Pantoea agglomerans JHS0317 and application thereof in tobacco growth promotion

Abstract

The content of IAA produced by the pantoea agglomerans JHS0317 can reach 26.81 mg / L, the content of ACC deaminase produced by the pantoea agglomerans JHS0317 is 470.34 U / L, the pantoea agglomerans JHS0317 has multiple functions of dissolving phosphorus, dissolving potassium, fixing nitrogen, producing siderophore and the like, meanwhile, the pantoea agglomerans JHS0317 has inhibition effects on tobacco fusarium oxysporum and fusarium solani, the inhibition rates are 42.98% and 41.20%, the germination rate of tobacco seeds is increased through treatment of the strain JHS0317, 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 tobacco seedlings treated by the strain JHS0317 with the bacterium content of 1 * 10 < 5 > cfu / mL in a seedling culture substrate are obviously higher than those of the tobacco seedlings treated by a contrast, and the strain can be used as a plant growth-promoting rhizobacteria and has a good application prospect in research and development of microbial fertilizers and biological prevention and control of tobacco fusarium root rot.

Description

Technical Field

[0001] The invention relates to a Pantoea agglomerans JHS0317 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 promote plant growth continuously and efficiently. Plant growth-promoting rhizobacteria often have 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 plant growth-promoting rhizobacteria can produce active and rich metabolites, which can inhibit the growth of pathogenic fungi and control their cell apoptosis and necrosis. By inoculating plant growth-promoting rhizobacteria, plant biomass and nutrient content can be increased, and the soil can be improved to a certain extent.

[0004] In this regard, the present invention provides rhizosphere soil of healthy crops collected from tobacco fields and surrounding farmlands in central Henan tobacco-growing areas, and isolates, screens and identifies a strain of Pantoea agglomerans JHS0317 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 a strain of Pantoea agglomerans JHS0317 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 implemented as follows: a strain of Pantoea agglomerans JHS0371, the preservation number of the strain is CCTCC NO: M2024739.

[0007] Application of Pantoea agglomerans JHS0371 in promoting tobacco growth.

[0008] A strain of Pantoea agglomerans JHS0371 was isolated from the rhizosphere soil of sweet potato by using the gradient dilution spread plate method.

[0009] A method for isolating Pantoea agglomerans JHS0371 is provided, wherein the rhizosphere soil of sweet potatoes is air-dried, the soil on the rhizosphere is gently brushed off with a small brush, a gradient dilution plate method is used, the soil suspension is diluted in sequence according to aseptic operation, the soil suspension is evenly spread on a NA plate to culture bacterial colonies, and the bacterial colonies are purified and cultured.

[0010] The method for promoting tobacco growth comprises the following steps: soaking sterilized naked tobacco seeds in a suspension of Pantoea agglomerata JHS0371, taking them out after 16 hours and rinsing them three times with sterile water.

[0011] The method for promoting tobacco growth comprises the following steps: inoculating Pantoea agglomerans JHS0371 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 placing the mixture 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 IAA content of the Pantoea agglomerans of the present invention can reach 26.81 mg / L, the ACC deaminase content is 470.34 U / L, and it has multiple functions such as phosphate solubilization, potassium solubilization, nitrogen fixation, and siderophore production. At the same time, it has an inhibitory effect on tobacco oxysporum and solani, with inhibition rates of 42.98% and 41.20%. Treatment with strain JHS0317 increased the germination rate of tobacco seeds and significantly promoted root growth; the bacterial content of strain JHS0317 in the seedling medium was 1×10 5 cfu / mL, the root activity of tobacco seedlings, the fresh weight of the whole plant, the aboveground fresh weight, the root fresh weight, the maximum leaf length and the maximum leaf width were significantly higher than those of the control treatment, which were increased by 54.81%, 153.73%, 171.93%, 40.00%, 54.06% and 56.17% respectively compared with the control. This strain can be used as a plant rhizosphere growth-promoting bacterium in the research and development of microbial fertilizers and the biological control of tobacco Fusarium root rot. It has multiple growth-promoting characteristics such as producing IAA, ACC deaminase, siderophore, cellulase, nitrogen fixation, phosphorus and potassium solubilization, and has a certain antagonistic effect on the main tobacco pathogens. It can improve the germination rate and root length of tobacco seeds, and at the same time improve the root activity and fresh weight of tobacco seedlings, etc., and has good application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1This is a schematic diagram of the JHS0317 colony morphology of the present invention.

[0014] Figure 2 It is a schematic diagram of the bacterial cell morphology corresponding to the present invention.

[0015] Figure 3 This is a phylogenetic tree constructed based on 16S rDNA in the present invention.

[0016] Figure 4 Schematic diagram of the effect of the rhizosphere bacteria fermentation liquid of the present invention on the root activity of tobacco seedlings.

[0017] Figure 5 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

[0018] 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.

[0019] 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.

[0020] like Figure 1 , 2 , 3, 4, and 5 show a strain of Pantoea agglomerans JHS0371, the deposit number of the strain is CCTCCNO: M2024739. The deposit date is April 22, 2024, and the Latin classification name is Pantoea agglomerans strain JHS0317.

[0021] Application of Pantoea agglomerans JHS0371 in promoting tobacco growth.

[0022] A strain of Pantoea agglomerans JHS0371 was isolated from the rhizosphere soil of sweet potato by using the gradient dilution spread plate method.

[0023] A method for isolating Pantoea agglomerans JHS0371 is provided, wherein the rhizosphere soil of sweet potatoes is air-dried, the soil on the rhizosphere is gently brushed off with a small brush, a gradient dilution plate method is used, the soil suspension is diluted in sequence according to aseptic operation, the soil suspension is evenly spread on a NA plate to culture bacterial colonies, and the bacterial colonies are purified and cultured.

[0024] The method for promoting tobacco growth comprises the following steps: soaking sterilized naked tobacco seeds in a suspension of Pantoea agglomerata JHS0371, taking them out after 16 hours and rinsing them three times with sterile water.

[0025] The method for promoting tobacco growth comprises the following steps: inoculating Pantoea agglomerans JHS0371 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 placing the mixture into a floating seedling tray to prepare a bacterial medium, and then carrying out floating seedling cultivation.

[0026] Specific implementation and verification: Rhizosphere soil samples of healthy sweet potato plants were collected around tobacco fields in Jiaxian County, Pingdingshan 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.

[0027] 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.

[0028] Qualitative screening: inoculate the strain to be tested into NA liquid culture medium containing 100 mg / L L-tryptophan, and culture at 28°C 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 and 33 mL of 0.5 mol / L FeCl, prepared immediately before use), and observe the color change after 30 min of color development in the dark. 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.

[0029] Quantitative determination rescreening: re-culture the IAA-positive strains and dilute the bacterial solution to OD 600 ≈1, take 2.5 mL of the supernatant of the strain and mix with an equal volume of Salkowski colorimetric solution to develop color for 30 min in the dark, and measure its absorbance at 530 nm. 530 An IAA standard curve was prepared as the ordinate, and the IAA content in the fermentation broth of the strain was calculated by comparing the IAA standard curve.

[0030] 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.

[0031] 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°C 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) are measured respectively, and the ratio of transparent circle to colony diameter (D / d) is calculated. The larger the ratio, the stronger the ability to solubilize phosphorus and potassium.

[0032] 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.

[0033] 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 bacteria were punched into cakes 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 pathogen cakes was used as the control. The treatment was repeated 3 times and inverted at a constant temperature of 25°C for 3-6 days. The diameter of the pathogen colony was measured by the cross method, and the antibacterial rate was calculated. Inhibition rate (%) = (control colony diameter-treatment colony diameter) / (control colony diameter-bacteria cake diameter) × 100.

[0034] 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.

[0035] 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.

[0036] Sterilized naked tobacco seeds of Zhongyan 100 were immersed in a suspension of Pantoea agglomerans JHS0371 (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 process 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 seedlings were measured, and the seed germination rate and growth rate were calculated.

[0037] Growth rate (%) = (root length of treatment group - root length of control group) / root length of control group × 100.

[0038] 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 culture 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. 45 ml of fermentation liquid was added to 450 g of sterilized seedling medium, mixed evenly, and then loaded into floating seedling trays (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 seedling tray was cultured for each treatment, and the blank control was mixed with the matrix with an equal volume of liquid culture medium, which was repeated 3 times. The temperature and water management were kept consistent during the period, 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 by referring 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).

[0039] 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 JHS0317 isolated from the sweet potato rhizosphere soil around the tobacco fields in Jiaxian County, Pingdingshan City, produced IAA at a content of 26.81 mg / L.

[0040] As shown in Table 1, the experimental determination showed that the ACC deaminase content in strain JHS0317 was 470.34 U / L, and it had growth-promoting effects such as siderophore production, phosphate and potassium solubilization, and nitrogen fixation.

[0041] 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.

[0042] As shown in Table 2, strain JHS0317 has an inhibitory effect on Fusarium oxysporum and Fusarium solani, with the inhibition rates being 42.98% and 41.20%, respectively.

[0043] Table 2 Inhibitory effects of strains on common tobacco pathogens like Figure 1 and 2 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 JHS0317 is yellow in color with a smooth surface. Microscopic observation shows that the bacterial cells are straight rod-shaped and move with peritrichous flagella. The size is (0.5-1.0) μm × (1.0-3.0) μm, which is consistent with the characteristics of Pantoea agglomerans.

[0044] Table 3 The results of physiological and biochemical tests show that strain JHS0317 is a facultative anaerobic bacterium, and the results of malonate, citrate utilization, catalase, gelatin liquefaction and MR tests are positive. It has a certain degree of motility and can utilize glucose, sucrose and mannitol. Combined with the "Common Bacteria System Identification Manual", the physiological and biochemical characteristics of rhizosphere growth-promoting bacteria JHS0317 are consistent with those of Pantoea agglomerans.

[0045] Table 3 Physiological and biochemical test results of strains “+” 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 “A” indicates an acid-producing reaction.

[0046] The 16S rDNA sequence of the strain to be tested obtained by PCR was compared with the GenBank database by BLAST. The results showed that the sequence similarity between strain JHS0317 (PP549618) and P. agglomerans Bel 1-1 (OQ979271.1) reached 99.51%. As can be seen from Figure 3, JHS0317 and P. agglomerans are clustered in the same branch. Combining morphological and physiological and biochemical identification, strain JHS0317 was identified as P. agglomerans.

[0047] The results of the culture dish filter paper moisturizing method (Table 4) showed that the strain JHS0317 showed a significant growth-promoting effect after seed soaking treatment. The germination rate of the strain JHS0317 treatment group increased by 2.68%; the root length and stem length were higher than the control group, and the root length of the tobacco seedlings was significantly higher than the control treatment, with a growth rate of 45.35%. This shows that the strain JHS0317 has a significant growth-promoting effect on tobacco seeds.

[0048] 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 treatment in each column indicate that there are statistically significant differences among the treatments (P<0.05), the same below.

[0049] As shown in Figure 4, the root activity of tobacco seedlings treated with strain JHS0317 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 51.80μg / h / g fresh weight, 54.81% higher than that of the control group, and there was no significant difference compared with the other two concentrations.

[0050] As shown in Table 5, the agronomic traits of tobacco seedlings were improved after treatment with strain JHS0317, among which the fresh weight of the whole plant, the fresh weight above ground, the fresh weight of the root system and the maximum leaf width were significantly increased. 5 cfu / mL, the whole plant fresh weight, aboveground fresh weight, root fresh weight, maximum leaf length and maximum leaf width of tobacco seedlings were significantly higher than those of the control treatment, which were increased by 153.73%, 171.93%, 40.00%, 54.06% and 56.17% respectively compared with the control treatment; in addition, the whole plant fresh weight, aboveground fresh weight and maximum leaf width of tobacco seedlings in this treatment group were significantly higher than those of the treatment group with a bacterial content of 1×10 7 cfu / mL and 1×10 6 cfu / mL treatment group, increased by 18.06%, 20.62%, 20.53% and 48.47%, 67.57%, 17.63% ( Figure 5 ).

[0051] Table 5 The growth-promoting effect of rhizosphere bacteria on tobacco seedlings The strain of the present invention produces an IAA content of 26.81 mg / L, which can promote tobacco seed germination and seedling growth. The JHS0317 of 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 common tobacco pathogenic fungi Fusarium oxysporum and Fusarium solani, and can inhibit the growth and development of pathogens by inhibiting the growth of pathogenic hyphae.

[0052] After inoculation with JHS0317, 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 153.73%, 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 Pantoea agglomerans JHS0371, characterized in that: The deposit number of the strain is CCTCC NO: M2024739.

2. An application of Pantoea agglomerans JHS0371 as claimed in claim 1 in promoting tobacco growth.

3. The Pantoea agglomerata JHS0371 according to claim 1, characterized in that: It was isolated from the rhizosphere soil of sweet potato using the gradient dilution spread plate method.

4. A method for isolating Pantoea agglomerata JHS0371, characterized in that: The method is as follows: after the sweet potato 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 Pantoea agglomerata JHS0371, 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: Pantoea agglomerans JHS0371 is inoculated into LB liquid culture medium, cultured in a constant temperature shaking incubator at 28°C and 180 r / min for 24 h, the fermentation liquid is added into the sterilized seedling medium, mixed evenly, and then loaded into a floating seedling tray to prepare a bacterial matrix, and then floating seedling culture is carried out.

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