Neorhizobium SP. and use thereof

Abstract

A Neorhizobium huautlense strain CNBG-PGPR-21 and uses thereof. The Neorhizobium sp. CNBG-PGPR-21 was deposited in China General Microbiological Culture Collection Center (CGMCC) on August 1st, 2022,

Description

[0001] The present invention pertains to the field of microbial technology, and particularly relates to a Neorhizobium sp. and uses thereof. BACKGROUND OF THE INVENTION

[0002] The healthy growth of plants cannot be separated from various nutrient elements provided by soil, wherein nitrogen is one of the essential nutrient elements, which plays a key role in the synthesis of proteins, nucleic acids and other various nutrient elements, as well as the growth and health of plants. Applying chemical nitrogen fertilizers to poor soil can effectively supplement nitrogen nutrient elements, so as to improve soil nutrient structure, promote plant growth rate and increase crop yield. However, excessive application of chemical fertilizers will also bring a series of problems such as soil hardening, nutrient imbalance, intensified acidification and decreased activity of beneficial microorganisms. One of the effective ways to solve these crises is to widely plant leguminous forages as green manure crops. Leguminous plants and nitrogen-fixing bacteria of Rhizobiaceae form root nodules or stem nodules with efficient nitrogen-fixing functions, which can fix and transform nitrogen in the atmosphere into ammonium nitrogen that can be directly utilized by plants, and part of the ammonium nitrogen can also be separated from the root nodules or stem nodules and enter the soil.

[0003] The leguminous plant, Sesbania herbacea, is a common excellent summer green manure crop in the fields in China. The plant is rich in nutrients and can adapt to warm and humid environments. At the same time, it has a strong tolerance to salt, waterlogging, barren, drought, diseases and pests, and wind, and can grow on saline soil with 0.3% soil salt content or alkaline soil with pH 9.5. The diversity of endogenous nitrogen-fixing Rhizobium species in Sesbania herbacea is very limited, and it is reported in the literature that Neorhizobium huautlense is the main one. The root nodule or stem nodule symbiont produced by nodulation of Neorhizobium huautlens with roots or stems of Sesbania herbacea plays an important role in the growth of Sesbania herbacea. However, the endogenous nitrogen-fixing bacteria in Sesbania herbacea currently reported can only tolerate 1.0% NaCl on the medium, and there is no report on the growth promoting function of this strain to other plants. SUMMARY OF THE INVENTION 17 07 24

[0004] Purpose of the present invention: In order to solve the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a Neorhizobium sp. with salt tolerance and plant growth promoting capacity, which is further identified as Neorhizobium huautlense.

[0005] The technical problem to be solved by the present invention is to provide the fermentation broth prepared by Neorhizobium huautlense.

[0006] The technical problem to be solved by the present invention is to provide use of Neorhizobium huautlense or the fermentation broth in nitrogen fixation of a plant or degradation of organic phosphorus or inorganic phosphorus.

[0007] The technical problem to be solved by the present invention is to provide use of Neorhizobium huautlense or the fermentation broth in salt tolerance of a plant.

[0008] The final technical problem to be solved by the present invention is to provide use of Neorhizobium huautlense or the fermentation broth in plant growth promoting.

[0009] Technical scheme: In order to solve the above technical problems, the present invention provides a Neorhizobium sp. CNBG-PGPR-21, and the Neorhizobium sp. CNBG-PGPR-21 was deposited in China General Microbiological Culture Collection Center on August 1st, 2022, with the deposit number of CGMCC No. 25336.

[0010] The present invention also includes a fermentation broth containing the Neorhizobium sp. CNBG-PGPR-21 or a bacterial suspension thereof.

[0011] The present invention also includes use of the Neorhizobium sp. CNBG-PGPR-21 or the fermentation broth or a bacterial suspension thereof in nitrogen fixation of a plant or degradation of organic phosphorus or inorganic phosphorus.

[0012] The present invention also includes use of the Neorhizobium sp. CNBG-PGPR-21 or the fermentation broth or a bacterial suspension thereof in salt tolerance of a plant.

[0013] Wherein, the use includes culturing the Neorhizobium sp. CNBG-PGPR-21 in a medium containing 0-2% NaCl.

[0014] Wherein, the medium is an R2A medium or a YMA medium.

[0015] Wherein, the R2A medium includes an R2A solid medium and an R2A liquid medium; the R2A solid medium includes 0.25 g of tryptone, 0.5 g of acid hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5 g of glucose, and 15.0 g of agar; and in the R2A liquid medium, the agar in the R2A solid medium is removed, and the remaining ingredients are the same. 17 07 24

[0016] Wherein, the YMA medium includes a YMA solid medium and a YMA liquid medium; the YMA solid medium includes 0.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of sodium chloride, 10.0 g of mannitol, 1.0 g of yeast extract, 0.025 g of Congo red, 15.0 g of agar, and 1000 mL of distilled water; and in the YMA liquid medium, the agar in the YMA solid medium is removed, and the remaining ingredients are the same.

[0017] The present invention also includes use of the Neorhizobium sp. CNBG-PGPR-21 or the fermentation broth or a bacterial suspension thereof in plant growth promoting.

[0018] Wherein, the plant includes, but not limited to soybeans.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the Neorhizobium huautlense CNBG-PGPR-21 CGMCC No. 25336 of the present invention has fast growth and good stability, and can grow and pass on a Rhizobium medium YMA containing 2.0% NaCl, thereby showing its unique salt tolerance and nitrogen fixation capacities. The Neorhizobium huautlense CNBG-PGPR-21 of the present invention can degrade phosphorus in an inorganic phosphorus plating medium within 6 d; the Neorhizobium huautlense CNBG-PGPR-21 can grow normally on a solid medium only containing lecithin as the phosphorus source in terms of degradation of organic phosphorus. The Neorhizobium huautlense CNBG-PGPR-21 of the present invention can significantly increase the fresh weight of overground parts of soybean seedlings by 75.0%. At the same time, the average plant length can be increased by 7.5% and the fresh weight of underground parts can be increased by 13.8% via the bacterial liquid of the Neorhizobium huautlense CNBG-PGPR-21. Brief Description of the Drawings

[0020] Fig. 1 shows the colony morphology of the Neorhizobium huautlense CNBG-PGPR-21 on a plate;

[0021] Fig. 2 shows the cell morphology of the Neorhizobium huautlense CNBG-PGPR-21;

[0022] Fig. 3 shows the position of the Neorhizobium huautlense CNBG-PGPR-21 in the phylogenetic tree;

[0023] Fig. 4 shows the growth of the Neorhizobium huautlense CNBG-PGPR-21 on the YMA solid medium containing 2% NaCl;

[0024] and Fig. 5 shows the experiment results of promoting soybean seedling growth via the Neorhizobium huautlense CNBG-PGPR-21. 17 07 24 DETAILED DESCRIPTION

[0025] The present invention will be described in detail in combination with the accompanying drawings and examples.

[0026] Example I Isolation and identification of Neorhizobium huautlense CNBG-PGPR-21

[0027] (1) Sample dilution and coating

[0028] A Sesbania herbacea sample was collected from the beach of Linhong Estuary (34°45’ north latitude and 119° 13’ east longitude) in Lianyun New Town, Lianyungang, Jiangsu Province. The root was taken, washed in running water first to wash the surface soil off, and then shaken and washed in sterile normal saline for about 30 min. The root was moved step by step into alcohol with a gradient of 50%, 70%, 90% and 100%, vibrated and washed for 15 minutes, respectively. The root was placed in a sterile culture dish and put in a sterile bechtop until the alcohol was well volatilized. The middle part of the root was taken and cut into several sections with a length of 0.3-0.5 cm. The treated root was placed in a 50 mL centrifuge tube, into which was added an appropriate amount of normal saline and a small amount of zircon beads with a diameter of 0.5 cm, placed in a nucleic acid extractor (MP FastPrep-24), and vibrated once at the highest frequency for 45 s. 100 pL of the supernatant was absorbed, and coated on an R2A solid medium plate (the R2A medium: 0.25 g of tryptone, 0.5 g of acid hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5 g of glucose, and 15.0 g of agar, pH 7.2 ± 0.2 (25°C), which was autoclaved at 121°C for 20 min).

[0029] The supernatant was diluted in 10-fold to prepare another one in parallel. The plates were put upside down in a constant temperature incubator at 30°C and cultured for at least 24 h, and observed regularly until colonies appeared.

[0030] (2) Isolation and purification of strains

[0031] All the plates with colonies using the same sample were taken out, and the dilution plates with obvious single colonies and moderate quantity were selected. The colonies with different morphologies on the plates were picked out onto new R2A plates, purified by zoning, and purified repeatedly until single colonies appeared, and then streaking cultured repeatedly for at least 5 times.

[0032] (3) Dyeing and preservation

[0033] The colony of the strain to be detected was picked out onto the central area of a glass slide, and after four steps of primary dyeing with the ammonium oxalate-crystal violet dye 17 07 24 solution, mordant dyeing with the iodine solution, decoloring with 75% ethanol, and secondary dyeing with safranin, observed by microscopic examination, and the dyeing result was recorded; and the purified and dyed single colony from the R2A solid medium plate was picked out into a sterile R2A liquid medium, and cultured at 200 r / m and 30°C for 24±2 h. After being shaken homogeneously, the bacterial liquid was mixed with 60% sterile glycerol in V:V of 1:1, subpackaged into sterile bacteria-preserving tubes, labeled and preserved in a refrigerator at -80°C.

[0034] (4) Identification of strains

[0035] The plate containing strains with the corresponding number was taken out of the refrigerator at 4°C. A single colony was picked out as the DNA template for PCR amplification, 16S rDNA sequencing and sequence alignment. The specific operation was as follows:

[0036] 1) 50 pL of amplification system:

[0037] The 50 pL system consisted of the following five parts: the Taq enzyme Mix (25 pL), the upstream primer 27F (1 pL), the downstream primer 1492R (1 pL), the DNA template (1 pL) and ddH2O (22 pL). Wherein, the sequence of 27F was: 5’-AGA GTT TGA TCM TGG CTC AG-3’, and the sequence of 1492R sequence was: 5 -GGY TAC CTT GTT ACG ACT T-3’. The length of the amplified fragment was about 1413 bp, and the sequence of the amplified nucleotide was shown as SEQ ID NO.l in the sequence list.

[0038] 2) Amplification condition

[0039] Pre-denaturation temperature: 94°C

[0040] Step 1, denaturation: 94°C for 30 s

[0041] Step 2, annealing: 56°C for 60 s

[0042] Step 3, extension: 72°C for 45 s

[0043] Number of cycles: 30 times

[0044] Step 4, the final extension: 72°C for 10 min

[0045] Step 5, storage: 4°C

[0046] 3) Agarose gel electrophoresis

[0047] 1 g of agarose was weighed and dissolved in 100 mL of a TBE electrophoresis buffer, heated by microwave until the solution was clear, into which was added l%o of a GelRed nucleic acid dye, shaken uniformly, stood until there were no bubbles, and slowly poured into a gel plate. After standing for about 1 h, the solidified gel mass was taken out and put into an 17 07 24 electrophoresis tank. 4 pL of a PCR amplification product was added into each gel hole, and the gel was run at 120 V for 20 min. The gel mass was taken out, put into a gel imager with UV light selected, and photographed. The number of the corresponding samples with a clear band at 1500 bp on the gel mass was recorded, and the remaining PCR amplification product of this sample was placed in a refrigerator at 4°C.

[0048] 4) Purification of PCR products

[0049] The PCR amplification product stored in the refrigerator at 4°C was taken out, and the PCR product was purified by the AxyPrep™ PCR Cleanup Kit (a nucleic acid purification kit) to obtain purified DNA.

[0050] (5) Sequence identification

[0051] The resuspended DNA obtained in the above steps was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing result was aligned with the National Center for Biotechnology Information (NCBI) GeneBank. The result shows that the strain provided by the present invention is Neorhizobium huautlense. The strain has been deposited in China General Microbiological Culture Collection Center (CGMCC) since August 1st, 2022, with the deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101, and the deposit number of the strain is CGMCC No.25336.

[0052] The Neorhizobium huautlense CNBG-PGPR-21 CGMCC No.25336 is aerobic, Gram-negative and does not form spores. Referring to Fig. 1, colonies form on the surface of the plating medium within 1-2 days. The colonies are small, round, and convex lens shaped as a whole. They are milky white with surfaces smooth and translucent, and slimy. Referring to Fig. 2, the thalli of CNBG-PGPR-21 observed under the oil immersion lens of a microscope are long rod shaped, with a length of 1-3 pm and a width of 0.3-1 pm. After being cultured on a YMA solid medium containing 2% NaCl for several days, the colonies are light pink. With regard to the position of the Neorhizobium huautlense CNBG-PGPR-21 in the phylogenetic tree, we can see Fig. 3.

[0053] Example 2 Growth of Neorhizobium huautlense CNBG-PGPR-21 on Ashby nitrogen-free medium

[0054] 1) Ashby nitrogen-free solid medium (Catalogue of agricultural strains in China, 2001): 10 g of glucose, 0.2 g of potassium dihydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.2 g of sodium chloride, 0.1 g of calcium sulfate dihydrate, 5 g of calcium carbonate, 20 g of agar, and 1000 mL of distilled water, which was autoclaved at 121°C for 20 min. In the Ashby nitrogen-free liquid medium, the agar was removed, and the remaining ingredients were the same. 17 07 24

[0055] 2) Plate streaking culture: A single colony was picked out from the plate of the correctly identified strain, inoculated on the Ashby nitrogen-free solid medium, and cultured while standing at the constant temperature of 30°C for 48 h to observe the growth on the plate. The nitrogen fixation capacity could be determined only when the colony passed at least three times and could be cultured normally in the liquid medium.

[0056] 3) Summary of results: the Neorhizobium huautlense CNBG-PGPR-21 can grow rapidly on the Ashby nitrogen-free solid medium and liquid medium, thereby demonstrating that it has a strong nitrogen fixation capacity. A single colony was picked out from the plate, inoculated into 5 ml of the Ashby nitrogen-free liquid medium, and cultured under vibrating for 72 h at 30°C and 120 rpm / min. The number of cells in the bacterial liquid was confirmed to be 1.0* 108 / mLby microscopic examination.

[0057] Example 3 Growth of Neorhizobium huautlense CNBG-PGPR-21 on Rhizobium medium YMA

[0058] 1) YMA Rhizobium solid medium: 0.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of sodium chloride, 10.0 g of mannitol, 1.0 g of yeast extract, 0.025 g of Congo red, 15 0 g of agar, and 1000 mL of distilled water, the pH of which was adjusted to 7.0=0.2 (25°C), and which was autoclaved at 121°C for 20 min. In the YMA Rhizobium liquid medium, the agar was removed, and the remaining ingredients were the same.

[0059] 2) Plate streaking culture: A single colony was picked out from the plate of the correctly identified strain, inoculated on the YMA nitrogen-free solid medium, and cultured while standing at the constant temperature of 30°C for 48 h to observe the growth on the plate. The nitrogen fixation capacity could be determined only when the colony passed at least three times.

[0060] 3) Summary of results: the Neorhizobium huautlense CNBG-PGPR-21 can grow on the YMA nitrogen-free solid medium and liquid medium.

[0061] Example 4 Experiment on salt-tolerant growth of Neorhizobium huautlense CNBG-PGPR-21

[0062] 1) R2A medium: 0.25 g of tryptone, 0.5 g of acid hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5 g of glucose, and 15.0 g of agar, pH 7.2 ± 0.2 (25°C), which was autoclaved at 121°C for 20 min. In the R2A liquid medium, the agar was removed, and the remaining ingredients were the same.

[0063] 2) Preparation of liquid medium: The R2A liquid media containing 0%, 1%, 3%, 4%, 6% and 8% NaCl were formulated respectively, and sterilized at a high temperature for later use. 17 07 24

[0064] Preparation of solid medium: The R2A and YMA solid media containing 0%, 1%, 3% and 5% NaCl were formulated respectively, and sterilized at a high temperature for later use.

[0065] 3) Strain activation: The medium plate grown with the required strain was taken out from the refrigerator at 4°C. The single colony on the plate was dipped with an inoculation ring for aseptic operation into a centrifuge tube filled with 5 ml of the R2A or YMA liquid medium, and cultured overnight at the condition of 30°C.

[0066] 4) Salt tolerance experiment:

[0067] Liquid medium: The bacterial liquid of the Neorhizobium huautlense CNBG-PGPR-21 cultured overnight (the concentration of the bacterial liquid was about 5><108CFU / ml) was inoculated respectively into 5 ml of the R2A liquid medium containing 0%, 1%, 3%, 4%, 6% and 8% NaCl respectively in an inoculation amount of 5%, and cultured under shaking at the condition of 30°C. After that, for the strains at a different gradient, the growth of each strain was observed and determined at 16 h and 23 h of culture, respectively.

[0068] Solid medium: The activated strain was plate streaking cultured on the surface of the R2A and YMA solid medium plates containing 1%, 2%, 3% and 5% NaCl. The strain was cultured overnight at the condition of 30°C.

[0069] 5) Summary of results:

[0070] The Neorhizobium huautlense CNBG-PGPR-21 can grow normally in the R2A liquid medium containing 1% NaCl, but cannot grow normally in the R2A liquid medium with salinity higher than 1% NaCl.

[0071] The Neorhizobium huautlense CNBG-PGPR-21 can grow normally on the R2A solid medium containing 1% NaCl, but cannot grow normally in the R2A solid medium with salinity higher than 1% NaCl.

[0072] The Neorhizobium huautlense CNBG-PGPR-21 can grow normally on the YMA solid medium containing 2% NaCl, but cannot grow normally on the YMA solid medium with salinity higher than 2% NaCl. Referring to Fig. 4, after being cultured for about one month on the YMA solid medium with 2% NaCl, the colony was changed from milky white to light pink.

[0073] Example 5 Inorganic phosphorus degradation capacity of Neorhizobium huautlense CNBG-PGPR-21

[0074] 1) Preparation of medium

[0075] The inorganic phosphorus solid medium plate and inorganic phosphorus liquid medium were prepared respectively, wherein the ingredients of the inorganic phosphorus solid 17 07 24 medium were 10 g of glucose, 5 g of tricalcium phosphate, 0.1 g of calcium carbonate, 0.5 g of magnesium sulfate, 0.5 g of ammonium sulfate, 0.1 g of calcium sulfate, 0.005 g of ferric chloride, 20 g of agar, and 1000 mL of distilled water, and autoclaved at 121°C for 20 min. In the inorganic phosphorus liquid medium, the agar ingredient was removed, and the remaining components were the same as the inorganic phosphorus solid medium (note: calcium carbonate needed to be sterilized and added separately, then poured into the plate).

[0076] 2) Qualitative experiment of inorganic phosphorus degradation capacity

[0077] The bacteria-preserving tubes of the strain CNBG-PGPR-21 frozen in the refrigerator at -80°C were taken out, thawed to a fluid state on the ice, and shaken to be mixed well. An appropriate amount of the bacterial liquid was picked out with an inoculation ring and streaked within three zones on the Nutrient Agar (NA) solid medium plate. The plate was cultured at the constant temperature of 30°C for 24±2 h. The formula of the NA culture medium was as follows (per liter): 10.0 g of peptone, 3.0 g of beef powder, 5.0 g of sodium chloride, and 15.0 g of agar, pH 7.3±0.1. The single colony was picked out and purified for 1-2 times, and identified and confirmed. After confirmation, the single colony was picked out onto the inorganic phosphorus solid medium plate. The plate was cultured at the constant temperature of 30°C for 1-9 d, and observed whether there was a phosphorus dissolving circle around the colony.

[0078] According to the experiment, the strain CNBG-PGPR-21 grows well on the inorganic phosphorus solid medium, and the phosphorus dissolving circle can appear at 6 d.

[0079] Example 6 Organic phosphorus degradation capacity of Neorhizobium huautlense CNBG-PGPR-21

[0080] 1) Preparation of medium

[0081] The organic phosphorus solid medium plate and organic phosphorus liquid medium were prepared respectively, wherein the ingredients of the organic phosphorus solid medium were 10 g of glucose, 5 g of lecithin, 5 g of calcium carbonate, 0.5 g of magnesium sulfate, 0.5 g of ammonium sulfate, 0.1 g of calcium sulfate, 0.005 g of ferric chloride, 20 g of agar, and 1000 mL of distilled water, and autoclaved at 121°C for 20 min. In the organic phosphorus liquid medium, the agar ingredient was removed, and the remaining components were the same as the organic phosphorus solid medium (note: calcium carbonate needed to be sterilized and added separately).

[0082] 2) Qualitation of organic phosphorus degradation capacity

[0083] The bacteria-preserving tubes of the strain CNBG-PGPR-21 frozen in the refrigerator at -80°C were taken out, thawed to a fluid state on the ice, and shaken to be mixed well. An appropriate amount of the bacterial liquid was picked out with an inoculation ring and 17 07 24 streaked within three zones on the NA solid plate. The plate was cultured at the constant temperature of 30°C for 24±2 h. The single colony was picked out and purified for 1-2 times, and identified and confirmed. After confirmation, the single colony was picked out onto the organic phosphorus solid plate. The plate was cultured at the constant temperature of 30°C for 1-9 d, and observed whether there was a phosphorus dissolving circle around the colony.

[0084] According to the experiment, the strain CNBG-PGPR-21 grows well on the organic phosphorus solid plate.

[0085] Example 7 Experiment on promoting soybean seedling growth via Neorhizobium huautlense CNBG-PGPR-21

[0086] 1) Preparation of fermentation broth: The Neorhizobium huautlense CNBG-PGPR-21 of the present invention was inoculated into a sterilized YMA culture solution, and cultured for 24 hours at the condition of 30°C and 200 r / min to obtain a fermentation broth. The bacterial liquid was diluted to a concentration of 5xlO6CFU / ml for later use, while sterile water was set as a control.

[0087] 2) Experiment design: The soybean variety Heijiao 00-1152 was used, which was from Nanjing Agricultural University. A bacterial liquid treatment group and a control group were provided, with 8-9 replicates for each treatment. Each seedling was treated by root irrigation with 50 mL of the bacteria, and was treated by irrigation with the bacteria during the period of the seedling growth to two leaves and one heart. After two weeks, the growth and development indexes during seedling stages were determined, including plant height, root length and fresh weight of overground parts / underground parts.

[0088] Experimental result: As shown in Fig.5, the Neorhizobium huautlense CNBG-PGPR-21 of the present invention can significantly increase the fresh weight of overground parts of soybean seedlings by 75.0%. At the same time, the average plant length can be increased by 7.5% and the fresh weight of underground parts can be increased by 13.8% via the bacterial liquid of the Neorhizobium huautlense CNBG-PGPR-21. The strain has no significant effect on the root length of soybean seedlings, but it can be observed that the strain can promote the abundance of plant lateral roots to some extent.

Claims

17 07 241. A Neorhizobium sp. CNBG-PGPR-21, characterized in that the Neorhizobium sp. CNBG-PGPR-21 was deposited in China General Microbiological Culture Collection Center (CGMCC) on August 1st, 2022, with the deposit number of CGMCC No. 25336.

2. A fermentation broth comprising the Neorhizobium sp. CNBG-PGPR-21 according to claim 1 or a bacterial suspension thereof.

3. Use of the Neorhizobium sp. CNBG-PGPR-21 according to claim 1 or the fermentation broth or a bacterial suspension thereof according to claim 2 in nitrogen fixation of a plant or degradation of organic phosphorus or inorganic phosphorus.

4. Use of the Neorhizobium sp. CNBG-PGPR-21 according to claim 1 or the fermentation broth or a bacterial suspension thereof according to claim 2 in plant growth promoting.

5. The use according to claim 4, wherein the plant is soybeans.

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