Enterobacter ludwigii XL0404 and application thereof in tobacco growth promotion

Abstract

The content of IAA produced by the enterobacter ludwigii XL0404 can reach 18.94 mg / L, the content of ACC deaminase produced by the enterobacter ludwigii XL0404 is 484.48 U / L, and the enterobacter ludwigii XL0404 has multiple functions of dissolving phosphorus, dissolving potassium, fixing nitrogen, producing siderophore and the like, has an inhibition effect on main root and stem disease pathogenic bacteria such as phytophthora nicotianae, rhizoctonia solani, rhizoctonia tectorum, fusarium oxysporum, fusarium solani and the like, and has an inhibition rate of 41.20-55.88%; the germination rate of the tobacco seeds is increased through treatment of the strain XL0404, and the root length is remarkably promoted; the root activity, the whole plant fresh weight, the overground fresh weight, the root fresh weight, the maximum leaf length and the maximum leaf width of the tobacco seedlings treated by the strain XL0404 with the bacterium content of 1 * 10 < 7 > cfu / mL in a seedling culture substrate are obviously higher than those of the tobacco seedlings treated by contrast treatment, and the strain XL0404 can be used as plant growth-promoting rhizobacteria and has a good application prospect in research and development of microbial fertilizers and biological prevention and control of tobacco rhizome diseases.

Description

Technical Field

[0001] The invention relates to a strain of Enterobacter Ludwigii XL0404 and application thereof in promoting tobacco growth. Background Art

[0002] Tobacco is an important cash crop in my country. Henan Province is a major tobacco-producing province with a long history of tobacco cultivation. However, with the increase in the number of years of tobacco cultivation, problems such as soil environmental deterioration, imbalance in soil nutrient supply, decline in tobacco leaf quality, and decline in flue-cured tobacco production efficiency have frequently occurred. In addition, tobacco seedlings are prone to die during the seedling stage, which affects the uniformity and strength of the seedlings, has a serious impact on field production, and to a certain extent restricts the increase in tobacco leaf yield. Therefore, cultivating high-quality, high-yield, disease-resistant and stress-resistant seedlings is an important prerequisite for producing high-quality flue-cured tobacco.

[0003] Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms that colonize plant roots and have a positive impact on plant growth. They serve as a bridge between plants and soil, establish a healthy rhizosphere system through interactions with soil and plants, and continuously and efficiently promote plant growth. PGPR usually has a variety of characteristics that directly or indirectly alleviate stress and promote plant growth. The direct growth-promoting mechanism includes improving the ability of crops to absorb nitrogen, improving the ability of roots to dissolve phosphorus and potassium, producing iron carriers, producing indole-3-acetic acid, synthesizing ACC-deaminase, and producing plant hormone substances. The indirect growth-promoting mechanism is mainly reflected in the fact that PGPR can produce active and rich metabolites, which can inhibit the growth of pathogenic fungi and control their cell apoptosis and necrosis. By inoculating PGPR, plant biomass and nutrient content can be increased, and the soil can be improved to a certain extent. Therefore, selecting PGPR that matches the local conditions according to local conditions provides excellent strains for further research and development of microbial fertilizers specifically for tobacco seedlings, and is also an effective way to ensure the "quality and efficiency improvement" of tobacco-growing soil.

[0004] In this regard, the present invention provides the method of collecting rhizosphere soil of healthy crops in tobacco fields and surrounding farmlands in central Henan Province, isolating, screening and identifying a strain of Enterobacter Ludwigii XL0404 that has multiple growth-promoting mechanisms for tobacco growth and has biocontrol potential, and its application in tobacco growth promotion, to provide bacterial strain resources for the development of economical and environmentally friendly microbial fertilizers. Summary of the invention

[0005] In view of the above problems, the present invention provides Enterobacter ludwigii XL0404 and its application in promoting tobacco growth, so as to overcome the defects of the prior art.

[0006] The technical solution of the present invention is achieved as follows: a strain of Enterobacter Ludwigii XL0404, the preservation number of the strain is CCTCC NO: M2024738.

[0007] Application of Enterobacter ludwigii XL0404 in tobacco growth promotion.

[0008] A strain of Enterobacter ludwigii XL0404 was isolated from the rhizosphere soil of peanut using the gradient dilution spread plate method.

[0009] A method for isolating Enterobacter Ludwigii XL0404 comprises the following steps: air-drying peanut rhizosphere soil, gently brushing the soil on the rhizosphere with a small brush, using a gradient dilution plate method to dilute the soil suspension in sequence according to aseptic operation, evenly coating the soil suspension on a NA plate to culture bacterial colonies, and purifying and culturing the bacterial colonies.

[0010] A method for promoting tobacco growth comprises: soaking sterilized naked tobacco seeds in a bacterial suspension of Enterobacter Ludwigii XL0404, taking them out after 16 hours and rinsing them with sterile water for 3 times.

[0011] A method for promoting tobacco growth, comprising: inoculating Ludwig Enterobacter XL0404 into LB liquid culture medium, culturing in a constant temperature shaking incubator at 28°C and 180 r / min for 24 h, adding the fermentation liquid into a sterilized seedling medium, mixing evenly, and then loading into a floating seedling tray to prepare a bacterial medium, and then carrying out floating seedling cultivation.

[0012] The present invention has the following positive effects: the Ludwig Enterobacter XL0404 of the present invention can produce IAA content of up to 18.94 mg / L, and the ACC deaminase content is 484.48 U / L, and has multiple functions such as phosphate solubilization, potassium solubilization, nitrogen fixation, and siderophore production. At the same time, it has an inhibitory effect on major root and stem disease pathogens such as tobacco phytophthora, Rhizoctonia solani, Acanthocystis iris, Fusarium oxysporum and Fusarium solani, and the inhibition rate is 41.20% to 55.88%. The root vitality of tobacco seedlings treated with strain XL0404, the fresh weight of the whole plant of tobacco seedlings, the fresh weight above ground, the fresh weight of the root system, the maximum leaf length and the maximum leaf width are significantly higher than those of the control treatment, and are increased by 43.40%, 66.42%, 65.79%, 70.00%, 33.19% and 26.17% respectively compared with the control. The strain can be used as a plant rhizosphere growth-promoting bacterium in the research and development of microbial fertilizers and the biological prevention and control of tobacco root and stem diseases, and has good application prospects. It has multiple growth-promoting properties such as producing IAA, ACC deaminase, siderophore, cellulase, nitrogen fixation, and phosphate and potassium solubilization, and has a certain antagonistic effect on the main tobacco pathogens. It can increase the germination rate and root length of tobacco seeds, as well as the root vitality and fresh weight of tobacco seedlings, and has good application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the morphological characteristics of the strain of the present invention.

[0014] Figure 2 This is a phylogenetic tree constructed based on 16S rDNA in the present invention.

[0015] Figure 3 Schematic diagram of the effect of the rhizosphere bacteria fermentation liquid of the present invention on the root activity of tobacco seedlings.

[0016] Figure 4 This is a schematic diagram of the growth-promoting effect of the rhizosphere bacterial fermentation liquid of the present invention on tobacco seedlings during the seedling raising period. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0018] In the following description of the invention, it should be noted that the terms "upper", "lower", "left", "right", "inner", "outer", etc., indicating directions or positional relationships, are based on directions or positional relationships shown in the drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction or be constructed and operated in a specific direction. The term "connection" only indicates the connection between devices and has no special meaning.

[0019] like Figure 1 , 2 As shown in , 3, and 4, a strain of E. ludwigii XL0404, the deposit number of the strain is CCTCCNO: M2024738, the deposit date is April 22, 2024, and the result is survival. The depository is China Center for Type Culture Collection, and the Latin classification name of the culture requested for deposit is: Emerobacter ludwigii strain XL0404.

[0020] Application of Enterobacter ludwigii XL0404 in tobacco growth promotion.

[0021] A strain of Enterobacter ludwigii XL0404 was isolated from the rhizosphere soil of peanut using the gradient dilution spread plate method.

[0022] A method for isolating Enterobacter Ludwigii XL0404 comprises the following steps: air-drying peanut rhizosphere soil, gently brushing the soil on the rhizosphere with a small brush, using a gradient dilution plate method to dilute the soil suspension in sequence according to aseptic operation, evenly coating the soil suspension on a NA plate to culture bacterial colonies, and purifying and culturing the bacterial colonies.

[0023] A method for promoting tobacco growth comprises: soaking sterilized naked tobacco seeds in a bacterial suspension of Enterobacter Ludwigii XL0404, taking them out after 16 hours and rinsing them with sterile water for 3 times.

[0024] A method for promoting tobacco growth, comprising: inoculating Ludwig Enterobacter XL0404 into LB liquid culture medium, culturing in a constant temperature shaking incubator at 28°C and 180 r / min for 24 h, adding the fermentation liquid into a sterilized seedling medium, mixing evenly, and then loading into a floating seedling tray to prepare a bacterial medium, and then carrying out floating seedling cultivation.

[0025] Specific implementation and verification: Rhizosphere soil samples of healthy peanut plants were collected around tobacco fields in Jian'an District, Xuchang City. Tobacco pathogens tested: Phytophthora nicotianae, Fusarium solani, Fusarium oxysporum, Rhizoctonia solani, Aphanomyces iridis, and tobacco varieties (Zhongyan 100) were provided by the Tobacco Research Institute of Henan Academy of Agricultural Sciences.

[0026] After the collected rhizosphere soil samples were air-dried, the soil on the rhizosphere was gently brushed off with a small brush and collected for later use. The gradient dilution plate method was used to dilute the samples into 10 -3 , 10 -4 , 10 -5 Take 100 μL of the soil suspension and spread it evenly on the NA plate to culture bacterial colonies. Pick bacterial colonies of different colors, shapes and sizes for purification and culture, and store them in a 4°C refrigerator for later use. Qualitative screening: inoculate the strain to be tested into NA liquid culture medium containing 100 mg / L L-tryptophan, culture at 28℃ and 180 r / min for 48 h, take 100 μL of the supernatant after centrifugation at 8000 r / min for 10 min, add an equal volume of Salkowski colorimetric solution (150 mL of 35% perchloric acid, 3 mL of 0.5 mol / L FeCl 3, prepared freshly), protect from light and color for 30 min, then observe the color change. If pink appears, it is positive, indicating that the strain can secrete IAA. The darker the color, the greater the intensity of secretion. No color change is negative, indicating that the strain cannot secrete IAA.

[0027] Quantitative determination rescreening: IAA-positive strains were re-shaken and the bacterial solution was diluted to OD 600 ≈ 1. 2.5 mL of the supernatant of the strain was mixed with an equal volume of Salkowski colorimetric solution for color development in the dark for 30 min, and its absorbance at 530 nm was measured. An IAA standard curve was prepared with IAA concentration as the horizontal axis and OD 530 as the vertical axis, and the IAA content in the fermentation broth of the strain was calculated by comparing the IAA standard curve. The ELISA quantitative detection kit (Microorganism ACCD ELISA KIT) was used with a microplate reader (Tecan Infinite F50) to determine the content of ACC deaminase produced by the strain.

[0028] The selected strains were inoculated on Ashby nitrogen-free solid medium, Monjinna organic phosphorus, inorganic phosphorus solid medium and potassium-solubilizing medium, respectively, and cultured at 25℃ for 3 days. The growth state of the bacteria in different identification medium and the presence or absence of transparent circles were observed. If the strain grows normally on the nitrogen-fixing medium, it means that the strain has the ability to fix nitrogen; if the strain grows normally on the phosphorus-solubilizing and potassium-solubilizing medium and a transparent circle appears around the strain, it means that the strain has the ability to solubilize phosphorus and potassium, and the transparent circle diameter (D) and colony diameter (d) were measured respectively, and the ratio of transparent circle to colony diameter (D / d) was calculated. The larger the ratio, the stronger the ability to solubilize phosphorus and potassium.

[0029] The strain to be tested was inoculated on CAS medium, with the medium without bacteria as the control. Each strain was repeated 3 times and cultured at 25℃ for 5 days. The growth of the strain and the changes around the colony were observed. If the strain grew normally on CAS solid medium and an orange halo appeared around the colony, it indicated that the strain had the ability to produce siderophore.

[0030] The plate confrontation method was used to determine the antibacterial rate of the strain against Fusarium oxysporum, Fusarium solani, Phytophthora, Rhizoctonia solani and Aphanomyces iris. The pathogens were used as targets. After 5-7 days of culture, the bacterial cakes were punched out with a 6 mm puncher and inoculated in the center of the PDA medium. The bacteria to be tested were inoculated at symmetrical positions 2.5 cm away from the center of the plate. The treatment group inoculated with only the pathogenic bacterial cake was used as the control. The treatment was repeated 3 times and inverted at a constant temperature of 25℃ for 3-6 days. The diameter of the pathogenic colony was measured by the cross method, and the antibacterial rate was calculated. Inhibition rate (%) = (control colony diameter-treatment colony diameter) / (control colony diameter-bacterial cake diameter) × 100.

[0031] The rhizosphere growth-promoting bacteria were inoculated on NA plates and cultured at 25°C for 2 days to observe the colony culture characteristics of the strains. The physiological and biochemical characteristics of the rhizosphere growth-promoting bacteria strains were analyzed, including aerobic and anaerobic tests, citrate utilization test, malonate utilization test, motility test, oxidase production test, catalase production test, sugar alcohol fermentation test, gelatin liquefaction test, methyl red (MR) test, acetylmethyl carbinol (VP) test, nitrate reduction test, and litmus milk test, with 3 replicates per treatment.

[0032] The bacterial genomic DNA was extracted using the Ezup column bacterial genomic DNA extraction kit (Shanghai Biotech Co., Ltd.) as a template, and a 25 μL PCR reaction system was prepared using bacterial universal primers 27F and 1492R for amplification. The PCR product was commissioned to Shanghai Biotech Co., Ltd. for sequencing, and the 16S rDNA sequence obtained by sequencing was compared with BLAS on NCBI, and the phylogenetic tree was constructed using the software MEGA7.0 to determine the taxonomic status of the strain.

[0033] Sterilized naked tobacco seeds of Zhongyan 100 were immersed in a suspension of Enterobacter Ludwigii XL0404 (1×10 8 cfu / mL) and were taken out after 16 h and rinsed three times with sterile water. Sterilized seeds treated with sterile water were used as control. There were 25 seeds in each dish (5×5) and the experiment was repeated three times. The germination and growth of tobacco seeds were observed on the 14th day, the root length, stem length and total length of the tobacco seedlings were measured, and the seed germination rate and growth rate were calculated.

[0034] Growth rate (%) = (root length of treatment group - root length of control group) / root length of control group × 100.

[0035] The growth-promoting effect of the strain on tobacco was determined by the floating seedling method by mixing the substrate with bacteria. The growth-promoting bacteria were inoculated into LB liquid medium and cultured in a constant temperature shaking incubator at 28°C and 180 r / min for 24 h. The concentration of the fermentation liquid was adjusted to 1×10 8 cfu / mL, 1×10 7 cfu / mL, 1×10 6 cfu / mL. Add 45 mL of fermentation liquid to 450 g of sterilized seedling medium, mix well, and then put into floating seedling tray (6×10 holes) to prepare a bacterial content of 1×10 7 cfu / mL, 1×10 6 cfu / mL, 1×10 5 cfu / mL of the contaminated matrix. One tray of seedlings was cultured for each treatment, and the blank control was an equal volume of liquid culture medium mixed with the matrix, which was repeated 3 times. During the period, the temperature and water management were kept consistent, and no fertilizer was applied. During the root extension period of tobacco seedlings (43 days after sowing), 5 tobacco seedlings with uniform growth were selected from each treatment, and their root activity was determined according to the method of the plant root activity kit (Suzhou Grace Biotechnology Co., Ltd.). 50 days after sowing, 10 tobacco seedlings with uniform growth were selected from the floating tray of each treatment, and their root length, leaf number, maximum leaf length, maximum leaf width, whole plant fresh weight, aboveground tobacco seedling fresh weight, and underground root fresh weight were measured according to the agronomic trait measurement method in the Tobacco Industry Standard of the People's Republic of China (YC / T 142-2010).

[0036] Data processing was performed using Excel and DPS9.01 software, and variance analysis was performed using Duncan's new multiple range method. The results are shown below: The strain XL0404 isolated from the peanut rhizosphere soil around the tobacco fields in Jian'an District, Xuchang City produced IAA at a content of 18.94 mg / L.

[0037] As shown in Table 1, the experimental determination showed that the ACC deaminase content in strain XL0404 was 484.48 U / L, and it had growth-promoting effects such as siderophore production, phosphorus and potassium solubilization, and nitrogen fixation.

[0038] Table 1 Growth-promoting ability of rhizosphere bacteria "D / d" indicates the ratio of the diameter of the clear zone to the diameter of the colony. ACC: ACC deaminase; NPA: inorganic phosphorus decomposition; OPA: organic phosphorus decomposition; NFb: nitrogen fixation; Sil: potassium silicate decomposition; Sid: siderophore production.

[0039] As shown in Table 2, strain XL0404 has different degrees of inhibition on the five major tobacco rhizome disease pathogens, with inhibition rates ranging from 42.28% to 55.88%, among which the inhibition rate on tobacco phytophthora was the highest, at 55.88%.

[0040] Table 2 Inhibitory effects of strains on common tobacco pathogenic fungi like Figure 1 As shown, the strain of the present invention is a Gram-negative bacterium. When cultured on NA medium at 25°C for 2 days, the colonies are small and nearly round. The strain XL0404 is light yellow in color, convex in the middle, and bright and smooth on the surface. Microscopic observation shows that the bacterial cells are rod-shaped, arranged individually, and are (0.6-1.0) μm×(0.8-1.8) μm in size, which is consistent with the characteristics of Enterobacter Ludwigii.

[0041] Table 3 The results of physiological and biochemical tests show that strain XL0404 is a facultative anaerobic bacterium, and the results of malonate, citrate utilization, catalase, gelatin liquefaction and MR tests are positive. It has certain motility and can utilize glucose, sucrose and mannitol. Combined with the "Common Bacterial System Identification Manual", the physiological and biochemical characteristics of rhizosphere growth-promoting bacteria XL0404 are consistent with those of Enterobacter Ludwigii.

[0042] Table 3 Physiological and biochemical test results of bacteria : “+” indicates a positive reaction; “++” indicates an acid- and gas-producing reaction; “-” indicates a negative reaction; “±” indicates a facultative reaction; “V” indicates a weak positive reaction; and “B” indicates a reduction reaction.

[0043] The 16S rDNA sequences of the strains obtained by PCR were compared with the GenBank database by BLAST. The results showed that the sequence similarity between strain XL0404 (OR752044) and E. ludwigii YC-IL1 (MT002728.1) reached 99.86%. As can be seen from Figure 2, XL0404 and E. ludwigii were clustered in the same branch. Combining morphological and physiological and biochemical identifications, strain XL0404 was identified as E. ludwigii.

[0044] The results of the culture dish filter paper moisturizing method (Table 4) showed that the strain XL0404 showed a significant growth-promoting effect after seed soaking. The germination rate of the strain XL0404 treatment group increased by 4.00%; the root length and stem length were higher than those of the control group, and the root length of the tobacco seedlings was significantly higher than that of the control treatment, with a growth rate of 43.60%. This shows that the strain XL0404 has a significant growth-promoting effect on tobacco seeds.

[0045] Table 4 Effects of rhizosphere bacteria soaking on seed germination and growth The data in the table are Mean±SE. Different lowercase letters in each column and each treatment indicate that there are statistically significant differences among the treatments (P<0.05), the same below.

[0046] As shown in Figure 3, the root activity of tobacco seedlings treated with strain XL0404 in floating seedling culture was significantly higher than that of the blank control group. 5 cfu / mL, the root activity of tobacco seedlings was the highest, which was 47.98μg / h / g fresh weight, 43.40% higher than that of the control group, and there was no significant difference compared with the other two concentrations.

[0047] From Table 5, we can see that the agronomic traits of tobacco seedlings were improved after being treated with strain XL0404. 7 cfu / mL and 1×10 6 cfu / mL, the fresh weight of the whole plant, aboveground weight, root fresh weight, maximum leaf length and maximum leaf width of tobacco seedlings were significantly higher than those of the control treatment, increasing by 66.42%, 65.79%, 70.00%, 33.19%, 26.17% and 59.70%, 57.02%, 80.00%, 30.44%, 24.47% respectively compared with the control treatment, while there was no significant difference between the two treatments. When the bacterial content of strain XL0404 in the seedling medium was 1×10 5 cfu / mL, the whole plant fresh weight, aboveground fresh weight, root fresh weight, maximum leaf width and root length of tobacco seedlings were significantly higher than those of the control treatment, which were increased by 37.31%, 27.19%, 95.00%, 10.21% and 44.60%, respectively compared with the control treatment (Figure 4).

[0048] Table 5 The growth-promoting effect of rhizosphere bacteria on tobacco seedlings The strain of the present invention produces an IAA content of 18.94 mg / L, which can promote tobacco seed germination and seedling growth. The present invention has multiple growth-promoting properties such as nitrogen fixation, phosphorus and potassium dissolution, iron carrier production, ACC deaminase, etc. It also has a biological control function, has a certain antagonistic effect on several common tobacco pathogenic fungi, and can inhibit the growth and development of pathogens by inhibiting the growth of pathogenic hyphae.

[0049] After inoculation with XL0404, the germination rate, root length and stem length of tobacco seeds were all improved, the fresh weight of the whole plant of tobacco seedlings increased by 66.42%, and the root activity, maximum leaf length, maximum leaf width and total root length were also significantly improved. The obtained IAA-producing strain has both biological control and multiple growth-promoting functions, showing the diversity of life-promoting properties, which also shows that the strain has the potential to improve nutrient utilization, promote plant growth and root elongation, and has the potential to be further developed as a microbial fertilizer for agricultural production.

Claims

1. A strain of Enterobacter ludwigii XL0404, characterized in that: The deposit number of the strain is CCTCC NO: M2024738.

2. A use of the Ludwig bacterium XL0404 as claimed in claim 1 in promoting tobacco growth.

3. The Enterobacter Ludwigii XL0404 according to claim 1, characterized in that: It was isolated from peanut rhizosphere soil using the gradient dilution spread plate method.

4. A method for isolating Enterobacter Ludwigii XL0404, characterized in that: The method is as follows: after the peanut rhizosphere soil is air-dried, the soil on the rhizosphere is gently brushed off with a small brush, and the gradient dilution plate method is used to dilute it into a soil suspension in sequence according to sterile operations. The soil suspension is evenly spread on the NA plate to culture bacterial colonies, and the bacterial colonies are purified and cultured.

5. A method for promoting tobacco growth, characterized in that: The method is as follows: the sterilized naked tobacco seeds are immersed in a suspension of Enterobacter Ludwigii XL0404, and after 16 hours, they are taken out and rinsed three times with sterile water.

6. A method for promoting tobacco growth, characterized in that: The method is as follows: Ludwig Enterobacter XL0404 is inoculated into LB liquid culture medium, cultured in a constant temperature shaking incubator at 28°C and 180 r / min for 24 hours, the fermentation liquid is added to the sterilized seedling medium, mixed evenly, and then loaded into a floating seedling tray to prepare a bacterial matrix, and then floating seedling is carried out.

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