CN113736687B

CN113736687B - Disease-resistant growth-promoting salt-tolerant bacillus and application thereof

Info

Publication number: CN113736687B
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: 2022-12-06
Anticipated expiration: 2041-07-08
Also published as: CN113736687A

Abstract

The invention discloses a salt-tolerant Bacillus (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 microorganisms for biological control of plant diseases, 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 preservation, health care, oxidation resistance, beauty treatment and the like. At present, oil peonies are planted in large areas in Shandong, henan, shanxi, anhui, gansu and other places in China. With the expansion of planting area and the increase of planting years, various diseases of oil peonies also increase year by year, so that the yield of peony seeds is reduced, and the quality of peony seed oil is influenced, wherein root rot, which is a soil-borne disease caused by Fusarium solani, is a serious harm.

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 the 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 (Bacillus spp.) in bacteria has the advantages of capability of producing spores to resist severe conditions, high propagation speed, easiness in soil colonization and the like, and is widely applied. Currently, bacillus capable of inhibiting plant pathogenic microorganisms selected 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: CN 109825457A) in 2019 discloses that a strain of bacillus halodurans (B.halolerans) E40207A2 has a remarkable antagonistic effect on virus diseases caused by potato virus Y; and Chinese patent (publication No. CN 110343641A) discloses that a salt-tolerant bacillus (B.halotolerants) SYW-1 can effectively prevent and treat cotton verticillium wilt, and is favorable for improving the yield and quality of cotton; in addition, a strain of bacillus halotolerans (B.halotolerans) BW9 disclosed in Chinese patent (publication No. CN 110819565A) has strong antagonistic activity, siderophore activity and nitrogen fixation effect, and can effectively inhibit various plant pathogenic bacteria, thereby showing that the bacillus halotolerans has the potential of being 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 significant soil environment affected by plant roots, so that the number and types of microorganisms are also obviously higher than those of 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 (Wang Xueshan and the like, screening and identification of antagonistic bacteria against peony root rot, shandong agricultural science, vol.44, no. 7 in 2012, wuyuzhu and the like, research on 6 biocontrol fungi and bacteria for preventing and treating peony root rot, shandong forestry science and technology, no. 6 in 2004), but research objects are ornamental peonies, and research on screening of biocontrol bacteria against oil 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 control effect on the root rot of peony for oil, and can promote the growth of peony plants 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.

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 Hengzhi 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) can reach 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 salt-tolerant bacillus liquid is a thallus suspension, and the preparation method is preferably as follows: liquid culture is carried out on the halotolerant bacillus, preferably in LB liquid culture medium, in a shaking table at 28 ℃ and 140rpm for 48-96 h, fermentation liquor is obtained as thallus suspension, and more preferably, the fermentation liquor is diluted to 10 ℃ by sterile distilled water ⁸ cfu·mL ^-1 The suspension of the cells was prepared.

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 control 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 at the same time. 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 agent for preventing and treating plant root rot, particularly the root rot of peony for oil, 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 in the number of double-resistant marker strains colonized 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 μ 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). Culturing in an inverted culture chamber at 37 deg.C for 24-36h, observing the plate, selecting colonies with different shapes, colors and sizes, and streaking on 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, decocting in distilled water, filtering with 8 layers of gauze when water is boiled, supplementing 1L distilled water, and adding 20g glucose and 16g agar powder. 3) Separation and purification of pathogenic bacteria: 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 and healthy junction, sequentially treating the diseased and healthy roots with 75% alcohol for 30s and 0.1% mercuric chloride for 30-60 s, washing the diseased and healthy roots with sterile water for 3 times, cutting the washed Paeonia ostii roots into 4-5 mm squares, putting the squares into a PDA culture medium added with streptomycin (1%), culturing the square blocks in an incubator at 28 ℃, and storing the strains subjected to monospore separation and purification in a refrigerator at 4 ℃ for later use. 4) Pathogenicity determination 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 sterilized inoculation needle is used for pricking the epidermis of the root of the paeonia ostii, then the root is placed in a spore suspension of 1 x 108. Multidot.ml < -1 > and soaked in sterile water for 30min as a blank control. 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: 200g of peeled potatoes are cut into small pieces, then the small pieces are placed in 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: (ITS 1: 5) 'TCCGTAGGTGAACCTGCGG-3', ITS4: 5-. 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, and 12.5. Mu.L of 2 XTaq PCR Super Mix. PCR amplification procedure: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing (ITS: 55 ℃ 1min, mt SSU 60 ℃ 1.5 min), extension at 72 ℃ for 1min,35 cycles; further extension was carried out at 72 ℃ for 10min. The amplified products were checked by 1% agarose gel electrophoresis, and then sent to Biotechnology engineering (Shanghai) Ltd for sequencing, and the sequencing results were 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 plant to have serious disease, and can be separated into the connected strains, the numbers of the strains are FD1, FD8, FD10 and FD13 respectively, the disease incidence is 100 percent, and the disease indexes are 86.67 percent, 85.00 percent, 36.67 percent and 71.67 percent respectively. The main symptoms of the diseased plants are that the roots turn black and rot, which is consistent with the field fruiting. And inoculating the roots which have been diseased in the pathogenicity experiment into a PDA (personal digital assistant) plate for strain separation, wherein the isolated strain and the inoculated strain have the same shape and form under the same nutrition and culture conditions, and the inoculated strain is proved to be the 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 the pathogenicity is different. 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 figure 1 and the figure 2 for phylogenetic trees), 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 modified PDA culture medium (namely a PDA culture medium and an LB culture medium 1 are mixed according to a ratio of 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 peony root rot for oil) are selected, circular pathogenic bacteria blocks with the diameter of 0.5cm are taken by a sterilization puncher and placed in the center of a flat plate, the bacillus to be detected is inoculated at a position 2.5cm away from the pathogenic bacteria blocks, each flat plate is inoculated with 3 kinds of bacillus, 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 (4) rescreening the bacillus which has the inhibiting effect on pathogenic bacteria (FD 1, FD8, FD10 and FD 13) in the primary screening. Re-screening antagonistic bacteria: selecting a single colony, inoculating the single colony in an LB liquid culture medium, culturing the single colony for 24-48h at 37 ℃ and 150rpm, and using the cultured bacterial liquid for the subsequent plate confronting experiment. Inoculating activated pathogenic bacteria block with diameter of 0.5cm into improved PDA plate center, 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 5-7d, measuring pathogenic bacteria growth condition and antagonistic radius 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 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 strain has homology with Bacillus halotolerans (NR 115283.1) of 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 morphological characteristics and physiological and biochemical determination results of LS147 Strain

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 the microorganisms on plants is judged under laboratory conditions by determining whether the microorganisms produce Indole-3-acetic acid (IAA), produce siderophores 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 and inoculating 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 ability

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 up to 1L, and the pH value is 7.0-7.5;

DF Medium: KH2PO 4g, na2HPO4 g, mgSO4.7H2O 0.2g, feSO4.7H2O 0.2g, glucose 2g, gluconic acid 2mL, citric acid 2g, (NH 4) 2SO4 2g, constant volume to 1L, pH 7.2.

ADF culture medium: 3mmol/LACC is used as a unique nitrogen source instead of (NH 4) 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 ensure that the two solutions are uniformly mixed with each other to obtain a dye solution c;

10 × MM9 salt solution: 20mL (Na 2HPO4, 30g, KH2HPO4,1.5g, naCl,2.5g, NH4Cl,5g; double distilled water, 500 mL) and 6.04g of piperazine diethanol sulfonic acid were added into a clean triangular flask with 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 culture medium d.

The dye solution c, the culture medium d, 1mmol/L CaCl2,1mmol/L MgSO4.7H2O (xylonite, 2014) and 20% glucose are respectively sterilized (115 ℃,20 min), 10% acid casein hydrolysate is filtered and sterilized, and then all the components are placed in a 50 ℃ water bath kettle for heat preservation and standby.

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

Ashby nitrogen-free solid medium: 10g of mannitol; naCl 0.2g; 5g of CaCO 3; KH2PO 40.2g; mgSO40.2g; 0.1g of CaSO 4; 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 biocontrol microbial inoculum, the biocontrol microbial inoculum needs to have strong growth rate in addition to disease resistance, and ensure that the biological activity of the halotolerant bacillus LS147 can be maintained for a long time after the biocontrol microbial inoculum is 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 (OD 590) at 590nm by using a microplate reader, and drawing a growth curve.

2. Determination of colonization ability: first, two antibiotics, rifampicin and ampicillin, were labeled on Bacillus halodurans LS147, and rifampicin solution was added to the 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. Sucking 100 mul of activated bacterial liquid, adding it to the culture medium containing rifampicin 10 mul g/mL, culturing at 28 deg.C for 36-48 h, sucking 100 mul of bacterial liquid from the bottle, transferring it to the culture medium containing rifampicin 20 mul g/mL, and sequentially culturing to screen out the mutant strains with rifampicin resistance. Ampicillin resistance was given to the mutant strain having rifampicin resistance by 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 marker. The inhibition effect of the double-resistant marker strain is verified by using fusarium solani (FD 1, FD8, FD10 and FD 13) 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 10 ⁸ And (3) pouring CFU/mL bacterial suspension into a pot plant which is used for root irrigation and planting 2-year-old oil peonies, wherein the adding amount of bacterial liquid in each pot is 50mL, and each pot is treated for 3 pots. Respectively at 1 week, 4 weeks and 7 weeks after root irrigationSampling rhizosphere soil in week, 10 week and 15 week, and taking 10 weeks by gradient dilution method ^-3 、10 ^-4 And 10 ^-5 And (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 live bacteria shows a certain descending trend after the root irrigation treatment of the bacteria liquid is carried out for different time, but the number of the live bacteria can be stabilized at a certain level after the treatment for 7 weeks, and the number of the live bacteria can still be maintained at 6 multiplied by 10 per gram of soil even after the treatment for 15 weeks ⁶ Above CFU, certain viable bacteria number ensures the exertion of disease resistance of the strains, and provides guarantee for the application of the strains as the biocontrol microbial inoculum (see figure 7).

TABLE 3 change of bacteriostasis rate of LS147 double-antibody labeled strain and original strain

Example 4 antagonistic spectrum 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 (Pectibacter 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 <3mm; "+ +"3< antagonistic radius <8mm; "+ + + +" antagonizes a radius >8mm.

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

The bacillus halodurans LS147 cultured in an LB liquid culture medium for 18 hours is used as a seed solution, the seed solution is inoculated into the LB liquid culture medium in an inoculation amount of 1 percent, the mixture is cultured in a shaking table at the temperature of 28 ℃ and the rpm of 140 for 48 to 96 hours, and the bacteria depth of a fermentation liquid is diluted to 108cfu/mL by sterile distilled water for later use. Beating pathogenic bacteria Fusarium solani FD1, FD8, FD10, and FD13 into blocks with diameter of 7mm with a puncher, mixing, inoculating into PD culture medium, inoculating 8 blocks of bacterial sheets per 250ml, culturing in shaking table at 28 deg.C and 160rpm for 7d, centrifuging, filtering with gauze to obtain mycelium, adding into a blender, adding sterile water, breaking, diluting with distilled water until spore is 10% ⁶ one/mL for use. Selecting 2-year-old peony plants as test material, selecting healthy plants with consistent size, washing with clear water to remove floating soil, washing with 75% alcohol, and transferringAnd (4) planting. 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 salt-tolerant bacillus LS147, and respectively inoculating the biocontrol bacterium suspension and clear water; treatment 3 (P + R): simultaneously inoculating 50mL of salt-tolerant bacillus LS147 and pathogenic bacteria, wherein the biocontrol bacteria suspension and the pathogenic bacteria spore suspension are respectively; treatment 4 (P): inoculating pathogenic bacteria, wherein the pathogenic bacteria spore suspension and clear water are respectively 50mL; treatment 5 (P + C): inoculating pathogenic bacteria, and simultaneously adding chemical pesticide hymexazol, the pathogenic bacteria spore suspension and 500 times hymexazol dilution each 50mL. 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 time of root irrigation by the biological control solution, seedlings are harvested after 2 months of continuous planting, and the disease occurrence condition of the plants is counted, wherein the results are shown in table 5, in the treatment CK and the treatment R without adding the pathogenic bacteria, the plants do not have disease, in the treatment P only with adding the pathogenic bacteria, the plants are all diseased, the root rot is blackened, the disease is 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 and the treatment of the P + R and the P + C are not obviously different, and the halophilic bacillus LS147 has a better prevention and treatment effect on the peony root rot for oil.

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 the pathogenic bacteria, the fresh weight, the dry weight, the height of the overground part plants and the root length of the plants can be obviously improved by adding the pathogenic bacteria and the treatment P + R of the halotolerant bacillus LS 147; compared with the treatment P + C by adding pathogenic bacteria and medicaments, all 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. Salt-tolerant bacillus strain: (a)Bacillus halotolerans) The preservation number is CGMCC No.19502.

2. The Bacillus halodurans (b) of claim 1Bacillus halotolerans) The application in preventing and treating plant root rot.

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

4. Use according to claim 2, characterized in that: the root rot is caused by fusarium solani (F.), (Fusarium solani) Root rot caused.

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 bacterial liquid of the salt-tolerant bacillus is a thallus suspension.

7. The use of claim 6, wherein: the preparation method of the thallus suspension comprises the following steps: and (3) culturing the salt-tolerant bacillus in an LB liquid culture medium for 48-96 hours in a shaking table at the temperature of 28 ℃ and the rpm of 140 to obtain fermentation liquor as a thallus suspension.

8. The use of claim 7, wherein: diluting the fermentation broth with sterile distilled water to 10 deg.C ⁸ cfu·mL ^-1 As a suspension of the cells.

9. The Bacillus halodurans (b) of claim 1Bacillus halotolerans) Application in promoting plant growth.

10. The use of claim 9, wherein: the plant is oil peony.

11. The Bacillus halodurans (b) of claim 1Bacillus halotolerans) In the prevention and treatment of pathogenic bacteria of chestnut blightCryphonectria parasitica) Bacterial Populus tremulus ulcer pathogenic bacteria: (Botryosphaeria dothidea) Pathogenic bacteria of phytophthora capsici (Leonian Bombycis) ((II))Phytophthora capsici) Pathogenic bacteria of cabbage root rot: (A), (B)Fusarium oxysporum) Bacterial spot pathogens of tomato (A)Pseudomonas syringae) Carrot soft rot pathogen (A)Pectobacterium carotovorum) Or/and tomato bacterial wilt bacterium (Ralstonia solanacearum) The application of the plant disease caused by the plant disease.