CN114854627B - Pseudomonas fluorescens for preventing and treating bacterial wilt and application thereof - Google Patents
Pseudomonas fluorescens for preventing and treating bacterial wilt and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/27—Pseudomonas
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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Abstract
The invention relates to a Pseudomonas fluorescens strain, which is identified as Pseudomonas fluorescens (Pseudomonas fluorescens) LSW-4 and is preserved in China general microbiological culture Collection center, with a preservation date: 2022, 4, 11, deposit number: CGMCC 24668. The bacterial strain is gram negative bacteria, the form of single bacterial colony is circular, the color is milky white, the surface is smooth, the edge is neat, the bacterial strain has good inhibition and antagonism effects on bacterial wilt, has good direct disease control effects on tobacco bacterial wilt, and in field experiments, fluorescent pseudomonas LSW-4 also shows excellent resistance to tobacco bacterial wilt, the morbidity is only 10.00%, the disease index is 5.78, the control effect is 79.97%, and the effect of effectively controlling tobacco bacterial wilt by fluorescent pseudomonas LSW-4 in fields is fully proved.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganism control, relates to pseudomonas fluorescens for controlling bacterial wilt, and further relates to application of pseudomonas fluorescens.
Background
The environment suitable for tobacco growth is influenced by factors such as altitude, air temperature, soil conditions and the like, the tobacco growth is mostly concentrated in a certain area, continuous cropping phenomenon is common, the pH of soil of tobacco is reduced, effective nitrogen, phosphorus and potassium in the soil are obviously increased, and the quality and yield of tobacco are influenced to a certain extent. In addition, the continuous cropping phenomenon can also cause a large amount of accumulation of soil-borne pathogenic bacteria, so that a large outbreak of soil-borne diseases is caused, serious economic loss is caused, and especially tobacco bacterial wilt caused by the large accumulation of tobacco bacterial wilt is an obvious example.
The most typical symptom of tobacco bacterial wilt (Tobacco bacterial wilt) is leaf blight, black streak appears on stems, often called half-crazy and tobacco blast. The pathogenic bacteria inducing tobacco bacterial wilt is Ralstonia solanacearum (Ralstonia solanacearum), the distribution range is wide, and the host plants can reach up to 50 families and more than 200 genera. The ralstonia solanacearum mainly invades from three parts of root wounds of tobacco, root tips of tobacco plants or secondary roots of tobacco plants, cannot invade from air holes, finally colonizes on xylem of the tobacco, and proliferates in a large quantity, extracellular polysaccharide produced by the ralstonia solanacearum can block vascular bundle tissues, so that nutrition supply is blocked, the tobacco plants cannot normally obtain nutrition substances to ensure normal growth of overground parts, later tobacco plant leaves are yellowing and necrosis, single-side leaves are withered, tobacco plant growth is blocked, and finally the whole plant dies.
At present, measures such as fallow rotation, screening and breeding disease-resistant varieties, chemical control and the like exist for preventing and treating the tobacco bacterial wilt, but in practical application, a plurality of limitations still exist. If the tillage area is relatively tension, the fallow wheels cannot be popularized on a large scale. The variety with tobacco bacterial wilt resistance is limited, and is influenced by bacterial wilt differentiation seeds in different areas, so that the difficulty of screening and cultivating disease-resistant varieties is increased. In addition, the resistance effect is poor, and the resistance is easy to be restricted by conditions such as environment and the like when the plant is applied in the field, so that the resistance is lost. The chemical agent is used for preventing and controlling tobacco bacterial wilt, and in the environment of tobacco planting soil for long-term continuous cropping, the nutrient proportion of the soil is regulated, so that the growth and development of tobacco plants are hindered, plant diseases and insect pests occur greatly, the quality of tobacco leaves is influenced to a certain extent, and the problems of exceeding of pesticide residues of the tobacco leaves, soil environmental pollution and the like can be caused by using the tobacco leaves with increased concentration. From the aspect of sustainable development, the search for an efficient and green method for preventing and controlling tobacco bacterial wilt becomes a difficult problem that researchers need to break through.
Disclosure of Invention
In view of the above, the invention aims to provide the Pseudomonas fluorescens LSW-4 for preventing and treating bacterial wilt, and also relates to the application of the Pseudomonas fluorescens in inhibiting and/or preventing and treating the bacterial wilt, so as to reduce the problems of exceeding of pesticide residues of tobacco leaves and soil environmental pollution and the like caused by chemical agents.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. a pseudomonas fluorescens strain which is pseudomonas fluorescens (Pseudomonas fluorescens) LSW-4 deposited at the chinese microbiological bacterial culture collection center, address: beijing city, chaoyang district, north Chen Xili No. 1, 3, date of preservation: 2022, 4, 11, deposit number: CGMCC 24668.
Furthermore, the Pseudomonas fluorescens strain is a gram negative bacterium, the single colony is in a colony circular shape, the color is milky white, the surface is smooth, the edge is neat, and the middle is slightly raised.
2. The invention also provides a biological microbial agent, which comprises a pseudomonas fluorescens LSW-4 strain with a preservation number of CGMCC 24668, which is preserved in the China general microbiological culture Collection center.
Furthermore, the biological agent also comprises a carrier attached with pseudomonas fluorescens LSW-4 strain, wherein the carrier is bran, straw powder or diatomite.
Preferably, chaff is used as a carrier.
3. The invention also provides a preparation method of the biological agent, which comprises the steps of activating the pseudomonas fluorescens LSW-4, inoculating the activated pseudomonas fluorescens LSW-4 into an LB liquid culture medium for culture, and culturing for 6-72h at the temperature of 28-30 ℃.
4. The application of the pseudomonas fluorescens LSW-4 strain as a biological agent in inhibiting and/or preventing bacterial wilt,
further, the Pseudomonas fluorescens LSW-4 strain is used as a biological agent for inhibiting and/or preventing bacterial wilt, and the use concentration of the Pseudomonas fluorescens LSW-4 in the biological agent is 1 multiplied by 10 6 cfu/mL-1×10 12 cfu/mL。
Further, the Pseudomonas fluorescens LSW-4 strain is used as a biological agent for application in inhibiting and/or preventing bacterial wilt, after the Pseudomonas fluorescens LSW-4 is activated, the bacterial wilt is inoculated into an LB liquid culture medium for culture, and the bacterial wilt is cultured for 6-72 hours at the temperature of 28-30 ℃ to obtain the liquid bacterial agent.
The invention has the beneficial effects that: the fluorescent pseudomonas LSW-4 has good inhibition and antagonism on the bacterial wilt, the inhibition zone of the bacterial wilt can reach 12.30mm-33.76mm under different concentrations, a potted plant preliminary test shows that the fluorescent pseudomonas LSW-4 has good direct disease control effect on tobacco bacterial wilt, in further field experiments, the fluorescent pseudomonas LSW-4 also shows excellent resistance on tobacco bacterial wilt, the morbidity is only 10.00%, the disease index is 5.78, the control effect is 79.97%, and the fluorescent pseudomonas LSW-4 can be fully proved to be capable of effectively controlling tobacco bacterial wilt in fields and also has a certain growth promoting effect. The microbial inoculum reaches the growth log phase 10-12 h after inoculation and does not start to drop until 72h after inoculation.
Preservation of biological materials
Pseudomonas fluorescens (Pseudomonas fluorescens) LSW-4 of the invention is deposited at China general microbiological culture Collection center, address: beijing city, chaoyang district, north Chen Xili No. 1, 3, date of preservation: 2022, 4, 11, deposit number: CGMCC 24668.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a partial planogram showing the bacteriostatic effect of rescreening antagonistic bacteria on bacterial wilt.
FIG. 2 shows the results of a pre-test screening section of a pot.
FIG. 3 shows the effect of rhizosphere regulation of different microbial agents on tobacco plant bacterial wilt resistance.
FIG. 4 shows the effect of rhizosphere regulation of different microbial agents on tobacco plant bacterial wilt resistance index.
FIG. 5 is a morphology of LSW-4 single colonies.
FIG. 6 is a view showing the morphology of LSW-4 transmission electron microscope bacteria.
FIG. 7 is a schematic representation of the creation of an LSW-4 phylogenetic tree based on 16S rDNA.
FIG. 8 shows the growth curve of Pseudomonas fluorescens LSW-4.
FIGS. 9-13 are graphs showing the number of live bacteria sampled at different time points for the microbial inoculum prepared from different carriers; wherein the carrier of fig. 9 is diatomite, the carrier of fig. 10 is straw powder, the carrier of fig. 11 is bran, the carrier of fig. 12 is talcum powder, and the carrier of fig. 13 is oyster shell powder.
FIGS. 14 to 16 are sequentially showing the culture charts of the number of viable bacteria sampled at different time points of the microbial inoculum obtained in each microbial inoculum medium of example 7.
FIG. 17 shows the results of potting experiments with different solid Pseudomonas fluorescens LSW-4 bacteria.
Detailed Description
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 specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Example 1
1. Soil sample collection: soil samples were collected on day 29, 8 in 2019, and the soil sample collection information is shown in table 1. Pulling off 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 of the tobacco roots with rhizosphere soil of 3 healthy tobacco plants in a collection bag, repeating for 3 times, and sealing to make marks.
Table 1 soil sample collection information table
2. Test pathogen activation: the pathogenic bacteria ralstonia solanacearum (Ralstonia solanacearum) CQPS-1 separated from the Chongqing peng water tobacco bacterial wilt disease pathogenic tobacco strain has been subjected to research on identification and characteristics of the pathogenic bacteria ralstonia solanacearum (Ralstonia solanacearum). Activating tobacco bacterial wilt CQPS-1, and propagating in liquid culture medium B under the conditions of constant temperature shaking table 30deg.C, 180rpm/min for about 12 hr, and spectrophotometry to detect OD value=0.8-1.0, wherein the tobacco bacterial wilt concentration is about 1×10 9 cfu/mL. The culture medium used in this example and its specific formulation are as follows:
NA medium: 5.0g of bactopeptone, 1.0g of yeast powder, 10.0g of glucose, 3.0g of beef powder, 20.0g of agar powder, 1000mL of distilled water and pH of 6.8-7.2; sterilizing at 121deg.C for 20min.
LB medium: 5.0g of yeast extract, 10.0g of sodium chloride, 10.0g of tryptone and 1000mL of distilled water; sterilizing at 121deg.C for 20min.
Liquid medium B: 1.0g of casein amino acid, 5.0g of glucose, 10.0g of bactopeptone, 1.0g of yeast extract and 1000mL of distilled water; sterilizing at 121deg.C for 20min.
3. Isolation and purification of strains
The test adopts a dilution flat plate coating method to separate and purify bacteria in a soil sample, and comprises the following specific operations: weighing 10g of soil sample, adding 90mL of sterile water, slightly vibrating, fully mixing, vibrating for 30min on a constant-temperature shaking table of 220r/min, and standing for 10min; taking supernatant of the soil fungus suspension after standing, and preparing 10 by adopting a gradient dilution method -2 、10 -3 、10 -4 And 10 -5 Is a diluent of (a); taking 100 mu L of the diluent with each concentration gradient, adding the diluent onto a NA plate with coating beads, and uniformly oscillating back and forth to uniformly coat bacterial suspension, wherein each concentration gradient is repeated for 3 times; culturing the coated NA plate in an inverted manner in a constant temperature incubator at 30 ℃; taking out NA board after 2d, observing the growth condition of colony in ultra clean bench, picking different single colony according to the colony form and color as judging basis, culturing in LB liquidCulturing at 30deg.C for 12 hr/min, culturing at 30deg.C for 48 hr, repeating each streak plate for 3 times, purifying the separated bacteria, and repeating the step of picking single colony on NA plate for three times until all colonies on NA plate are consistent.
After separating and purifying bacteria in the soil sample, intuitively judging the shape and color difference of the bacteria, separating 184 strains of bacteria altogether, and further evaluating antagonism of the separated 184 strains of bacteria on tobacco bacterial wilt by adopting a flat plate counter method for primary screening, wherein the specific operation method comprises the following steps: taking bacterial suspension (10) of activated tobacco bacterial wilt 6 cfu/mL) of 100 mu L, adding the mixture to an NA plate of the sterilized coated beads, and uniformly coating; placing a 6mm filter paper sheet in the center of the NA plate coated with the tobacco bacterial wilt; each purified test bacteria was taken in 5. Mu.L and attached to a filter paper sheet, and each treatment was repeated 3 times. After being placed in a constant temperature incubator at 30 ℃ for inverted culture for 48 hours, the bacteria inhibition zone is observed to be formed.
The 49 bacteria with antagonism to tobacco bacterial wilt are separated by primary screening, obvious inhibition zones are formed, but the inhibition effects are different, and the diameters of the inhibition zones are different, and the results are shown in table 2.
Table 2 diameter meter of primary screening bacteriostasis ring
The antagonistic bacteria of the primary screening are further screened again, and the specific operation method comprises the following steps: taking a bacterial suspension (10) of activated tobacco bacterial wilt in advance 6 cfu/mL) of 100 mu L, adding the mixture to an NA plate of the sterilized coated beads, and uniformly coating; placing a 6mm filter paper sheet in the center of the NA plate coated with the tobacco bacterial wilt; transferring the bacteria with antagonism to tobacco bacterial wilt in the primary screening result to LB liquid culture medium to culture until the bacterial wilt is 10 9 cfu/mL (OD=0.8-1), ensuring the consistency of the concentration of the bacteria to be detected, taking 5 mu L of the bacteria to be detected, and connecting the bacteria to be detected on a filter paper sheet, wherein each treatment is repeated for 3 times. After being placed in a constant temperature incubator at 30 ℃ for inverted culture for 48 hours, the bacteria inhibition zone is observed to be formed or not, and the cross method is adopted for measurementThe size of each inhibition zone is measured. 24 bacteria still having antagonism to the bacterial wilt are re-screened from the 49 antagonistic bacteria of the primary screening, fig. 1 is a partial planogram of the antibacterial effect of the re-screened antagonistic bacteria to the bacterial wilt, and fig. 3 is a diameter table of the re-screened antibacterial zone.
Table 3 diameter meter of re-screening bacteriostasis ring
Example 2
In order to effectively prevent and control the tobacco bacterial wilt, screening of the tobacco bacterial wilt antagonistic bacteria is not limited to evaluating the direct inhibition effect of the antagonistic bacteria on the tobacco bacterial wilt, but also confirms the direct disease control effect of the antagonistic bacteria on the tobacco bacterial wilt through a potting pre-test.
Selecting four-leaf and one-heart tobacco seedlings with consistent and healthy sizes for potting test, transferring the activated antagonistic bacteria to LB liquid culture medium for culture, placing the culture medium at 30 ℃, culturing at 170r/min for 12 hours, detecting the OD=0.8-1 by a spectrogradiometer, inoculating 10mL of bacterial liquid to each tobacco seedling, treating 10 tobacco seedlings, and inoculating 10mL of sterile water to blank control. The cultivation conditions of the greenhouse were set as follows: the mixture is irradiated for 12 hours at the temperature of 28 ℃ and the humidity of 85 percent. During the growth of tobacco seedlings, the tobacco seedlings are regularly irrigated by sterile water to ensure the normal growth of the tobacco seedlings, the incidence rate of the tobacco bacterial wilt is investigated according to national standard GB/T2322-2008 of the bacterial wilt and indoor grading standards from the beginning of the tobacco bacterial wilt, the screening part results of a potted plant pre-test are shown in fig. 2, and the influence of antagonistic bacteria on the tobacco bacterial wilt is shown in table 4.
TABLE 4 Table 4
Example 3
In order to clearly determine the influence of the concentration of LSW-4 and the inoculation time on tobacco bacterial wilt, four-leaf and one-core Yunyan 87 tobacco seedlings are selected for laboratory experiments by adopting a common seedling tray seedling culture mode, and the concentrations are respectively 1 multiplied by 10 8 cfu/mL、1×10 7 cfu/mL、1×10 6 Root irrigation was performed at cfu/mL antagonistic bacteria concentration.
Each tobacco strain is inoculated with antagonistic bacteria LSW-4 according to different treatment concentration of 10mL, after the antagonistic bacteria LSW-4 is inoculated for 3d, each tobacco strain is irrigated with root and inoculated with bacterial wilt CQPS-1 (1 multiplied by 10) 7 cfu/mL 10 mL). 10 tobacco seedlings are treated each time, the tobacco seedlings are repeated for 3 times, the greenhouse culture condition is that the illumination is carried out for 12 hours, the temperature is 28 ℃, the humidity is 85%, the investigation is carried out according to national standard GB/T2322-2008 and indoor grading standards from the occurrence of the tobacco bacterial wilt, and the occurrence of the bacterial wilt is recorded once every 24 hours. The antagonistic bacteria LSW-4 is found to have concentration effect on prevention and control of tobacco bacterial wilt, namely the higher the concentration of the antagonistic bacteria LSW-4 is, the better the prevention and control effect on tobacco bacterial wilt is. When the incidence rate of the blank control is 66.66%, 1×10 8 cfu/mL、1×10 7 cfu/mL、1×10 6 The morbidity of cfu/mL is 11.11%,25.00%,44.45%, the disease indexes are 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 potting pre-test of example 2, several antagonistic bacteria LSW-4, LSW-32 and PSG-26 which did not attack the disease were selected, and a field test was performed in Chongqing unitary Yang Cangling Zhen-Xa dam village in 2019 (the village of Qing mountain city, qing Ling Qing Cheng village with the altitude 1143m, the longitude and latitude of which is east longitude E108 degrees 34 '22.99', north latitude N29 degrees 0 '42.83', and the average transplanted tobacco seedling per mu was about 1200 strains), the test variety was Yunyan 87, and the test material: (1) Self-extracting antagonistic bacteria (LSW-4, PSG-26, LSW-32); (2) trichoderma harzianum; (3) bacillus subtilis; (4) Miao Jiangzhuang; (5) Paenibacillus polymyxa. And (2) to (5) are purchased in the market, and each microbial inoculum is root-irrigated and applied. Treatment 1: paenibacillus polymyxa; treatment 2: trichoderma harzianum agent; treatment 3: a seedling strengthening compound microbial inoculum; treatment 4: a bacillus subtilis agent; treatment 5: LSW-4; treatment 6: PSG-26; treatment 7: LSW-32; treatment 8 (CK): clear water control, each microbial inoculum concentration is 1×10 8 cfu/mL. After the antagonistic bacteria of the re-screening are activated, transferring to LB liquid medium for culture, placing at 30 ℃ and 170r/min for culture for 12 hours, detecting the OD=0.8-1 by a spectrogradiometer, and adopting a root irrigation mode.
Cultivation conditions: in the early stage, floating seedling raising is adopted for cultivation of tobacco seedlings, unified field management is carried out on tobacco plant growth management according to relevant technical standards, transplanting time is 4 months and 30 days, axillary buds are controlled by 12.5% flumetralin EC when central flowers bloom, topping is carried out 7 months and 7 days, and harvesting is carried out 7 months and 18 days. The effect of rhizosphere regulation of different microbial agents on 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 in the present invention have good effects on agronomic traits in the tobacco plant group (according to YC/T142-1998, standard of investigation of agronomic traits of tobacco).
TABLE 5
The plot selected in the test is a perennial bacterial wilt high-incidence plot, the effect result of rhizosphere regulation of different microbial agents on tobacco plant resistance to tobacco bacterial wilt is shown in fig. 3 (morbidity) and fig. 4 (disease index), the morbidity and all of rhizosphere regulation treatment of different microbial agents are lower than those of control treatment, and significant difference exists between the rhizosphere regulation treatment and clear water control, so that the resistance of tobacco plants to tobacco bacterial wilt can be affected to a certain extent through the rhizosphere regulation mode. The results show that: the best disease control effect is antagonizing bacteria LSW-4, the later disease rate is only 10.00 percent, the later disease rate is 15.00 percent, the disease rates of Paenibacillus polymyxa, strong seedlings, PSG-26 and LSW-32 are 43.33 percent, 38.33 percent and 41.67 percent respectively, and the disease rate of blank control is 61.67 percent. The disease index is consistent with the trend of the incidence, LSW-4 is 5.78, the disease index of bacillus subtilis is 7.22, the disease indexes of paenibacillus polymyxa, strong seedlings, PSG-26 and LSW-32 are 16.13, 18.44, 20.46 and 17.96 respectively, the disease index of blank control is 28.85, the control effect of LSW-4 is 79.97%, the LSW-4 is fully proved to be capable of effectively controlling tobacco plant bacterial wilt in a field, the tobacco plants irrigated by LSW-4 enter a mass period 2d earlier than the control, and the tobacco plants irrigated by LSW-4 are superior to the control in plant height, effective leaf number, leaf length, leaf width and leaf area.
Example 4
Identification of antagonistic bacteria LSW-4, FIG. 5 is a single colony morphology of LSW-4, and FIG. 6 is a morphology observation of LSW-4 transmission electron microscope bacteria. After the LSW-4 was cultured in TSA medium for 48 hours, the morphology of single colony was observed and gram-stained, and it was observed that LSW-4 was gram-negative bacteria, the morphology of single colony was colony-circular, the color was milky white, the surface was smooth, the edges were clean, and the middle was slightly raised. The result of the transmission electron microscope shows that the bacterial form is rod-shaped and has terminal flagella.
Bacterial DNA (DNA extraction kit of Beijing Soxhlet technology Co., ltd.) was extracted and PCR amplification was performed on the 27F/1492R region of the bacterial DNA according to amplification primers (27F and 1492R). 27F: agagttttgatcctggcttag; 1492R: ggttacttgttacgactt. Sequencing PCR amplified products by Hua big gene company, BLAST comparing sequencing results in GenBank, selecting a 16S rDNA sequence with higher homology as a reference object, constructing a phylogenetic tree by adopting a neighbor-joining method, and establishing an LSW-4 phylogenetic tree based on the 16S rDNA in FIG. 7. The strain LSW-4 was identified as Pseudomonas fluorescens (Pseudomonas fluorescens). The growth condition of antagonistic bacteria LSW-4 is clarified by measuring the OD value of the antagonistic bacteria LSW-4 by a spectrophotometer, the growth rule is known, the antagonistic bacteria LSW-4 is totally measured for 72 hours, the obvious decline trend of the OD value is observed, and the LSW-4 reaches the growth log phase between 10 hours and 12 hours. FIG. 8 shows the growth curve of Pseudomonas fluorescens LSW-4.
The strain LSW-4 is preserved in China general microbiological culture Collection center, address: beijing city, chaoyang district, north Chen Xili No. 1, 3, date of preservation: 2022, 4, 11, deposit number: CGMCC 24668.
Example 5
Soil after application of antagonistic bacteria LSW-4 was collected in two times, the period of collection was 6 months 23 days (Wanglong period), 7 months 18 days (topping late period) was compared before and after the two data, 3 replicates, and changes in the soil microbial community after application of antagonistic bacteria LSW-4 were analyzed.
The following conclusion was reached by high throughput sequencing from DNA samples of soil treated with LSW-4 at different times and analysis by means of the american Ji Yun platform:
(1) By analyzing the composition of the collected soil samples of 6 months 23 and 7 months 18, comparing the soil samples of 6 months 23 and 7 months 18, after antagonistic bacteria LSW-4 treatment, the proportion of the green curved fungus door (chlorflexi) in the bacterial community shows a growing trend, the Ascomycota (Ascomycota) in the fungal community is reduced by 11.41%, the Mortierella (Mortierella) is increased by 3.54%, and the Basidiomycota (Basidiomycota) is increased by 3.76%.
(2) Statistical analysis of β diversity revealed that at OTU level, the difference between the treated LSW-4 and the blank control was not very significant in the soil at day 6 months 23, whether bacterial or fungal. In the soil of 7 months and 18 days, the effect on bacterial communities is large after LSW-4 treatment, and obvious difference appears.
(3) By discriminant analysis of the soil sample LEfSe after LSW-4 treatment, the soil sample collected on day 6 and 23 shows that the soil sample is significantly enriched as Luo He bacillus (Rhodanobacter) after LSW-4 treatment at the level of bacterial community genus; the fungi treated by LSW-4 are obviously enriched to Becky Eurotium (Pseudomonas) and Setophoma, rickenella, cotylidia. The soil sample was significantly enriched in bacterial communities after LSW-4 treatment for 7 months and on fungus levels was significantly enriched for obscurbibateralles, and was predominantly Codinaea, scytalidium after LSW-4 treatment.
LSW-4 is used as a biocontrol strain, can directly inhibit the proliferation of pathogenic bacteria, induces plants to generate disease resistance, and can play a role in regulating and controlling the micro-ecological structure of soil in soil. Soil is an environment in which plants directly grow, and is also an important medium for pathogenic bacteria and plant interaction. Whether soil is healthy or not directly affects the health of plants. The growing severity of soil-borne diseases is often caused by the disruption of the microecological balance of soil caused by 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 soil is healthy. The change of the microbial composition of soil and the occurrence of soil-borne diseases are closely related to plant health. After antagonistic bacteria LSW-4 are applied to Pseudomonas fluorescens (Pseudomonas fluorescens), the microbial community structure of tobacco rhizosphere soil is affected, and the fluorescent Pseudomonas fluorescens LSW-4 can regulate and control the abundance of certain specific microbial populations in the microbial community composition, so that the occurrence of soil-borne disease bacterial wilt is reduced. The soil samples collected on day 6 and 23 showed that, at the level of the bacterial community, they were significantly enriched in Luo He bacillus (rhodobacter) after LSW-4 treatment; the fungi treated by LSW-4 are obviously enriched to Becky Eurotium (Pseudomonas) and Setophoma, rickenella, cotylidia. The soil sample was significantly enriched in bacterial communities after LSW-4 treatment for 7 months and on fungus levels was significantly enriched for obscurbibateralles, and was predominantly Codinaea, scytalidium after LSW-4 treatment.
Example 6
In order to further apply the strain LSW-4 to commercial products, on the basis of earlier research, the preparation carrier and the solid microbial agent suitable for the pseudomonas fluorescens are continuously examined so as to solve the problems of transportation and storage of the pseudomonas fluorescens and facilitate application to field prevention and treatment of tobacco bacterial wilt.
1. Preparation of liquid culture 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: 15% -20% (w/v) of agar is added into LB liquid culture medium.
2. Carrier body
Carrier material: oyster shell powder (500 g/bag of the beginner flower electronic commerce Limited company in the millizhou city), diatomite (500 g/bag of the fine chemical industry of the deng peak), talcum powder (500 g/bottle of the fine chemical industry of the Fuchen), corn straw powder (250 g/20 meshes of the deep processing of the agricultural products of the Lifeng) and Gu Kangfen (250 g/20 meshes of the deep processing of the agricultural products of the Lifeng).
Respectively taking oyster shell powder, diatomite, talcum powder, straw powder and bran powder, sterilizing with high-pressure steam at 121 ℃ for 30 minutes, and then drying in a constant-temperature oven at 60 ℃ to a constant state. Table 6 shows the pH of the different carriers.
TABLE 6 pH of different Carriers
3. Bacterial culture
Inoculating Pseudomonas fluorescens LSW-4 into LB liquid medium according to 1% -1.5% of inoculation amount, placing in a 30 ℃ constant temperature shaking table for 180r/min, and culturing for 24-48 h.
4. Centrifuging
The cultured bacterial fermentation broth was centrifuged at 8000r for 5min with a 50ml centrifuge tube previously sterilized.
5. Suspending
The centrifuged bacteria were added to LB liquid medium of about one tenth of the original bacteria volume to suspend and mix gently. The concentration of the mixed bacterial liquid is about 1 multiplied by 10 12 cfu/ml。
6. Mixing
Adding the suspended bacterial liquid into carriers such as sterile oyster shell powder, diatomite, talcum powder, straw powder, chaff and the like according to the volume-to-mass ratio (ml: g), uniformly mixing for 30-40min under the condition of 180r/min of a constant temperature shaking table at 30 ℃, finally ventilating and airing in a sterile workbench to constant weight, respectively filling into a sterilized glass bottle, and uniformly stirring by using a sterile glass rod.
7. Preservation of
Sealing with sealing film, storing at normal temperature, sampling for 10d, 30d and 90d, measuring viable cell number, and screening out carrier with maximum viable cell number. Samples were taken for gradient dilution coating and the viable count was counted (LB solid medium, 3 samples were taken each time, 1g was weighed, 3 concentrations per sample, 3 plates per concentration).
The ratio of the bacterial liquid to the carrier is 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 bacterial agent to the carrier is more than 1.5:1, the viable count of the solid bacterial agent prepared by the same carrier is not greatly changed when the ratio of the bacterial agent to the carrier is more than 1.5:1, and when the ratio of the solid bacterial agent prepared by diatomite is more than 2.5:1, the viable count is not greatly changed when the test is carried out on the 10 th day. As proved by experiments, the carrier suitable for preparing the Pseudomonas fluorescens LSW-4 is bran, straw powder and diatomite, and the bran is preferred. The bran is a raw material, so the bran is a more convenient and cheap source, and the number is large, so the bran is selected as an optimal carrier for further testing and research.
Fig. 9-13 show the number of viable bacteria at different time points when the ratio of the bacterial liquid to the carrier is 1:1, wherein the carrier of fig. 9 is diatomite, the carrier of fig. 10 is straw powder, the carrier of fig. 11 is bran, the carrier of fig. 12 is talcum powder, and the carrier of fig. 13 is oyster shell powder. Table 7 shows the number of viable bacteria obtained by sampling at different time points when the ratio of the bacterial liquid to the carrier was 1:1.
Table 7 viable count (cfu/g) was measured for each vector at different time points
| Carrier body | 10d | 30d | 90d |
| Chaff | 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 |
| Talc 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 an inoculum size of 1% -1.5%, placing the LB liquid culture medium in a shaking table at a constant temperature of 30 ℃ for 180r/min, culturing for 24h-48h, and centrifuging the cultured bacterial fermentation liquor with a sterilizing centrifuge tube for 8000r and 5min; the centrifuged thalli is suspended by a microbial inoculum culture medium with the volume of 1/8-1/12 of the original bacterial liquid, and is gently mixed by shaking.
Microbial inoculum 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, 20-30 g of sodium caseinate, 1000ml of constant volume and natural pH.
Microbial inoculum medium 2: the carrier is chaff, 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.
Microbial inoculum medium 3: the carrier is chaff, 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, 1000ml of constant volume and natural pH.
Adding bacterial solutions suspended by using culture mediums of different bacterial agents into carriers with the volume-mass ratio of 0.5-1.5:1 (ml: g), respectively adding the bacterial solutions into the carriers subjected to aseptic treatment, uniformly mixing for 30-40min under the condition of 180r/min of a constant temperature shaking table at 30 ℃, and finally ventilating and airing in an aseptic workbench to constant weight. In order to accelerate the drying speed, the pseudomonas fluorescens LSW-4 solid microbial inoculum can be obtained by uniformly mixing, sterile filtering and then ventilating and drying under the sterile condition to constant weight. It may also be freeze-dried or spray-dried.
The microbial inoculum culture medium of the invention is tested by a plurality of component combinations and different concentrations, cereal bran is used as a carrier, the effective viable bacteria number of 10 days, 30 days and 90 days is inspected under the condition that the ratio of microbial inoculum to the carrier is 1:1, the optimal microbial inoculum culture medium is confirmed, the viable bacteria count statistics are obtained by sampling the microbial inoculum obtained by each microbial inoculum culture medium at different time points, and fig. 14-16 are sequentially and respectively obtained by sampling the viable bacteria count culture diagrams of the microbial inoculum obtained by each microbial inoculum culture medium at different time points. Finally, using bran as a carrier, screening LB liquid culture medium under the conditions of bacterial liquid and carrier 1:1, adding 0.01-0.02% of sodium carboxymethylcellulose, 0.5-1.5% of dextrin and 2-3% (w/v) of sodium caseinate into the microbial inoculum culture medium, and improving the loading rate and effective viable bacteria quantity of pseudomonas fluorescens LSW-4 on the bran carrier, wherein the rest data are more, and only the content and data related to the invention are shown.
TABLE 8 viable count statistics (cfu/g)
| Carrier+microbial inoculum culture medium | 10d | 30d | 90d |
| Chaff + microbial inoculum 1 | 4.8×10 11 | 1.9×10 11 | 5.7×10 10 |
| Chaff + microbial inoculum 2 | 9.7×10 10 | 1.3×10 10 | 7.3×10 9 |
| Chaff + microbial inoculum 3 | 7.5×10 9 | 3.6×10 8 | 8.3×10 7 |
Example 8
Through screening the carrier and the microbial inoculum culture medium, the Pseudomonas fluorescens LSW-4 solid microbial inoculum prepared by different methods is subjected to an indoor potting test, and further comparison and confirmation are carried out. Four-leaf and one-core Yunyan 87 tobacco seedlings are selected for laboratory experiments in a mode of common seedling tray seedling culture, three replicates are arranged for each treatment, and 12 plants are repeated for each treatment. And (3) applying 2g of microbial inoculum to each plant of tobacco substrate at about 60g during transplanting, culturing the transplanted tobacco plants at 30 ℃, illuminating for 16 hours and 8 hours at night, and watering in time under the constant temperature greenhouse condition with the air humidity of 85%. After 3 days of seedling recovery, the bacterial concentration is 1 multiplied by 10 7 cfu/ml of bacterial wilt root irrigation, 10ml of root irrigation per plant of tobacco. Disease investigation was performed from the initial stage of onset, and data were recorded and analyzed.
The following preparation methods of different Pseudomonas fluorescens LSW-4 solid microbial agents are the same as in example 2, and the carrier and microbial agent culture medium are examined under the same conditions that the ratio of the microbial liquid to the carrier is 1:1.
Microbial inoculum 1: the carrier is straw powder, 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.
Microbial inoculum 2: the carrier is 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.
Microbial inoculum 3: the carrier is straw powder, 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, 20-30 g of sodium caseinate, 1000ml of constant volume and natural pH.
Microbial inoculum 4: the carrier is straw powder, 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, 1000ml of constant volume and natural pH.
Microbial inoculum 5: the carrier is 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, 1000ml of constant volume and natural pH.
Microbial inoculum 6: the carrier is 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.
Microbial inoculum 7: the carrier is 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, 20-30 g of sodium caseinate, 1000ml of constant volume and natural pH.
CK: and (5) comparing with clear water.
As a result, FIG. 17 shows that A1 to A7 correspond to the same numbers of microbial agents, respectively.
A1: the morbidity is 66.67 percent, the disease index is 52.08, and the relative control effect is 12.79 percent;
a2: the morbidity is 58.33 percent, the disease index is 49.31, and the relative control effect is 17.44 percent;
a3: the morbidity is 52.78 percent, the disease index is 42.36, and the relative control effect is 29.07 percent;
a4: the morbidity is 66.67 percent, and the disease index 51.04 is 14.53 percent relative to the control effect;
a5: the morbidity is 58.33 percent, the disease index is 50.00, and the relative control effect is 16.28 percent;
a6: the morbidity is 44.44 percent, the disease index is 38.89, and the relative control effect is 34.88 percent;
a7: the incidence rate is 29.17 percent, the disease index 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 living bacteria number and the indoor potting test data show that the effect of taking chaff as a carrier of the bacterial strain LSW-4 is better than that of other carriers, the chaff is taken as the carrier, 0.01-0.02% of sodium carboxymethyl cellulose and 0.5-1.5% of dextrin are added, the bacterial activity of the fluorescent pseudomonas LSW-4 solid bacterial agent prepared by a bacterial agent culture medium with 2-3% (w/v) of sodium caseinate is highest, the long-time normal-temperature preservation condition can be met, the storage and transportation of the bacterial strain are facilitated, the effect of relative prevention effect on tobacco bacterial wilt is best, the bacterial strain is suitable for industrial production, and the separated bacterial strain can be fully applied to field prevention and treatment of the tobacco bacterial wilt and fully plays the actual effect and effect.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (7)
1. A Pseudomonas fluorescens strain is characterized in that the strain is Pseudomonas fluorescens @Pseudomonasfluorescens) LSW-4, deposited at China general microbiological culture Collection center, address: beijing city, chaoyang district, north Chen Xili No. 1, 3, date of preservation: 2022, 4, 11, deposit number: CGMCC No.24668.
2. A biological agent comprising the pseudomonas fluorescens LSW-4 strain of claim 1.
3. The biological agent according to claim 2, comprising the pseudomonas fluorescens LSW-4 strain of claim 1 and a carrier, wherein the carrier is bran, straw powder or diatomaceous earth.
4. The method for preparing the biological agent according to claim 2 or 3, wherein after the Pseudomonas fluorescens LSW-4 is activated, the bacterial agent is inoculated into LB liquid medium for culture and is placed under the condition of 28-30 ℃ for 6-72 hours.
5. Use of the pseudomonas fluorescens strain according to claim 1 as a biological agent for inhibiting and/or controlling tobacco bacterial wilt.
6. The use of a Pseudomonas fluorescens strain according to claim 5 as a biological agent for inhibiting and/or controlling tobacco bacterial wilt, wherein Pseudomonas fluorescens LSW-4 is used in a concentration of 1X 10 in the biological agent 6 cfu/mL-1×10 12 cfu/mL。
7. The use of the Pseudomonas fluorescens strain according to claim 5 as a biological agent for inhibiting and/or preventing tobacco bacterial wilt, wherein the liquid bacterial wilt is obtained by activating Pseudomonas fluorescens LSW-4, inoculating the activated Pseudomonas fluorescens LSW-4 into LB liquid culture medium, culturing, and culturing at 28-30 ℃ for 6-72h.
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