxymethylcellulose, 0.5 to 1.5 percent of dextrin and 2 to 3 percent 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 the step (2) is 0.5-3 ml: 1g of the total weight of the composition.

9. The method for producing a microbial agent according to claim 6, wherein the concentration of Pseudomonas fluorescens LSW-4 in the bacterial solution is 1X 10 ¹⁰ cfu/ml～1×10 ¹² cfu/ml。

10. The use of the microbial agent of any one of claims 3-5 for controlling tobacco bacterial wilt.