CN111718874B

CN111718874B - Phosphorus-solubilizing bacterium RP22, fermentation product, microbial inoculum and application thereof

Info

Publication number: CN111718874B
Application number: CN202010569008.5A
Authority: CN
Inventors: 焦如珍; 韦宜慧; 陈嘉琪; 董玉红; 厚凌宇; 赵光宇; 王超群
Original assignee: Research Institute of Forestry of Chinese Academy of Forestry
Current assignee: Research Institute of Forestry of Chinese Academy of Forestry
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2022-02-11
Anticipated expiration: 2040-06-19
Also published as: CN111718874A

Abstract

The invention relates to the field of microbiology, and particularly discloses a phosphorus-solubilizing bacterium RP22, a fermentation product, a microbial inoculum and application thereof. The phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 contains a nucleotide sequence shown in SEQ ID NO.1, and the preservation number is CGMCC No. 19866. The phosphorus-solubilizing bacterium (Pseudomonas grimontii) RP22 has the functions of solubilizing phosphorus, producing iron carriers and promoting plant growth, and can improve soil environment and promote the utilization rate of phosphate fertilizer. The application of the Chinese fir compound in plant seedlings has the effect of promoting growth, improves the plant height, the ground diameter and the root growth, and particularly can remarkably promote the growth of Chinese fir seedlings. Compared with chemical fertilizers, the phosphorus-dissolving microorganisms are more environment-friendly and ecological, have more lasting effect, and have important value for improving the growth of the fir seedlings and further improving the survival rate of forestation and the growth after forestation.

Description

Phosphorus-solubilizing bacterium RP22, fermentation product, microbial inoculum and application thereof

Technical Field

The invention relates to the field of microbiology, and particularly relates to a phosphorus-solubilizing bacterium RP22, a fermentation product, a microbial inoculum and application thereof.

Background

Most of phosphorus in soil is insoluble phosphate which is difficult to utilize, the content of water-soluble phosphorus which can be directly absorbed and utilized by plants is low, and the utilization rate of the soil phosphorus is usually only 10-15%, so that the phosphorus often becomes a key factor for limiting the productivity of the cedarwood. At present, phosphate fertilizers are mainly water-soluble fertilizers, such as superphosphate, heavy superphosphate and citrate-soluble phosphate fertilizers, such as calcium magnesium phosphate fertilizers, although the phosphate content of soil can be rapidly increased and phosphorus available for plants can be increased, the utilization rate of the applied fertilizers is low, soil hardening, acidification, harmful substance accumulation, serious unbalance of nutrient element and microbial population structures and deterioration of soil ecological environment are caused after long-term application.

The phosphorus-dissolving microorganism converts insoluble phosphate in soil into water-soluble phosphorus which can be absorbed and utilized by plants through the metabolism of the phosphorus-dissolving microorganism, can improve the utilization rate of the fertilizer, can also prevent soil hardening, avoids soil acidification and harmful substance accumulation, and has the advantages of safety, environmental protection and the like.

The fir is a fast-growing wood species, and the demand for wood is high. However, leaching in common growing areas is strong, the content of metal ions in soil is high, phosphorus is easily fixed, the utilization rate of the phosphorus in the soil is extremely low, and the sustainable development of the fir wood artificial forest is severely restricted. At present, no phosphorus-dissolving microorganism for promoting the growth of the fir and a technical method for inoculating the fir seedlings are available. Therefore, it is required to provide a phosphate solubilizing bacterium RP22, and a fermentation product, a microbial inoculum and an application thereof to solve the above problems.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a phosphorus-dissolving bacterium RP22 with the functions of dissolving phosphorus, producing siderophores and promoting plant growth, and a fermentation product, a microbial inoculum and application thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

the phosphate solubilizing bacterium (Pseudomonas grimontii) RP22 contains a nucleotide sequence shown in SEQ ID NO. 1.

The phosphorus-solubilizing bacteria (Pseudomonas grimontii) RP22 is preserved in China general microbiological culture Collection center (CGMCC for short; No.3 of Beijing university Hokko No.1 of the morning area, China academy of sciences, and postal code 100101) within 5-month and 22-day 2020 of 2020, is classified and named as Pseudomonas grimontii with the preservation number of CGMCC No. 19866.

The phosphorus solubilizing bacteria (Pseudomonas grimontii) RP22 is obtained by separating endophyte from roots of 1-year-old fir seedlings and screening the endophytes, and the growth promoting capability of the strain is judged according to the plant height, ground diameter, root growth amount and activity of the fir seedlings through a fir seedling potting test.

(1) Screening method

a. Quantitative determination of phosphorus dissolving capacity of the strain: the effective phosphorus content of the selected bacterial liquid is more than 70 mg.L^-1The strain of (1).

b. And (3) measuring the activity of ACC deaminase of the strain: ACC deaminase activity of greater than 0.5. mu. mol is selected_·mg^-1·h^-1The strain of (1).

c. And (3) measuring the IAA production capacity of the strain: the IAA content in the selective bacterial liquid is more than 15 mu g/mL^-1The strain of (1).

d. And (3) measuring the siderophore produced by the strain: strains producing yellow halos on CAS agar plates were selected.

(2) Pot experiment

a. And (3) strain expansion culture: inoculating the activated strain into a beef extract peptone culture medium to prepare a seed liquid. And inoculating the seed liquid into a beef extract peptone liquid culture medium for amplification culture.

b. And (3) fir wood inoculation: diluting the cultured bacteria liquid with sterile water, and applying the diluted bacteria liquid to seedlings by using a sterile injector in a mode of 1/2 leaf surface spraying and 1/2 root irrigation. Samples were taken after 90 days for assay.

c. And (4) determining the result: measuring the height and the ground diameter of the seedling, calculating the growth amount, measuring the root biomass and the root activity, and comparing with a control.

Further, the present invention provides a fermentation product of the above-mentioned phosphate solubilizing bacterium (Pseudomonas grimontii) RP 22.

The invention provides a microbial inoculum, which contains the phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 or the fermentation product.

Preferably, the microbial inoculum is a solid microbial inoculum or a liquid microbial inoculum.

The invention provides a plant fertilizer which contains the phosphorus-solubilizing bacteria (Pseudomonas grimontii) RP22 or the fermentation product.

Preferably, the plant is fir.

The invention provides a soil ecological environment regulator, which contains the phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 or the fermentation product.

The soil ecological environment regulator can relieve the soil ecological environment deterioration problems of soil hardening, acidification, harmful substance accumulation, nutrient element and microorganism population structure unbalance and the like caused by long-term application of phosphate fertilizer.

The invention also provides application of the phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 or the fermentation product or the microbial inoculum in any one of the following aspects:

(1) the application in dissolving phosphorus and/or producing iron carrier;

(2) the application in promoting the growth of plants; preferably, the plant is fir;

(3) the application in improving the utilization rate of phosphate fertilizer;

(4) the application of the phosphate fertilizer in preventing soil hardening, acidification and harmful substance accumulation caused by applying the phosphate fertilizer;

(5) the application in the preparation of plant fertilizer and/or soil ecological environment regulator.

In the application of promoting plant growth, preferably, the specific mode of application is as follows: adding the phosphorus-solubilizing bacteria (Pseudomonas grimontii) RP22, the fermentation product or the microbial inoculum to root soil of the plant and/or spraying the phosphorus-solubilizing bacteria (Pseudomonas grimontii) on leaf surfaces of the plant.

Preferably, the phosphate solubilizing bacteria (Pseudomonas grimontii) RP22, the fermentation product or the microbial inoculum are diluted and applied to plant seedlings in a mode of 1/2 foliage spraying and 1/2 root irrigation.

The invention has the beneficial effects that:

the phosphorus-solubilizing bacterium (Pseudomonas grimontii) RP22 has the functions of solubilizing phosphorus, producing iron carriers and promoting plant growth, and can improve soil environment and promote the utilization rate of phosphate fertilizer. The compound fertilizer is used for plant seedlings, has the growth promoting effect, improves the plant height, the ground diameter and the root growth amount, and has great potential particularly for promoting the growth of seedlings. Compared with chemical fertilizers, the phosphorus-dissolving microorganisms are more ecological and environment-friendly, and have important promotion effects on improving the outplanting rate of the first-class seedlings of the seedlings, improving the survival rate of forestation and facilitating the growth of forests after the forestation.

Drawings

FIG. 1 is a photograph showing the experimental results of the group treated with root irrigation and foliage spray (T2) of the present invention example 1 after 90 days of cultivation of the seedlings of Cunninghamiae Lanceolatae as compared with the experimental results of the CK group.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

1. Separating and screening strains

After cleaning fir seedlings, cutting roots into small sections of 2-3 cm, soaking in 75% alcohol for 3min, then soaking in 6% sodium hypochlorite solution for 6min, washing with sterile water for 7 times, placing in a sterile mortar, grinding into homogenate, taking 0.05mL of homogenate, coating the homogenate on a Pikovskaya (PVK) agar plate, culturing at 28 ℃ for 5-7 d, and checking whether the plate has bacterial colonies generating transparent halos. Colonies with obvious phosphorus-dissolving rings are picked and further purified by a continuous scribing method. After purification, single colony is selected and inoculated on a slant culture medium and preserved at 4 ℃.

Quantitative determination of phosphorus dissolving capacity of the strain: a250 mL Erlenmeyer flask was charged with 100mL of PVK liquid medium (PVK medium: 10g of glucose, (NH)₄)₂SO₄ 0.5g，MgSO₄·7H₂O 0.3g，NaCl 0.3g，KCl 0.3g，FeSO₄·7H₂O 0.03g，MnSO₄·H₂O 0.03g，Ca₃(PO4)₂ 5.0g，CaCO₃1.0g of agar, 18g of agar and distilled water till the volume is 1000mL, and the pH value is 7.0; calcium phosphate is added after sterilization), the strain is inoculated into a shake flask for culture after lineation activation, a single colony is scraped by a sterilization inoculating ring and inoculated into a shake flask containing 200mL of the cooled sterilization culture solution, the shake flask is placed in a constant temperature shaking table at 28 ℃ and cultured for 5d (the viable count is 5.3 x 10) at 180r/min⁸CFU/mL). And 5d, measuring the content of available phosphorus in the bacterial liquid, repeating the measurement for 3 strains, inoculating 1ml of the culture liquid sterilized at 121 ℃ into a control, and centrifuging the culture. Taking the supernatant, and determining the content of available phosphorus in the culture solution by a molybdenum-antimony colorimetric method. The available phosphorus content of phosphorus-solubilizing bacteria (Pseudomonas grimontii) RP22 is 78.86 mg.L^-1。

And (3) measuring the activity of ACC deaminase of the strain: the strain ACC deaminase activity was determined using the ACC kit (purchased from Qiuchongui Bio Inc. Shanghai) with a phosphate solubilizing bacterium (Pseudomonas grimontii) RP22 ACC deaminase activity of 0.50. mu. mol mg^-1·h^-1。

And (3) measuring the IAA production capacity of the strain: using an IAA kit (Shanghai enzyme-linked biosciences, Ltd.), the IAA producing ability of phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 is 15.46. mu.g.mL^-1。

And (3) measuring the siderophore produced by the strain: inoculating phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 strainCAS agar plates, a yellow halo was produced around the strain. The method for measuring the siderophore comprises the following steps: taking out the preserved strain, scraping a small amount of the strain, inoculating the strain into an LB culture medium, placing the strain in an incubator at 28 ℃ for 24h, selecting a single colony, inoculating the single colony onto a CAS plate culture medium, placing the single colony in the incubator at 28 ℃ for 48h, and observing and recording the color change around the colony. CAS plate medium: solution A: 60.5mg CAS, 50mL distilled water, 10mL ferric chloride solution (containing 1mM FeCl)₃·6H₂O, 10mM HCl); solution B: 72.9mg HDTMA (cetyltrimethylammonium bromide), 40mL distilled water; solution C: adding the solution A into the solution B, mixing uniformly, and sterilizing at 121 ℃ for 15 min; 2mL of 1mM calcium chloride solution and 2mL of 1mM magnesium sulfate solution, and adjusting the pH value of the solution to 6.8-7.0 by using biological buffer solution Pipes (sigma). Adding distilled water to 1000mL, adding 18g agar, sterilizing at 121 deg.C for 15min, cooling to below 60 deg.C, adding 50mL solution C, mixing, and making into plate. The colony diameter ratio (D/D) of the siderophore to the phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 is 1.8.

2. Identification of strains

16SrDNA sequencing: extracting DNA of each strain by water boiling method, performing PCR amplification with bacterial universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', nucleotide sequence shown in SEQ ID NO. 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', nucleotide sequence shown in SEQ ID NO. 3), and sequencing the amplified product with Huada gene. The determined 16SrDNA sequence (the nucleotide sequence is shown as SEQ ID NO. 1) is searched in GenBank, EzTaxon and BIGSdb databases, and the sequence search result shows that RP22 is homologous with Pseudomonas grimontii, and the similarity is up to 100%.

3. Preparation of inoculated bacterial liquid

Streaking the preserved phosphate solubilizing bacteria (Pseudomonas grimontii) RP22 on beef extract peptone agar plate, selecting single colony to perform shake flask culture in beef extract peptone liquid culture medium, culturing at 28 deg.C for 24 hr at 180r/min, adjusting the bacteria liquid to OD with non-inoculated culture medium₆₀₀The seed solution was prepared as 1 for use. Inoculating the seed liquid into a beef extract peptone liquid culture medium according to the inoculation amount of 1%, and culturing at 28 ℃ and 180r/min for 48h for later use.

4. Inoculating Chinese fir seedling

The inoculation design is shown in table 1, the inoculated bacterial liquid is set to 3 dilution times, namely 30 times, 60 times and 90 times, the contrast is tap water, 4 treatments are carried out, 16 strains are treated, and the treatments are repeated for 5 times. Namely, the number of each treated nursery stock is 16 multiplied by 5 which is 80; control is equivalent tap water (CK). And (3) test summation: the seedlings of 16 x 5 x 4-320 fir trees were inoculated 3 times in 8, 9 and 10 months, respectively, each 30mL each time. The inoculation mode comprises root irrigation, leaf surface spraying, root irrigation and leaf surface spraying. And (3) irrigating roots, namely absorbing diluted bacteria liquid by using an injector, uniformly injecting the diluted bacteria liquid into the periphery of seedlings, spraying the diluted bacteria liquid to the leaves by using the injector, uniformly spraying the diluted bacteria liquid to the seedlings, wherein the inoculation amount of irrigating roots and spraying the leaves is 15mL for irrigating roots, and spraying the leaves is 15 mL. Control (CK) was applied by root irrigation with tap water at the same amount as above (30 mL per plant per time). During the period, automatic sprinkling irrigation equipment is adopted for irrigation according to the dry and wet conditions of the matrix, and manual weeding is performed regularly. And 6, ending the pot experiment in 2019, 11 and 15 days, and performing sample collection and index measurement.

TABLE 1

5. Determination of the Effect of inoculation

The photographs of the experimental results of the root drenching + foliar spray treatment group (T2) and the CK group after 90d inoculation are shown in fig. 1.

The data were measured 90d after inoculation. The results of the experiments for each treatment group are shown in tables 2-8. In each table, the difference in letters between treatments represents significant differences.

The specific determination method is as follows:

the measurement of the fir growth index (the results are shown in tables 2 and 6):

during the test, the height of each seedling was measured with a ruler before each group began inoculation, and the ground diameter of the seedling was measured with a vernier caliper. After the test is finished, the seedlings are cleaned by clear water and surface impurities are removed, the filter paper absorbs the water, the roots, stems and leaves are respectively put into envelope bags after the water is absorbed, the water is removed for 0.5h at 105 ℃, the seedlings are dried to constant weight at 70 ℃, and then the biomass of each part is weighed and recorded. The calculation formula of the seedling quality index is QI (total biomass of seedlings (g)/[ (seedling height cm/ground diameter mm) + (stem weight g/root dry weight g) ].

And (3) measuring the root system indexes of the fir (see the results in table 3):

cutting the whole plant root system, cleaning with clear water, rapidly scanning with a root system scanner, and analyzing and calculating the root length, root surface area, root volume, root diameter and root tip number of the seedling by WinRHIO (root system analysis system). The root activity is measured by TTC (triphenyltetrazolium chloride) method.

Determination of nutrient content of fir leaves (see table 4 for results):

after crushing and sieving the dried fir leaf sample, the total nitrogen content of the leaf is measured by a Kjeltec (FOSS, Sweden) Kjeltec instrument.

And (3) total determination of phosphorus, potassium, magnesium and iron elements: subjecting the sample to HNO₃After digestion, measurement was performed by inductively coupled plasma mass spectrometry (ICP-MS).

Measurement of physiological indexes of fir leaves (see table 5 for results):

sample preparation: cleaning fir plant leaves, drying with filter paper, wrapping with tinfoil paper, placing in liquid nitrogen for 30s, preparing into powder at low temperature, and placing in a refrigerator at-80 deg.C to be tested.

Measuring the chlorophyll content by acetone-ethanol extraction method, measuring the soluble protein content of the leaves by Coomassie brilliant blue method, and measuring the soluble sugar content of the leaves by anthrone colorimetric method. The nitrate reductase activity (NR) is determined by a double antibody sandwich method, and the determination kit is purchased from Shanghai enzyme-linked biotechnology limited.

Matrix nutrient assay (results see table 7):

total nitrogen was measured by Kjeltec method and analyzed by 2300Kjeltec analyzer (FOSS, Sweden). And (3) total determination of phosphorus and potassium elements: subjecting the sample to HNO₃After digestion, measurement was performed by inductively coupled plasma mass spectrometry (ICP-MS). Determination of available phosphorus and available potassium: sample is subjected to NaHCO₃The leaching was followed by measurement using an inductively coupled plasma mass spectrometer (ICP-MS).

Determination of matriptase activity (results see table 8):

urease, cellulase, sucrase, dehydrogenase, acid phosphatase and nitrate reductase are all determined by a double-antibody sandwich method, and the determination kit is purchased from Shanghai enzyme-linked biotechnology limited.

TABLE 2 plant height and ground diameter growth of Chinese fir seedlings in different application modes

TABLE 3 root morphology and vitality of Chinese fir seedlings in different application modes

TABLE 4 nutrient contents of leaves of fir seedlings in different application modes

TABLE 5 physiological indices of partial Chinese fir seedlings in different application modes

TABLE 6 Chinese fir seedling biomass and seedling quality in different application modes

TABLE 7 nutrient content of the substrate for different modes of application

TABLE 8 matriptase Activity with different modes of application

As can be seen from the data in the tables, the phosphorus-solubilizing bacteria (Pseudomonas grimontii) RP22 has a good growth promoting effect on the fir, and has strong effects of improving soil properties and improving soil quality.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. The phosphate solubilizing bacterium strain RP22 is characterized in that the phosphate solubilizing bacterium strain RP22 is pseudomonas grignard (Pseudomonas grisea)Pseudomonas grimontii) The biological preservation number is CGMCC No. 19866.

2. The fermentation product of the phosphate solubilizing bacteria strain RP22 of claim 1.

3. A microbial preparation comprising the phosphorus-solubilizing bacterium strain RP22 according to claim 1 or the fermentation product according to claim 2.

4. A plant fertilizer comprising the phosphate solubilizing bacterium strain RP22 according to claim 1 or the fermentation product according to claim 2.

5. A soil ecological environment regulator comprising the phosphorus solubilizing bacterium strain RP22 according to claim 1 or the fermentation product according to claim 2.

6. Use of the phosphorus solubilizing bacterial strain RP22 according to claim 1 or the fermentation product according to claim 2 or the microbial inoculum according to claim 3 in solubilizing phosphorus and/or producing siderophores.

7. Use of the phosphate solubilizing bacterium strain RP22 according to claim 1 or the fermentation product according to claim 2 or the microbial agent according to claim 3 for promoting plant growth.

8. The application according to claim 7, characterized in that it is applied in such a way that: adding the phosphate solubilizing bacterium strain RP22, the fermentation product or the microbial inoculum to root soil of the plant and/or spraying the phosphate solubilizing bacterium strain RP22 and/or the microbial inoculum to leaf surfaces of the plant.

9. Use of the phosphate solubilizing bacterium strain RP22 according to claim 1 or the fermentation product according to claim 2 or the microbial inoculum according to claim 3 for improving the utilization rate of phosphate fertilizer.

10. Use of the phospholytic strain RP22 of claim 1 or the fermentation product of claim 2 or the microbial inoculum of claim 3 for preventing soil hardening, acidification, and accumulation of harmful substances caused by application of phosphate fertilizers.

11. Use of the phosphate solubilizing bacterium strain RP22 according to claim 1 or the fermentation product according to claim 2 or the microbial agent according to claim 3 for the preparation of a plant fertilizer and/or a soil ecological environment regulator.