A corn rhizobium strain NF11 and its application

Abstract

This invention discloses a strain of maize rhizobium NF11 and its applications, which is classified and named maize rhizobium. Rhizobium zea The rhizobium NF11 provided by this invention is an endophytic bacterium isolated from catalpa tissue culture seedlings. It has a positive effect on promoting the growth of catalpa tissue culture seedlings and potted catalpa seedlings, and can be used as a special live bacteria preparation for the propagation of catalpa seedlings. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27776, on July 3, 2023, in Beijing, China.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a corn rhizobium strain NF11 and its applications. Background Technology

[0002] The catalpa tree is a precious timber and ornamental tree species unique to China, with a cultivation history of over 2000 years. Its well-developed root system gives it strong wind resistance and soil-fixing ability, making it an excellent species for soil and water conservation. The catalpa tree grows rapidly and has strong resistance to environmental pollutants, also possessing the potential for remediating heavy metal pollution in soil. Its dense canopy provides excellent sound absorption and dust retention capabilities, making it an outstanding species for landscaping and street planting. The catalpa tree also has certain medicinal value; its leaves and seed oil extracts have been shown to contain antioxidant and anti-tumor functional components.

[0003] Although the catalpa tree has been cultivated in China for many years, previous research has focused primarily on physiological ecology, genotypes, environmental responses, and functional development, with limited research on the interaction between the catalpa tree and microorganisms. Plant endophytic bacteria are bacteria that live in the intercellular spaces or within the cells of various tissues and organs of healthy plants. Plants and endophytic bacteria coexist in their natural habitats for a long time, and some endophytic bacteria have special applications for plants; to a certain extent, the two have a harmonious symbiotic relationship. Plants experience various environmental stresses over a long period, ultimately leading to a decline in productivity. Obtaining stress-resistant varieties using genetic engineering is an important means of improving their productivity. However, since stress-resistant varieties do not provide stress tolerance and are not ecologically sound, utilizing potentially beneficial endophytic bacteria can be an alternative strategy to improve plant resistance.

[0004] Compared to exogenous microorganisms, the catalpa tree has adapted to its endophytic flora during its long-term growth and evolution, establishing a symbiotic relationship. Its abundant endophytic resources may be more suitable for development into functional microbial agents specifically for catalpa cultivation. It is noteworthy that no research reports on catalpa endophytic flora have been found to date. Therefore, this invention provides an endophytic rhizobium isolated from catalpa tissue culture seedlings, which exhibits outstanding nitrogen fixation, phosphorus solubilization, and disease inhibition effects, promoting the healthy growth of tissue culture seedlings and potted seedlings. It has significant application value in promoting the growth of catalpa tissue culture seedlings and rooted seedlings. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a maize rhizobium strain NF11 and its applications.

[0006] The technical solution provided by this invention is as follows: a strain of maize rhizobium NF11, characterized in that it is classified and named as maize rhizobium. Rhizobium zeaeIt is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27776, deposited on July 3, 2023, and located in Beijing, China.

[0007] Another objective of this invention is to use the maize rhizobium NF11 to promote the growth of catalpa tissue culture seedlings or potted catalpa seedlings.

[0008] The beneficial effects of this invention are as follows:

[0009] 1. Maize rhizobium NF11 is an endophytic bacterium isolated from catalpa tissue culture seedlings. In the long process of natural evolution, it has formed a mutually beneficial symbiotic relationship with the catalpa host, and has a positive effect on promoting the growth and propagation of catalpa seedlings.

[0010] 2. Currently, there is no known application of maize rhizobium in promoting the growth of catalpa seedlings. Through the analysis of the characteristics of maize rhizobium NF11, this invention found that it has a strong nitrogen-fixing effect and outstanding phosphorus-solubilizing ability. It also has a certain inhibitory effect on plant pathogens such as Alternaria and Allium spp., and has the potential to be used as an agricultural input for the large-scale cultivation of catalpa.

[0011] 3. The rhizobium NF11 of maize has a significant effect on promoting the growth of catalpa tissue culture seedlings. Compared with the blank control group, the average plant height of the experimental group increased by 39.6% and the average ground diameter increased by 36.05%. The use of rhizobium NF11 of maize to promote the growth of catalpa tissue culture seedlings can facilitate subsequent transplanting and greatly improve their survival rate.

[0012] 4. The rhizobium NF11 of maize has a good growth-promoting effect on potted catalpa seedlings. Compared with the blank control group, the average plant height of the potted catalpa seedling experimental group increased by 25.63% and the average ground diameter increased by 16.34%, which improved the survival rate of potted catalpa seedlings and enhanced the absorption of soil nutrients that are difficult to degrade and utilize.

[0013] 5. In addition to its good growth-promoting effect on tissue-cultured and potted seedlings of Catalpa macrophylla, the corn rhizobium NF11 can also be used for large-scale field cultivation of Catalpa macrophylla, thereby increasing the yield of Catalpa macrophylla trees and bringing significant economic and social benefits. Attached Figure Description

[0014] Figure 1 This is a colony morphology diagram of *Rhizobium NF11* in maize.

[0015] Figure 2 This is a microscopic morphological image of NF11, a rhizobium in maize. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0017] Example 1: Screening of endophytic bacteria in catalpa tissue culture seedlings

[0018] Catalpa tree tissue culture seedlings were obtained from the National Rare Tree Species Breeding Base in Zaoyuan Town, Zhangqiu City, Shandong Province. The seedlings were handled with sterile forceps and placed in sterile petri dishes, rinsed three times with sterile water, and then cut into small pieces with sterile scissors. The pieces were transferred to Erlenmeyer flasks containing 100 mL of sterile water. After incubation at 28℃ and 180 rpm for 1 hour, the mixture was serially diluted, and 0.1 mL was spread onto solid plates of LB, BRP, and PDA media, respectively, and incubated at 28℃ for 5 days. Single colonies were picked from the solid plates of LB, BRP, and PDA media and purified 2-3 times in their corresponding media. The strains were then isolated by preservation on test tube slant culture.

[0019] Twenty-four endophytic bacteria strains were isolated and screened from catalpa tissue culture seedlings. All strains grew well on LB medium, grew slowly on PDA medium, and did not grow on red arbutin medium. Among them, the endophytic bacteria NF11 isolated and screened from catalpa tissue culture seedlings on LB medium showed a significant growth-promoting effect after nitrogen fixation, phosphorus solubilization, and pathogen inhibition characteristics analysis, indicating a positive effect on the expansion of catalpa seedlings.

[0020] The LB medium (g / L) contained: peptone 10, yeast extract 5, NaCl 10, agar 20, pH 7.0.

[0021] The PDA culture medium (g / L) consisted of 5g potato starch, 20g glucose, 20g agar, and pH 5.6.

[0022] The Bengal Red medium (g / L) contains: peptone 5, glucose 10, KH2PO4 1, MgSO4·7H2O 0.5, Bengal Red 0.033, chloramphenicol 0.1, agar 20, pH 6.5.

[0023] Example 2: Genus identification of endophytic fungus NF11 in catalpa tissue culture seedlings

[0024] Endophytic bacteria NF11 from catalpa tissue culture seedlings were purified multiple times using the streak plating method. After culturing on LB agar for 2 days, the colonies were pale yellow, round, opaque, with a smooth and moist surface and regular edges (e.g., ...). Figure 1(As shown). The endophytic bacteria NF11 in catalpa tissue culture seedlings, after Gram staining, were observed under an optical microscope. The individual bacteria are rod-shaped and stain red with Gram stain, belonging to Gram-negative bacteria.

[0025] The LB solid medium (g / L) contained: peptone 10, yeast extract 5, NaCl 10, agar 20, pH 7.0.

[0026] The genomes of the selected strains were extracted using a bacterial genome extraction kit. PCR amplification was performed using universal primers for 16S rDNA: the forward primer was 27F: 5′AGAGTTTGATCCTGGCTCA3′; the reverse primer was 1492R: 5′GGTTACCTTGTTACGACTT3′. The amplification program was: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, repeated 30 times, with a final extension at 72℃ for 10 min. The PCR product was sequenced, and its nucleotide sequence length was 1375 bp. Blast sequence alignment analysis revealed that the endophytic bacterium NF11 in the catalpa tissue culture seedlings was related to... Rhizobium zeae The 16S rDNA sequence similarity of RZM18R (KX932068) reached 99.56%, indicating that they are the same species, belonging to the rhizobium family of maize (Maize rhizobium). Rhizobium zeae The endophytic fungus NF11 in the above-mentioned catalpa tissue culture seedlings is the same as the corn rhizobium NF11.

[0027] The 16S rDNA sequence of *Rhizobium NF11* in maize is as follows:

[0028] Example 3: Functional characteristics of maize rhizobium NF11

[0029] Nitrogen fixation effect: When the test tube slant culture of Rhizobium maize NF11 was spotted into nitrogen-free solid medium and cultured in the dark at 28℃ for 4 days, Rhizobium maize NF11 grew well on nitrogen-free solid medium, with a colony diameter of 3.75 mm, indicating that it has good nitrogen fixation ability. The culture medium of *Rhizobium maize* NF11, preserved in test tube slant culture, was inoculated into LB liquid medium and cultured at 28℃ and 180 rpm for 24 h. 2 mL of fermentation broth was then transferred to a centrifuge tube and centrifuged at 10000 rpm for 10 min to collect the cells. 1.5 mL of sterile water was added to prepare a bacterial suspension. 1 mL of the bacterial suspension was inoculated into nitrogen-free liquid medium and cultured with an equal volume of sterile water as a control. The culture was carried out at 28℃ and 180 rpm for 4 days. The nitrogen content in the fermentation broth of strain NF11 was 38.51 mg / L using the alkaline potassium persulfate-salicylic acid method, demonstrating that *Rhizobium maize* NF11 possesses good nitrogen-fixing ability to meet the needs of its growth and reproduction.

[0030] Phosphorus solubilization effect: The culture medium of *Rhizobium maize* NF11, preserved in test tube slant agar, was incubated at 28℃ for 7 days. The ratio of the phosphorus-solubilizing clear zone to the colony size was 1.89, indicating good phosphorus solubilization ability. The culture medium of *Rhizobium maize* NF11 was inoculated into LB liquid medium and incubated at 28℃ and 180 rpm for 24 hours. 2 mL of fermentation broth was centrifuged at 10000 rpm for 10 minutes to collect the cells. 1.5 mL of sterile water was added to prepare a bacterial suspension. 1 mL of the bacterial suspension was incubated in phosphorus-solubilizing liquid medium, with an equal volume of sterile water as a control. After 7 days of incubation at 28℃ and 180 rpm, the available phosphorus content in the *Rhizobium maize* NF11 culture medium was determined using the molybdenum antimony colorimetric method to be 1007.95 mg / L, demonstrating that *Rhizobium maize* NF11 possesses good phosphorus solubilization efficiency.

[0031] Antibacterial effect: Using *Alternaria alternata* and *Sterilaria spp.* as indicator fungi of plant pathogens, *Rhizobium maize* NF11 was cultured in LB liquid medium for 24 h. 100 μL of the culture was then spread onto PDA agar plates. A 5 mm sterile perforator was used to punch a hole in the center of each plate. Colonies of *Alternaria alternata* and *Sterilaria spp.* of equal size were then placed in the holes using the same sterile perforator. The plates were incubated at 28°C for 6 days, and the antibacterial effect was observed. Solid plates containing neither the tested strain nor *Sterilaria spp.* were used as controls. The antibacterial effect was calculated as: Inhibition rate (%) = (Coronation diameter of control group - Colony diameter of test group) / Colony diameter of control group × 100%. The results showed that *Rhizobium maize* NF11 had a certain ability to inhibit plant pathogens, with inhibition rates of 11.4% and 8.89% against *Alternaria alternata* and *Sterilaria spp.*, respectively.

[0032] The LB liquid culture medium (g / L) contained: 10g peptone, 5g yeast extract, 10g NaCl, and pH 7.0.

[0033] The nitrogen-free solid culture medium (g / L) is as follows: mannitol 10, KH2PO4 0.2, MgSO4·7H2O 0.2, NaCl 0.2, CaSO4·2H2O 0.1, CaCO3 5, agar 20, pH 7.0.

[0034] The nitrogen-free liquid culture medium (g / L) is: mannitol 10, KH2PO4 0.2, MgSO4·7H2O 0.2, NaCl 0.2, CaSO4·2H2O 0.1, CaCO3 5, pH 7.0.

[0035] The phosphorus-solubilizing solid culture medium (g / L) is as follows: glucose 10, Ca3(PO4)2 5, MgCl·6H2O 5, MgSO4·7H2O 0.25, KCl 0.2, (NH4)2SO4 0.1, agar 20, pH 7.5.

[0036] The phosphorus-solubilizing liquid culture medium (g / L) is as follows: glucose 10, Ca3(PO4)2 5, MgCl·6H2O 5, MgSO4·7H2O 0.25, KCl 0.2, (NH4)2SO4 0.1, pH 7.5.

[0037] The PDA culture medium (g / L) is: potato starch 5, glucose 20, agar 20, pH 5.6.

[0038] Example 4: Application of Maize Rhizobium NF11 in Promoting the Growth of Catalpa Tissue Culture Seedlings

[0039] Strain activation: A slant culture of *Rhizobium maize* NF11 was transferred to LB broth for activation. The culture was incubated at 30℃ and 200 rpm for 20 h on a rotating shaker until the bacterial cells became turbid. The viable count was determined to be 3.7 × 10⁻⁶. 9 cfu / mL.

[0040] Growth promotion of Catalpa tissue culture seedlings: The activated corn rhizobium NF11 suspension was inoculated into the rhizosphere of Catalpa tissue culture seedlings using a sterile syringe at a dosage of 2 mL / seedling. A control group was set up, and an equal volume of sterile physiological saline was added as a blank control. Each treatment group had 10 replicates. The Catalpa tissue culture seedlings were placed in a light incubator to simulate sunlight, and were periodically immersed in sterile water. The seedling height and ground diameter were measured 30 days after inoculation. Table 1 shows that the seedling height and ground diameter of the Catalpa tissue culture seedlings in the corn rhizobium NF11 treatment group were significantly higher than those in the blank control group, with an average increase of 39.6% in plant height and 36.05% in average ground diameter. Therefore, corn rhizobium NF11 has a good growth-promoting effect on Catalpa tissue culture seedlings.

[0041] The LB liquid culture medium (g / L) contained: 10g peptone, 5g yeast extract, 10g NaCl, and pH 7.0.

[0042] Table 1. Growth-promoting effect of maize rhizobium NF11 on catalpa tissue culture seedlings

[0043]

[0044] Example 5: Growth-promoting effect of maize rhizobium NF11 on potted catalpa seedlings

[0045] Strain activation: A slant culture of *Rhizobium maize* NF11 was transferred to LB broth for activation. The culture was incubated at 30°C and 200 rpm for 20 h on a rotary shaker until the bacterial cells became turbid. The culture was then centrifuged at 10,000 rpm for 5 min. The bacterial pellet was washed three times with sterile water, and the viable count was adjusted to 10⁻⁶ cells / mL with sterile water. 8 cfu / mL.

[0046] Growth Promotion of Potted Catalpa Seedlings: The activated corn rhizobium NF11 suspension was inoculated into the rhizosphere of one-year-old potted Catalpa seedlings at a rate of 10 mL per seedling. A control group and an equal volume of sterile physiological saline were added as a blank control. Each treatment group had 10 replicates. The potted Catalpa seedlings were placed in a greenhouse under natural light and watered as needed. The seedling height and ground diameter were measured 30 days after inoculation. As shown in Table 2, the plant height and ground diameter of the potted Catalpa seedlings in the corn rhizobium NF11 treatment group were significantly higher than those in the blank control group, with an average increase of 25.63% in plant height and 16.34% in average ground diameter. Therefore, corn rhizobium NF11 has a good growth-promoting effect on potted Catalpa seedlings.

[0047] The LB liquid culture medium (g / L) contained: 10g peptone, 5g yeast extract, 10g NaCl, and pH 7.0.

[0048] Table 2. Growth-promoting effect of maize rhizobium NF11 on potted seedlings of Catalpa bungei.

[0049]

[0050] It should be understood that any parts not described in detail in this specification belong to the prior art. The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements to the technical solutions of the present invention made by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of maize rhizobium NF11, characterized in that, Classified as maize rhizobium Rhizobium zeae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27776, deposited on July 3, 2023, and located in Beijing, China.

2. The application of the maize rhizobium NF11 strain as described in claim 1 in promoting the growth of catalpa tissue culture seedlings or potted catalpa seedlings.

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