A self-generating nitrogen-fixing strain and its application

Abstract

The present invention provides a self-generated nitrogen-fixing strain and its application, belonging to the field of microbial technology. The self-generated nitrogen-fixing strain of the present invention is BWLY3X‑6, which belongs to Klebsiellavariicola, and the deposit number is GDMCCNO: 63038. The present invention also provides a self-generated nitrogen-fixing bacterial agent, including a bacterial suspension of the strain BWLY3X‑6. The strain BWLY3X‑6 and its bacterial suspension of the present invention can effectively increase the root length, plant height, fresh weight and total nitrogen content of banana plants, and have a good growth-promoting effect on bananas.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a self-generating nitrogen-fixing strain and an application thereof. Background Art

[0002] The banana industry is a pillar industry of agriculture in tropical and subtropical regions of my country. In these advantageous areas of banana cultivation, the development of bananas has driven the development of industries such as pesticides, fertilizer supply, packaging materials, and transportation. Banana is one of the fruit varieties with the highest demand for nutrients. Among the nutrients it needs, nitrogen occupies an important position. Nitrogen can effectively affect the photosynthesis process of plants and the distribution of photoassimilates in various organs of plants, leading to the accumulation of dry matter, the development of plants and fruits, etc. Banana growers often apply nitrogen fertilizers to maximize productivity, such as applying a large amount of nitrogen fertilizers during the seedling stage. However, excessive use of nitrogen fertilizers will not only increase soil acidification and salinization, but also make plants more susceptible to pathogens and increase the severity of plant diseases, such as banana wilt. The main forms of nitrogen fertilizers are ammonium, urea, and nitrates. Existing research results show that nitrate fertilizers reduce the severity of Fusarium wilt, while ammonium increases its severity. All these current situations show that relying entirely on chemical nitrogen fertilizers is not a long-term solution after all.

[0003] Nitrogen-fixing bacteria can improve soil fertility, convert carbon monoxide into fuel, use molecular nitrogen as nitrogen nutrition, reduce it to NH3, and then synthesize amino acids and proteins. There are three types of nitrogen-fixing bacteria: autotrophic nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria, and associated nitrogen-fixing bacteria. Among them, the nitrogen-fixing symbiotic system formed by symbiotic nitrogen-fixing bacteria and corresponding plants is considered to be the most efficient natural nitrogen-fixing system in nature. However, it can only fix molecular nitrogen in the air when it is mutually beneficial with plants, which has certain limitations. Associated nitrogen-fixing bacteria must live in the rhizosphere, leaf surface, or animal intestines to fix nitrogen. Autotrophic nitrogen-fixing bacteria are widely found in soil and water in nature, including species of various physiological types, such as photoautotrophic, aerobic Nostoc, photoautotrophic, obligately anaerobic Chromatium, photoheterotrophic, facultatively anaerobic Rhodospirillum, chemoheterotrophic, aerobic Azotobacter, chemoheterotrophic, obligately anaerobic Clostridium pasteurianum, and chemoheterotrophic, facultatively anaerobic Klebsiella, etc. Compared with other types of nitrogen-fixing bacteria, autotrophic nitrogen-fixing bacteria have a lower nitrogen fixation efficiency, but they have strong adaptability to the environment, do not depend on plants to survive, can absorb nitrogen from the air and reproduce, and after death, the cell protein is mineralized in the soil, which still helps plants to utilize nitrogen. As plant growth-promoting bacteria, autogenous nitrogen-fixing bacteria can not only improve nitrogen utilization to promote plant growth through nitrogen fixation, but also improve phosphorus utilization. Studies have shown that the presence of nitrogen-fixing bacteria can affect the content ratio of carbon and sulfur in the soil, accelerate the mineralization of residual organic matter in the soil, and thus reduce the absorption of heavy metal ions in the soil by plant roots. In addition, autogenous nitrogen-fixing bacteria can also directly affect plant growth by synthesizing plant-related growth hormones. These hormones can not only promote plant growth and nutrient absorption, but also indirectly protect host plants from plant pathogens and stimulate other beneficial rhizosphere microorganisms, which play an important role in improving the content of soil organic nitrogen fertilizer and increasing crop yields. Therefore, screening efficient autogenous nitrogen-fixing bacteria and synthesizing biological organic fertilizers have become the key to research in the field of banana cultivation. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the object of the present invention is to provide a self-generating nitrogen-fixing strain BWLY3X-6, which belongs to Klebsiella variicola and has a good growth-promoting effect on banana.

[0005] The invention provides a self-generating nitrogen-fixing strain. The self-generating nitrogen-fixing strain is BWLY3X-6, belongs to Klebsiella variicola, and has a preservation number of GDMCC NO: 63038.

[0006] The present invention also provides application of the above-mentioned autogenous nitrogen-fixing strain in promoting the growth of bananas.

[0007] The present invention also provides an autogenous nitrogen-fixing bacterial agent, comprising a bacterial suspension of the autogenous nitrogen-fixing strain BWLY3X-6, wherein the deposit number of the autogenous nitrogen-fixing strain BWLY3X-6 is GDMCC NO: 63038.

[0008] Preferably, the concentration of the autogenous nitrogen-fixing strain BWLY3X-6 in the bacterial agent is 1×10 4 ~9×10 8 cfu / mL.

[0009] The present invention also provides a method for preparing the above-mentioned bacterial agent. The self-generated nitrogen-fixing strain BWLY3X-6 is inoculated into a liquid culture medium for cultivation, the culture solution is centrifuged, and the bacterial precipitate is resuspended to obtain a bacterial suspension of the self-generated nitrogen-fixing strain BWLY3X-6.

[0010] Preferably, the liquid culture medium is LB liquid culture medium.

[0011] Preferably, the culture temperature is 36-38° C., and the culture time is 9-11 hours.

[0012] Preferably, the centrifugation condition is: 3000-5000 rpm for 5-15 min.

[0013] The present invention also provides application of the autogenous nitrogen-fixing bacteria agent in promoting the growth of bananas.

[0014] Preferably, the dosage of the bacterial agent is 10 to 20 mL / strain.

[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0016] The ammonia nitrogen content secreted by the self-generated nitrogen-fixing strain of the present invention is 17.98 μg / mL, and the nitrogenase activity is 220.51 ng / L, and the self-generated nitrogen-fixing strain has good nitrogen fixation ability. The self-generated nitrogen-fixing strain of the present invention can effectively improve the plant height, stem height, stem girth, root length, aboveground fresh weight and underground fresh weight of banana within a certain concentration range, and has a good growth-promoting effect on banana.

[0017] Self-generating nitrogen-fixing strain BWLY3X-6 (Latin name Klebsiella variicola BWLY3X-6), depository: Guangdong Microbial Culture Collection Center, depository address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC NO: 63038, deposit date December 12, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Plate morphology of strain BWLY3X-6;

[0019] Figure 2 : Transmission electron microscopy photo of strain BWLY3X-6;

[0020] Figure 3 : Electrophoresis of strain 16SrRNA, gyrB and nitrogenase gene nifH detection;

[0021] Figure 4 : Phylogenetic tree of strain BWLY3X-6 based on 16SrRNA sequence;

[0022] Figure 5 : Phylogenetic tree of strain BWLY3X-6 based on gyrB sequence;

[0023] Figure 6 : Ammonia nitrogen standard curve;

[0024] Figure 7 : Comparison of plant heights of Nandina chinensis and Banana oleracea under different treatments;

[0025] Figure 8 : Comparison of stem height of Nandina chinensis and Banana oleracea with different treatments;

[0026] Fig. 9 : Comparison of stem girth of Nandina chinensis and Banana cinerea under different treatments;

[0027] Fig.10 : Comparison of root length of Nandina chinensis and Banana oleracea with different treatments;

[0028] Fig.11 : Comparison of fresh weight of aboveground parts of Nandina chinensis and Brazilian banana under different treatments;

[0029] Fig.12 : Comparison of fresh weight of underground parts of Nandina domestica and Banana with different treatments. DETAILED DESCRIPTION

[0030] The invention provides a self-generating nitrogen-fixing strain, which is BWLY3X-6 (Klebsiellavariicola BWLY3X-6), which is isolated from the rhizosphere soil of wild bananas in Bawangling, Hainan Province. It is proved by identification that it belongs to Klebsiella variicola, and was preserved in Guangdong Provincial Microbiological Culture Collection Center on December 12, 2022, with a preservation number of: GDMCC NO: 63038, and the address of the preservation unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City.

[0031] The present invention provides an application of a self-generating nitrogen-fixing strain BWLY3X-6 in promoting the growth of bananas, wherein the banana varieties include but are not limited to Musa acuminata AAA Cavendish cv. Bax and M. acuminata AAA Cavendish cv. 'Nantianhuang'. The present invention finds that the self-generating nitrogen-fixing strain BWLY3X-6 can increase the plant height, stem height, stem girth, root length, aboveground fresh weight and underground fresh weight of banana plants, and has a good growth-promoting effect on bananas.

[0032] The present invention also provides an autogenous nitrogen-fixing bacterial agent, comprising a suspension of autogenous nitrogen-fixing strain BWLY3X-6, wherein the deposit number of the autogenous nitrogen-fixing strain BWLY3X-6 is GDMCC NO: 63038. The concentration of the autogenous nitrogen-fixing strain BWLY3X-6 in the bacterial agent is 1×10 4 ~9×10 8 cfu / mL, preferably 1×10 6 ~1×10 8 cfu / mL.

[0033] The present invention also provides a method for preparing the above-mentioned self-generated nitrogen-fixing bacteria agent, comprising: inoculating the self-generated nitrogen-fixing strain BWLY3X-6 in a liquid culture medium for cultivation, centrifuging the culture solution, resuspending the bacterial precipitate, and obtaining a bacterial suspension of the self-generated nitrogen-fixing strain BWLY3X-6. In the present invention, the liquid culture medium is an LB liquid culture medium; the inoculation amount of the self-generated nitrogen-fixing strain BWLY3X-6 is 0.5-2%, preferably 1%; the culture temperature is 36-38°C, preferably 37°C; the culture time is 9-11h, preferably 10h; the culture method is preferably 180rpm / min shaking culture; the centrifugation method is preferably refrigerated centrifugation, the centrifugation condition is 3000-5000rmp / min centrifugation for 5-15min, the centrifugation speed is preferably 4000rmp / min, and the centrifugation time is preferably 10min; the bacterial precipitate is preferably resuspended with sterile water. As an optional embodiment, the centrifugation and bacterial precipitate resuspension operations of the present invention are repeated 2-3 times.

[0034] The present invention also provides the use of the above-mentioned autogenous nitrogen-fixing bacteria agent in promoting banana growth, wherein the dosage of the bacteria agent is 10-20 mL / plant, preferably 12-16 mL / plant, and more preferably 14 mL / plant. The use period of the bacteria agent of the present invention is the whole growth period of banana, preferably the seedling stage; the use method is to directly absorb the autogenous nitrogen-fixing bacteria agent and irrigate it to the roots of the plants.

[0035] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the embodiment of the present invention, LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar powder, and ddH2O were fixed to 1000 mL, and the pH was adjusted to 7.0-7.2.

[0037] Ashby nitrogen-free medium: mannitol 10g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, CaCO3 5g, agar powder 15-20g, ddH2O, make up to 1000mL, and adjust the pH to 7.2-7.4.

[0038] The culture medium used for physiological and biochemical identification was based on the book “Microbiology Experiments” co-authored by Zhao Bin and He Shaojiang.

[0039] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] Example 1

[0042] 1. Isolation and screening of the self-generating nitrogen-fixing strain BWLY3X-6

[0043] The rhizosphere soil of wild banana in Bawangling, Hainan Province was dug using the Z-shaped sampling method.

[0044] Prepare several triangular flasks filled with 90 mL of sterile water in advance, accurately weigh 10 g of the excavated root soil, place them in the triangular flasks, shake at room temperature and 180 rpm / min for 20 to 30 min, take them out after shaking, and dilute the suspension to 10 -1 , 10 -2 , 10 -3 For three orders of magnitude, 100 μL of suspension with different dilution multiples was taken from the clean bench and dropped into Ashby nitrogen-free fixed medium. The suspension was spread evenly with a spreader stick. Each dilution multiple was repeated three times and the suspension was cultured in a 28°C incubator for 2 to 3 days before observation.

[0045] The solid plate after culture was taken out, and the single colony with the growth advantage was picked out and purified on LB solid medium. The plate streak method was used to repeatedly streak, culture and observe the morphology of the single colony. After obtaining the pure colony, it was preserved in glycerol for subsequent experimental use.

[0046] The target strain BWLY3X-6 isolated and screened was proved to be the self-nitrogen-fixing strain BWLY3X-6 (Klebsiella variicola BWLY3X-6) through physiological and biochemical tests and molecular identification. It was deposited in the Guangdong Provincial Microbiological Culture Collection on December 12, 2022, with the deposit number: GDMCC NO: 63038, and the address of the deposit unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0047] 2. Morphological characteristics of the autotrophic nitrogen-fixing strain BWLY3X-6

[0048] The selected single colony was inoculated into liquid LB and cultured in a shaking incubator at 37°C and 180 rpm / min for 10 h. 5 μL of the bacterial solution was adsorbed on a copper sheet to prepare a slide, and the morphology of the self-generating nitrogen-fixing bacteria was observed under a transmission electron microscope.

[0049] The plate morphology of the self-generating nitrogen-fixing strain BWLY3X-6 is shown in Figure 1 , transmission electron microscopy photos see Figure 2 .Depend on Figures 1-2 It can be seen that the colony on the LB plate is light yellow and opaque, with a round and moist appearance, clear edges, and easy to pick up. Under the transmission electron microscope, the bacteria are observed to be rod-shaped, with capsules and no flagella, and the size of the bacteria is about 2.78μm×1.14μm.

[0050] 3. Physiological and biochemical identification of the autotrophic nitrogen-fixing strain BWLY3X-6

[0051] The isolated self-generating nitrogen-fixing strain BWLY3X-6 was subjected to glucose oxidation and fermentation test, citrate test, methyl red (MR) test, indole test, nitrate reduction test, catalase test, and gelatin liquefaction test. The tests were conducted with reference to the second edition of "Microbiology Experiments". The results are shown in Table 1.

[0052] Table 1 Physiological and biochemical characteristics of strains

[0053]

[0054] Note: + is positive; - is negative

[0055] Physiological and biochemical identification of the strain showed that the glucose oxidation and fermentation test was positive and produced gas; the citrate test was positive; the methyl red (MR) test was negative; the indole production test was positive; the nitrate reduction test was negative; the catalase test was positive; and the gelatin liquefaction test was positive.

[0056] 4. Identification of nitrogenase gene nifH, 16S rRNA sequence and gyrB sequence

[0057] DNA was extracted from the nitrogen-fixing strain BWLY3X-6. The primers for amplifying the nitrogenase gene nifH were nifHP1 (SEQ ID NO: 1: 5′-GGCTGCGATCCVAAGGCCGAYTCVACCCG-3′) and nifHP2 (SEQ ID NO: 2: 5′-CTGVGCCTTGTTYTCGCGGATSGGCATGGC-3′). The primers used for PCR amplification of 16S rRNA sequence were 27F (SEQ ID NO: 3: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO: 4: 5'-TACGGCTACCTTGTTACGACTT-3'); the primers used for PCR amplification of gyrB gene sequence were UP-1 (SEQ ID NO: 5: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP-2r (SEQ ID NO: 6: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3').

[0058] The reaction system is: template 1 μL, 10 mmol·L -1 1 μL of each primer, 12.5 μL of 2×TaqPCRMix, and ddH2O were used to make up to 25 μL system. The PCR amplification program for the 16S rRNA sequence was as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, 35 cycles; and extension at 72°C for 10 min. The PCR amplification program for the gyrB sequence was as follows: pre-denaturation at 94°C for 4 min; 94°C for 30 s, 60°C for 45 s, 72°C for 1 min, 35 cycles; and extension at 72°C for 10 min. The PCR amplification program for the nifH sequence was as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 120 s, 35 cycles; and extension at 72°C for 10 min. See the test results. Figure 3 After the amplification reaction, the products were recovered and sequenced by Shanghai Bioengineering. The returned sequences were compared with NCBI. At the same time, the phylogenetic tree was constructed for the strain using MEGA7.0.26 software. The strain species was determined by combining the morphological observation results with the results of physiological and biochemical identification.

[0059] The DNA sequences were compared by BLAST in NCBI, and the 11 most homologous complete sequences were selected. After analyzing and aligning the sequences in MEGA7, the phylogenetic tree was constructed by the neighbor method. Figures 4-5 In the phylogenetic tree constructed based on the 16S rRNA sequence of strain BWLY3X-6, the strain with the highest homology is Klebsiella variicola strain 03-311-0071, with a similarity of 99.65%; in the phylogenetic tree constructed based on the gyrB sequence, it can be seen that the strain with the highest homology is Klebsiella variicola strain LMG 23571, with a similarity of 99.83%. Based on the morphological characteristics, physiological and biochemical characteristics, nifH gene detection, and 16S rRNA and gyrB sequence analysis results, the strain was identified as Klebsiella variicola.

[0060] 5. Determination of nitrogen fixation ability of strains by indophenol blue-spectrophotometry

[0061] (1) Preparation of detection reagents

[0062] Ammonia nitrogen standard solution: Accurately weigh 0.2358g (NH4)2SO4 and dissolve it in 100mL of water to obtain an ammonia nitrogen stock solution with an ammonia nitrogen content of 500μg / mL, and then dilute it 10 times to obtain an ammonia nitrogen standard solution with a content of 50μg / mL.

[0063] 1.25% sodium nitrosoferricyanide solution: Accurately weigh 0.3622 g Na2Fe(CN)5NO·2H2O, add water to make up to 25 mL, and store at 4°C. All reagents used are domestic analytical grade.

[0064] Solution A: Accurately weigh 5.00 g of phenol and 2 mL of 1.25% sodium nitrosoferricyanide solution, mix and dissolve in 500 mL of water, and store at 4°C in a dark place to avoid exposure to light to turn reddish brown.

[0065] Solution B: Accurately weigh 2 g C6H5Na3O7, 2.50 g NaOH and 3.5 mL NaClO, mix and dissolve in 500 mL water, and store at 4°C in a dark place.

[0066] (2) Development of ammonia nitrogen standard curve

[0067] Take 10 test tubes, number them in sequence, add the corresponding reagents as listed in Table 2, vortex mix evenly, place in a 37°C constant temperature water bath, color reaction for 20 minutes, take out and cool to room temperature, measure the OD value at a wavelength of 637nm, and draw the ammonia nitrogen standard curve.

[0068] Table 2 Development of ammonia nitrogen standard curve

[0069]

[0070]

[0071] The experiment was repeated three times, and the results were plotted with the standard ammonia nitrogen content (μg) as the horizontal axis and the corresponding optical density as the vertical axis. Figure 6 As shown in the figure, there is a good linear relationship in the range of 0.5 to 5 μg, and the regression equation is y = 0.0633x + 0.0649, R 2 =0.9973, which can be used to determine the ammonia nitrogen content.

[0072] (3) Sample determination

[0073] The nitrogen-fixing bacteria were inoculated into Ashby liquid culture medium at a 1% inoculation rate. In a nitrogen-free environment, the nitrogen-fixing bacteria will use the nitrogen in the air to fix nitrogen and reduce the nitrogen in the atmosphere to ammonia, which is highly soluble in water. After the bacteria were cultured in a shaker at 28°C and 180 r / min for about 20 hours, they were taken out, part of the liquid was aspirated and centrifuged at 8000 rpm for 10 minutes, 100 μL of the supernatant was collected in a sterile EP tube as the sample to be tested, and the experiment was repeated three times.

[0074] As described in (2), take the sample to be tested, add the corresponding reagent, and determine its OD 637 The values ​​were read on a spectrophotometer, the obtained values ​​were substituted into the ammonia nitrogen standard curve, and the average was taken. The results are shown in Table 3.

[0075] Table 3 Determination of extracellular ammonia nitrogen secretion of strains

[0076]

[0077]

[0078] As shown in Table 3, the ammonia nitrogen content of the strain was about 17.98 μg / mL, indicating that it had good nitrogen fixation ability.

[0079] 6. Determination of nitrogenase activity

[0080] The nitrogenase activity of the strain was determined by a microbial nitrogenase ELISA kit (enzyme-labeled biological) according to the instructions, and the nitrogenase activity of the strain BWLY3X-6 was 220.51 ng / L, indicating that the strain has a strong self-generated nitrogen fixation ability.

[0081] Example 2

[0082] Preparation of the self-generating nitrogen-fixing bacteria BWLY3X-6 inoculum: The BWLY3X-6 strain was inoculated into sterilized liquid LB at an inoculum volume of 1% and placed in a shaker at 37°C and 180 rpm / min for about 10 h (the concentration calculated by coating the LB plate was about 3.73×108 cfu / mL), and then centrifuged at 4000 rpm / min in a refrigerated centrifuge for 10 min. The supernatant was discarded and the bacterial pellet was resuspended in sterile water. The operation was repeated twice. Finally, the resuspended bacterial solution was diluted to 5×10 4 spare.

[0083] Example 3

[0084] The difference between this embodiment and embodiment 2 is that the resuspended bacterial solution is diluted to 1×10 6 spare.

[0085] Example 4

[0086] The difference between this embodiment and embodiment 2 is that the resuspended bacterial solution is diluted to 1×10 8 spare.

[0087] Example 5

[0088] The growth-promoting effect of spontaneous nitrogen-fixing bacteria BWLY3X-6 on bananas:

[0089] In the Seedling Tissue Culture Center of the Tropical Crops Institute of the Chinese Academy of Tropical Agricultural Sciences in Danzhou City, Hainan Province, cup seedlings of Nantianhuang (M. acuminata AAA Cavendish cv. 'Nantianhuang') and Brazilian banana (Musa acuminata AAA Cavendish cv. Bax) with five leaves and one heart were selected and cultured in coconut bran substrate. When the seedlings grew to five leaves, seedlings with basically the same growth conditions were selected for experiments.

[0090] The bacterial suspensions prepared in Examples 2 to 4 were used.

[0091] Five treatments were set up for single factor experiments between different banana varieties, including blank control, 0.5% urea treatment, three different concentration gradient treatments of strains (10 4 cfu / mL, 10 6 cfu / mL, 10 8 cfu / mL), with five seedlings per treatment, repeated three times. During the treatment period, appropriate amount of water was added to the cup seedlings on time every day to ensure a moist environment of the soil. The nitrogen-fixing bacteria were applied to the stem base of the test banana once every 3 days, with each addition of 1 mL / plant. The blank control was applied with the same amount of sterile water, and the urea treatment was applied with the same amount of 0.5% urea, for a total of 14 inoculations.

[0092] After 60 days of treatment, the plant height, stem height, stem girth, root length, aboveground fresh weight and underground fresh weight of each banana plant were measured, and data analysis was performed using Excel, SPSS 26 and GraphPad Prism8. The results are shown in Tables 4-5 and Figures 7 to 12 .

[0093] Table 4 Biomass of banana variety Nantianhuang under different treatments

[0094]

[0095] Table 5 Biomass of banana variety Brazil banana under different treatments

[0096]

[0097] From Tables 4 to 5 and Figures 7 to 12 It can be seen that after 60 days of different treatments, the plant height, stem height, stem girth, root length, aboveground fresh weight and underground fresh weight of the experimental banana were measured respectively. The strain test group was compared with the blank treatment and urea treatment. The experimental groups showed significant differences as a whole. The overall growth condition of Nandina domestica was better than that of Brazilian banana, but the strain had a more advantageous growth-promoting effect on Brazilian banana than Nandina domestica.

[0098] For Nantian Huang, the concentration of the applied bacterial solution was 1×10 6 cfu / mL, the growth-promoting effect was most obvious. Compared with the CK treatment, the plant height increased by 25.9%, the stem height increased by 7.2%, the stem girth increased by 18.2%, the root length increased by 43.5%, the aboveground fresh weight increased by 46.3%, and the underground fresh weight increased by 22.9%; compared with the urea treatment, the plant height increased by 25.5%, the stem height increased by 2.2%, the stem girth increased by 19.0%, the root length increased by 53.8%, the aboveground fresh weight increased by 54.7%, and the underground fresh weight increased by 37.7%. The growth-promoting effect decreased at high concentrations.

[0099] For Brazilian banana, the growth-promoting effect was not obvious at low concentrations, and even inhibited growth to a certain extent compared with the blank treatment. However, when the concentration of the bacterial solution was 1×10 6 cfu / mL and 1×10 8 cfu / mL had a significant growth-promoting effect, especially 1×10 8 cfu / mL, compared with the CK treatment, the plant height increased by 37.8%, stem height increased by 26.3%, stem girth increased by 28.5%, root length increased by 54.6%, aboveground fresh weight increased by 68.6%, and underground fresh weight increased by 84.3%; compared with the urea treatment, the plant height increased by 6.1%, stem height increased by 9.0%, stem girth increased by 11.7%, root length increased by 69.6%, aboveground fresh weight increased by 29.6%, and underground fresh weight increased by 49.7%.

[0100] The above results show that the self-generated nitrogen-fixing bacteria BWLY3X-6 has a good growth-promoting effect on bananas. Among the various biomass changes, the root system has the most significant change. It is speculated that the growth-promoting effect of the strain on banana varieties is mainly achieved by colonizing the banana roots.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A self-generating nitrogen-fixing strain, characterized in that: The self-generating nitrogen-fixing strain is BWLY3X-6, belonging to Klebsiella variocola , the deposit number is GDMCC NO:63038.

2. Use of the spontaneous nitrogen-fixing strain according to claim 1 in promoting banana growth.

3. A self-generating nitrogen-fixing bacterial agent, characterized in that: The invention comprises a bacterial suspension of the self-generated nitrogen-fixing strain BWLY3X-6, and the deposit number of the self-generated nitrogen-fixing strain BWLY3X-6 is GDMCC NO:63038.

4. The autogenous nitrogen-fixing bacterial agent according to claim 3, characterized in that: The concentration of the autogenous nitrogen-fixing strain BWLY3X-6 in the bacterial agent is 1×10 4 ~9×10 8 cfu / mL.

5. The method for preparing the autogenous nitrogen-fixing bacterial agent according to any one of claims 3 to 4, characterized in that: The self-generated nitrogen-fixing strain BWLY3X-6 was inoculated into a liquid culture medium for cultivation, the culture solution was centrifuged, and the bacterial precipitate was resuspended to obtain a bacterial suspension of the self-generated nitrogen-fixing strain BWLY3X-6.

6. The preparation method according to claim 5, characterized in that: The liquid culture medium is LB liquid culture medium.

7. The preparation method according to claim 5, characterized in that: The culture temperature is 36-38°C and the culture time is 9-11 hours.

8. The preparation method according to claim 5, characterized in that: The centrifugation conditions are: 3000-5000 rpm for 5-15 min.

9. Use of the spontaneous nitrogen-fixing bacteria agent according to claim 3 or 4 in promoting the growth of bananas.

10. The use according to claim 9, characterized in that: The dosage of the bacterial agent is 10 to 20 mL per plant.

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