CN112111427A

CN112111427A - Bacillus subtilis PA8 and application thereof

Info

Publication number: CN112111427A
Application number: CN202011023243.9A
Authority: CN
Inventors: 马连杰; 廖敦秀; 张慧; 卢文才; 杭晓宁; 胡留杰; 张健; 李燕; 余端; 梁涛
Original assignee: Chongqing Academy of Agricultural Sciences
Current assignee: Chongqing Academy of Agricultural Sciences
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-12-22
Anticipated expiration: 2040-09-25
Also published as: CN112111427B

Abstract

A bacillus subtilis PA8 is characterized in that: the Bacillus subtilis PA8 is preserved in China center for type culture collection, is classified as Bacillus subtilis, has a preservation date of 2020, 8 months and 15 days, and has a preservation number of CCTCC M2020387. The bacillus subtilis PA8 has the capability of efficiently synthesizing IAA, and the yield of the IAA can be as high as 187 mg/L; the plant growth promoting agent has excellent siderophore production capacity and strong inhibition effect on fusarium oxysporum, can synergistically promote growth of vegetable plants through the multiple effects, can obviously improve seedling emergence amount when applied to a seedling raising substrate, and has excellent growth promoting effect on the vegetable plants.

Description

Bacillus subtilis PA8 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a bacillus subtilis PA8 and application thereof.

Background

The Plant Growth Promoting Rhizobacteria (PGPR) generally refers to a type of Rhizobacteria which grow in the rhizosphere soil environment or inside the root system, have the capability of Promoting Plant Growth or increasing crop yield and inhibiting diseases and pests, and have special significance for overcoming soil stress and improving the micro-ecological environment. The rhizosphere growth-promoting bacteria increase nutrient absorption of plants and promote plant growth mainly by synthesizing plant hormones such as indoleacetic acid (IAA) and the like and changing element forms by dissolving phosphorus, dissolving potassium, fixing nitrogen, producing siderophores and the like. PGPR found at home and abroad at present comprises more than 20 species such as pseudomonas, bacillus, agrobacterium, flavobacterium, serratia and the like, and the research on the PGPR is more and more emphasized, and a professional workshop is opened every 3 years internationally.

The vegetable plug seedling is an important link for the scale and intensive development of vegetable production, and is the first choice for vegetable planting households and industrial seedling raising fields due to the advantages of improving the seedling quality, increasing the transplanting and colonization rate and the like. With the wide application of plug seedling technology, the market demand of seedling substrate is increasing. The stable and coordinated water, air and fertilizer in the seedling raising substrate become a nutrition bank of the seedling root system, and the nutrients and water needed by the root system are ensured. At present, the research on the seedling substrate mainly focuses on the aspects of raw material selection and proportion, and few researches on how to cultivate disease-resistant, growth-promoting and healthy seedlings and improve the quality and disease resistance of the seedlings are carried out. The invention takes a common seedling substrate as a material, and prepares a functional biological seedling substrate by adding biocontrol bacteria with obvious growth promotion effect, thereby achieving the purposes of improving the quality of seedlings, strengthening the seedlings and strengthening the seedlings.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a bacillus subtilis strain which can efficiently produce IAA, has excellent siderophore production capacity and can efficiently inhibit fusarium oxysporum.

The purpose of the invention is realized by the following technical scheme:

the invention provides a Bacillus subtilis PA8 which is preserved in China center for type culture collection, the address is Wuhan university, Wuhan, China, and the classification is Bacillus subtilis PA8, the preservation date is 2020, 7 and 31 days, and the preservation number is CCTCC NO: m2020387.

The colony morphology of the bacillus subtilis PA8 is called as crinkled and round, the surface is rough and lusterless, the lawn is white, and the lawn is reddish brown after being cultured for a period of time, and the result of the biochemical determination is as follows: gram-positive bacteria.

The bacillus subtilis has strong capability of synthesizing IAA, has excellent capability of producing siderophore, and can efficiently inhibit the survival of fusarium oxysporum.

Further, the bacillus subtilis PA8 is applied to a microbial inoculum for promoting IAA synthesis, siderophore synthesis and inhibiting one or more functions of fusarium oxysporum.

The application of the bacillus subtilis PA8 is characterized in that: in particular to the application in a functional vegetable seedling substrate.

The bacillus subtilis PA8 can be used in a seedling biological matrix, and specifically, the bacillus subtilis PA8 is inoculated into a common seedling matrix and uniformly mixed to form a new seedling matrix, the ratio of the inoculation amount of the bacillus subtilis PA8 to the dry weight of the matrix is 5mL/100g, and the concentration of the bacillus subtilis PA8 is 10⁸CUF/mL。

The invention has the following technical effects:

the bacillus subtilis PA8 separated and screened by the method has the capability of efficiently synthesizing IAA, and the yield of the IAA can be as high as 187 mg/L; the plant growth promoting agent has excellent siderophore production capacity and strong inhibition effect on fusarium oxysporum, can synergistically promote growth of vegetable plants through the multiple effects, can obviously improve seedling emergence amount when applied to a seedling raising substrate, and has excellent growth promoting effect on the vegetable plants.

Drawings

FIG. 1: colony morphology of PA8 on LB plates.

FIG. 2: strain PA8 was cultured on a CAS plate (right and left panels, respectively).

FIG. 3: PA8 shows the bacteriostasis effect of Fusarium oxysporum GF12 (the left is a comparison graph, and the right is a bacteriostasis graph).

FIG. 4: the effect of 5mL of functional bacteria PA8 in 100g of substrate on the emergence of cucumber seedlings when being inoculated into the biological substrate (the left is CK group, and the right is PA8 group).

FIG. 5: the growth promoting effect of cucumber seedlings by inoculating 5mL of functional bacteria PA8 in 100g of matrix into the biological matrix (the left is PA8 group, and the right is CK group).

FIG. 6: the growth promoting effect of the pakchoi by inoculating 5mL of functional bacteria PA8 in 100g of the matrix into the biological matrix (the upper is CK group, and the lower is PA8 group).

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations to the present invention based on the above-described disclosure.

Example 1: separation and purification of bacterial strains

1. Culture medium

LB culture medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 20g of agar powder and 1L of distilled water, and sterilizing at 120 ℃ for 20 minutes.

2 test procedure

2.1 preparation of the culture Medium

LB medium as described in 1 was prepared, and liquid medium and slant medium were prepared.

2.2 preparation of soil Diluent

The soil sample is collected from a paddy field in Ping Anxiang, Fengjie, Chongqing city, 5g of the soil sample is weighed, placed into a triangular flask filled with 45mL of sterile water, shaken at 180rpm for 30min by a shaking table, and placed into a water bath kettle at 80 ℃ for boiling for 20min to kill other bacteria. Preparing soil suspension, and preparing concentration gradient of 10 times by 10-fold dilution method^-2、 10^-3、10^-4The soil suspension sample of (2).

2.3 dilution coating

Get 10^-2、10^-3、10^-4The soil dilution is 100 mu L, the soil dilution is coated on an LB culture medium, each gradient is repeated for 3 times, the culture is carried out at the temperature of 30 ℃, single colonies with different phenotypes are picked after the culture is carried out for 1 to 2 days, and the single colonies are purified and stored by a flat plate.

Example 2: determination of auxin and screening and identification of the Strain PA8

1 culture Medium

King medium (King): 20g of egg white and MgSO₄·7H₂O 1.5g，K₂HPO₄1.15g, 15mL of glycerin, 1L of distilled water and pH6.8 are used for measuring the IAA production capacity.

S2 colorimetric solution: FeCl₃ 4.5g，10.8mol/L H₂SO₄1L of the reagent is measured in the range of 5-200ug/mL, and generally more than 10ug/mL requires dilution of the colorimetric solution.

2 test procedure

The method for measuring the auxin by adopting a Salkowski colorimetric method comprises the following specific steps:

(1) qualitative determination of plant growth hormone IAA: inoculating the separated and purified bacterial suspension into the King culture medium (King) according to 1% of the volume of the culture medium, carrying out shake culture on a constant-temperature shaking table at 30 ℃ and 180r/min for 7d, centrifuging at 12000r/min for 10min, taking 0.1mL of supernatant, dripping the supernatant onto a detection plate, adding an equal amount of S2 colorimetric solution, taking 0.1mL of S2 colorimetric solution, respectively adding 50 mg/L, 30 mg/L and 10mg/L of standard plant growth hormone IAA (IAA) as gradient control, standing at room temperature for 15min, observing color change, and determining whether the strain has the capability of secreting IAA.

(2) Quantitative determination of plant growth hormone IAA: and (3) adding the supernatant capable of secreting IAA strains into an equal amount of S2 colorimetric solution, standing in the dark for 30min, measuring absorbance at 535nm by using an ultraviolet spectrophotometer, and calculating the IAA secretion amount of each strain according to an IAA standard curve. A strain PA8 with excellent growth promoting capability is obtained through qualitative measurement and quantitative measurement, in general, the L-Trp can be added to promote the Bacillus subtilis to synthesize IAA, in the case of no L-Trp, the IAA production of the strain PA8 is 187mg/L, and the strain is reserved for further study. In the measurement process, the detection is carried out after the inoculation culture is carried out by using other culture media, the detection result is not obviously changed, and the IAA production amount is maintained at 187 +/-2 mg/L.

3 identification of the Strain

Bacterial colony cultured by the strain PA8 through a plate is crumpled and round, has rough and lusterless surface and white lawn, and is reddish brown after being cultured for a period of time. The strain is subjected to biochemical identification, and the determination result is as follows: gram-positive bacteria, positive in catalase test, starch hydrolysis, cellobiose, fructose, mannose, glucose, maltose, sucrose and salicin test; the citrate test, the tyrosine hydrolysis, the xylose, the lactose, the galactose and the test are all negative.

The result of the 16SrDNA gene sequence determination of the strain PA8 is shown in a figure 2, the 16SrDNA gene sequence is subjected to Blast result analysis, the similarity of the strain and the Bacillus subtilis strain is up to 99%, and the strain can be classified as Bacillus subtilis PA8 according to the result of the 16SrDNA gene sequence determination.

Example 3: determination of siderophore production Capacity

1 culture Medium

CAS medium: each 100mL of the solution containing 20% of sucrose and 3mL of 10% acid-hydrolyzed casein, 1mmol/L of CaCl₂100μL，1mmol/L MgSO₄2mL of agar and 1.8g of agar were added slowly at about 60 ℃ to 5mL each of the phosphate buffer and the CAS stain to obtain CAS blue medium.

Table 1: 0.1mol/L phosphate buffer (pH6.8), diluted 10-fold at the time of use.

NaH₂PO₄·2H₂O	0.5905g
		KH₂PO₄	0.075g
NH₄Cl	0.250g
		NaCl	0.125g
Deionized water	The volume is up to 100mL

Detection of siderophore CAS dye liquor:

solution A: 0.079g CAS (chrome azure) is dissolved in 50mL deionized water, and 10mL of 1mmol/L FeCl is added₃Solution (containing 12 mmol/LHCl);

solution B: 0.069g of cetyltrimethylammonium bromide (HDTMA) was dissolved in 40mL of deionized water;

CAS blue assay (100 mL): slowly adding the solution A into the solution B along the wall of the beaker, and uniformly stirring to obtain the product.

2 test procedure

Selecting a single bacterial colony from a PA8 strain activated on an LB solid slant culture medium, inoculating the single bacterial colony into an LB deferrization liquid culture medium, repeating for 3 times, placing the single bacterial colony in a 28 ℃ microbial incubator for culturing for 2-3 days, selecting the single bacterial colony, transferring the single bacterial colony into the LB liquid culture medium, carrying out shake culture at 180rpm for 48 hours, sucking a bacterial liquid by a liquid transfer gun, inoculating the bacterial liquid on a CAS detection plate, carrying out inversion culture at a constant temperature of 30 ℃, inoculating 4 bacterial colonies/dish, repeating for 3 times, and culturing for 5 days at a temperature of 28 ℃. Observing the cultured CAS test plates, a distinct orange to yellow siderophore halo appears around the siderophore-secreting bacterial colonies. The strength of the siderophore production ability of each strain is determined by comparing the size of the transparent ring on the flat plate.

3 product siderophore functional assay

3.1 the functional qualitative determination of the siderophore: after the strain PA8 was grown on the CAS detection plate for 5D, a yellow transparent ring was clearly observed around the strain, as shown in FIG. 3, the diameter (D) of the transparent ring was 1.8cm, the diameter (D) of the strain was 1.1cm, and the D/D was 1.64. Therefore, the strain PA8 has stronger siderophore production capacity.

3.2 quantitative determination of siderophores: the relative content of the siderophore is A/Ar measured by adopting a quantitative detection method of the siderophore of the water-soluble pigment producing bacterium described by Wangping et al, and the smaller the value is, the stronger the siderophore synthesis ability is.

The PA8 lawn is taken and transferred into a sterilized MKB liquid culture medium (5 g of casamino acid, 15mL of glycerol, K2HPO42.5g, MgSO4 & 7H2O 2.5.5 g, 1L of double distilled water, pH value of 7.2), is subjected to shake cultivation at 28 ℃ for 48H and then centrifugation, 3mL of supernatant is added with a certain amount of CAS detection solution with the volume ratio of 1:1, the mixture is fully mixed and stood for 1H, and then the absorbance value (A) at the wavelength of 630nm is measured by a spectrophotometer. And taking double distilled water for adjusting zero by contrast. Another 3mL of the uninoculated medium was mixed with 3mL of CAS detection solution, and absorbance at a wavelength of 630nm was measured by the same method as a reference value (Ar), the value of a/Ar was 0.38, and the active carrier unit of PA8 was 61.94% by calculating the siderophore activity unit Su ═ [ (Ar-a)/Ar ] × 100.

Example 4: determination of the ability to inhibit Fusarium oxysporum

1 culture Medium

A PDA culture medium is selected for carrying out a bacteriostatic test, and the formula of the culture medium is as follows: 200g of potato, 20g of glucose, 15-20g of agar, 1000mL of distilled water and natural pH.

2 test procedure

Inoculating activated soybean root rot pathogen fusarium oxysporum to the center of a PDA (personal digital assistant) plate, uniformly punching 4 holes at a position 2.5cm away from the center of the plate, respectively adding 50 mu L of PA8 bacterial suspension with OD600 of 0.1, adding an equal amount of sterile water in a contrast, transferring the plate to a 28 ℃ incubator for about 5 days, and observing the bacteriostatic effect.

Determination of bacteriostatic ability

As shown in FIG. 4, a significant inhibitory effect of strain PA8 on Fusarium oxysporum can be seen on the plates.

Example 5: biological matrix with strain PA8 as functional bacteria

1 Strain culture

Culturing the strain: PA8 was inoculated into a 500ml Erlenmeyer flask containing 300ml LB medium and shake-cultured at 30 ℃ and 180rpm for 48 hours for use.

After shaking culture of strain PA8, centrifuging at 8000rpm and 4 deg.C for 10min, washing with tap water for 2 times, resuspending strain PA8 in sterile water, adjusting concentration to 10⁸CUF/mL。

2 development of the substrate

2.1 seedling raising effect of biological matrix using strain PA8 as functional bacteria on cucumber

The seedling test treatments were as follows: (1) a normal seedling substrate (CK); (2) the bacterial strain PA8 after being resuspended in sterile water is inoculated into a common seedling substrate (PA8) according to the addition of 5mL per 100g of dry substrate weight, each treatment is repeated for 25 times, and the related growth indexes are respectively measured after about 20 days: plant height, root length, stem thickness, leaf length, leaf width, fresh weight of overground part, root weight.

Table 2 shows the effect of 20 days of biological substrate seedling on the growth of cucumber in seedling stage

Note: different letters in the same column indicate significant differences at the 0.05 level.

In a seedling culture test, compared with a common seedling culture substrate (CK), the seedling culture biological substrate (PA8) prepared by adding the functional bacteria PA8 has a remarkable influence on seedling emergence of cucumber seedlings, as shown in figure 4, a plurality of plug trays do not have seedling emergence in a substrate plug tray of a control group, and the seedling culture biological substrate added with the functional bacteria PA8 has seedling emergence phenomenon in each plug tray, the seedling emergence amount of the cucumber seedlings is remarkably superior to that of the control group, the seedling culture biological substrate has a remarkable growth promoting effect on the growth of the cucumber seedlings, indexes such as plant height, root length, stem thickness, fresh weight of overground parts, root weight, leaf area and the like are remarkably increased, and the indexes are respectively increased by 86.23%, 25.92%, 77.5%, 64.21%, 211.8% and 120.93% in sequence.

2.2 seedling raising effect of biological matrix using strain PA8 as functional bacteria on Chinese cabbage

The seedling test treatments were as follows: (1) a normal seedling substrate (CK); (2) the strain PA8 after being resuspended in sterile water was inoculated into a normal seedling substrate (PA8) in an amount of 5mL per 100g of the dry weight of the substrate. The cucumber seeds are sterilized, soaked and germinated, and are embedded into a treated substrate after white exposure, and each treatment is repeated for 50 times. The plant height, stem thickness, leaf number, root length and fresh weight of overground part of the plant are respectively measured about 12 days.

Table 3: the influence of the biological matrix seedling culture for 12 days on the growth of the cabbage seedling stage.

Treatment of	Plant height (cm)	Stem diameter (mm)	Number of blades (sheet)	Root length (cm)	Fresh weight on ground (g)
						CK	11.71a	2.75a	5.3a	5.81a	6.60a
PA8	15.26b	3.20b	5.7a	7.06b	9.57b

In the cabbage seedling tray seedling test: compared with the common seedling raising substrate (CK), the seedling raising biological substrate (PA8) prepared by adding functional bacteria has obvious growth promoting effect on the growth of the cabbage seedlings, and other growth indexes except the number of leaves have obvious difference (Table 3 and figure 6).

2.3 seedling raising effect of biological matrix using strain PA8 as functional bacteria on tomato

The seedling test treatments were as follows: (1) a normal seedling substrate (CK); (2) the strain PA8 after being resuspended in sterile water was inoculated into a normal seedling substrate (PA8) in an amount of 5mL per 100g of the dry weight of the substrate. Tomato seeds were sterilized, soaked for germination, exposed to white, and embedded in treated substrate, with 50 replicates per treatment. The plant height, stem thickness, leaf number, root length and fresh weight of overground part of the plant are measured after about 40 days.

Table 4: the influence of the biological matrix seedling culture on the growth of the tomato in the seedling stage.

In the tomato seedling tray seedling test: compared with the common seedling raising substrate (CK), the seedling raising biological substrate (PA) developed by adding the functional bacteria has significant differences on the plant height, stem thickness and underground fresh weight of tomato seedlings (Table 4).

To sum up, in the seedling tray seedling test: compared with the common seedling substrate (CK), the seedling biological substrate (PA) developed by adding functional bacteria has obvious growth promotion effect on the growth of cucumber seedlings, Chinese cabbage and tomatoes, and the growth indexes of parts of the seedlings are obviously different.

Sequence listing

<110> agriculture academy of sciences of Chongqing City

<120> bacillus subtilis PA8 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1454 bp

<212> DNA

<213> Bacillus subtilis

<400> 1

gggcatggcg ggtgctataa tgcgatcgag cggacagatg ggagcttgct ccctgatgtt 60

agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120

aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata aaaggtggct 180

tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240

ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360

ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420

aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540

gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600

gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720

ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta wttcraagca acgcgaagaa cccttaccag 960

gtcttgacat cctctgacaa tcctagaaga taggacgtcc ccttcggggg cagagtgaca 1020

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200

cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca 1260

aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320

taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttagga gccagccgcc 1440

gaagtgacag atgt 1454

Claims

1. A bacillus subtilis PA8 is characterized in that: the bacillus subtilis PA8 is preserved in China center for type culture Collection and is classified as bacillus subtilis (A)Bacillus subtilis) The preservation date is 2020, 8, 15 days, and the preservation number is CCTCC M2020387.

2. The use of Bacillus subtilis PA8 of claim 2 in a microbial inoculum for promoting IAA synthesis, siderophore synthesis and inhibiting one or more effects of Fusarium oxysporum.

3. The use of bacillus subtilis PA8 as set forth in claim 1, wherein: in particular to the application in a functional vegetable seedling substrate.

4. The use of bacillus subtilis PA8 as claimed in claim 2 wherein: specifically, bacillus subtilis PA8 is inoculated into a common seedling substrate and uniformly mixed to form a new seedling substrate, the ratio of the inoculum size of the bacillus subtilis PA8 to the dry weight of the substrate is 5mL/100g, and the concentration of the bacillus subtilis is 10⁸ CUF/mL。