Phosphorus-solubilizing and / or growth-promoting cellulose-decomposing strain NJAU-PS1 and application thereof

Abstract

The application provides a cellulose-decomposing strain NJAU-PS1 with phosphorus solubilization and / or growth promotion and application thereof. The cellulose-decomposing strain NJAU-PS1 is deposited in the China General Microbiological Culture Collection Center, located at No. 1, Xibei Road, Yard 3, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on September 25, 2023, and has a deposit number of CGMCC No. 28551. The strain has the functions of active phosphorus, cellulose degradation and growth promotion, can effectively promote straw decomposition and improve the phosphorus content in soil, and has a significant growth promotion function on crops such as rice.

Description

Technical Field

[0001] This invention relates to a biological and environmentally friendly method, which studies a cellulose-decomposing strain NJAU-PS1 with phosphorus solubilization and / or growth-promoting functions and its applications. Background Technology

[0002] Data shows that my country produces approximately 700 million tons of agricultural waste straw annually, accounting for 20% to 30% of the world's total, making it the world's largest straw producer. Straw resources are rich in carbon, nitrogen, potassium, phosphorus, and other trace elements, and returning straw to the field is an effective agronomic measure for soil improvement and fertilization in agricultural production. However, due to its complex molecular structure, straw is difficult to degrade naturally, and its cellulose utilization rate is generally low. The degradation of straw cellulose plays a crucial role in the carbon cycle of agricultural soils, particularly in effectively increasing soil organic matter content. Cellulose-degrading microorganisms effectively degrade cellulose by secreting cellulase, which is not only highly effective but also environmentally friendly. The population of cellulose-degrading microorganisms is extensive, and their application prospects are broad.

[0003] Phosphorus is a common limiting nutrient element in soil, and phosphorus deficiency in rice can cause stunted growth and other problems. Currently, adding chemical fertilizers is the most direct and quickest way to supplement phosphorus, but the excessive use of phosphate fertilizers inevitably leads to soil compaction, soil depletion, and environmental pollution. Activating and fully utilizing soil phosphorus through phosphorus-solubilizing microorganisms can alleviate the problem of soil phosphorus deficiency and is widely considered a green, environmentally friendly, and efficient method. The rational utilization of phosphorus-solubilizing microorganisms and the development of microbial fertilizers have significant research value for reducing chemical fertilizer input and increasing crop yields.

[0004] Currently, isolating and screening single-function bacterial strains from the environment is a common method, but these methods often suffer from limitations in cellulose degradation and phosphorus activation capabilities. Furthermore, they primarily explore one aspect of the strain's function, with limited development of other potential functions. Therefore, there is significant research potential in identifying and developing strains that can efficiently produce plant hormones to promote rice growth, particularly in rice paddies where straw has been returned to the field. Summary of the Invention

[0005] To address the above problems, this invention provides a cellulose-decomposing bacterium, NJAU-PS1, which solubilizes phosphorus and promotes cellulosic growth, and its applications.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A strain of Bacillus cereus NJAU-PS1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 25, 2023, and the accession number is CGMCC No. 28551.

[0008] Furthermore, the application of the aforementioned NJAU-PS1 in the degradation of cellulose.

[0009] Furthermore, the application of the aforementioned NJAU-PS1 in promoting rice growth.

[0010] The beneficial effects of this invention are:

[0011] This invention provides a cellulose-degrading strain NJAU-PS1 that can solubilize phosphorus and / or promote growth. This strain has the functions of activating phosphorus, degrading cellulose, and promoting growth. It can effectively promote the decomposition of straw and improve the phosphorus content in the soil, and has a significant growth-promoting function for crops such as rice. Attached Figure Description

[0012] Figure 1 This is a colony diagram of the organophosphate culture medium for the initial screening of Monkina.

[0013] Figure 2 This is a colony diagram of the inorganic phosphorus culture medium for the initial screening of Monkina.

[0014] Figure 3 This is a colony diagram of the isolated and purified strain.

[0015] Figure 4 Quantitative test for IAA production.

[0016] Figure 5 This is a diagram of bacterial cell structure under an optical microscope after Gram staining.

[0017] Figure 6 Phylogenetic tree of NJAU-PS1.

[0018] Figure 7 The image shows the results of using PS1 bacterial solution to promote rice seedling growth.

[0019] Certificate of Preservation of Biological Materials

[0020] NJAU-PS1, classified as Bacillus cereus, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 25, 2023, and the accession number is CGMCC No. 28551. Detailed Implementation

[0021] Example 1: Screening and Identification of Strains

[0022] 1. Initial screening process

[0023] Sampling and bacterial extraction were performed on soil samples from the straw return layer of a long-term rice-wheat rotation system at an agricultural research institute's experimental base in the Yangtze River region of Jiangsu Province. 10g of soil sample was added to an Erlenmeyer flask containing 100ml of sterile water, shaken at 30℃ and 120r / min for 30min, and allowed to stand. The supernatant was then serially diluted 10⁻⁶ times. -1 Up to 10 -6 10 times, use a pipette to aspirate 10 -4 10 -5 10 -6 200 μL of each diluted solution was spread onto Monkina organic phosphorus medium and Monkina inorganic phosphorus medium and incubated at 28°C for 3-4 days, with 3 replicates. Colonies with different morphologies were picked, isolated, and purified. The colony diameter (d) and the diameter of the clear zone (D) were measured and recorded. The phosphate solubility index of phosphate-solubilizing bacteria was expressed as D / d. The initial screening colonies are shown in the attached figure. Figure 1 , 2 The culture media consisted of organic phosphorus and inorganic phosphorus, respectively, and the three purified colonies are shown in the attached figures. Figure 3 From left to right, they are NJAU-PS1, NJAU-PS2, and NJAU-PS3.

[0024] The initial screening media were Monkina inorganic phosphorus medium and Monkina organic phosphorus medium. The Monkina inorganic phosphorus medium formula was: glucose 10g, (NH4)2SO4 0.5g, KCl 0.3g, NaCl 0.3g, FeSO4·7H2O 0.03g, MgSO4·7H2O 0.3g, MnSO4·4H2O 0.03g, Ca3(PO4)2 10g, agar powder 18g, distilled water 1000mL, pH 7.0-7.5, sterilized by steam at 115℃ for 20min. The Monkina organic phosphorus medium formula was: glucose 10g, (NH4)2SO4 0.5g, KCl 0.3g, NaCl 0.3g, FeSO4·7H2O 0.03g, MgSO4·7H2O 0.3g, MnSO4·4H2O 0.03g, CaCO3 5g, lecithin 0.2g, agar powder 18g, distilled water 1000mL, pH 7.0-7.5, steam sterilized at 115℃ for 20min.

[0025] 2. Quantitative experiment on IAA production

[0026] The preliminarily purified bacteria were inoculated with a solution containing 100 mg·L⁻¹ -1L-tryptophan was added to LB broth (20 mL LB + 2 mg L-tryptophan) and cultured on a shaker at 30°C and 180 rpm for 1 day. The bacterial suspension was centrifuged at 8000 rpm for 10 min, and the supernatant was added to an equal volume of Salkowski colorimetric solution. The mixture was incubated in the dark for 30 min, and the OD530 value was measured. A 50 mg / L IAA stock solution was prepared using analytical grade IAA, and serially diluted to obtain a series of IAA standard solutions. Sterile LB broth was used as a blank control to measure the OD530 value, and a standard curve was plotted. The IAA production of this strain was calculated based on the standard curve.

[0027] IAA stock solution: Accurately weigh 50 mg of indoleacetic acid into a 2 mL centrifuge tube, add 1 mL of anhydrous ethanol to dissolve it completely to obtain a 50 mg / L IAA stock solution.

[0028] S2 colorimetric solution: Weigh 4.5g FeCl3 and dissolve it in 300mL distilled water. Slowly add 587.4mL 98% H2SO4. After the solution cools, accurately dilute to 1L and store for later use. The range of IAA determination is 5-20μg / mL.

[0029] from Figure 4 As can be seen from the results, compared with CK and other strains, NJAU-PS1 has the strongest IAA production capacity. The IAA production of NJAU-PS1 gradually increases and stabilizes from the first day. Therefore, NJAU-PS1 can produce IAA efficiently and with relatively stable yield. The IAA concentration reaches a maximum of 22.25 mg / L on the 5th day of fermentation, and then stabilizes above 20 mg / L on the 6th and 7th days.

[0030] 2. Phosphorus dissolution test

[0031] The purified NJAU-PS1 strain was picked up with an inoculation loop and inoculated into LB seed culture medium. The culture was incubated at 30°C for 36 hours on a shaker at 180 rpm. A 2% inoculum was then transferred to a 250 mL Erlenmeyer flask containing 100 mL of secondary screening medium and incubated at 28°C for 7 days on a shaker at 160 rpm. Repeats and blanks were performed. The secondary screening medium formula was: glucose 10 g, Ca3(PO4)2 5 g, MgCl2 5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, and distilled water 100 mL. The medium was sterilized by steam sterilization at 120°C for 20 minutes. Approximately 10 mL of the culture medium was centrifuged at 10000 rpm to obtain the supernatant. The soluble phosphorus content in the culture medium was determined using the molybdenum antimony colorimetric method. The results are shown in Table 1 below.

[0032] Table 1 Results of secondary screening of phosphate-solubilizing strains

[0033]

[0034] Compared to the control (CK) blank treatment, the soluble phosphorus content increased by 85.7%, thus demonstrating a certain phosphorus solubility. Therefore, further tests were conducted to assess its performance.

[0035] 4. Filter paper strip disintegration test

[0036] The bacterial strain was inoculated onto filter paper strip culture medium and shaken on a shaker at 30℃ and 120 rpm for 4 days. The weight of the filter paper strips before and after inoculation was measured, and the degradation rate of the filter paper was calculated. The filter paper strip culture medium formula was: (NH4)2SO4 1g, MgSO4·7H2O 0.5g, KH2PO4 1g, yeast extract 0.1g, filter paper strips (1cm×4cm) 3 per Erlenmeyer flask, distilled water 1000mL, and sterilized at 120℃ for 20min.

[0037] 5. Carboxymethyl cellulase activity assay

[0038] ① Crude enzyme solution: Take the supernatant of the double-screened fermentation broth (day 4) and centrifuge it (4℃, 4000r / min, 15min).

[0039] ② Construction of the standard curve: The reducing sugar content was determined using the DNS method. Eight 25 mL colorimetric tubes were used. 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mL of standard glucose solution (6 mol / L) were added to each tube, respectively. The solutions were diluted to 2 mL with distilled water, and 1.5 mL of 3,5-dinitrosalicylic acid was added to each tube. The tubes were then heated in a boiling water bath for 5 minutes, cooled, and brought to a final volume of 25 mL with distilled water. The tubes containing 0 mL of standard glucose solution were used as blank controls. The absorbance of each tube was measured at 540 nm. A standard curve was plotted with glucose content and the corresponding absorbance on the x-axis and y-axis, respectively.

[0040] ③ Definition of carboxymethyl cellulase activity: One unit of enzyme activity, expressed as U / mL, is the amount of enzyme required to produce 1 μmol of glucose by hydrolyzing CMC-Na (sodium carboxymethyl cellulose) substrate in 1 mL of enzyme solution under specified temperature and pH conditions for 0.5 h. The formula for calculating enzyme activity is as follows:

[0041] Enzyme activity (U / mL) = Glucose content (mg) * Dilution factor * 5.56 / Enzyme volume added to reaction solution (mL) * Time (h)

[0042] ④ Carboxymethyl cellulase activity assay (reducing sugar method): Place 1 mL of crude enzyme solution diluted 8 times for each strain into a 25 mL colorimetric tube, preheat in a constant temperature water bath (60℃) for 2 min, add 4 mL of CMC-Na solution preheated in a constant temperature water bath at 60℃ (3 parallel test tubes for each sample), incubate in a constant temperature water bath (60℃) for 30 min, add 1 mL of sodium hydroxide (2 mol / L) and shake well, add 3 mL of DNS reagent, place in a boiling water bath for 5 min, cool, and bring to a final volume of 25 mL with distilled water. Measure the absorbance at a wavelength of 540 nm. Calculate the CMC enzyme activity based on the standard curve.

[0043] Table 2. Filter paper disintegration test and CMC enzyme activity test of NJAU-PS1

[0044]

[0045] Note: "***" indicates that most of the filter paper has been broken and degraded, "**" indicates that some of the filter paper has been degraded, and "*" indicates that the filter paper is relatively intact.

[0046] As shown in Table 2, compared with the control group, the degradation rate of the NJAU-PS1 inoculated filter paper was about 10.78 times that of the control group, and the CMC enzyme activity was 15.79±0.16U / ml.

[0047] 6. Strain identification

[0048] 6.1 Colony morphology characteristics

[0049] NJAU-PS1 colonies grow rapidly on LB agar plates, forming dense, smooth, pale yellow, and opaque colonies that gradually turn yellowish-brown over time. The reverse side of the medium remains pale yellow. Under a microscope, the stained bacterial cells appear oval. (See attached image) Figure 3 , 5 The images shown are colony diagrams and Gram staining diagrams of bacterial cells.

[0050] 6.2 Physiological and Biochemical Characteristics

[0051] Perform the methyl red test, starch hydrolysis test, citrate test, catalase test, and Gram staining test. The specific procedures are as follows:

[0052] Methyl red test: Pick a small amount of the test bacteria from the agar slant and incubate in glucose peptone water medium at 37°C for 36 hours. Add 3 drops of methyl red reagent to each culture medium, mix well, and observe. If the culture medium turns red, the MR test is positive, indicated by "+". If the culture medium remains yellow, the MR test is negative, indicated by "-".

[0053] Starch hydrolysis test: Pick test cells and inoculate them onto a starch agar plate. Incubate upside down in a 37°C incubator for 24 hours. Then remove the plate, open the lid, and add a small amount of iodine solution to the plate. Gently rotate the plate until the iodine solution evenly covers the entire surface. If a colorless transparent ring appears around the colony, it indicates that the starch has been hydrolyzed, which is a positive result ("+"). Otherwise, it is a negative result ("-").

[0054] Citrate test: Pick test bacteria from the agar slant and inoculate them onto citrate slant medium. Incubate at 37°C for 36 hours, then observe the color change of the medium. If it turns blue, it is positive ("+"), otherwise it is negative ("-").

[0055] Catalase test: Add 3% hydrogen peroxide directly to the test strain and observe immediately. If a large number of bubbles are produced, it is positive ("+"), otherwise it is negative ("-").

[0056] The measurement results are shown in Table 3 below.

[0057] Table 3. Results of NJAU-PS1 physiological and biochemical tests

[0058]

[0059] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0060] 6.3 Species identification based on 16S rRNA sequencing

[0061] The purified strain was sequenced by Shanghai Meiji Biopharmaceutical Co., Ltd., and the 16S rRNA sequence is shown in SEQ ID NO.1. Low-quality bases were removed from both ends of the raw sequences obtained by Sanger sequencing to obtain clean sequences, which were then assembled to obtain the assembled sequence. The sequence was compared with the NT database BLAST to obtain information on the top 10 species with the highest similarity, and the species with the highest similarity was selected as the bacterial species identification result. The comparison results showed that the gene sequence of NJAU-PS1 had a similarity of 98.58% with Bacillus cereus; therefore, NJAU-PS1 was identified as Bacillus cereus.

[0062] The NJAU-PS1 strain was identified based on 16S rRNA gene sequence analysis, and a phylogenetic tree of NJAU-PS1 was constructed using MEGA 7. Figure 6 As shown in the phylogenetic tree, strain NJAU-PS1 is most closely related to Bacillus cereus. Therefore, strain NJAU-PS1 is identified as belonging to the genus Bacillus cereus.

[0063] 6.4 Preservation of microbial strains

[0064] Glycerin can be stored at -20°C for 1 year, and slant can be stored at 4°C for 1 month.

[0065] Example 2: Effects of NJAU-PS1 bacterial culture on rice growth

[0066] Preparation method of NJAU-PS1 bacterial suspension: Inoculate activated colonies into LB liquid medium, and incubate at 30℃ and 180 rpm for 24 hours to obtain a turbid liquid, which is the bacterial suspension.

[0067] A rice germination experiment was conducted. Nangeng 50 was selected as the test rice variety. Plump, uniformly sized seeds were selected. Five treatments were set up: control (CK) with water, and dilutions of the original bacterial solution at 20, 50, 100, and 200 times, soaked for 2 days. Seeds were then disinfected by immersing them in 70% ethanol for 15 minutes, followed by rinsing three times with distilled water to remove the ethanol. After disinfection, the seeds were placed in 100mL Erlenmeyer flasks, and 50mL of the corresponding concentration solution was added. The flasks were incubated at 28℃ for 2 days, followed by two days of germination. Seeds with uniform growth from each treatment were then placed in hydroponic boxes and cultured at 28℃ with normal water irrigation for 7 days, maintaining 60% humidity. Figure 7 This describes the germination status of rice seeds after soaking and normal growth for 7 days. Root length, total number of roots, aboveground plant height, and fresh weight were measured after cultivation.

[0068] Table 4. Effects of bacterial culture concentration on rice growth

[0069]

[0070] The table shows that rice treated with 50-fold and 100-fold dilutions of the bacterial solution exhibited better growth. The rice plants reached a height of over 5.9 cm, with a maximum root length of 4.29 cm and a total of 9 or 10 roots. Due to limitations in hydroponics, the difference in fresh weight was not significant, but plant height, root length, and total root number showed significant differences compared to other treatments. The 50-fold or 100-fold dilution of the bacterial solution had a particularly significant promoting effect on rice growth.

Claims

1. A strain of Bacillus cereus NJAU-PS1, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on September 25, 2023, with accession number CGMCC No. 28551.

Images