CN111484944A - Growth-promoting bacillus and application thereof - Google Patents

Abstract

The invention provides growth-promoting bacillus and application thereof, wherein the bacillus is bacillus subtilis FJAT-10626, which is preserved in China general microbiological culture Collection center (CGMCC NO. 16408). The bacillus of the invention can obviously promote the growth of crops, develop the root systems of the crops and enhance the stress resistance of the crops.

Description

Growth-promoting bacillus and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a growth-promoting bacillus strain and application thereof in plant growth.

Background

Plant growth-promoting rhizobacteria (PGPR) are a class of bacteria that colonize the Plant root system and promote Plant growth. In recent years, PGPR has been a research hotspot in the fields of microbiology, phytopathology and the like, and is also a technical place for high-tech enterprises of microbial fertilizers to compete for robbery. By inoculating PGPR, the ineffective nutrition in soil can be validated, crop diseases can be prevented and controlled, the use of pesticides and fertilizers can be reduced, and the method is also a fundamental way for solving the pollution of soil, water sources and foods.

The bacterial strain with the growth promoting function is used as the microbial fertilizer, and has the advantages of full utilization of soil elements, environmental safety and the like. However, growth promoting functional strains are deficient, and screening functional strains is a problem to be solved urgently at present.

Disclosure of Invention

Therefore, it is needed to provide a strain with growth promoting function to solve the problem of lack of the strain with growth promoting function.

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

a growth promoting bacillus, characterized by: the Bacillus is Bacillus subtilis FJAT-10626 with the scientific name of Bacillus subtilis subsp. Inaquosum FJAT-10626, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.16408, the preservation date of 2018, 9 and 3 days, and the preservation address of the institute of microorganisms and research of China academy of sciences, Beijing, China.

The colony morphology of the bacillus FJAT-10626 is as follows: round, smooth surface, rough edges, dry, yellow, opaque, flat larger colonies.

The specific method is that the bacillus fermentation liquor is prepared into the concentration of 1 × 10⁵-2×10⁵cfu/m L, soaking the strain for 2-3 days, and placing at 25-30 deg.C under illumination for 16-20 h/day.

Furthermore, the growth-promoting bacillus is applied to promoting the growth of plant roots.

A plant growth promoting microbial inoculum comprises the bacillus.

The invention has the beneficial effects that:

(1) the bacillus of the invention can improve the activity of plant seeds, promote the seeds to take root and sprout, and effectively shorten the growth cycle of plants.

(2) The bacillus of the invention can obviously promote the growth of crops, develop the root systems of the crops and enhance the stress resistance of the crops.

Drawings

FIG. 1 is a colony morphology of Bacillus FJAT-10626, the left image is the colony growth state of FJAT-10626 strain on the whole plate, and the right image is a partial enlarged view of the colony on the left image.

FIG. 2 is a tree of evolutionary trees of the 16S rRNA sequence identification results of Bacillus FJAT-10626 according to an embodiment.

FIG. 3 shows the effect of Bacillus FJAT-10626 on the growth of tomato seeds.

FIG. 4 shows the growth promoting effect of Bacillus FJAT-10626 on tomato seeds.

FIG. 5 shows the effect of Bacillus FJAT-10626 on the growth of garlic root, wherein the garlic on the left is a bacterial solution group and the garlic on the right is a control group.

Detailed Description

To explain technical contents, achieved objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in combination with specific embodiments.

Example 1 identification of Bacillus

1. Test materials

1.1 test strains

Test strains: the bacillus FJAT-10626 is separated from leaf endophyte of Changchang kumquat in Fujian province, is frozen and preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.16408 at the temperature of-80 ℃.

1.2 culture Medium

L B solid culture medium (purchased from the manufacturer) formula, i.e. 10g tryptone, 10g sodium chloride, 5g yeast powder, 15g agar, 1000m L water, pH 7.0, (2) L B liquid culture medium (purchased from the manufacturer) formula, i.e. 10g tryptone, 10g sodium chloride, 5g yeast powder, 1000m L water, pH 7.0.

2. Test method

2.1 activation of the test strains

Taking out the test strain from a refrigerator at the temperature of minus 80 ℃, after the test strain is warmed to room temperature, streaking the test strain in an ultra-clean bench into a L B solid agar medium plate, inversely placing the test strain in a biological incubator for 2 d.2d at the temperature of 30 ℃, observing the colony morphology, selecting a single colony for secondary streaking culture, ensuring that the activated colony morphology is single, selecting a proper amount of the single colony in a L B liquid medium, and performing shake culture at the temperature of 30 ℃ for 2d to obtain a seed solution.

2.2 Strain morphology and 16s rRNA identification

And (3) strain morphology, namely inoculating the purified bacillus onto an L B plate by streaking, culturing at the constant temperature of 30 ℃ for 48h, and observing the characteristics of the size, the color, the edge uniformity, the wettability and the like of a bacterial colony after the bacterial colony grows out.

The molecular identification is that pure strains are inoculated to L B liquid culture medium, the shaking table is placed at 30 ℃, after the strains are cultured to a logarithmic phase, the genome DNA of the strain FJAT-10626 is extracted by adopting a Tris-saturated phenol method, 16S rRNA gene universal primers 27F and 1492R are adopted for PCR amplification, the PCR reaction program refers to the literature of Zhengxuefang and the like (Zhengxuefang, Liubo, Zhuyanqing, and the like, the screening and identification of the tomato bacterial wilt biocontrol bacillus is [ J ]. Chinese biological prevention and control institute, 2016,32(5):657 and 665.), the PCR products are sent to Shanghai Boshang biotechnology Limited for Sanger sequencing, EZBIOCou is used for completing sequence homology comparison, and the sequence is analyzed by MEGA 6.0.6 software and the system development tree is constructed.

3. Results of Strain identification test

3.1 Strain morphology

The colony morphology of FJAT-10626 is as follows: round, smooth surface, rough edges, dry, yellow, opaque, flat larger colonies. The colony morphology is shown in FIG. 1.

3.216 identification of the S rRNA Gene

The 16S rRNA gene of the strain FJAT-10626 has the following nucleic acid sequence:

ACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAG(SEQ ID NO:1)

comparing the 16S rRNA gene sequence SEQ ID NO 1 of the strain FJAT-10626 with an EZBioCloud gene database, the strain FJAT-10626 has the closest genetic relationship with Bacillus subtilis sp. Downloading 16S rRNA gene sequences of strains with higher homology, carrying out comparative analysis, constructing a phylogenetic tree, and forming the phylogenetic tree as shown in figure 2 when a neighbor-Joining method is adopted and the Bootstrap value is 1000 times. In the constructed phylogenetic tree, the strain FJAT-10626 and Bacillus subtilis sp.

EXAMPLE 2 growth promoting Effect of Bacillus

1. Growth promotion test for tomato

Selecting single colony of test strain, inoculating into 250m L conical flask containing 100m L L B liquid culture, and culturing at 30 deg.C for 48 hr (counting bacteria under microscope, bacterial density is 10%⁸cfu/m L above) the test strain fermentation broth was diluted 1000-fold and clear water was used as control (ck).

Selecting tomato seeds with relatively consistent growth vigor, placing the tomato seeds in a transparent culture box of 9cm with 2-3 layers of filter paper laid at the bottom, placing 15 mung bean seeds in a constant-temperature artificial climate box at 27 ℃, and illuminating for 16h and dark for 8 h. Tracking and observing the tomato germination condition, recording the germination number until no new germination grains appear in 3d continuously, measuring the germ length of the tomato germination radicle machine, and analyzing the promoting effect of the test bacteria on tomato seed germination.

The germination percentage (number of germinated seeds on specified days/number of test seeds) is × 100%

The germination index is ∑ (Gt/Dt), wherein Gt is the number of seeds to be germinated at t d, and Dt is the corresponding number of days to be germinated.

Vigor index (germination index × embryo root length (cm)

The test data adopts DPS software and a new double-polarization method (Duncan) to carry out the significance test of the data difference among treatments.

2. Garlic growth promotion test method

Inoculating a test strain into L B liquid culture medium, culturing for 48h in a shaking table at 30 ℃ and 170rpm to obtain fermentation liquor, diluting the fermentation liquor by 1000 times with clear water, then putting the diluted fermentation liquor into a 150m L conical flask, putting garlic on a bottle mouth to ensure that the bottom of the garlic is soaked by bacterial liquid, taking the clear water as a Control (CK), replenishing water at proper time, culturing for 14d in a laboratory greenhouse at 25 ℃, and measuring the length, fresh weight and dry weight of roots.

3. Test results

3.1 tomato growth promoting Effect

The comparison between the test group and the ck group is shown in Table 1, FIG. 3 and FIG. 4. the experimental results show that the growth effect of tomato seeds in the ck control group is compared, the bacillus FJAT-10626 is diluted 1000 times, namely the concentration is about 1 × 10⁵-2×10⁵The germination rate, the germination index, the root length, the stem length and the seed vitality index of cfu/m L are respectively 107.90%, 83.26%, 318.20%, 134.06% and 264.88% of ck control, the five indexes have significant difference with the ck control, and rhizomes grown from seeds soaked by the bacterial liquid are thicker, so that the tomato seeds are soaked by the fermentation liquid of bacillus FJAT-10626, and the germination and growth of the seeds can be obviously promoted.

TABLE 1 growth promoting ability of Bacillus FJAT-10626 on tomato seeds

Note: in the above table, ab represents significant difference (P <0.05)

3.2 growth promoting Effect of Garlic

The growth of garlic roots is shown in table 2 and fig. 5. The experimental result shows that compared with the CK group, the fresh weight of the root of the garlic in the FJAT-10626 bacteria liquid soaking group is increased by 12.92%, and the dry weight of the root is increased by 14.99%. Experimental results show that the FJAT-10626 bacterial liquid has an obvious effect of promoting the growth of plant roots.

TABLE 2 growth promoting effect of Bacillus FJAT-10626 on garlic root

In conclusion, the bacillus FJAT-10626 has a remarkable growth promoting effect. In the practical application process, spores can be addedThe bacillus fermentation liquor is diluted by at least 1000 times, namely the bacillus concentration is 1 × 10⁵-2×10⁵cfu/m L, soaking seeds for 2-3 days, placing at 25-30 deg.C under illumination for 16-20 h/day to promote plant seed germination, or pouring prepared bacterial suspension into soil to promote plant root growth, or diluting 1000 times fermentation broth, i.e. bacterial concentration 1 × 10⁵-2×10⁵The cfu/m L is used as a plant rooting microbial inoculum to promote the growth of plant roots, the bacillus of the invention has only growth promotion effect and no phosphate solubilizing effect, and can be combined with other functional strains to be prepared into a microbial compound fertilizer.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

Sequence listing

<110> institute of agricultural biological resources of academy of agricultural sciences of Fujian province

<120> growth promoting bacillus and application thereof

<130>10626

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1425

<212>DNA

<213> Bacillus subtilis subsp. inaquosum

<400>1

acatgcagtc gagcggacag atgggagctt gctccctgat gttagcggcg gacgggtgag 60

taacacgtgg gtaacctgcc tgtaagactg ggataactcc gggaaaccgg ggctaatacc 120

ggatggttgt ttgaaccgca tggttcaaac ataaaaggtg gcttcggcta ccacttacag 180

atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc aacgatgcgt 240

agccgacctg agagggtgat cggccacact gggactgaga cacggcccag actcctacgg 300

gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga 360

gtgatgaagg ttttcggatc gtaaagctct gttgttaggg aagaacaagt accgttcgaa 420

tagggcggta ccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc 480

gcggtaatac gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc 540

ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat tggaaactgg 600

ggaacttgag tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga 660

tgtggaggaa caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga 720

aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc 780

taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg 840

gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900

agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcctctgac 960

aatcctagag ataggacgtc cccttcgggg gcagagtgac aggtggtgca tggttgtcgt 1020

cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt 1080

tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg 1140

gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacag 1200

aacaaagggc agcgaaaccg cgaggttaag ccaatcccac aaatctgttc tcagttcgga 1260

tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc 1320

cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa 1380

cacccgaagt cggtgaggta accttttagg agccagccgc cgaag 1425

Claims (5)

1. A growth promoting bacillus, characterized by: the Bacillus is Bacillus subtilis FJAT-10626 with the scientific name of Bacillus subtilis subsp. Inaquosum FJAT-10626, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.16408, the preservation date of 2018, 9 and 3 days, and the preservation address of the institute of microorganisms and research of China academy of sciences, Beijing, China.

2. Use of the growth promoting bacillus of claim 1 to promote germination of a plant seed.

3. The use of Bacillus cereus in promoting germination of plant seed according to claim 2, wherein the Bacillus cereus fermentation broth is prepared to have a concentration of 1 × 10⁵-2×10⁵cfu/m L, soaking the strain for 2-3 days, and placing at 25-30 deg.C under illumination for 16-20 h/day.

4. Use of the growth promoting bacillus of claim 1 for promoting root growth in a plant.

5. A plant growth promoting microbial inoculum is characterized in that: the microbial inoculum comprises the bacillus of claim 1.

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