Pseudomonas oryzae NKP20, microbial agent and application of pseudomonas oryzae NKP20

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses pseudomonas oryzae NKP20, a microbial agent and application of the pseudomonas oryzae NKP20. The pseudomonas oryzae NKP20 (CCTCC NO: M 2024730) provided by the invention can be used for quickly converting mineral phosphorus stably existing in rock or soil into an effective form which can be absorbed by plants in a large proportion and at low cost. After the pseudomonas oryzae NKP20 is cultured in a phosphorus-containing rock liquid culture medium for 3 days, the phosphorus dissolving amount can reach 300 mg / L, and the phosphorus dissolving amount can reach 30%; the phosphate solubilizing amount can reach 400 mg / L after 7 days of culture, and the phosphate solubilizing amount can reach 40%. According to the invention, the pseudomonas oryzae NKP20 is combined with the phosphate solubilizing capacity of soil and the phosphate solubilizing capacity of rock, and reference is provided for later research of wild strains. When the microbial fertilizer is applied to crops, the phosphorus dissolving effect can be achieved, the crop yield can be obviously increased, and meanwhile the yield can be increased in successive years.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Pseudomonas oryzae NKP20, a microbial agent and applications thereof. Background Art

[0002] Most of the agricultural land in my country is deficient in phosphorus, because most of the phosphorus in the soil exists in the form of insoluble phosphorus, mainly due to the low utilization rate of chemical fertilizers. When soluble fertilizers are applied to the soil, the utilization rate in the season is generally only 10% to 25%. The remaining elements in acidic soils are easily chelated and fixed by the Fe and Al elements in the soil to form water-insoluble Fe-P and Al-P; the remaining elements in alkaline soils are easily chelated and fixed by the Ca and Mg elements in the soil to form water-insoluble Ca-P and Mg-P, making it difficult for plants to absorb and utilize. In response to the problems of agricultural fertilizer abuse and low utilization rate, the country advocates the development of green ecological organic fertilizers to ensure the green development of agriculture. It is necessary to improve the fertilization method, increase the fertilizer utilization rate, implement green energy-saving actions for agricultural machinery, and promote the increase of organic fertilizers and the reduction and efficiency of chemical fertilizers.

[0003] There are many types of microorganisms in nature that have the effects of dephosphorization, etc. The use of microbial dephosphorization methods has the characteristics of low cost and simple methods, and has a good industrial development prospect. However, different bacteria have very different abilities to decompose nutrient elements. At present, the strains used in industrial production are single, mostly rhizobia, and there is a lack of basic research on other functional strains. There are few strain library resources, most of which are bacteria, and these bacteria have strong spontaneous mutation, which leads to the weakening or disappearance of functional characteristics during the passage process, resulting in obvious crop yield increase effects but no good dephosphorization effect, or good dephosphorization effect but no significant increase in crop yield, or continuous inoculation of functional bacteria but no year-on-year increase in yield.

[0004] Based on this, the development of a biological agent with efficient phosphorus dissolution effect is of great significance for the resource utilization of insoluble phosphorus and agricultural production. Summary of the invention

[0005] In view of the above problems, the present invention provides a Pseudomonas oryzae NKP20, a microbial agent and applications thereof. Pseudomonas oryzae NKP20 is a new type of efficient phosphate-solubilizing strain that can convert mineral phosphorus stably present in rocks or soil into an effective form that can be absorbed by plants.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is: In a first aspect, the present invention provides a Pseudomonas oryzihabitans NKP20, whose deposit number is CCTCC NO: M 2024730. The 16S rDNA of the Pseudomonas oryzihabitans NKP20 is shown in SEQ ID NO.1.

[0007] Compared with the prior art, the Pseudomonas oryzae NKP20 provided by the present invention is a natural strain isolated from the rhizosphere soil of plants. Compared with the mutant strains induced in the laboratory, it is easier to colonize, safer and more environmentally friendly, and has the potential to be developed and applied as a new type of microbial strain resource.

[0008] Phosphorus-rich rocks are widely distributed in my country, and more than 220 kinds of phosphorus-containing minerals have been found in the rocks of geological strata. The rice-dwelling Pseudomonas NKP20 provided by the present invention can quickly, in large proportions, and at low cost convert the mineral phosphorus that is stably present in rocks or soil into an effective form that can be absorbed by plants. The results of the embodiment show that the amount of phosphorus solubilized by the rice-dwelling Pseudomonas NKP20 after culturing in a phosphorus-rich liquid culture medium for 3 days can reach 300 mg / L, and the amount of phosphorus solubilized can reach 30%; the amount of phosphorus solubilized after culturing for 7 days can reach 400 mg / L, and the amount of phosphorus solubilized can reach 40%. The present invention combines the rice-dwelling Pseudomonas NKP20 with the phosphorus solubilization ability of soil and rock, provides a reference for the later research of wild strains, and has important value for the development of microbial fertilizers such as phosphorus-solubilizing bacterial fertilizers.

[0009] In a second aspect, the present invention provides an application of the Pseudomonas oryzae NKP20 in rock or soil phosphate solubilization.

[0010] In a third aspect, the present invention provides a microbial agent, comprising the Pseudomonas oryzae NKP20.

[0011] Preferably, the microbial agent is a liquid agent, and the number of viable bacteria of Pseudomonas oryzae NKP20 in the microbial agent is not less than 10 8 CFU / mL.

[0012] In a fourth aspect, the present invention provides an application of the microbial agent in rock or soil phosphate solubilization.

[0013] In a fifth aspect, the present invention provides a method for dephosphorization, comprising the following steps: S1, activating the Pseudomonas oryzae NKP20 to obtain an activated bacterial solution; S2, inoculating the activated bacterial liquid into a phosphorus-containing rock liquid culture medium, culturing, and obtaining phosphorus-solubilizing substances.

[0014] Preferably, in S1, the culture medium used for the activation is LB liquid culture medium.

[0015] Preferably, in S1, the activation culture time is 20h~24h.

[0016] Preferably, in S2, the phosphorus-containing rock liquid culture medium comprises the following raw materials in parts by weight: 8 to 12 parts of glucose, 3 to 7 parts of phosphorus-containing rock, and MgCl 2 6H 2 O 4.8~5.2 parts, MgSO 4 7H 2 O 0.2~0.3 parts, KCl 0.15~0.25 parts, (NH 4 ) 2 SO 4 0.08~0.12 parts and 950~1020 parts of water, pH=7.0~7.5.

[0017] The amount of the phosphorus-containing rock added to the phosphorus-containing rock liquid culture medium of the present invention can be adjusted according to the phosphorus content of the phosphorus-containing rock. Generally, the phosphorus content of the phosphorus-containing rock is 10wt% to 30wt%. In the present invention, the phosphorus-containing rock can be apatite, and its phosphorus content is about 20wt%.

[0018] For example, the water is distilled water or deionized water, and the particle size of the phosphorus-containing rock is 10 μm to 18 μm.

[0019] Preferably, in S2, the amount of the activated bacterial solution added is 3.5% to 4.5% of the mass of the phosphorus-containing rock liquid culture medium.

[0020] For example, in S2, the culture may be performed by shaking culture.

[0021] Preferably, in S2, the culture temperature is 28°C to 32°C, and the culture time is 6 to 7 days.

[0022] The microbial agent (Pseudomonas oryzae NKP20) provided by the present invention can quickly, in large proportion and at low cost convert the mineral phosphorus stably present in rocks into an effective form that can be absorbed by plants; at the same time, the microbial agent can also increase the functional bacterial population, dissociate the insoluble elements such as phosphates in the rocks, form soluble nutrients, and effectively improve the absorption efficiency of the nutrients.

[0023] In a sixth aspect, the present invention provides a microbial fertilizer, comprising the Pseudomonas oryzae NKP20 or the microbial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of the phosphate solubilization ability of each strain in Example 1 of the present invention; Figure 2 is a phylogenetic tree of Pseudomonas oryzae NKP26 in Example 1 of the present invention; Figure 3 is a SEM image of the apatite surface not acted upon by Pseudomonas oryzae NKP26 in Example 2 of the present invention; Figure 4 This is a SEM image of the apatite surface acted upon by Pseudomonas oryzae NKP26 in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] The culture medium used in the examples is: PDA liquid culture medium: 200 g potato, 20 g glucose, add distilled water to 1000 mL, and adjust pH to 7.0~7.5.

[0027] PDA solid medium: Add 15g~20g agar to PDA liquid medium.

[0028] Phosphorus rock liquid culture medium: glucose 10.0 g, 12 μm apatite powder 5.0 g, MgCl 2 6H 2 O 5.0g, MgSO 4 7H 2 0.25 g, KCl 0.2 g, (NH 4 ) 2 SO 4 0.1g, add deionized water to 1000mL, and adjust the pH to 7.0~7.5.

[0029] Phosphorus rock solid culture medium: Add 15g~20g agar to the phosphorus rock liquid culture medium.

[0030] LB liquid culture medium: peptone 10g, yeast extract 5g, NaCl 10g, add deionized water to 1000mL, adjust pH = 7.0~7.5.

[0031] LB solid medium: Add 15g~20g agar to LB liquid medium.

[0032] In order to better illustrate the present invention, further examples are given below.

[0033] Example 1 This example is used to illustrate the acquisition, identification and preservation of Pseudomonas oryzae NKP20.

[0034] (1) Isolation and purification of strains.

[0035] Source of bacteria: Phosphate mine area in a village in Yiling District, Yichang City, Hubei Province (31°18″N, 111°12″E, 1108m above sea level). Five sampling points were selected in each place to collect rhizosphere soil (depth 0~15cm, 2mm around the root) of wild plants during the vegetative growth period. The five sampling points were mixed in sterile bags, and after picking out debris such as wood chips and stones, the collected soil samples were mixed evenly, passed through a 2 mm soil sieve, and stored in a refrigerator at 4°C.

[0036] Phosphate-solubilizing bacteria were screened from soil samples using dilution plates, and phosphate-solubilizing bacteria were isolated using the separation method. After removing obvious stones and residual roots from the soil in the clean bench, 10 g of fresh rhizosphere soil sample from the phosphate mine area was weighed and placed in a conical flask containing 90 mL of sterile water to obtain 10 -1 Soil sample dilution. Oscillate the dilution in a high-speed constant temperature incubator at 28°C and 150 rpm for 20 min. After standing for 30 s, take 1 mL of the supernatant and add it to a test tube containing 9 mL of sterile water to obtain 10 -2 Then, the soil sample dilution was performed in sequence according to the 10-fold dilution method to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 Soil sample dilution. Use a pipette to take 0.1 mL of each sample and dilute it by 10 times. -2 , 10 -3 , 10 -4 , 10 -5 Four groups of soil sample dilutions with different concentrations were spread on phosphorus-containing rock solid culture media, and each concentration was repeated 5 times (i.e., one group of concentrations corresponded to 5 plates). After the plates were sealed, they were placed upside down in a constant temperature and humidity incubator at 28°C for 5 to 7 days to observe whether a phosphate-solubilized zone could be formed around the colonies.

[0037] The strains were screened by measuring the phosphate solubilization ability of phosphate-solubilizing bacteria. On the solid culture medium containing phosphate rock, phosphate-solubilizing bacteria will dissolve apatite to form a transparent or turbid phosphate solubilization ring. 52 colonies with obvious phosphate solubilization rings were selected in the initial screening. The bacteria were dipped on the PDA plate with an inoculation needle, streaked and cultured and numbered NKP1~NKP52. The representative strains obtained above were inoculated in 50mL LB liquid culture medium and enriched and cultured in a high-speed constant temperature culture shaker at 30℃ and 120rmp for 24h. After the purified bacteria were activated and cultured, 2mL of the bacterial solution in the liquid culture medium was inoculated in a conical flask containing 50mL of phosphate rock liquid culture medium, and an equal amount of sterile water was inoculated as the control group. Three replicates were set for each treatment and cultured on a shaker at 30℃ and 180rpm for 7 days. The supernatant of the bacterial solution was taken to detect the soluble phosphorus content, and the pH value of the supernatant was measured at the same time. The soluble phosphorus content in each culture medium was compared, and the phosphate-solubilizing bacteria were rescreened.

[0038] To evaluate the ability of the strain to dissolve phosphorus from apatite, the strain was cultured in a liquid medium containing phosphorus rock at 30°C with shaking for 7 days. The culture was then centrifuged at 12,000 rpm for 5 min to remove apatite and bacterial cells. The phosphorus content in the supernatant was determined by inductively coupled plasma mass spectrometry. The results showed that (see Figure 1 ), the phosphorus solubilization efficiency of NKP13, NKP20, NKP24, NKP18, NKP15, NKP27, NKP23, and NKP21 reached more than 14% within 3 days, and the phosphorus solubilization efficiency of NKP13 and NKP20 reached more than 30%. Among them, the change data of the available phosphorus content and pH value of the NKP20 strain during the dissociation process are shown in Table 1. As can be seen from Table 1, within one week of culture, the phosphorus release rate of the NKP20 strain showed an upward trend with the extension of the culture time. After 7 days of culture, the phosphorus release amount can reach more than 400 mg / L, and the phosphorus release rate reaches more than 40%; the pH value of the culture solution showed a downward trend, and after 7 days of culture, the pH dropped to 4, indicating that under the conditions of insoluble inorganic phosphorus, NKP20 bacteria need to adapt to the environment and decompose insoluble phosphorus into fast-acting phosphorus for their own growth and development. It has a strong acid production ability and dissolves insoluble phosphates by secreting organic acids, thereby accompanied by efficient release of phosphorus; and the lower the pH value in the culture solution, the greater the content of fast-acting phosphorus, showing a certain negative correlation. Therefore, among the above strains, we purified and screened the target strain NKP20 as a representative strain for subsequent experiments.

[0039] Table 1 Changes in available phosphorus content and pH value of NKP20 strain during dissociation

[0040] (2) Identification of strains.

[0041] NKP20 was cultured in 50 mL LB liquid medium for 3 days, 5 mL of the culture medium was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the precipitate was used for strain identification. Genomic DNA was extracted from the cells using a soil DNA extraction kit (Biomarker Technologies, Beijing, China). The 16S rDNA of the strain was PCR amplified using universal primers 27F (5'-agagtttgatcctgtgctcag-3') and 1492R (5'-ggttacctttgttacgactt-3'). The amplified gene sequence of 16S rDNA is shown in SEQ ID NO.1: agtttgatca tggctcagat tgaacgctgg cggcaggcct aacacatgca agtcgagcgg atgagaagag cttgctcttc gattcagcgg cggacgggtg agtaatgcct aggaatctgc ctggtagtgg gggacaacgt ttcgaaagga acgctaatac cgcatacgtc ctacgggaga aagcaggga ccttcgggcc ttgcgctatc agatgagcct aggtcggatt agctagttgg tggggtaatg gctcaccaag gcgacgatcc gtaactggtc tgagaggatg atcagtcaca ctggaactga gacacggtcc agactcctac gggaggcagc agtgggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt gtgttaagaa ggtcttcgga ttgtaaagca ctttaagttg ggaggaaggg cattaagcga ataccttgct gttttgacgt taccgacaga ataagcaccg gctaactctg tgccagcagc cgcggtaata cagagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtggg tggtttgtta agttggatgt gaaagccccg ggctcaacct gggaactgca tccaaaactg gcaagctaga gtacggtaga gggtggtgga atttcctgtg tagcggtgaa aagcgtagat ataggaag acaccagtgg cgaaggcgac cacctggacc gatactgaca ctgaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg gaatccttga gattttagtg gcgcagctaa cgcattaagt tgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac atgcagagaa ctttccagag atggattggt gccttcggga actctgacac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct tgtccttagt taccagcacg ttatggtggg cactctaagg agactgccgg tgaaaaccg gaggaaggtg gggatgacgt caagtcatca tggcccttac ggcctgggct acatacgtgc tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg agctaatctc acaaaaccga tcgtagtccg gatcgcagtc tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaccagaa gtagctagtc taaccttcgg gaggacggtt accacggtgt gattcatgac tggggtgaag tcgtaacaag gtagccgtag gggaacctgc ggctggatca cctcctt。

[0042] The obtained PCR products were sequenced by Zero2IPO Biotechnology Co., Ltd., China. The sequence of NKP20 16S rDNA was input into the NCBI BLAST system. Similar sequences were downloaded by the neighbor-joining method using MEGA11.0 software to construct the phylogenetic tree of NKP20. The results are shown in Figure 2 As shown. Figure 2 As can be seen from the figure, NKP20 strain and Pseudomonas oryzae ( Pseudomonas oryzihabitans ) had the highest homology, with a homology rate of 100.00%. Therefore, the NKP20 strain was identified as Pseudomonas oryzae.

[0043] (3) Preservation and storage of strains.

[0044] 25% glycerol liquid culture medium tube storage method: Inoculate NKP20 strains in LB liquid culture medium, culture to the logarithmic growth phase, take 1mL bacterial culture solution and 1mL sterile glycerol with a volume concentration of 50% and add them to the sterilized 2mL centrifuge tube in a 1:1 ratio. At this time, the volume concentration of glycerol in the tube is 25%. Then place the centrifuge tube in a -20℃ refrigerator for storage. This storage method is valid for 1 year. When taking bacteria again, place the centrifuge tube in a 28℃ water bath, wait for it to melt, and inoculate it until the culture medium is activated for 2~3 generations before use.

[0045] The strain NKP20 obtained above was deposited in the China Center for Type Culture Collection on April 19, 2024, located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with the deposit number of CCTCC NO: M 2024730.

[0046] Example 2 This example is used to illustrate the rock phosphate solubilization effect of Pseudomonas oryzae NKP20.

[0047] The rock used in this example is apatite, which was collected from a phosphate mine in a village in Yiling District, Yichang City, Hubei Province (31°18″N, 111°12″E, 1108m above sea level), placed in a sterile kraft paper bag, and stored in a refrigerator. Before use, it needs to be rinsed with distilled water, naturally air-dried, ground, passed through a 1000 mesh sieve (particle size <15μm), and sterilized under high pressure at 121°C for 20min before use.

[0048] The phosphate solubilization method of Pseudomonas oryzae NKP20 comprises the following steps: S1, activating Pseudomonas oryzae NKP20 in LB liquid culture medium and culturing for 22 hours to obtain an activated bacterial solution.

[0049] S2, the activated bacterial solution was inoculated into 50 mL of phosphate rock liquid culture medium at a 4% inoculum volume, and cultured on a shaker at 180 rpm at 30 °C for 7 days. At this time, the number of viable bacteria of Pseudomonas oryzae NKP20 was about 10 8 CFU / mL, and the phosphate solution was obtained.

[0050] The surface morphology and structural changes of apatite after 3 days of leaching (shaking culture) of NKP20 strain and metabolites were observed. The apatite residue was dried at 50℃, fixed and sprayed with gold, and its surface characteristics on the 3rd day of leaching were observed using a scanning electron microscope. Figure 3 This is the surface morphology of apatite after leaching without NKP20 strains and metabolites. It can be seen that the surface of the rock particles is smooth, with clear edges and corners, and obvious unevenness; Figure 4 This is the surface appearance of apatite after leaching by NKP20 strain and its metabolites. Figure 3 In comparison, the surface of apatite becomes rough and uneven, and the large angles and protrusions are split into finer edges and small particles. It is also observed that the rock surface is entangled with many hyphae, and penetrates into the cracks of the rock, increasing the contact area with the rock surface and forming hyphae-mineral aggregates, making the apatite rock surface rougher. This indicates that the hyphae have mechanical destructive force on the rock, making the rock structure loose. Pseudomonas oryzae NKP20 and its metabolites have obvious weathering effects on apatite.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Pseudomonas oryzae NKP20, characterized in that: Its deposit number is CCTCC NO: M 2024730.

2. Use of the Pseudomonas oryzae NKP20 according to claim 1 in phosphate solubilization in rock minerals or soil.

3. A microbial agent, characterized in that: The invention comprises the Pseudomonas oryzae NKP20 as described in claim 1.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a liquid agent, and the number of live bacteria of Pseudomonas oryzae NKP20 in the microbial agent is not less than 10 8 CFU / mL.

5. Use of the microbial agent according to claim 3 or 4 in rock or soil phosphate solubilization.

6. A method for dephosphorization, characterized in that: The following steps are involved: S1, activating the Pseudomonas oryzae NKP20 according to claim 1 to obtain an activated bacterial solution; S2, inoculating the activated bacterial liquid into a phosphorus-containing rock liquid culture medium, culturing, and obtaining phosphorus-solubilizing substances.

7. The method for solubilizing phosphate by Pseudomonas oryzae NKP20 according to claim 6, characterized in that: In S1, the culture medium used for activation is LB liquid culture medium; and / or In S1, the activation culture time is 20h~24h.

8. The method for solubilizing phosphate by Pseudomonas oryzae NKP20 according to claim 6, characterized in that: In S2, the phosphorus-containing rock liquid culture medium includes the following raw materials in parts by weight: 8 to 12 parts of glucose, 3 to 7 parts of phosphorus-containing rock, 4.8 to 5.2 parts of MgCl2·6H2O, 0.2 to 0.3 parts of MgSO4·7H2O, 0.15 to 0.25 parts of KCl, 0.08 to 0.12 parts of (NH4)2SO4 and 950 to 1020 parts of water, pH=7.0 to 7.

5.

9. The method for solubilizing phosphate by Pseudomonas oryzae NKP20 according to claim 6, characterized in that: In S2, the amount of the activated bacterial solution added is 3.5% to 4.5% of the mass of the phosphorus-containing rock liquid culture medium; and / or In S2, the culture temperature is 28°C to 32°C, and the culture time is 6 to 7 days.

10. A microbial fertilizer, characterized in that: It includes the Pseudomonas oryzae NKP20 described in claim 1 or the microbial agent described in claim 3 or 4.

Images