CN113801818A

CN113801818A - Bacillus and application thereof

Info

Publication number: CN113801818A
Application number: CN202111129194.1A
Authority: CN
Inventors: 陈玉蓝; 向泉桔; 王勇; 辜运富; 罗琳; 陈强; 赵珂
Original assignee: SICHUAN TOBACCO Corp LIANGSHANZHOU BRANCH
Current assignee: SICHUAN TOBACCO Corp LIANGSHANZHOU BRANCH
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2021-12-17
Anticipated expiration: 2041-09-26
Also published as: CN113801818B

Abstract

The invention discloses bacillus and application thereof, wherein the bacillus is Bacillus pumilus PSB25, and the preservation number is CCTCC NO: the M2020851, 16SrRNA gene sequence is: 1 in SEQ ID NO. The Bacillus pumilus PSB25 can promote the growth of corn plants, increase the number of corn roots, the root length, the plant height, the number of leaves and the leaf area, inhibit plant pathogenic bacteria, increase the phosphorus content in the plants, and has good application prospect in increasing the phosphorus content in tobacco leaves and the growth of the plants.

Description

Bacillus and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to bacillus and application thereof.

Background

The tobacco is an important economic crop in China, and besides being used as a raw material of cigarettes, a large amount of natural chemical substances contained in the tobacco, such as nicotine, protein, solanesol and nicotine, are high-quality chemical and medical raw materials. The microbial agent suitable for tobacco cultivation is excavated, so that the yield and the quality of tobacco leaves can be effectively improved. Phosphorus is closely related to photosynthesis of crops, phosphorus participates in compound processes formed in the photosynthesis, sucrose and starch are synthesized by phosphorus, and substances such as nicotine and oil in tobacco are not synthesized by phosphorus. Therefore, the content and supply conditions of phosphorus and potassium elements in the soil of the tobacco zone have great influence on the growth, yield and quality of tobacco, and the development of the tobacco industry is severely restricted by the lack of available nutrients.

Phosphorus element is one of three essential nutrients for plant growth, 75% of soil is deficient in phosphorus in China, particularly northern calcareous soil, although the total phosphorus content is high and is between 0.57 and 0.79g/kg, most of phosphorus exists in the soil as large amount of free calcium carbonate, most of phosphorus exists in insoluble calcium phosphate salt, the content of effective phosphorus which can be absorbed and utilized by crops is low, 95% of phosphorus in the soil is in an ineffective form, and most of phosphorus is in a form of Ca in the soil²⁺、Fe²⁺、Fe³⁺、Al³⁺Binding forms a poorly soluble phosphate. Therefore, how to develop and effectively utilize the part of the phosphorus fixed by the soil isAt present, the problem to be solved in agricultural production in China is urgently needed.

The phosphate solubilizing microorganism is a special microorganism functional group capable of converting insoluble combined phosphorus in soil into soluble phosphorus which can be absorbed and utilized by plants, and has obvious effects on converting the insoluble phosphorus in the soil, improving the effective phosphorus content in the soil, improving the utilization rate of phosphate fertilizer, promoting the growth of crops and the like. Some plant rhizosphere growth promoting bacteria can secrete organic acid to dissolve insoluble inorganic phosphate or secrete extracellular phosphatase to digest organic phosphorus such as insoluble phosphate ester and the like, so that biologically available phosphorus is released, the content of soluble phosphorus in soil is increased, and plant growth is promoted.

The above background is for the purpose of facilitating an understanding of the present application and is not a prior art which has been disclosed to the general public prior to the present application.

Disclosure of Invention

In view of the above problems, the present invention provides a bacillus strain which can effectively decompose organic and inorganic phosphorus, promote plant growth, and inhibit plant pathogenic bacteria.

The bacillus of the application is named as Bacillus pumilus PSB25, the strain is preserved in China Center for Type Culture Collection (CCTCC) (address: in Wuhan university, Bayihui 299 in Wuhan district, Wuhan City, Hubei province) 12/4 days 2020, and the preservation number is CCTCC NO: m2020851.

The sequence of the 16S rRNA of the Bacillus pumilus PSB25 strain is as follows: 1 in SEQ ID NO.

The microbiological characteristics of the bacillus pumilus PSB25 are: the colony has irregular shape, wrinkle, wavy edge, grey white color, gram positive stain, and the characteristics of dissolving phosphorus, dissolving potassium, producing IAA, and biologically preventing and treating plant diseases.

On one hand, the application also provides a microbial inoculum.

A microbial inoculum, which contains the Bacillus pumilus PSB 25.

In one or more specific embodiments of the present application, the above-described microbial agents are applied to plant growth or plant propagation.

In one aspect, the present application also provides a fertilizer.

A fertilizer contains the Bacillus pumilus.

In one or more specific embodiments of the present application, the fertilizer described above is applied to corn, rice, wheat, or tobacco planting.

In one aspect, the present application also provides a plant growth agent.

A plant growth promoter for promoting plant growth comprises the Bacillus pumilus PSB 25.

In one aspect, the present application also provides a medicament.

A medicament comprising the Bacillus pumilus PSB 25.

In one or more specific embodiments of the present application, the above-described medicament is applied to plant growth or plant propagation.

In one or more specific embodiments of the present application, the above-mentioned medicament is applied to plant diseases caused by late blight bacteria, ralstonia solanacearum, and pyricularia oryzae.

In one or more specific embodiments of the present application, the above-described medicament is for use in inhibiting staphylococcus aureus.

In one aspect, the present application also provides progeny, mutants, and derivatives.

Progeny, mutants and derivatives of the above-mentioned Bacillus pumilus PSB 25.

In one aspect, the application also provides a preparation method of the bacillus pumilus PSB 25.

A method for preparing the Bacillus pumilus PSB25 comprises the following steps:

first tobacco plant selection

Collecting the roots of a plurality of well-grown tobacco plants, putting the tobacco roots into a sterilized sealed plastic bag, attaching a label, and freezing and storing;

separation of phosphorus-dissolving endophytic bacteria of tobacco

Preparing an inorganic phosphorus culture medium and an organic phosphorus culture medium, sterilizing the surface of a tobacco root sample, grinding, coating grinding fluid on a flat plate of the inorganic phosphorus culture medium and the organic phosphorus culture medium, culturing at constant temperature of 30 ℃ for 4-5 days, and selecting a strain with a hydrolysis ring for streak purification;

thirdly, the strain is point-connected to the corresponding inorganic phosphorus culture medium and organic phosphorus culture medium flat plates, the conditions of the transparent rings are observed, the diameter D of the bacterial colony and the diameter D of the transparent ring are measured, and the phosphorus dissolving capacity of the strain is determined according to the D/D value.

In one or more specific embodiments of the present application, the tobacco plant is selected from the Puge town, Puge county, Liangshan, Sichuan.

The invention principle and the beneficial effects are as follows:

the bacillus pumilus PSB25 has high organophosphorus dissolution rate, effective phosphorus content of 176.56mg/L, potassium dissolving amount of 4.94mg/L and IAA yield of 308.84mg/L, and can promote plant growth. Meanwhile, the pesticide composition has antagonistic effect on late blight bacteria, fusarium wilt bacteria, ralstonia solanacearum and staphylococcus aureus of crops. The strain is used as a biological preparation material for crops, especially for tobacco, and has good application value no matter a new biological control microbial inoculum or a growth regulator is developed.

The strain PSB25 can promote plant growth, inhibit plant pathogenic bacteria, and increase the number of plant roots, root length, plant height, leaf number and leaf area.

The PSB25 strain can promote the growth of corn plants, improve the number of corn roots, the root length, the plant height, the number of leaves and the leaf area, inhibit plant pathogenic bacteria, improve the phosphorus content in the plants, and has better application prospect in improving the phosphorus content in tobacco leaves and the growth of the plants.

Drawings

FIG. 1 is a graph showing the phosphate solubilizing effect of a partial phosphate solubilizing bacterium plate;

FIG. 2 is a phylogenetic tree map based on the rRNA gene of tobacco endophyte PSB 2516S according to the present invention;

FIG. 3 is a plate bacteriostasis experiment chart;

FIG. 4 is a PSB25 biochemical profile;

FIG. 5 is a graph of PSB 25-treated maize plant growth, where CK is non-inoculated maize and 25 is PSB 25-inoculated maize.

Detailed Description

The invention will be further explained with reference to the drawings.

Example 1 isolation and characterization of Bacillus pumilus PSB25

1.1 tobacco plant selection

The root of the tobacco in this example was collected from Puji Town, Puge county, Liangshan, Sichuan province. Randomly collecting multiple well-grown tobacco plants, placing into a sterilized sealed plastic bag, labeling, timely taking back to the laboratory in an ice box, and storing in a refrigerator at 4 deg.C.

1.2 isolation of tobacco phosphorus-solubilizing endophytic bacteria

Preparing an inorganic phosphorus culture medium and an organic phosphorus culture medium. Sterilizing the surface of a tobacco root system sample preserved in a refrigerator (4 ℃) of 5g, transferring the tobacco root system sample to a sterile mortar, fully grinding the tobacco root system sample, smearing grinding fluid on inorganic phosphorus culture medium and organic phosphorus culture medium flat plates, culturing the tobacco root system sample at the constant temperature of 30 ℃ for 4-5 days, selecting bacterial strains with hydrolysis rings (shown in figure 1 and shown in table 1) for scribing and purifying, wherein 17 bacterial strains with hydrolysis rings exist.

The 17 strains were further spotted on respective inorganic phosphorus medium and organic phosphorus medium plates, cultured for 7D, and the hydrolysis ring condition was observed, and the colony diameter (D) and the transparent ring diameter (D) were measured, and the phosphorus solubilizing ability was determined from the D/D values (Table 1).

TABLE 117 isolate numbering and its phosphate solubilizing ability prescreening

1.3 identification of Bacillus pumilus PSB25

Endophyte PSB25 isolated from tobacco roots has irregular colony shape, wrinkles, wavy edge, grey white color and gram positive staining. Agarose gel electrophoresis results show that the size of the 16S rRNA gene amplification fragment of the strain is about 1300bp, and the detected sequence is shown as SEQ ID No. 1.

Phylogenetic analysis showed that strain PSB25 has 100% homology to both Bacillus pumilus MF062630 and MF187639, which have been included in NCBI (FIG. 2), and the strain was determined to be Bacillus pumilus based on its physicochemical properties and phylogenetic tree of the 16S rRNA gene.

Based on the above characteristics, strain PSB25 was identified as Bacillus pumilus (Bacillus pumilus PSB 25). The strain is preserved in China Center for Type Culture Collection (CCTCC) (address: Wuhan university, eight paths 299 in Wuchang district, Wuhan City, Hubei province) in 12 months and 4 days in 2020, and the preservation number is CCTCC NO: m2020851.

EXAMPLE 2 Indolylacetic acid (IAA) production assay by endophytic bacteria

Culturing the 17 separated bacteria to logarithmic phase, taking 50 μ L of bacteria liquid in beef extract liquid culture medium (about 3mL) containing 0.5g/L tryptophan (each bacteria liquid corresponds to beef extract liquid culture medium containing 0.5g/L tryptophan), repeating the steps for 3 times, carrying out shaking culture at 28 ℃ and 140r/min for 1.5 days, taking 50 μ L of bacteria liquid in a cleaned white porcelain plate, adding 100 μ L of color developing agent, keeping dark at 25 ℃ for 30min, if pink appears, indicating that IAA is generated, and taking beef extract liquid culture medium containing 0.5g/L tryptophan which is not inoculated as a control.

Continuously culturing the endophytic bacteria producing the indoleacetic acid in a beef extract liquid culture medium containing 0.5g/L tryptophan for 24-48h, centrifuging at 8000r/min for 5min, taking 2ml of supernate, adding 4ml of Salkowski color developing agent, keeping out of the sun at 25 ℃ for 30min, and measuring the absorbance value at 530 nm. The yield (mg/L) of IAA was calculated by using a blank medium as a control and an optical density corresponding to a standard sample of IAA as a standard curve, and the IAA production amounts of different strains are shown in Table 2.

TABLE 2 ability of tobacco rhizosphere to culture endophytic phosphate-solubilizing bacteria to produce IAA

The results showed that 4 of the 17 isolated nicotiana tabacum endophytes had IAA yields of 100mg/L and above, with the PSB25 strain having the highest IAA yield of 308.8 mg/L.

Example 3 Potassium solubilizing ability assay

The results of potassium-solubilizing growth-promoting capability tests on the phosphate solubilizing strains show that the potassium-solubilizing effects of 17 phosphate solubilizing strains are significant (table 3), the potassium-solubilizing rates are all between 0.20% and 0.29%, and the potassium-solubilizing rates of PSB219 and PSB 154 are all over 0.25%, respectively 0.27% and 0.29%.

TABLE 3 determination of the Potassium solubilizing Capacity of the phosphate solubilizing bacteria

Example 4 antibacterial Activity assay of Bacillus pumilus PSB25

The antibacterial activity test of PSB25 is further analyzed by integrating the test data of phosphorus and potassium dissolving and IAA production capability. The plate confronting method is adopted to determine the bacteriostasis of the separated strains to late blight bacteria and rice blast bacteria. The diameter of the zone of inhibition (HD) and the diameter of the Colony (CD) were determined, and the ratio HD/CD was calculated to screen out the strains with inhibitory effect (Table 4). The bacteriostatic action of the isolated strains on escherichia coli, pseudomonas solanacearum and staphylococcus aureus was determined by a blank drug sensitive strip method (fig. 3).

TABLE 4 ability of endophytic bacteria in tobacco roots to combat pathogens

Note: the HD/CD value is between 3 and 4; ++: the HD/CD value is between 2 and 3; +: the HD/CD value is between 1 and 2; -: HD/CD values less than 1.

The results show that the PSB25 strain has better antagonistic effect on late blight bacteria, rice blast bacteria, ralstonia solanacearum and staphylococcus.

EXAMPLE 5 Biochemical characterization of Bacillus pumilus PSB25

Further examination of the growth of PSB25 under different fermentation conditions showed (FIG. 4) that among the selected carbon and nitrogen sources, Bacillus brevis PSB25, which is the best carbon and nitrogen source, was better grown, wherein the best carbon and nitrogen source was corn meal and peptone, respectively. Has better growth in the pH range of 5-8, and the growth is inhibited when the NaCl concentration is higher than 7, and the optimal growth temperature is about 25 ℃.

Example 6 corn potting application

The method comprises the steps of utilizing the internal phosphorus-solubilizing bacteria PSB25 which are obtained by separation and screening from the root of tobacco in the early stage of the experiment and have the functions of resisting diseases and promoting growth and are activated (LB solid culture medium is cultured for 2d at 25 ℃), then inoculating the activated bacteria PSB25 into LB broth culture solution, culturing the activated bacteria PSB in a constant-temperature shaking table at 30 ℃ and 120r/min for 36 hours, adding the activated bacteria PSB into the LB broth culture medium containing glucose as seed solution in a ratio of 1:1, culturing the bacteria PSB in the constant-temperature shaking table at 30 ℃ and 120r/min for 36 hours to enable the number of viable bacteria to reach more than 10 hundred million, and preparing bacterial suspension for later use.

The corn seeds to be tested are subjected to surface sterilization by 0.4 percent sodium hypochlorite, then are washed by sterile water for 4 times to remove sodium hypochlorite, are soaked in sterile water, are cultured in an incubator at 30 ℃ overnight, then are transferred to a sterile gauze for accelerating germination, five uniform corn seeds with uniform growth vigor are planted in 9 flowerpots, 2kg of soil (with the effective phosphorus content of 7.95mg/kg) is respectively filled in each flowerpot, and 100ml of PSB25 microbial inoculum is inoculated to the roots of the tobaccos in a pouring mode. Repeatedly adding the bacterial suspension once every month, irrigating for 4 times totally, culturing in a light culture chamber with sterile water as a Control (CK) until seedlings emerge, transferring to the outdoor, periodically watering for management, and harvesting the plant sample after 45 days. The growth of the plants (e.g., corn plant height, total tobacco leaf count, stem thickness, fresh weight, dry weight) was determined and the results are shown in FIG. 5 and Table 5.

TABLE 5

Note: wherein CK is non-inoculated corn.

As can be seen from FIG. 5 and Table 5, the plant height, stem thickness, dry and fresh weight and leaf number of the tobacco inoculated with the PSB25 microbial inoculum are increased to some extent compared with those of the blank control. The Bacillus pumilus (Bacillus subtilis) PSB25 obtained in the patent has obvious growth promotion effect on corn plants. The total phosphorus content of the tobacco plant inoculated with the PSB25 microbial inoculum is increased compared with that of a control, which shows that the phosphorus content of the plant can be promoted by the obtained Bacillus pumilus PSB 25.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Liangshan corporation, tobacco corporation, Sichuan province

<120> bacillus and application thereof

<160> 1

<210> 1

<211> 1446

<212> DNA

<213> artificial

<400> 1

TCGTCGCGGC TATAATGCAG TCGAGCGGAC AGAAGGGAGC TTGCTCCCGG ATGTTAGCGG 60

CGGACGGGTG AGTAACACGT GGGTAACCTG CCTGTAAGAC TGGGATAACT CCGGGAAACC 120

GGAGCTAATA CCGGATAGTT CCTTGAACCG CATGGTTCAA GGATGAAAGA CGGTTTCGGC 180

TGTCACTTAC AGATGGACCC GCGGCGCATT AGCTAGTTGG TGAGGTAACG GCTCACCAAG 240

GCGACGATGC GTAGCCGACC TGAGAGGGTG ATCGGCCACA CTGGGACTGA GACACGGCCC 300

AGACTCCTAC GGGAGGCAGC AGTAGGGAAT CTTCCGCAAT GGACGAAAGT CTGACGGAGC 360

AACGCCGCGT GAGTGATGAA GGTTTTCGGA TCGTAAAGCT CTGTTGTTAG GGAAGAACAA 420

GTGCAAGAGT AACTGCTTGC ACCTTGACGG TACCTAACCA GAAAGCCACG GCTAACTACG 480

TGCCAGCAGC CGCGGTAATA CGTAGGTGGC AAGCGTTGTC CGGAATTATT GGGCGTAAAG 540

GGCTCGCAGG CGGTTTCTTA AGTCTGATGT GAAAGCCCCC GGCTCAACCG GGGAGGGTCA 600

TTGGAAACTG GGAAACTTGA GTGCAGAAGA GGAGAGTGGA ATTCCACGTG TAGCGGTGAA 660

ATGCGTAGAG ATGTGGAGGA ACACCAGTGG CGAAGGCGAC TCTCTGGTCT GTAACTGACG 720

CTGAGGAGCG AAAGCGTGGG GAGCGAACAG GATTAGATAC CCTGGTAGTC CACGCCGTAA 780

ACGATGAGTG CTAAGTGTTA GGGGGTTTCC GCCCCTTAGT GCTGCAGCTA ACGCATTAAG 840

CACTCCGCCT GGGGAGTACG GTCGCAAGAC TGAAACTCAA AGGAATTGAC GGGGGCCCGC 900

ACAAGCGGTG GAGCATGTGG TTTAATTCGA AGCAACGCGA AGAACCTTAC CAGGTCTTGA 960

CATCCTCTGA CAACCCTAGA GATAGGGCTT TCCCTTCGGG GACAGAGTGA CAGGTGGTGC 1020

ATGGTTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG AGCGCAACCC 1080

TTGATCTTAG TTGCCAGCAT TCAGTTGGGC ACTCTAAGGT GACTGCCGGT GACAAACCGG 1140

AGGAAGGTGG GGATGACGTC AAATCATCAT GCCCCTTATG ACCTGGGCTA CACACGTGCT 1200

ACAATGGACA GAACAAAGGG CTGCGAGACC GCAAGGTTTA GCCAATCCCA CAAATCTGTT 1260

CTCAGTTCGG ATCGCAGTCT GCAACTCGAC TGCGTGAAGC TGGAATCGCT AGTAATCGCG 1320

GATCAGCATG CCGCGGTGAA TACGTTCCCG GGCCTTGTAC ACACCGCCCG TCACACCACG 1380

AGAGTTTGCA ACACCCGAAG TCGGTGAGGT AACCTTTATG AGCCAGCCGC CGAAGTGACA 1440

GAGTCA 1446

Claims

1. The bacillus pumilus PSB25 has the preservation number of CCTCC NO: m2020851.

2. The Bacillus pumilus PSB25 of claim 1, wherein the 16S rRNA sequence of Bacillus pumilus PSB25 is: 1 in SEQ ID NO.

3. The Bacillus pumilus PSB25 of claim 1, wherein the Bacillus pumilus PSB25 has the following properties: the colony is irregular in shape, has wrinkles, is wavy at the edge, is gray white, and is gram-positive.

4. The Bacillus pumilus PSB25 of claim 1, wherein the Bacillus pumilus PSB25 has phosphate solubilizing, potassium solubilizing, and IAA producing properties.

5. The Bacillus pumilus PSB25 of claim 1, wherein the Bacillus pumilus PSB25 has phytosanitary properties.

6. A microbial preparation comprising the Bacillus pumilus PSB25 of any one of claims 1 to 5.

7. The use of the inoculant of claim 6 for plant growth or plant propagation.

8. A fertilizer comprising the Bacillus pumilus PSB25 of any one of claims 1-2.

9. The fertilizer of claim 8 is applied to corn, rice, wheat or tobacco planting.

10. A plant growth promoting agent comprising Bacillus pumilus PSB25 as defined in any one of claims 1 to 5.

11. A medicament comprising Bacillus pumilus PSB25 as described in any one of claims 1-5.

12. The use of the medicament of claim 11 for plant growth or plant propagation.

13. The medicament of claim 11 is applied to plant diseases caused by late blight bacteria, ralstonia solanacearum and magnaporthe grisea.

14. The use of the medicament of claim 11 for inhibiting staphylococcus aureus.

15. Progeny, mutants and derivatives of Bacillus pumilus PSB25 according to any of claims 1-5.

16. A method of preparing the Bacillus pumilus PSB25 of any one of claims 1-5, comprising the steps of:

first tobacco plant selection

separation of phosphorus-dissolving endophytic bacteria of tobacco

Preparing an inorganic phosphorus culture medium and an organic phosphorus culture medium, sterilizing the surface of a tobacco root sample, grinding, coating a grinding fluid on the inorganic phosphorus culture medium and an organic phosphorus culture medium flat plate, culturing at a constant temperature of 30 ℃ for 4-5 days, and selecting a strain with a hydrolysis ring for streak purification;

17. The method of preparing the bacillus pumilus PSB25, according to claim 16, wherein the tobacco plant is selected from the townsk, pregen, pregabane, liangshan, sichuan.