CN113736687A

CN113736687A - Disease-resistant growth-promoting salt-tolerant bacillus and application thereof

Info

Publication number: CN113736687A
Application number: CN202110774467.1A
Authority: CN
Inventors: 彭霞薇; 董益豪; 贾曼曼; 张敬涛; 周金星
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2021-07-08
Filing date: 2021-07-08
Publication date: 2021-12-03
Anticipated expiration: 2041-07-08
Also published as: CN113736687B

Abstract

The invention discloses a strain of Bacillus halotolerans (Bacillus halotolerans) with the preservation number of CGMCC No. 19502. The salt-tolerant bacillus has a strong inhibiting effect on root rot caused by fusarium solani, has a good preventing and treating effect on the root rot of the peony for oil by using the bacillus liquid, and can promote the growth of peony for oil, so that the fertilizer and pesticide investment can be reduced, the environmental pollution can be reduced, and the sustainable development of agriculture can be realized.

Description

Disease-resistant growth-promoting salt-tolerant bacillus and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, particularly relates to the field of plant disease biological control microorganisms, and particularly relates to disease-resistant growth-promoting salt-tolerant bacillus and application thereof.

Background

The peony (Paeonia suffruticosa Andr) for oil is a novel woody oil crop originally produced in China, has strong seed production capacity and high seed oil yield, contains more than 90 percent of unsaturated fatty acid in peony seed oil, particularly has the alpha-linolenic acid which is more than 40 percent of that of olive oil and has the effects of health preserving, health care, oxidation resistance, beauty treatment and the like, and the peony seed oil is more than 40 percent of unsaturated fatty acid. At present, oil peonies are planted in large areas in Shandong, Henan, Shanxi, Anhui, Gansu and other places in China. With the enlargement of planting area and the increase of planting years, the occurrence of various diseases of oil peonies also rises year by year, the yield reduction of peony seeds is caused, and the quality of peony seed oil is influenced, wherein the serious harm is root rot, which is a soil-borne disease caused by Fusarium solani.

At present, in production practice, soil-borne diseases are mainly prevented and controlled by using some chemical pesticides for soil disinfection or crop rotation, but the use of pesticides can cause environmental pollution and food safety problems, increase production cost and induce pathogenic bacteria to generate drug resistance. The biocontrol soil-borne disease prevention and control method is a green, environment-friendly and economic method, is safe and harmless to human beings, animals and plants while effectively controlling diseases, reduces environmental pollution, meets the national development requirements on sustainable agriculture and environment-friendly society, and becomes a research hotspot for disease prevention and control in recent years.

The biocontrol microorganism mainly comprises fungi (such as trichoderma, penicillium and the like), actinomycetes (such as streptomyces) and bacteria (such as bacillus, pseudomonas and the like). And Bacillus spp in bacteria can generate spores to resist severe conditions, has high propagation speed, is easy to colonize soil and the like, and is widely applied. Currently, bacillus capable of inhibiting plant pathogenic microorganisms mainly include bacillus subtilis (b.subtilis), bacillus amyloliquefaciens (b.amyloliquefaciens), bacillus megaterium (b.megaterium), bacillus belgii (b.velezensis), bacillus siamensis (b.siamensis), and the like. The Chinese patent (publication number: CN109825457A) in 2019 discloses that a strain of salt-tolerant bacillus (B.halotolerants) E40207a2 has a remarkable antagonistic effect on virus diseases caused by potato virus Y; and Chinese patent (publication number: CN110343641A) discloses that a strain of bacillus halotolerans (B.halotolerans) SYW-1 can effectively prevent and control cotton verticillium wilt, and is beneficial to improving the yield and quality of cotton; in addition, a strain of bacillus halotolerans (B.halotolerans) BW9 disclosed in Chinese patent (publication No. CN110819565A) has strong antagonistic activity, siderophore production activity and nitrogen fixation effect, and can effectively inhibit various phytopathogens, thereby indicating that the bacillus halotolerans has the potential to be used as a biological control microorganism. Meanwhile, the patent results also show that the antagonistic effects of the same kind of salt-tolerant bacillus on different pathogenic bacteria are greatly different, so that in the process of applying the salt-tolerant bacillus as a bio-control preparation, more strains capable of preventing and controlling other diseases need to be continuously screened, and more strain resources are provided for the application of the strain.

The screening environment of the biocontrol strain is mainly soil, including rhizosphere soil and non-rhizosphere soil. Rhizosphere soil is the most significantly affected soil environment by plant roots, so that the number and types of microorganisms are also significantly higher than non-rhizosphere soil. The rhizosphere microorganism has multiple functions, such as phosphate solubilizing bacteria, azotobacter and other plant growth promoting bacteria, disease resistance and the like, so the rhizosphere microorganism is a resource library for obtaining the microorganisms with multiple functions. According to research reports, researchers screen biocontrol bacteria with antagonistic action on peony root rot pathogenic bacteria (Wangxishan and the like, screening and identification of peony root rot antagonistic bacteria, Shandong agricultural science, vol.44, No. 7 in 2012, Yuzhu and the like, research on 6 biocontrol fungi and bacteria for preventing and treating peony root rot, Shandong forestry science, No. 6 in 2004), but research objects are ornamental peonies, and research on screening of biocontrol bacteria for oil-use peony root rot pathogenic bacteria aiming at seed setting is rarely reported. In order to ensure high and stable yield of the peony for oil, ensure the quality of peony seed oil and reduce the occurrence of root rot of the peony for oil, it is necessary to screen biocontrol microorganisms aiming at the disease and fully excavate and utilize beneficial microorganisms at the rhizosphere.

Disclosure of Invention

The invention aims to provide a strain of disease-resistant growth-promoting salt-tolerant Bacillus (Bacillus halotolerans) LS147 and application thereof, wherein the strain has a strong inhibiting effect on root rot caused by fusarium solani, the strain liquid has a good preventing and treating effect on the root rot of oil peony, and can promote the growth of oil peony plants, so that the fertilizer and pesticide investment can be reduced, the environmental pollution can be reduced, and the sustainable development of agriculture can be realized.

The salt-tolerant Bacillus (Bacillus halotolerans) LS147 disclosed by the invention is a strain separated and screened from rhizosphere soil of healthy 5-year-old oil peony plants in Shanxi Yangzhi area, and has a strong inhibition effect on fusarium solani. The strain grows rapidly on an LB solid culture medium, bacterial colonies are grey white and opaque, after being cultured for 36 hours, wrinkles appear, the bacterial bodies are rod-shaped, have spores and are gram-positive, the bacterial bodies can produce catalase by using starch and gelatin, ammonia is produced, and the V.P. determination is positive. 16S rRNA gene amplification and sequencing analysis are carried out on the strain LS147, and the result shows that the homology of the strain LS147 with Bacillus halotolrans (NR 115929.1 and NR 115063.1) is up to more than 99 percent, and the strain LS147 is far away from other Bacillus. Based on the characteristics, the LS147 strain is named as salt-tolerant Bacillus (Bacillus halotolerans), which is preserved in China general microbiological culture Collection center (address: Beijing city, Chaoyang district, North Chen Xilu No.1, institute of microbiology, China academy of sciences, postal code: 100101) within 3 months and 24 days in 2020, and the strain is detected to survive with the preservation number of CGMCC No. 19502.

The invention further provides application of the Bacillus halotolerans in preventing and treating plant root rot. Preferably, said plant is a peony for oil, further preferably said root rot is a root rot caused by Fusarium solani (Fusarium solani).

When the method is specifically applied, the bacterial liquid of the salt-tolerant bacillus is utilized to irrigate roots of plants. The liquid of the salt-tolerant bacillus is a thallus suspension, and the preparation method is preferably as follows: performing liquid culture on the salt-tolerant bacillus, preferably culturing in an LB liquid culture medium in a shaking table at 28 ℃ and 140rpm for 48-96 h to obtain a fermentation liquid as a thallus suspension, and more preferably diluting the fermentation liquid to 10 ℃ by using sterile distilled water⁸cfu·mL^-1As a suspension of the cells.

The invention also provides application of the Bacillus halotolerans in promoting plant growth. Preferably, the plant is oil peony.

The invention also provides application of the Bacillus halodurans (Bacillus halodurans) in preventing and treating plant diseases caused by Chinese chestnut epidemic pathogenic bacteria (cryptonectria parasitica), bacterial poplar ulcer pathogenic bacteria (Botryosphaeria dothidea), Phytophthora capsici (Phytophthora capsici), Chinese cabbage root rot pathogenic bacteria (Fusarium oxysporum), tomato bacterial spot pathogenic bacteria (Pseudomonas syringae), carrot soft rot pathogenic bacteria (Pectobacterium carotovorum) or/and tomato bacterial wilt pathogenic bacteria (Ralstonia solanacearum).

The implementation verification shows that the salt-tolerant bacillus LS147 has a good prevention and treatment effect on the root rot of the peony for oil, has a good prevention effect, and can promote the growth of the peony for oil. In addition, the salt-tolerant bacillus LS147 disclosed by the invention has a strong inhibiting effect on root rot caused by fusarium solani, also has a strong inhibiting effect on pathogenic bacteria of plague of Chinese chestnuts, pathogenic bacteria of tomato bacterial wilt and pathogenic bacteria of bacterial poplar ulcer, has a strong inhibiting effect on pathogenic bacteria of phytophthora capsici, pathogenic bacteria of root rot of Chinese cabbage and pathogenic bacteria of bacterial spots of tomatoes, has a certain inhibiting effect on pathogenic bacteria of soft rot of carrots, and has an inhibiting effect superior to that of a salt-tolerant bacillus LS10 stored in a laboratory, so that the salt-tolerant bacillus LS147 has a potential preventing effect on various diseases. Therefore, the salt-tolerant bacillus can be used as a biocontrol preparation for preventing and treating plant root rot, particularly oil peony root rot, so that the fertilizer and pesticide investment can be reduced, the environmental pollution is reduced, and the sustainable development of agriculture is realized.

Drawings

FIG. 1 construction of a phylogenetic tree of Fusarium solani based on the ITS sequence using the software MEGA X.

FIG. 2 construction of a phylogenetic tree of Fusarium solani based on mtSSU sequences using software MEGA X.

FIG. 3LS147 colony characteristics.

FIG. 4 LS147 strain 16S rDNA phylogenetic tree of the present invention.

FIG. 5 identification of siderophore production ability of salt tolerant Bacillus.

FIG. 6 growth curves of Bacillus halodurans LS147 strain.

FIG. 7 shows the change of the colonization amount of the double-resistant marker strain in the soil at different times after the root irrigation treatment.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

Example 1 isolation and identification of Bacillus halodurans LS147

1. Source of soil sample

The soil is taken from a peony planting base in Xiangyuan county of Changzhi, Shanxi, selecting healthy peony plants for 5 years of oil, pulling up the plants with roots, brushing the soil adhered to the roots (namely rhizosphere soil) by using a sterile brush after shaking gently, mixing samples of 5 plants into a soil sample, filling the soil into a sterile plastic bag, and taking the sterile plastic bag back to a laboratory to be stored in a refrigerator at 4 ℃.

2. Isolation of Bacillus strains

Placing 10g rhizosphere soil sample in 90mL sterile distilled water, uniformly oscillating for 30min, treating in a high-temperature water bath at 85 ℃ for 30min to kill most of non-spore bacteria, and diluting the supernatant to 10-3, 10-4 and 10-5 times respectively. 100. mu.L of the diluted solution was applied to LB solid medium (NaCl 10g, tryptone 10g, yeast extract 5g, agar powder 20g, distilled water 1000mL, pH 7.0-7.5). Inversely culturing in a constant temperature incubator at 37 ℃, observing the plate after 24-36h, and selecting bacterial colonies with different shapes, colors and sizes to streak on a solid LB plate. And storing the purified strain in a refrigerator at 4 ℃ for further screening of antagonistic bacteria.

3. Collection of pathogenic bacteria

The collection process of pathogenic bacteria is specifically as follows:

1) collecting the peony root rot for oil: the peony is collected from main planting bases of oil peonies in Shanxi Changzhi areas, the variety is Paeonia ostii, and oil peonies with typical root rot symptoms of withered overground parts and rotten and blackened underground parts are collected and taken back to a laboratory after being marked. 2) Culture medium: potato dextrose agar medium (PDA): cutting 200g peeled potato into small pieces, boiling in distilled water, filtering with 8 layers of gauze after the water is boiled, supplementing 1L with distilled water, and adding 20g glucose and 16g agar powder. 3) Separation and purification of pathogenic bacteria: the separation of pathogenic bacteria is carried out by adopting a conventional tissue method: firstly, cleaning the Paeonia ostii diseased roots with tap water, taking the diseased roots at the diseased junction, sequentially treating the diseased roots with 75% alcohol for 30s and 0.1% mercuric chloride for 30-60 s, washing with sterile water for 3 times, cutting the washed peony roots into 4-5 mm squares, putting the squares into a PDA culture medium added with streptomycin (1%), culturing in a 28 ℃ incubator, and storing the strains subjected to monospore separation and purification in a 4 ℃ refrigerator for later use. 4) Determination of pathogenicity of pathogenic bacteria: all the separated strains are subjected to pathogenicity experiments, 3-year-old paeonia ostii with consistent growth vigor and no diseases is used, the skin of the root of the paeonia ostii is punctured by a sterilized inoculation needle, then the root is soaked in a spore suspension of 1 x 108. multidot.ml < -1 > for 30min, and the blank is soaked in sterile water for 30 min. Then planting the paeonia ostii in matrix soil, 2 plants in each pot, treating 10 paeonia ostii seedlings each, repeating for 3 times, then placing in a greenhouse at 25 ℃, and watering every day after inoculation for 48 hours to ensure the disease condition. And (5) investigating the disease degree of the paeonia ostii plants 30 days after planting, and calculating the disease index. 5) And (3) identification of pathogenic bacteria: comprises the steps of observing morphology and identifying molecular biology, inoculating pathogenic bacteria in a potato sucrose agar culture medium (PSA) (the formula is that 200g of peeled potatoes are cut into small pieces, then the small pieces are put into distilled water for boiling, after the water is boiled, 8 layers of gauze are used for filtering, the distilled water is used for supplementing to 1L, 20g of sucrose and 16g of agar powder are added, and the characteristics of bacterial colonies are observed and recorded. Pathogenic bacteria are inoculated in a carnation agar culture medium (CLA) (formula: a plurality of dried and sterilized carnation leaves, agar 20g and distilled water 1L), and the morphological characteristics of conidia are observed by a microscope after the bacteria grow out. Extracting pathogenic bacteria genome DNA by using an OMEGA fungus extraction kit, and adopting a universal primer ITS: (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS 4: 5'-TCCTCCGCTTATTGATATGC-3') and primer mt SSU (NMS1a: 5'-CAGCAGTGAGGAATATTGGTCAATG-3', NMS2b: 5'-GCGGATCATCGAATTAAATAACAT-3') were subjected to PCR amplification. The PCR reaction system is 25. mu.L, 2. mu.L of DNA template, 1. mu.L of each of the upstream and downstream primers, 8.5. mu.L of ddH2O 8.5, and 12.5. mu.L of 2 XTaq PCR Super Mix. PCR amplification procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing (ITS: 55 ℃ for 1 min; mt SSU: 60 ℃ for 1.5min), extension at 72 ℃ for 1min, 35 cycles; further extension was carried out at 72 ℃ for 10 min. The amplification product was checked by 1% agarose gel electrophoresis, and then sent to Biotechnology engineering (Shanghai) Ltd for sequencing, and the sequencing result was subjected to BLAST analysis in NCBI and phylogenetic tree construction using MEGA X software.

As a result: in Shanxi province Long curing area, 22 oil peony 'Paeonia ostii' fields are collected together, 43 pure culture strains are obtained through co-separation, 41 isolates belong to Fusarium solani (F.solani) according to the morphological characteristics of bacterial colonies, the separation frequency is 95.35%, and the isolates are preliminarily identified as 8 Fusarium solani according to microscopic observation. The pathogenicity determination result shows that 4 fusarium solani cause the treated plants to have serious morbidity, and the fusarium solani can be re-separated into the inoculated strains, wherein the strains are respectively numbered as FD1, FD8, FD10 and FD13, the morbidity is 100 percent, and the disease indexes are respectively 86.67 percent, 85.00 percent, 36.67 percent and 71.67 percent. The main symptoms of the diseased plants are that the roots turn black and rot, which is consistent with the field results. And inoculating the roots which have been diseased in the pathogenicity experiment into a PDA (PDA) plate to separate strains, wherein the forms of the separated strains and the inoculated strains are the same under the same nutrition and culture conditions, and the inoculated strains are proved to be pathogenic bacteria of the Paeonia ostii root rot according to the Koehz rule. The above experiments show that the strains FD1, FD8, FD10 and FD13 are pathogenic bacteria of peony for oil 'Paeonia ostii' but have different pathogenicity. The 4 strains of bacteria are subjected to molecular identification, the sequencing results of the bacteria are compared in GenBank, the similarity between the sequence of the bacteria and fusarium solani (F.solani) reaches 98-100% (see the phylogenetic tree in figure 1 and figure 2), and the 4 strains of pathogenic bacteria causing the root rot of the peony for oil are all determined to be fusarium solani (F.solani) by combining morphological observation.

4. Primary screen for antagonistic bacteria

Screening the antagonistic effect of the separated strain by using a plate confronting culture method. In the center of a flat plate of an improved PDA culture medium (namely a mixture of the PDA culture medium and an LB culture medium in a ratio of 1: 18.5g of PDA, 5g of NaCl, 5g of tryptone, 2.5g of yeast extract powder, 10g of agar powder and 1000mL of distilled water), activated pathogenic bacteria (4 Fusarium solani FD1, FD8, FD10 and FD13 which cause the highest incidence rate of oil peony root rot) are selected, a circular pathogenic bacteria block with the diameter of 0.5cm is placed in the center of the flat plate by using a sterilization puncher, the bacillus to be detected is spotted at a position 2.5cm away from the pathogenic bacteria block, 3 types of bacillus are spotted on each flat plate, the bacillus is cultured in an incubator at 28 ℃ for 5-7d, and the inhibition condition of the bacillus to the pathogenic bacteria is observed and recorded every day.

5. Compound sieve for antagonistic bacteria

And (3) rescreening the bacillus which has the inhibiting effect on pathogenic bacteria (FD1, FD8, FD10 and FD13) in the primary screening. Re-screening antagonistic bacteria: and selecting a single colony, inoculating the single colony in an LB liquid culture medium, culturing at 37 ℃ and 150rpm for 24-48h, and using the cultured bacterial liquid for the next plate confrontation experiment. Inoculating activated pathogenic bacteria block with diameter of 0.5cm to the center of improved PDA plate, symmetrically punching holes at two sides 2.5cm away from the pathogenic bacteria block, adding 100 μ L of bacterial liquid into each hole, repeating each strain for 3 times, culturing in 28 deg.C incubator for 5-7 days, measuring the growth condition and antagonistic radius of pathogenic bacteria with vernier caliper, and calculating antibacterial rate. The primary screening result shows that 22 strains with bacteriostasis function on 4 fusarium solani are provided, and the inhibition effect of different antagonistic strains on 4 fusarium solani is different. The results of statistics of the inhibition rates of 4 fusarium solani with strong inhibition effect on 3 strains are shown in table 1. In conclusion, the best bacteriostatic effect is LS147, and the bacteriostatic rate of the LS147 on 4 strains of fusarium solani is 65.73-78.54%.

TABLE 1 inhibition ratio (%) of antagonistic strains against 4 pathogenic bacteria

6. Physical and chemical characteristics of antagonistic bacteria LS147 and 16S rRNA molecular identification

According to the manual of 'common bacteria system identification', morphological observation and physiological and biochemical identification are carried out on the strain LS147 with better effect of antagonizing fusarium solani, and meanwhile, 16S rRNA gene amplification and sequencing analysis are carried out on the strain LS 147. The strain grows rapidly on an LB solid culture medium, bacterial colonies are grey white and opaque, after being cultured for 36 hours, wrinkles appear, the bacterial bodies are rod-shaped, have spores and are gram-positive, catalase can be produced by using starch and gelatin, ammonia is produced, and V.P. determination is positive, and the result is shown in a figure 3 and a table 2. BLAST comparison analysis is carried out on NCBI after 16S rRNA gene sequencing, and the result shows that the homology of the Bacillus halolerans (NR 115283.1) with the Bacillus halolerans can reach up to 100 percent and is far away from other Bacillus. Based on the characteristics, the LS147 strain is named as salt-tolerant Bacillus (Bacillus halotolerans) (figure 4), and the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms 24 months and 24 days in 2020, and is abbreviated as CGMCC (Unit Address: No. 3 of West Lu 1 of Beijing Indoron the south of the morning, institute of microbiology, China academy of sciences, postal code: 100101), and the preservation number is CGMCC No. 19502.

TABLE 2 LS147 Strain morphological characteristics and physiological and biochemical assay results

Note: "+" indicates a positive result, and "-" indicates a negative result.

Example 2 determination of Nitrogen fixation and growth promotion function of Bacillus halodurans LS147

Microorganisms having plant growth promoting effects generally promote plant growth by producing certain growth promoting factors which are secreted into the environment. The growth promoting effect of microorganisms on plants was judged under laboratory conditions by determining whether they produce Indole-3-acetic acid (IAA), siderophore and ACC deaminase.

1. Identification of siderophore production ability

Inoculating the activated halotolerant bacillus LS147 into a CAS culture medium, culturing at 28 ℃ for 48-144 h, and judging whether the strain has siderophore production capacity according to whether orange transparent rings are generated around colonies.

2. ACC dehydrogenase Activity identification

Firstly, streaking activated halotolerant bacillus LS147 into a DF culture medium, carrying out inverted culture at 37 ℃ for 24-36h, transferring a strain in the DF culture medium into an ADF culture medium taking ACC as a unique nitrogen source for culture, and if the strain can normally grow, indicating that the strain can utilize ACC and has ACC dehydrogenase activity; otherwise, it is not.

3. Identification of Nitrogen fixation Capacity

The bacterial strain to be tested is picked into an Ashby nitrogen-free solid culture medium by using a clean toothpick, and the bacterial strain can grow well to indicate that the bacterial strain has nitrogen fixing capacity after passage for 6 generations, and cannot grow out to indicate that the bacterial strain does not have nitrogen fixing capacity.

The various culture media and the formula are as follows:

liquid LB medium: 10g of NaCl, 10g of tryptone and 5g of yeast extract powder, wherein the volume is constant to 1L, and the pH value is 7.0-7.5;

DF culture medium: KH2PO 44 g, Na2HPO 46 g, MgSO4 & 7H2O 0.2.2 g, FeSO4 & 7H2O 0.2.2 g, glucose 2g, gluconic acid 2mL, citric acid 2g, (NH4)2SO 42 g, to volume of 1L, pH 7.2.

ADF culture medium: 3mmol/LACC was used as the sole nitrogen source instead of (NH4)2SO4 in DF medium.

CAS assay medium, including the following solutions:

solution a: 0.012g CAS (chrome azure S) is dissolved in 10mL distilled water, and 2mL1mmol/L FeCl3 solution containing 10mmol/L HCl is added;

solution b: 0.015g of HDTMA (hexadecyltrimethylammonium bromide) was dissolved in 8mL of distilled water;

dye solution c: slowly adding the solution a into the solution b, and slightly shaking to uniformly mix the two solutions to obtain a dye solution c;

10 × MM9 salt solution: 20mL of (Na2HPO4, 30 g; KH2HPO4, 1.5 g; NaCl, 2.5 g; NH4Cl, 5 g; double distilled water, 500mL) and 6.04g of piperazine diethanol sulfonic acid were added to a clean Erlenmeyer flask containing 150mL of double distilled water, after mixing, the pH was adjusted to 6.8 with 50% NaOH, and 3.2g of agar powder was added to obtain a medium d.

The dye solution c, the culture medium d, 1mmol/L CaCl2, 1mmol/L MgSO 4.7H 2O (xylonite, 2014) and 20% glucose are respectively sterilized (115 ℃ and 20min), and after 10% acid hydrolyzed casein is filtered and sterilized, the dye solution, the culture medium d and the CaCl2 are all placed in a 50 ℃ water bath kettle for heat preservation and standby.

Respectively measuring the 0.2mL of 1mmol/L CaCl2, 4mL of 1mmol/L MgSO4 & 7H2O, 6mL of 10% casamino acid and 2mL of 20% glucose, adding the mixture into the culture medium d, adding the dye solution c along the bottle wall, fully and uniformly mixing (but not generating bubbles) to obtain a blue qualitative detection culture medium, pouring 30mL of the blue qualitative detection culture medium into each dish, and placing the blue qualitative detection culture medium in a sterile operation table for later use.

Ashby nitrogen-free solid medium: 10g of mannitol; NaCl 0.2 g; CaCO 35 g; KH2PO40.2g; MgSO40.2g; CaSO40.1g; 18g of agar powder; 1000mL of water.

The result is shown in FIG. 5, an orange-yellow transparent ring is generated around the colony of the bacillus halodurans LS147, which indicates that the bacillus halodurans LS147 can produce a pig carrier; in addition, the strain can grow in an ADF medium only containing ACC as a unique nitrogen source, and the ACC deaminase producing capability is shown; after 6 generations of continuous subculture on the Ashby nitrogen-free solid medium, the halotolerant bacillus LS147 can still grow on the nitrogen-free medium, which shows that the halotolerant bacillus LS147 has the nitrogen fixation capacity.

Example 3 growth and colonization Capacity of Bacillus halodurans LS147

As a bio-control agent, the bio-control agent needs to have a disease resistance capability and a strong growth rate, and can ensure that the bio-control agent can keep the biological activity for a long time after being applied to a field, so that the colonization condition of the halotolerant bacillus LS147 in a growth curve and soil is determined.

1. And (3) measuring growth characteristics: inoculating the seed solution of the halotolerant bacillus LS147 into 20mL LB liquid culture medium with the inoculation amount of 1%, culturing in a shaking table at 37 ℃ and 130r/min, respectively taking the bacterial solution at 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and 72h, measuring the light absorption value (OD590) at 590nm by using a microplate reader, and drawing a growth curve.

2. Determination of colonization ability: firstly, the salt-tolerant bacillus LS147 is marked with rifampicin and ampicillinRifampicin solution was added to a flask containing 20mL of LB liquid medium to give final concentrations of 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL, 120. mu.g/mL, 160. mu.g/mL, 200. mu.g/mL, and 300. mu.g/mL. And (3) sucking 100 mu L of activated bacterial liquid, adding the bacterial liquid into a culture medium containing 10 mu g/mL of rifampicin, culturing for 36-48 h at 28 ℃, sucking 100 mu L of bacterial liquid in the bottle, transferring the bacterial liquid into a culture medium containing 20 mu g/mL of rifampicin, and culturing in sequence, thus screening out the mutant strains with rifampicin resistance. Ampicillin resistance labeling was performed on the mutant strain having rifampicin resistance using the same method, and the strain was gradually cultured in a medium containing 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL of ampicillin, so that the strain was given a double resistance label. The inhibition effect of the double-resistant marker strain is verified by using fusarium solani (FD1, FD8, FD10 and FD13) as a material and adopting a plate confrontation method, so that the original disease resistance of the double-resistant marker strain is maintained. Culturing the above strains to obtain 10⁸And (3) pouring CFU/mL bacterial suspension into a pot plant planted with 2-year-old oil peonies, wherein the addition amount of bacterial liquid in each pot is 50mL, and each pot is treated by 3 pots. Sampling rhizosphere soil at 1 week, 4 weeks, 7 weeks, 10 weeks and 15 weeks after root irrigation, respectively, and taking 10 weeks by adopting a gradient dilution method^-3、10^-4And 10^-5And (3) coating the diluted soil diluent on an LB culture medium containing rifampicin and ampicillin resistance at the same time, culturing for 24-72 h in an incubator at 30 ℃, counting and calculating the number of strains in the soil, and detecting the bacteriostatic ability of the recovered strains to verify the colonization ability of the salt-tolerant bacillus LS147 in the soil.

The results show that the halotolerant bacillus LS147 grows rapidly, the inoculation starts to enter the logarithmic phase after 6h, the stationary phase is reached 18h after the inoculation, and the stable cell number is kept all the time thereafter (see figure 6). The bacterial strain still keeps the bacteriostatic effect after being marked by the double antibody, and the bacteriostatic ability is not obviously changed, and the result is shown in a table 3; the number of the viable bacteria presents a certain descending trend after the root-irrigation treatment of the bacteria liquid is carried out for different time, but the number of the viable bacteria can be stabilized at a certain level after the treatment for 7 weeks, and even if the number of the viable bacteria is maintained at 6 multiplied by 10 per gram of soil after the treatment for 15 weeks⁶A certain number of viable bacteria above CFUEnsures the exertion of disease resistance of the strain and provides guarantee for the application of the strain as a bio-control microbial inoculum (see figure 7).

TABLE 3 change of bacteriostatic rate between LS147 double-resistant labeled strain and original strain

Example 4 antagonistic spectra of Bacillus halodurans LS147

Pathogenic microorganisms of plant fungal diseases and bacterial diseases with relatively common morbidity are selected as experimental materials, the antagonistic bacterial spectrum of the salt-tolerant bacillus LS147 is measured by adopting a plate confrontation method, and another strain of disease-resistant and salt-tolerant bacillus LS10 stored in a laboratory is selected as a control strain. Wherein the pathogenic fungi are: 1. phytophthora capsici (Phytophthora capsicii); 2. pathogenic bacteria of chestnut blight (cryptonectria parasitica); 3. rhizoctonia solani poplarae (Rhizoctonia solani); chinese cabbage root rot pathogen (Fusarium oxysporum). The pathogenic bacteria are: 1. tomato bacterial spot pathogen (Pseudomonas syringae); 2. carrot soft rot pathogen (Pebacteroium carotovorum); 3. tomato bacterial wilt bacteria (Ralstonia solanacearum); 4. bacterial poplar canker (Botryosphaeria dothidea).

The results show (table 4) that the salt-tolerant bacillus LS147 has a strong inhibiting effect on root rot caused by fusarium solani, also has a strong inhibiting effect on pathogenic bacteria of plague of the Chinese chestnut, pathogenic bacteria of bacterial wilt of tomato and pathogenic bacteria of bacterial poplar ulcer, has a strong inhibiting effect on pathogenic bacteria of phytophthora capsici, pathogenic bacteria of root rot of Chinese cabbage and pathogenic bacteria of bacterial spots of tomato, has a certain inhibiting effect on pathogenic bacteria of soft rot of carrot, and has an inhibiting effect superior to that of a salt-tolerant bacillus LS10 stored in a laboratory, thereby showing that the salt-tolerant bacillus LS147 has a potential preventing effect on various diseases.

TABLE 4 salt tolerant Bacillus LS147 bacteriostasis profile

Note: "-" no antagonism; "+" 0< antagonistic radius <3 mm; "+ +" 3< antagonistic radius <8 mm; "+ + + +" antagonizes a radius >8 mm.

Example 5 controlling Effect of Bacillus halodurans LS147 on peony root rot

The method comprises the steps of taking bacillus halodurans LS147 cultured in an LB liquid culture medium for 18 hours as a seed solution, inoculating the seed solution into the LB liquid culture medium in an inoculation amount of 1%, culturing the seed solution in a shaking table at 28 ℃ and 140rpm for 48-96 hours, and diluting the thallus depth of a fermentation liquid to 108cfu/mL with sterile distilled water for later use. Beating pathogenic Fusarium solani FD1, FD8, FD10, FD13 into 7mm diameter blocks with a puncher, mixing, inoculating into PD culture medium, inoculating 8 blocks of bacterial slices per 250ml, culturing in a shaker at 28 deg.C and 160rpm for 7d, centrifuging, filtering with gauze to obtain mycelium, adding into a stirrer, adding sterile water, crushing, diluting with distilled water until spore is 10⁶one/mL for use. The 2-year-old peony plants for oil are taken as test materials, healthy plants with consistent sizes are selected, the loose soil is washed away by clear water, and the plants are transplanted after being washed once by 75% alcohol. 5kg of soil is filled in a plastic pot with the diameter of 24.5cm and the height of 20.5cm, 6 peony seedlings are uniformly planted, and watering is carried out regularly. After two months of seedling revival, the root irrigation method is adopted to inoculate the bacterial strain. A total of 5 treatment groups were set: process 1 (CK): control, 100mL tap water; treatment 2 (R): inoculating 50mL of halotolerant bacillus LS147 into each of the biocontrol bacterium suspension and the clear water; treatment 3(P + R): simultaneously inoculating 50mL of halotolerant bacillus LS147 and 50mL of pathogenic bacteria respectively; treatment 4 (P): inoculating pathogenic bacteria, wherein the pathogenic bacteria spore suspension and clear water are respectively 50 mL; treatment 5(P + C): inoculating pathogenic bacteria, and simultaneously adding 50mL of chemical pesticide hymexazol, pathogenic bacteria spore suspension and 500 times hymexazol dilution. Each treatment was repeated 3 times. Irrigating the roots of the biocontrol bacteria suspension and the hymexazol once every 7 days for 3 times, and harvesting the seedlings after the roots are irrigated for two months in the last time. Counting the morbidity and calculating the disease index; and (4) measuring the plant height, the fresh root weight, the fresh plant weight and other indexes of the plant.

After the 3 rd biological control solution is used for root irrigation, seedlings are harvested after 2 months of continuous planting, the disease occurrence condition of the plants is counted, the results are shown in table 5, the plants do not suffer from diseases in the treatment CK and the treatment R without adding pathogenic bacteria, the plants are completely suffered from diseases in the treatment P with only adding pathogenic bacteria, the root rot is blackened, the diseases are serious, and the disease index is 87.96%. In the treatment P + R added with pathogenic bacteria and halophilic bacillus LS147 and the treatment P + C added with pathogenic bacteria and medicament, the morbidity and disease index are obviously lower than those of the treatment P, the morbidity and disease index of a P + R treatment group are respectively reduced by 55.38% and 60.74%, although the effects are slightly lower than those of the P + C treatment group, the treatment of the P + R and the treatment of the P + C are not obviously different, and the fact that the halophilic bacillus LS147 has a better prevention and treatment effect on the peony root rot for oil is proved.

TABLE 5 prevention and treatment effects of Bacillus halodurans LS147 on peony root rot

The results of the growth promotion effect of the biocontrol bacteria liquid treatment on oil peony plants are shown in table 6, and the indexes of the plants P treated by only adding pathogenic bacteria are obviously lower than those of other treatment groups, which indicates that the occurrence of diseases retards the normal growth of the oil peony plants. The indexes of fresh weight, dry weight, overground plant height and root length of the treated R plant of the salt-tolerant bacillus LS147 are all larger than those of the treated CK, which shows that the salt-tolerant bacillus LS147 has the function of promoting plant growth. Compared with the treatment P added with pathogenic bacteria, the treatment P + R added with the pathogenic bacteria and the halotolerant bacillus LS147 can obviously improve the fresh weight, the dry weight, the height of the overground part plants and the root length of the plants; compared with the treatment P + C by adding pathogenic bacteria and medicaments, the indexes of the treatment P + R are all larger than those of the treatment P + C group, but the indexes of plants do not reach obvious difference. The results show that the salt-tolerant bacillus LS147 has good disease control effect and can promote the growth of oil-used peony plants.

TABLE 6 influence of salt tolerant Bacillus LS147 on oil peony

Claims

1. A strain of salt-tolerant Bacillus (Bacillus halotolerans) with the preservation number of CGMCC No. 19502.

2. Use of Bacillus halotolerans (Bacillus halotolerans) according to claim 1 for controlling root rot in plants.

3. Use according to claim 2, characterized in that: the plant is oil peony.

4. Use according to claim 1 or 2, characterized in that: the root rot is a root rot caused by Fusarium solani (Fusarium solani).

5. The use of claim 4, wherein: the salt-tolerant bacillus liquid is used for irrigating roots of plants.

6. The use of claim 5, wherein: the liquid of the salt-tolerant bacillus is a thallus suspension, and the preparation method is preferably as follows: performing liquid culture on the salt-tolerant bacillus, preferably culturing in an LB liquid culture medium in a shaking table at 28 ℃ and 140rpm for 48-96 h to obtain a fermentation liquid as a thallus suspension, and more preferably diluting the fermentation liquid to 10 ℃ by using sterile distilled water⁸cfu·mL^-1As a suspension of the cells.

7. Use of Bacillus halotolerans (Bacillus halotolerans) according to claim 1 for promoting plant growth.

8. The use of claim 6, wherein: the plant is oil peony.

9. Use of Bacillus halodurans (Bacillus halodurans) according to claim 1 for controlling plant diseases caused by chestnut blight pathogen (cryptonectria parasitica), bacterial poplar ulcer pathogen (Botryosphaeria dothidea), Phytophthora capsici (Phytophthora capsici), chinese cabbage root rot pathogen (Fusarium oxysporum), tomato bacterial spot pathogen (Pseudomonas syringae), carrot soft rot pathogen (Pectobacterium carotovorum) or/and tomato bacterial wilt pathogen (Ralstonia solanacearum).