Bacillus amyloliquefaciens for promoting plant growth and application of bacillus amyloliquefaciens

Abstract

The invention discloses bacillus soluble protein for promoting plant growth and application thereof, the bacillus soluble protein is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO: M20242764, and the preservation date is December 9, 2024. The bacillus amyloliquefaciens Z5078-1 strain provided by the invention is a bacterium separated from soil of a root system of a tea tree, and experiments show that the separated strain has a very strong promoting effect on root length and plant height of plants such as lettuce, Chinese cabbage, rice and soybean, and has a promoting effect on growth of the tea tree and formation of tea leaf quality; and the strain is safe and harmless to plants, can effectively promote plant growth, and is helpful for realizing safe and pollution-free production of the plants.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a proteolytic bacillus capable of promoting plant growth and application thereof. Background Art

[0002] Tea tree (Camellia sinensis L.) is a perennial woody plant and is the first beverage crop discovered and used in my country. At present, more than 50 countries on five continents in the world grow tea, and nearly 3 billion people in more than 160 countries and regions drink tea. The experience of growing and drinking tea was passed on from my country. Tea tree is an acidophilic crop. The soil in the tea garden is usually acidic, and the pH value is usually between 4.5-6.5. Due to its special growth environment, the physical and chemical properties of tea tree soil and the soil microecology undergo directional changes. Tea trees can selectively promote the growth and colonization of functional microorganisms that are beneficial to their own growth. There are many types of rhizosphere microorganisms and a large number of them. There is an exclusive relationship between them and the root system of tea trees based on co-evolutionary pressure. These changes in the microecology of tea tree soil undoubtedly provide the possibility for the screening of plant-beneficial microorganisms. Therefore, screening strains that can promote crop growth in the soil at the root of tea trees is not only a feasible method, but also has important practical application value. Summary of the invention

[0003] The object of the present invention is to provide a proteolytic Bacillus for promoting plant growth and application thereof, so as to solve the problems raised in the above background technology.

[0004] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a protein-soluble Bacillus for promoting plant growth, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO:M20242764 and the deposit date is December 9, 2024.

[0005] Preferably, the culture of Bacillus proteolyticus.

[0006] Preferably, the culture of the proteolytic Bacillus is used to promote plant growth.

[0007] The present invention provides a method for preparing a culture of proteolytic Bacillus, the method comprising:

[0008] Take 2g of fresh water and add 18g of sterile distilled water to make 10 -1 The soil suspension was shaken at 150 r / min for 25 min and then left to rest for 5 min;

[0009] Pipette 1 mL of the supernatant into 9 mL of sterile water and gradually dilute to 10 -4 gradient;

[0010] 300 μL was applied to a solid TSA medium with a diameter of 15 cm and cultured for 2 days;

[0011] A single colony was picked and purified three times using solid LB medium, and stored in a -20° refrigerator with 25% glycerol at a ratio of glycerol: bacterial liquid = 5:2.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The protein-soluble Bacillus Z5078-1 strain provided by the present invention is a bacterium isolated from the soil of the root system of tea trees. Experiments have shown that the isolated strain has a strong promoting effect on the root length and plant height of plants such as lettuce, cabbage, rice, and soybeans, and has a promoting effect on the growth of tea trees and the formation of green tea quality. In addition, the strain is safe and harmless to plants, can effectively promote plant growth, and is helpful to achieve safe and pollution-free production of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the effect of strain Z5078-1 on tea plant growth;

[0015] Figure 2 This is a schematic diagram of the effect of the bacterial solution of strain Z5078-1 on the growth of lettuce and soybean, where A is lettuce and B is soybean;

[0016] Figure 3 This is a schematic diagram of the growth of strain Z5078-1 in nitrogen-fixing medium;

[0017] Figure 4 Schematic diagram of the morphology of strain Z5078-1, wherein A is the Gram staining image of bacterial strain Z5078-1 (10×40), and B is the colony image of bacterial strain Z5078-1;

[0018] Figure 5 The figure is a schematic diagram of 16sRNA sequence amplification of strain Z5078-1, wherein 1 is the target band of 16sRNA of bacterial strain Z5078-1, 2 is DL2 000DNAMarker Lane, and 3 is the standard electrophoresis band of DL2 000DNAMarker Lane;

[0019] Figure 6 This is a schematic diagram of the developmental tree of strain Z5078-1 constructed using the NJ method (neighbor-joining method);

[0020] Figure 7 Schematic diagram of the effect of strain Z5078-1 on the root system of tea trees. DETAILED DESCRIPTION

[0021] In order to further explain the technical solution of the present invention, it is described in detail below through specific embodiments.

[0022] The present invention provides a Bacillus proteolyticus Z5078-1 for promoting plant growth, which is deposited in China Center for Type Culture Collection, with a deposit address of Wuhan University, Wuhan, China, a deposit number of CCTCC NO: M20242764, and a deposit date of December 9, 2024.

[0023] The method for preparing a culture of proteolytic Bacillus of the present invention comprises:

[0024] Take 2g of fresh soil sample, the root soil sample of the tea tree is from Jin Guanyin tea tree in Xibian Village, Xingcun Town, add 18g of sterile distilled water to 10 -1 The soil suspension was shaken at 150 r / min for 25 min and then left to rest for 5 min;

[0025] Pipette 1 mL of the supernatant into 9 mL of sterile water and gradually dilute to 10 -4 gradient;

[0026] 300 μL was applied to a solid TSA medium with a diameter of 15 cm and cultured for 2 days;

[0027] A single colony was picked and purified three times using solid LB medium, and stored in a -20° refrigerator with 25% glycerol at a ratio of glycerol: bacterial liquid = 5:2 to obtain a strain.

[0028] Embodiment 1

[0029] Fermentation broth preparation

[0030] The strain was inoculated into a sterilized 250 mL Erlenmeyer flask containing 100 mL LB medium, and then placed in a shaker at 28°C, 120 r, and cultured for 7 days. The bacterial liquid was then collected for subsequent biological tests.

[0031] Biological test of growth-promoting ability of bacterial strain fermentation broth

[0032] Put a piece of filter paper in the tissue culture bottle, take 5mL of fermentation liquid and 100 times diluted fermentation liquid and add them to the tissue culture bottle, sow 5 lettuces that have just turned white in each tissue culture bottle, use 100 times LB culture medium as control, and culture it in a 28℃ constant temperature incubator, and set up 3 replicates for each experiment. After 3 days of culture, measure the root length and plant height of lettuce. Promotion rate = (treatment-control) / control*100%. The steps of the biological test of weed suppression ability of cabbage and rice as receptors and 100 times diluted fermentation liquid are the same as above.

[0033] Three sprouting soybeans were placed in a planting pot with a diameter of 10.8 cm and a height of 9.8 cm containing vermiculite. Fermentation liquid with a coefficient of 100 was added every two days for three times. The root length and plant height of the soybeans were measured three days after the third addition.

[0034] Embodiment 2

[0035] Functional identification of strains

[0036] 1) Determination of nitrogen fixation ability of strains

[0037] The strain was inoculated in Axubei nitrogen-free medium (glucose 10.0 g, KH2PO4 0.2 g, CaCO3 5.0 g, MgS04·7H2O0.2 g, NaCl 0.2 g, CaSO4 0.2 g, agar 15 g, distilled water 1000 mL, pH 7.0). After 2 days, the growth ability of the strain was observed and the diameter of the strain growth was measured.

[0038] 2) Determination of the ability of strains to degrade inorganic phosphorus

[0039] The strain was inoculated in phosphate-solubilizing medium (glucose 10.0 g, (NH4)2SO4 0.5 g, KCl 0.3 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, MnSO4·4H2O 0.03 g, FeSO4·7H2O 0.03 g, Ca3(PO4)2 8.0 g, distilled water 1000 mL, agar 15 g, pH 7.0-7.5), and after 2 days, the strain was observed to see whether it had a phosphate-solubilizing circle.

[0040] 3) Determination of indoleacetic acid production by strains

[0041] Weigh indole-3-acetic acid (lAA) standard, prepare a mother solution with a concentration of 100 mg·L-1 with grade tertiary water, and dilute it to a standard solution with a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg·L-1 according to the corresponding multiples. Mix the standard solution with Fe-HClO4 colorimetric solution at a ratio of 1:2 (v·v), react in the dark for 30 minutes, and measure the OD550 value.

[0042] To determine the IAA production, pick fresh bacterial moss and place it in LB medium, culture it at 25℃, 180r·min for 7 days. Make the volume of the fermentation liquid in the triangular flask to 50mL, centrifuge it at 10000rpm for 10min, take 1mL of the supernatant and add 2mL of Salkowski's reaction solution. After mixing, react in the dark at room temperature for 30min, and use the blank culture medium after centrifugation as the control, and measure the absorbance of the reaction solution at 530nm. Draw a standard curve with the indoleacetic acid standard, and substitute the measured value into the standard curve to obtain the amount of indoleacetic acid produced.

[0043] Embodiment 3

[0044] Morphological identification

[0045] 1) Morphological observation of colonies

[0046] Inoculate the strain in the middle of the LB solid culture medium plate and culture it continuously at 28°C for 24 hours. Observe the morphological characteristics of the colonies such as shape, size, color, and protrusions every day.

[0047] 2) Microscopic morphological observation

[0048] Use a bacteria needle to pick out a colony that has been cultured on the LB solid medium plate for 2 days, place it on a slide with sterile water in the middle, first gram stain it, then observe its morphology under a microscope and take photos to record:

[0049] (1) Dry and wash the slide before staining, add a drop of sterile water, pick a small amount of culture from the colony with an inoculation loop and evenly spread it on the sterile water until the smear is dry;

[0050] (2) Primary staining: add a drop of crystal violet stain to the coated area, stain for 1 min, and then rinse with distilled water;

[0051] (3) Mordanting: Add a drop of Lugol's iodine solution to rinse away the residual water, cover the coated area for 1 minute, and rinse immediately;

[0052] (4) Decolorization: Dry the residual water and add 95% ethanol to the coated area. Wash until the purple color disappears for 22 seconds and then rinse with water immediately.

[0053] (5) Re-staining: add safranin dye, stain for 3 minutes, and rinse with water;

[0054] (6) After drying, observe with an oil immersion lens;

[0055] (7) Take photos using a microscope.

[0056] Embodiment 4

[0057] Molecular biological identification of bacterial strains

[0058] The genomic DNA of purified bacteria was extracted using the EX1150 bacterial genomic DNA extraction kit. According to the specific primers for bacterial identification, namely the upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', the downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', the strain was subjected to PCR. The PCR amplification system was 50 μL, including 1.0 μL of genomic DNA (20 ng / μL), 5 μL of 10×Buffer (containing 2.5 mM Mg2+), 1.5 μL of upstream and downstream primers (10 μM), 0.5 μL of DNA, 1.0 μL of Taq polymerase (5u / μL), 1.0 μL of dNTP (10 mM), and sterile deionized water was added to 50 μL. The reaction conditions were 95°C 5 min pre-denaturation and then entering the cycle, and the cycle parameters were 95°C 30 s, 58°C 30 s, 72°C 7 min, and 35 cycles. Take 3 μL of PCR product and run it on 1% (mass fraction) agarose gel electrophoresis. After the PCR product was purified by gel recovery kit, it was entrusted to Shanghai Paisono Biotechnology Co., Ltd. for sequence determination, and the sequence information was compared with the data in the NCBI16S database using the NCBI Blast program to obtain the species information with the greatest sequence similarity to the species to be tested, which was the identification result. Phylogenetic analysis was performed using MEGA 5.05 software. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method.

[0059] Embodiment 5

[0060] Strain pot experiment

[0061] Take the tea garden soil and dry it naturally. After grinding, remove the gravel and debris in the soil and sieve it. Weigh 3kg of the sieved fine soil and place it in a flowerpot with a diameter of 15cm and a height of 13cm. Plant 3 one-year-old cinnamon tea seedlings with the same growth in the soil. After 20 days of planting the tea trees, add 100mL of strain dilution adjusted to an OD value of 0.6 with distilled water. The control is 100mL of distilled water. The experiment was repeated 3 times. Add the diluted fermentation liquid once every 3 days, and add the LB culture medium coefficient liquid once for the control. After 30 days of planting, measure the root length and plant height of the tea tree, use a chlorophyll meter to measure the chlorophyll content of the penultimate new leaf of the tea tree, use a photosynthesis meter to measure the photosynthesis of the penultimate new leaf of the tea tree, and scan the root system with a root scanner to measure the biochemical components (water extract, tea polyphenols, caffeine, total catechins, total flavonoids, free amino acids, soluble sugars).

[0062] The biochemical component determination method is briefly described as follows: The water extract determination refers to the national standard GB / T 8305-2013, take 0.5g tea powder and add it to a 100mL conical flask, use a measuring cylinder to measure about 40mL of boiling distilled water and shake it well, and heat it in a boiling water bath for 45min (shake it well in the middle). After taking it out, filter it while it is hot, and then rinse the residue with distilled water 3 times. Put the tea residue and the weighed filter paper into an empty centrifuge tube of known weight, and then place it in a constant temperature drying oven at 85℃. After drying for 5 days, take it out and weigh it. Repeat the experiment three times.

[0063] The determination of tea polyphenols was carried out according to the national standard GB / T 8313-2002. The determination was carried out using the Folin phenol colorimetric method. 0.2g of tea powder was placed in a 100mL conical flask, 5mL of 70% methanol solution at 70℃ was added, and the mixture was extracted at 70℃ for 10min. After repeated extraction twice, the filtrates were combined and the volume was adjusted to 10mL. 1mL of tea soup was taken, 5mL of Folin phenol reagent was added and shaken, and 4mL of sodium carbonate solution was added. The blank solution of the reagent was used as the control, and the absorbance value was measured at a wavelength of 765nm. The experiment was repeated three times.

[0064] The free amino acid determination was carried out according to the national standard GB / T 8314-2013, and the ninhydrin colorimetric method was used. 0.3g of tea powder was added to 45mL of boiling distilled water, and then the mixture was placed in a boiling water bath for 10min. The filtrate was fixed to 50mL. 2mL of tea soup was taken, 0.5mL of phosphate buffer and 0.5mL of ninhydrin aqueous solution were added, and the mixture was heated in a boiling water bath. After 15min, the mixture was taken out, cooled, fixed to 25mL, and shaken. The reagent blank solution was used as a control, and the absorbance value was measured at a wavelength of 570nm. The experiment was repeated three times.

[0065] The determination of flavonoids refers to SN / T 4592-2016. Use a pipette to draw 2 mL of the mother liquor tea made from free amino acids, add 8 mL of 1% aluminum chloride aqueous solution, and shake well. Use the reagent blank solution as a control, measure the absorbance value at a wavelength of 420 nm, and repeat the experiment three times.

[0066] Caffeine determination was carried out according to the national standard GB / T 8312-2013. 2.5 mL of mother liquor tea prepared from free amino acids was pipetted with a pipette, 1 mL of 0.01 mol / L hydrochloric acid and 0.25 mL of alkaline lead acetate solution were added, and the volume was fixed to 25 mL with distilled water, and the volume was shaken and filtered; 5 mL of filtrate was pipetted, 0.02 mL of 4.5 mol / L sulfuric acid solution was added, and the volume was fixed to 10 mL with distilled water, and the volume was shaken and filtered. The reagent blank solution was used as a control, and the absorbance value was measured at a wavelength of 274 nm. The experiment was repeated three times.

[0067] The soluble sugar determination refers to GB / T 37493-2019. Use a pipette to dilute the tea soup made by free amino acids by 5 times, take 2mL and add it to a 50mL volumetric flask containing 16mL anthrone reagent, shake well, heat in a boiling water bath, and cool after 7 minutes. Use the reagent blank solution as a control, measure the absorbance value at a wavelength of 620nm, and repeat the experiment three times.

[0068] The total catechins determination reference determination was carried out by slightly modified GB / T 23193-2017 high performance liquid chromatography method. 1g tea sample was placed in a 200mL beaker, 100mL boiling distilled water was added, and the mixture was placed in a boiling water bath for 30min, filtered, transferred to a 100mL volumetric flask, fixed to volume, and filtered with a 0.45μm aqueous phase filter membrane after shaking. The chromatographic conditions were as follows: chromatographic column: C18 (particle size 5μm, 250mm×4.6mm); mobile phase A was 0.02mol / L potassium dihydrogen phosphate solution, and mobile phase B was 100% acetonitrile. The flow rate was 1.0mL / min; column temperature was 30±0.5℃; injection volume was 5μL; detection wavelength was 210nm. The gradient elution program was: 0-18 min, A:B=87:13; 18-40 min, A:B=70:30; 40-50 min, A:B=70:30. The experiment was repeated three times.

[0069] Experimental Results

[0070] The fermentation liquid of Z5078-1 strain diluted 100 times had a strong promoting effect on the root length and plant height of lettuce, which were 35.25% and 63.19% respectively. For the more sensitive cabbage, the fermentation liquid of the strain diluted 100 times also had a strong promoting effect on its root length and plant height, which were 55.02% and 75.41% respectively, as shown in Table 1.

[0071] The promotion rates of root length and plant height of rice were 59.73% and 10.50%, respectively, and those of soybean were 33.31% and 15.58%, respectively. Figure 1 and Figure 2 shown.

[0072] Table 1 Effects of dilution of Z5078-1 fermentation broth on recipient plants

[0073]

[0074]

[0075] Pot test

[0076] The effects of strain Z5078-1 on the growth of tea trees are shown in Table 2. The results show that the strain has an effect on the growth of tea trees. The strain promotes the root length of tea trees by 11.97%, the plant height by 9.63%, the chlorophyll content of tea leaves by 8.18, and the net photosynthetic rate of tea leaves by 26.23%.

[0077] Table 2 Effect of strain Z5078-1 on tea tree growth

[0078] Root length (cm) Plant height(cm) Chlorophyll content (SPAD) Net photosynthetic rate (μmol·m-1·s-1) Comparison 10.78±0.79 28.25±0.70 27.14±0.93 5.30±0.38 Z5078-1 strain 12.07±0.23 30.97±1.17 29.36±1.18 6.69±0.23 Promotion rate (%) 11.97 9.63 8.18 26.23

[0079] The effect of Z5078-1 strain on the growth of tea tree roots is shown in Table 3. The results show that the strain has an effect on the growth of tea tree roots. The strain increased the total length of tea tree roots by 60.51%, the surface area by 40.09%, the root volume by 22.22, and the root tip by 21.11%. Figure 7 shown.

[0080] Table 3 Effects of strain Z5078-1 on the root system of tea trees

[0081] Total length (cm) Surface area (cm2) Root volume (cm3) root tip Comparison 280.76±5.48 61.80±3.45 1.08±5.33 379±8.15 Z5078-1 450.66±6.36 86.58±3.32 1.32±4.82 459±3.49 Promotion rate (%) 60.51 40.09 22.22 21.11

[0082] The effect of strain Z5078-1 on the biochemical components of tea is shown in Table 4. The results show that the strain has an effect on the biochemical components of tea. The strain treatment increased the water extract by 3.67%, total catechins by 22.06%, total flavonoids by 15.74%, total flavonoids by 15.74%, free amino acids by 25.34%, caffeine by 4.66% and tea polyphenols by 31.55%. The strain treatment can increase the phenol-amino ratio of tea trees, which is beneficial for making oolong tea.

[0083] Table 4 Effects of strain Z5078-1 on the biochemical components of tea

[0084]

[0085]

[0086] Functional analysis of strains

[0087] The analysis of the strain's function showed that the strain could grow in nitrogen-fixing medium, indicating that the strain had nitrogen-fixing ability, but no obvious phosphate-solubilizing ring was seen in the phosphate-solubilizing medium, indicating that the strain's phosphate-solubilizing ability was insufficient. The analysis of the strain's IAA production ability showed that the strain had a strong IAA production ability, and the IAA content in the fermentation broth was as high as 18.27 mg / L.

[0088] Table 5 Functional analysis of strain Z5078-1

[0089] Nitrogen fixation Phosphate solubilization IAA(mg / L) Strain Z5078-1 have none 18.27±2.36

[0090] Strain identification

[0091] The colonies were cultured on LB medium at a constant temperature of 28°C for 2 days, with a diameter of 0.5-1 cm, white colonies, smooth edges, shiny colony surfaces, moist, nasal mucus. Gram staining results showed that the strain was rod-shaped and Gram-positive.

[0092] The results of 16sRNA sequencing of the strain showed that the sequence size was 1424 bp. By comparing homology with the data in NCBI (Blast), the sequence similarity between the strain and Bacillus proteolyticus was more than 100%, and it was inferred that the strain was Bacillus proteolyticus (protein soluble Bacillus).

[0093] The results of morphological and 16sRNA identification showed that the strain was a species of Bacillus proteolyticus. Figure 3 , Figure 4 , Figure 5 , Figure 6 shown.

[0094] The results of 16sRNA gene sequencing of strain Z5078-1 are as follows:

[0095]

[0096] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A proteolytic Bacillus for promoting plant growth, characterized in that: It is deposited in the China Center for Type Culture Collection, with the accession number being CCTCC NO:M20242764 and the deposit date being December 9, 2024.

2. A culture containing the proteolytic Bacillus according to claim 1.

3. Use of a culture containing the proteolytic Bacillus according to claim 2 in promoting plant growth.

4. The method for preparing the culture according to claim 2, characterized in that: The method comprises: Take 2g of fresh soil sample and add 18g of sterile distilled water to 10 -1 The soil suspension was shaken at 150 r / min for 25 min and then left to rest for 5 min; Pipette 1 ml of the supernatant into 9 ml of sterile water and gradually dilute to 10 -4 gradient; 300 μL was applied to a solid TSA medium with a diameter of 15 cm and cultured for 2 days; A single colony was picked and purified three times using solid LB medium, and stored in a -20° refrigerator with 25% glycerol at a ratio of glycerol: bacterial liquid = 5:2.

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