Low-temperature-resistant pseudomonas and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to low-temperature-resistant pseudomonas and application thereof. The technical problem to be solved by the invention is to develop pseudomonas with higher application value. According to the technical scheme, the pseudomonas sp. WL-1 is provided, and the preservation number of the pseudomonas sp. WL-1 is CCTCC (China Center for Type Culture Collection) NO: M20242311. The invention provides a strain of novel pseudomonas. The strain has IAA production activity and can promote the growth of tobacco seedlings. The tobacco treated by the strain is better in appearance growth vigor and dry and fresh weight, and meanwhile, the nitrogen metabolism and cold resistance of leaves are also improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a low-temperature resistant Pseudomonas strain and application thereof. Background Art

[0002] Pseudomonas is Gram-negative, non-spore-forming, aerobic, straight or slightly curved rod-shaped. The size of the bacteria is 0.5-1×1.5-4μm, most of which have polar flagella and can move. They are mostly distributed in soil, water and various plants. They have a strong ability to decompose organic matter and can use a variety of organic matter as energy sources. At present, there have been a large number of reports on the application research of Pseudomonas around the world, involving fields including agricultural biological control, plant growth regulation, environmental protection and pharmaceutical development. Among them, research and application in biological control and plant growth promotion are the most extensive. At present, the most common and widely used Pseudomonas species include Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas chlororaphis, Pseudomonas aeruginosa, etc. (Reference: https: / / mp.weixin.qq.com / s?__biz=Mzg5MzU0MzkyNQ==&mid=2247500261&idx=2&sn=0ab67b69640c09aca6fcd0ac9fcff5f5&chksm=c02fb57ff7583c6999a77c3c78b1592275ac9bab3e77d17ffc249142581610582ea414ddeb c5&scene=27). In agriculture, Pseudomonas, as one of the most widely used biocontrol bacteria, can inhibit the growth and reproduction of pathogens through antibiotics, competition (nutrient competition and site competition), secretion of secondary metabolites, improving plant nutrition and inducing plant systemic resistance, thereby reducing the occurrence of diseases, promoting plant growth and development, and product accumulation. In environmental protection, Pseudomonas is mainly used for the degradation of chemical pesticides, wastewater treatment, oil pollution treatment, etc. In medicine, anti-tumor active substances are mainly isolated from Pseudomonas. In short, Pseudomonas has a good application prospect in agriculture, environment and medicine ([1] Wang Zhirong. Research on biological control of post-harvest orange penicillium and green mold by fluorescent Pseudomonas ZX [D]. Southwest University, 2019. [2] Ning Shuang. Research on the growth-promoting and disease-resistant effects and mechanisms of endophytic Pseudomonas BTa14 and Bar25 [D]. Yantai University, 2019. [3] Yang Haijun, Tan Zhoujin, Xiao Qiming, et al. Research on the biological control effect of Pseudomonas [J]. Chinese Journal of Eco-Agriculture, 2004, 12(3): 158-161.).

[0003] At present, low temperature, high rainfall and low light weather are often encountered during tobacco seedling raising and transplanting, which leads to low quality of tobacco seedlings, slow growth in the early stage of tobacco and reduced quality of tobacco leaves. Therefore, there is an urgent need to develop local low-temperature-resistant growth-promoting bacteria. Through the collection and screening of a large amount of tobacco-growing soil, a highly efficient Pseudomonas sp.WL-1 was isolated. Through indoor experiments, seedling raising and field applications, it was found that it has a good promoting effect on the quality of tobacco seedlings and the growth and yield of tobacco leaves in the field, which provides practical technology and scientific basis for the high-quality production of Chongqing flue-cured tobacco. Summary of the invention

[0004] The technical problem to be solved by the present invention is to develop Pseudomonas with greater application value.

[0005] The technical solution of the invention is a strain of Pseudomonas sp. WL-1, whose preservation number is CCTCCNO: M20242311.

[0006] The invention also provides application of the Pseudomonas sp. WL-1 in producing IAA.

[0007] The invention also provides a product containing IAA, comprising Pseudomonas sp. WL-1, secretions of Pseudomonas sp. WL-1 or / and fermentation liquid of Pseudomonas sp. WL-1.

[0008] The invention also provides application of the Pseudomonas sp. WL-1 in promoting tobacco growth.

[0009] Specifically, the promoting tobacco growth is to promote the growth of the aboveground part of tobacco, promote the accumulation of dry matter in the middle leaves of tobacco, and increase plant height, stem girth, maximum leaf length, maximum leaf width and number of effective leaves.

[0010] Furthermore, the application is carried out under low temperature conditions.

[0011] The invention also provides a microbial preparation for promoting tobacco growth, comprising Pseudomonas sp. WL-1, secretions of Pseudomonas sp. WL-1 and / or fermentation liquid of Pseudomonas sp. WL-1.

[0012] The invention also provides application of the Pseudomonas sp. WL-1 in improving cold resistance or oxidation resistance of tobacco.

[0013] The invention also provides the use of the Pseudomonas sp. WL-1 in promoting the accumulation of NR, soluble sugar, soluble protein, CAT, POD or SOD in tobacco.

[0014] Beneficial effects of the present invention: The present invention provides a new strain of Pseudomonas. The strain has the activity of producing IAA and can also promote the growth of tobacco seedlings. The appearance growth and dry fresh weight of tobacco treated with the strain are better, and the nitrogen metabolism and stress resistance (mainly cold resistance) of the leaves are also improved. Inoculation of WL-1 can significantly improve the NR, soluble sugar, soluble protein, CAT, POD, and SOD of flue-cured tobacco leaves in the seedling stage in floating seedling cultivation, and has a significant promoting effect on the antioxidant and stress resistance of tobacco seedlings. The results of field tests show that the use of WL-1 can significantly promote the growth of the aboveground part of tobacco plants and the accumulation of dry matter in the middle leaves, and the plant height, stem girth, maximum leaf length, maximum leaf width and number of effective leaves are all improved; at the same time, it has a good effect of increasing production and income, and the increase in production and income can reach 10.58 kg / mu, 374.47 yuan / mu, and 3.8%. As for the upper and middle leaves, the chemical component content of the WL-1 treatment was appropriate and the internal chemical component coordination was better than that of the CK treatment; in particular, the nicotine and total sugar content was reduced, the nitrogen-alkali ratio and sugar-alkali ratio of the tobacco leaves were increased, and the reducing sugar content of the tobacco leaves was increased, indicating that the WL-1 treatment can significantly improve the economic benefits of flue-cured tobacco and improve the coordination of the chemical components of tobacco leaves.

[0015] The low-temperature-resistant Pseudomonas sp. WL-1 of the present invention was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, postal code 430072) on October 23, 2024, and its preservation number is CCTCC NO: M20242311. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , at 15℃ for 60 days, the growth-promoting bacteria WL-1 promoted the growth of tobacco seedlings.

[0017] Figure 2 , bar chart of the effect of inoculation with WL-1 on the microbial community in the rhizosphere soil of tobacco seedlings (genus level).

[0018] Figure 3, volcano plot of total ion mode metabolites in the treatment group compared with the control. The horizontal axis is the multiple change value of the metabolite expression difference between the two groups, that is, log2FC, and the vertical axis is the statistical test value of the difference in metabolite expression, that is, -log10 (pvalue). The higher the value, the more significant the expression difference. The values ​​of the horizontal and vertical axes have been logarithmically processed. Each point in the figure represents a specific metabolite, and the size of the point represents the Vip value (the contribution of the metabolite to the difference between the two groups). The points on the left are metabolites with downregulated expression differences, and the points on the right are metabolites with upregulated expression differences. The closer to the left and right and the top, the more significant the expression difference. Up is the number of significantly upregulated substances, and down is the number of significantly downregulated substances. Nosig is the number of substances with no significant changes.

[0019] Figure 4 , KEGG enriched pathways of differential metabolites.

[0020] Figure 5 , the colony morphology of strain WL-1 grown on LB plate (left) and the bacterial morphology observed under an optical microscope (right). DETAILED DESCRIPTION

[0021] Example 1 Isolation and Identification of Pseudomonas sp. WL-1

[0022] Soil samples were collected from the plots with good field growth in Chongqing's high-altitude tobacco area every year. After mixing, 10 g of soil was weighed and diluted with sterile water. The dilution multiple was 10. -4 , 10 -5 and 10 -6 The soil suspension was coated on LB solid plates and cultured at 10°C in the dark. When single colonies grew on the plates, colonies of different forms were picked and the strains were purified by plate streaking to obtain a single colony named WL-1. Since the culture temperature of the isolated bacteria was 10°C, the bacteria had the characteristics of rapid growth under low temperature conditions, but the optimal growth temperature was higher than 20°C, and they were all low-temperature resistant bacteria.

[0023] Tobacco seeds were sterilized with 0.1% CuSO4 for 20 minutes, rinsed with sterile water for 3 to 5 times, soaked at 40℃ for 30 minutes, soaked in cold water for 24 hours, and aseptically planted on MS medium. Germination and culture were carried out at room temperature of 25℃, light intensity of 8000lx, humidity of 70%, and light-dark cycle of 12:12. After 8 days of seedling cultivation, tobacco seedlings with relatively consistent germination were selected and transplanted to new MS medium. Ten tobacco seedlings were treated for each treatment. Single colonies of purified strains were picked out in advance in a test tube (LB liquid medium), and cultured at 28℃ and 180r for 1 to 3 days. 10uL of bacterial solution was absorbed for each treatment, and 1μL of bacterial solution was added to the middle of each tobacco seedling. LB liquid medium was added as a control. After adding bacterial solution, the tobacco seedlings were cultured for a certain period of time at a low temperature of 15℃, light intensity of 8000lx, humidity of 70%, and light-dark cycle ratio of 12:12. The strains with better growth-promoting effect were selected according to the growth conditions of the roots and aboveground parts of the tobacco seedlings. By comparing the growth of tobacco seedlings with the uninoculated control, 15 strains were screened for significant growth promotion of tobacco seedlings. The growth-promoting properties of these strains were screened three times repeatedly to confirm the stability of their activity. Among them, the strain numbered WL-1 had the most significant growth-promoting effect on tobacco seedlings and the most stable growth-promoting effect.

[0024] The formula of the above LB medium is: 10g of peptone, 5g of yeast powder, 5g of sodium chloride, 15g of agar added to the solid medium, 1000mL of deionized water, sterilized at 121°C for 25min and poured into plates for use.

[0025] The formula of the above-mentioned MS culture medium is: 1.9 g potassium nitrate, 1.65 g ammonium nitrate, 0.44 g calcium chloride, 0.83 mg potassium iodide, 8.6 mg zinc sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 2 mg glycine, 0.5 mg nicotinic acid, 0.17 g potassium dihydrogen phosphate, 0.37 g magnesium sulfate, 6.2 mg boric acid, 22.3 mg manganese sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.1 g inositol, 0.1 mg thiamine hydrochloride, 0.5 mg pyridoxine hydrochloride, 0.0373 g disodium ethylenediaminetetraacetic acid, 30 g sucrose, 8 g agar, 1000 mL deionized water, sterilize at 116°C for 30 min and pour into plates for use.

[0026] The results of WL-1 bacteria promoting tobacco seedling growth after 60 days of culture are shown in Figure 1 .Depend on Figure 1 It can be seen that when the number of true leaves of the tobacco seedlings in the WL-1 treatment group reached 5, the control group had only 3 true leaves, and the leaves of the treatment group were significantly larger than those of the control group. In addition, the root system of the tobacco seedlings in the treatment group was more developed, as shown by longer main roots, more developed lateral roots, and more abundant root hairs than that of the control group.

[0027] A series of morphological identifications were performed on the strain WL-1 isolated and purified above ( Figure 5), the bacteria formed translucent, waxy white, neatly edged colonies on LB solid plates, with abundant extracellular secretions, and the bacteria were short rod-shaped under optical microscope, and Gram staining was negative. The bacterial genomic DNA was extracted, and 16S rDNA was amplified by PCR and sequenced (detected by Shanghai Meiji Biological Company). After comparison with the GenBank database of NCBI, the strain was identified as Pseudomonas sp.

[0028] SEQ ID NO.1 16S rDNA

[0029]

[0030] Example 2 Indoleacetic acid (IAA) production activity of Pseudomonas sp. WL-1

[0031] IAA is a common plant growth hormone. Growth-promoting bacteria can secrete IAA to promote plant growth. Therefore, the ability of the isolated strain to secrete IAA was determined. The strain was cultured in KingB-Trp medium: peptone 20g, K2HPO4 1.15g, MgSO4·7H2O1.5g, glycerol 15mL, L-tryptophan 0.1g, deionized water 1000mL, pH = 7.0. Salkowski reagent: FeCl3·6H2O 1.015g, H2SO4 150mL, deionized water 250mL.

[0032] KingB-Trp medium: peptone 20 g, K2HPO4 1.15 g, MgSO4 7H2O 1.5 g, glycerol 15 mL, L-tryptophan 0.1 g, pH = 7.0

[0033] Salkowski reagent: prepare 10 mL of 0.5 mol / LFeCl3 solution, add it to 500 mL of 35% HClO4, mix well before use, and store in dark place;

[0034] 3. Steps: Pick a single colony from the plate and activate it in KingB-Trp medium for 24 hours. Transfer the test bacterial solution to new KingB-Trp medium at a 1% inoculation rate and incubate at 15°C and 180 r / min. -1 The culture was shaken for 72 h under the conditions;

[0035] Qualitative: Take 50μL of bacterial suspension and drop it on a white ceramic plate. At the same time, add 50μL of Salkowski colorimetric solution. Use blank culture medium as blank control and 50mg / L IAA standard colorimetric solution as positive control. Place the white ceramic plate at room temperature and avoid light for 30 minutes and then observe. If the color turns red, it means that IAA can be secreted.

[0036] Quantification: Take 1 mL of culture medium, centrifuge at 12000g for 5 minutes, take 500 μL of supernatant and add an equal volume of Salkowski reagent, color development in the dark at room temperature for 30 minutes, measure the optical density at 530 nm, use blank culture medium as control, and use the optical density corresponding to pure IAA as a standard curve to calculate the output of IAA (mg / L). Each treatment was repeated 3 times, and the average output was 32.25 μg / mL.

[0037] Example 3: WL-1 bacteria alleviates low temperature chilling injury in tobacco seedlings under low temperature conditions

[0038] 1. Effects of inoculation of cold-tolerant growth-promoting bacteria WL-1 on the microbial community in tobacco rhizosphere soil

[0039] A field experiment was conducted in Sanyi Township, Pengshui from April 20, 2023 to June 10, 2023. The average temperature varied between 15 and 22°C. Pseudomonas WL-1 was inoculated into LB liquid culture medium and cultured at 28°C and 120rpm for 18 hours to obtain WL-1 fermentation liquid. The fermentation liquid of Pseudomonas WL-1 was diluted 10 times with water and inoculated by root irrigation when the tobacco seedlings were transplanted. There were three replicates in the treatment group and the control group, with 30 tobacco seedlings in one replicate block. After 50 days, the topsoil of 5 tobacco seedlings roots was randomly taken from each replicate and mixed evenly. Then 2g of soil samples were taken to extract total DNA. The total DNA was sequenced using bacterial 16S rDNA universal primers and Illumina MiSeq platform. After quality control filtering of the sequencing results, community diversity analysis was performed. Results are shown in Figure 2 .Depend on Figure 2 It can be seen that after WL-1 treatment, the community diversity at the genus level increased significantly. For example, the abundance of Sphingomonas, norank_f_Gemmatimonadaccae, unclassified_f_Micrococcaceae, unclassified_f_Xanthobacteraceae, norank_f_Roseiflexaceae, Gemmatimonas and other genera were significantly higher than that of CK. According to the data, Sphingomonas is a rhizosphere microorganism that is beneficial to plants. It can degrade organic pollutants, promote plant growth and inhibit plant pathogens. The unknown genus of Gemmatimonadaceae can decompose organic matter, produce growth hormones and similar substances, fix nitrogen, and produce a clumping effect similar to slime mold to help bacteria find nutrients. The unknown genus of Micrococcaceae can promote plant growth, prevent soil-borne diseases, and participate in the decomposition of organic matter. Unclassified f Xanthobacteraceae is a genus of bacteria in the order Rhizobiales that may have nitrogen fixation. Unclassified f Roseiflexaceae is a genus of bacteria in the phylum Chloroflexi that has green pigment and can fix carbon dioxide via the 3-hydroxy-propionic acid pathway rather than the traditional Calvin pathway. Gemmatimonas contains bacterial chlorophyll and can photosynthesize and survive on light energy, which allows Gemmatimonas to survive in extreme environments, such as the Gobi Desert, where there is plenty of light but little nutrition.

[0040] 2. Effects of inoculation of growth-promoting bacteria on root secretions of tobacco seedlings under low temperature conditions

[0041] In order to analyze the active substances that may exist in the secretions of bacteria or plants and are beneficial to the growth of bacteria themselves and tobacco plants, metabolomics technology (LC-MS / MS) was used to determine the effects of the cold-resistant Pseudomonas strain WL-1 on the root secretions of tobacco seedlings under low temperature conditions.

[0042] Tobacco seedlings were cultured on plates. The roots were inoculated with strain WL-1 and then treated at low temperature of 15℃ for 20 days. The MS culture medium near the tobacco seedlings was sampled and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for metabolite determination. First, the sample was accurately weighed for desalting and grinding beads were added. Under low temperature, an extract containing an internal standard (L-2-chlorophenylalanine) (methanol: water = 4:1 (v:v)) was added to extract metabolites. The sample solution was ground in a frozen tissue grinder and then extracted by low temperature ultrasonic. The sample was placed at -20℃ for 30 minutes, then centrifuged and the supernatant was taken into a vial with an inner insert for analysis. Liquid chromatography was used to separate the components, and the single components were then ionized in the ion source of a high vacuum mass spectrometer, separated according to the mass-to-charge ratio (m / z) to obtain a mass spectrum. Finally, the qualitative and quantitative results of the sample were obtained by analyzing the mass spectrometry data of the sample. The instrument platform for LC-MS analysis was the UHPLC-Q Exactive system of Thermo Fisher Scientific. The sample mass spectrometry signal was collected in positive and negative ion scanning mode with a mass scanning range of m / z: 70-1050.

[0043] The preprocessed matrix files were analyzed for differences, and the main differential metabolites in the treatment group were obtained by comparing the relative contents of metabolites in the treatment group and the control group. Figure 3 ) showed that compared with the root secretions of tobacco seedlings not inoculated with bacteria, there were 65 identified differential metabolites in the WL-1 treatment (35 up-regulated metabolites and 30 down-regulated metabolites). The WL-1-inoculated group showed more differential metabolites than the uninoculated control group, indicating that the WL-1 strain may have a stronger regulatory effect on the growth of tobacco seedlings. Among them, the photopigment Lumichrome (LC) and Oseltamivir Oseltamivir are common components of the three types of differential metabolites. KEGG pathway enrichment was performed, and it was found that the pathway with significantly enriched metabolites was riboflavin metabolism, and the metabolite enriched in this pathway was LC, which was significantly upregulated in the WL-1 strain treatment. LC is one of the endogenous, lipophilic riboflavin (vitamin B2) photodegradation products, and plays an important role in cellular oxidation and energy metabolism. Studies have shown that riboflavin is related to the adaptation of plants to abiotic stresses, and has the effect of promoting growth and enhancing plant resistance.

[0044] The differential metabolites in the treatment groups were subjected to KEGG pathway enrichment analysis to obtain pathways with significantly enriched metabolites in the corresponding metabolites. Figure 4 As shown in the figure, the significantly enriched pathways include phenylpropanoid biosynthesis, arginine and proline metabolism, and furfural degradation in metabolism-related pathways (Metabolism, M), and plant hormone signal transduction in environmental information processing (EIP). The up-regulated substances enriched in these pathways include ferulic acid (FA), chavicol, L-glutamic gamma-semialdehyde, 5-hydroxymethyl-2-furaldehyde, and zeatin, and the down-regulated substances include N-acetyl-L-glutamate 5-semialdehyde.

[0045] Ferulic acid is a phenolic acid commonly found in plants and is an intermediate product of the phenylpropanoid biosynthesis pathway, which is one of the defense mechanisms of plants. In most plants, on the one hand, ferulic acid can cross-link with macromolecules such as polysaccharides and lignin in plant suberin and cutin in the form of monomers and dimers under the action of ferulic acid esterase, playing a certain role in the morphological construction and growth and development of plant cell walls. On the other hand, ferulic acid has good antioxidant activity and has a certain scavenging effect on reactive oxygen. Zeatin is a common cytokinin in higher plants and is an endogenous hormone in plants. It plays a key role in plant growth and development, and its expression is closely related to plant growth and stress resistance.

[0046] In addition to ferulic acid and zeatin, indole-3-acetamide (IAM) is also a common component in the differential metabolites treated with WL-1. Studies have shown that IAM is an intermediate in the biosynthesis of IAA. The IAM pathway is the most characteristic pathway for bacterial synthesis of IAA, and the synthesis of IAA is mainly achieved in two steps. Tryptophan is first converted to IAM by tryptophan-2-monooxygenase (IaaM) encoded by the iaaM gene, and then IAM is converted to IAA by IAM hydrolase (IaaH) encoded by iaaH. Cytosine and aspartic acid were also found in the differential metabolites treated with WL-1 as the main metabolites that increased significantly in the strain WL-1 treatment. In previous studies, cytosine was found to be one of the chemotactic signals of Pseudomonas. Chemotaxis refers to the chemotaxis and behavior of motile microorganisms that use flagella or pili to swim toward or away from certain chemicals in the external environment by sensing their stimulation, so that bacteria tend to beneficial stimuli and escape from harmful stimuli. Chemotaxis is an adaptive response of bacteria to changes in the microenvironment and is one of the key factors that determine the colonization ability of strains. Studies have shown that Pseudomonas putida F1 and PRS2000 are attracted by cytosine, but not by thymine or uracil. Aspartic acid is annotated as a metabolism of bacterial chemotaxis in KEGG functional pathway analysis, and aspartic acid belongs to non-aromatic amino acids. Moench et al. showed that the presence of amino groups seems to have a strong effect on the chemotaxis of P. aeruginosa. Sampedro and Oku et al. showed that Pseudomonas aeruginosa and Pseudomonas fluorescens showed positive chemotaxis to 20 common amino acids, among which P. fluorescens Pf0-1 showed a good chemotactic response to aspartic acid. Therefore, the spread and colonization of strain WL-1 in the roots of tobacco seedlings may be attracted by chemotactic signals such as cytosine and aspartic acid.

[0047] Example 4 Application effect of WL-1 in floating seedling cultivation

[0048] It was carried out in a floating seedling greenhouse. The low-temperature resistant bacteria agent test set up WL-1 and CK treatments; the concentration used was 100 times diluted after culture to the logarithmic phase, and the control (CK) was the culture medium plus an equal amount of clean water. The method of use was set to spray once, that is, spraying was carried out when the substrate was plated. Each treatment was repeated 3 times, and each treatment area had 100 tobacco seedlings. Except for the above measures, other seedling management measures were the same as usual.

[0049] 1 Determination of the effect of inoculation strain WL-1 on agronomic traits of flue-cured tobacco floating seedlings

[0050] At the seedling stage (55-60 days after sowing), 10 representative tobacco seedlings from each treatment were randomly selected using the five-point sampling method, and the agronomic traits of the tobacco seedlings were measured according to the Chinese tobacco industry standard YC / T142-2010.

[0051] Table 1 Effects of inoculated strain WL-1 on agronomic traits of flue-cured tobacco seedlings

[0052] deal with Stem height (cm) Stem circumference (cm) Number of blades Maximum leaf length (cm) Maximum leaf width (cm) CK 3.93±0.5b 1.31±0.23ab 2.7±0.48ab 13.43±0.79b 4.66±0.51b WL-1 5.07±0.71a 1.29±0.14a 3.1±0.57a 15.54±0.4a 5.6±0.44a

[0053] As shown in Table 1, compared with the uninoculated treatment (CK) group, inoculation of WL-1 can significantly increase the stem height, maximum leaf length and maximum leaf width of flue-cured tobacco in the floating seedling stage, and has an effective promoting effect on the agronomic traits of tobacco seedlings.

[0054] 2 Determination of the effect of inoculation strain WL-1 on the fresh weight and dry weight of flue-cured tobacco floating seedlings

[0055] At the seedling stage (55-60 days after sowing), 10 representative tobacco seedlings of each treatment were randomly selected by five-point sampling method for fresh weight and dry weight determination. The fresh (dry) weight was weighed on a balance, and the samples were washed with clean water and dried with absorbent paper before being weighed directly. The dry weight was first fixed at 105℃ in a ventilated drying oven for 30 minutes, and then dried at 80℃ to constant weight before weighing.

[0056] Table 2 Effects of inoculation with strain WL-1 on fresh weight and dry weight of flue-cured tobacco seedlings

[0057]

[0058]

[0059] As shown in Table 2, compared with the uninoculated treatment (CK) group, inoculation with WL-1 can significantly increase the fresh weight of flue-cured tobacco leaves, stems and roots in the seedling stage in floating seedling culture, and the dry weight of the stems and roots of the inoculated group is also increased, which has an effective promoting effect on the growth of tobacco seedlings.

[0060] 3. Determination of the effects of inoculated strain WL-1 on the physiological characteristics of flue-cured tobacco leaves grown in floating seedling culture

[0061] At the seedling stage (55-60 days after sowing), 10 representative fresh tobacco leaves of each treatment were randomly harvested by the five-point sampling method and stored in liquid nitrogen for determination of leaf enzyme activity and quality indexes, and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), nitrate reductase (NR) and malondialdehyde (MDA), soluble total sugar, free proline (FP) and soluble protein (SP) in the leaves. Each index was determined by the corresponding detection kit, and the test steps were consistent with the operating instructions.

[0062] Table 3 Effects of inoculation with strain WL-1 on leaf physiological characteristics of flue-cured tobacco during floating seedling cultivation

[0063]

[0064] As shown in Table 3, compared with the uninoculated treatment (CK) group, inoculation of WL-1 can significantly increase the NR, soluble sugar, soluble protein, CAT, POD, and SOD of flue-cured tobacco leaves in the seedling stage in floating seedling culture, and has a significant promoting effect on the antioxidant and stress resistance of tobacco seedlings.

[0065] Based on the results in Tables 1 to 3, it can be seen that the application of low-temperature-resistant bacteria WL-1 can significantly promote the appearance growth and dry and fresh weight of tobacco seedlings, and also improve the nitrogen metabolism and stress resistance of leaves.

[0066] Example 5 Application effect of WL-1 in the field

[0067] The test site was set up in a village (1243.3m above sea level, 107°44′11″ east longitude, 29°41′91″ north latitude). The soil physical and chemical properties of the test site are as follows: pH value is 5.40, and available potassium, available phosphorus, alkaline nitrogen, and organic matter are 275.00, 72.1, 90.63, and 18.44 g.kg, respectively. -1 . Set up two treatments, control (CK) and WL-1. Apply 50 ml of bacterial solution to each plant, using a concentration of 20 times diluted from the logarithmic phase. The control (CK) is the culture medium plus an equal amount of water. Immediately after transplanting, irrigate with bacterial solution. Each treatment was repeated 3 times, and the area of ​​each plot was 60 square meters. Randomly arranged in the field. Except for the above measures, other management measures are the same as usual.

[0068] The results showed that the use of low-temperature-resistant bacteria WL-1 can significantly promote the growth of the aboveground part of tobacco plants and the accumulation of dry matter in the middle leaves. It also has a good effect of increasing production and income, with an increase of 10.58 kg / mu, 374.47 yuan / mu, and 3.8%. As for the upper leaves and middle leaves, the chemical component content of WL-1 treatment is appropriate and the coordination of internal chemical components is better than that of CK treatment.

[0069] 1 Determination of the effect of inoculated strain WL-1 on agronomic traits of flue-cured tobacco

[0070] The agronomic traits of tobacco leaves under different treatments were measured at the tobacco cluster stage and flat-top stage according to the national standard YC / T 142 2010 “Investigation and measurement methods for agronomic traits of tobacco”.

[0071] Table 4 Effects of inoculated strain WL-1 on agronomic traits of flue-cured tobacco

[0072]

[0073] As shown in Table 4, the plant height, stem girth, maximum leaf length, maximum leaf width and number of effective leaves of tobacco inoculated with WL-1 at the cluster stage were significantly higher than those of the non-inoculated (CK) group; the stem girth and number of effective leaves of tobacco inoculated with WL-1 at the dome stage were significantly higher than those of the non-inoculated (CK) group, and the plant height, maximum leaf length and maximum leaf width of the inoculated group were also significantly improved, so it has an effective promoting effect on the agronomic traits of flue-cured tobacco.

[0074] 2 Effect of inoculation of strain WL-1 on dry matter accumulation in flue-cured tobacco leaves at maturity

[0075] The upper, middle and lower tobacco leaves of each treatment were collected at the maturity stage of flue-cured tobacco, and the dry weight was first fixed in a ventilated drying oven at 105°C for 30 minutes, and then dried at 80°C to constant weight for determination of the dry weight of the tobacco leaves.

[0076] Table 5 Effect of inoculation with strain WL-1 on leaf dry weight of various parts of Wulong test tobacco plants (maturity stage)

[0077] deal with Upper leaves (g / plant) Middle leaf (g / plant) Lower leaves (g / plant) CK 53.07±0.71a 81.46±4.55b 18.81±1.16a WL-1 50.37±13.36a 112.76±8.7a 21.39±1.31a

[0078] As shown in Table 5, the dry weight of the middle leaves of the flue-cured tobacco inoculated with WL-1 was significantly higher than that of the uninoculated treatment (CK) group, and the dry weight of the lower leaves of the inoculated group was also significantly increased, so the WL-1 strain can effectively increase the dry matter accumulation of flue-cured tobacco leaves during the mature stage.

[0079] 3 Effects of inoculation with strain WL-1 on yield, output value, quality and chemical composition of flue-cured tobacco

[0080] After the flue-cured tobacco leaves are taken off the poles, the yield, output value, quality and conventional chemical composition of the tobacco leaves are tested.

[0081] Table 6 Effect of inoculation strain WL-1 on the economic benefits of Wulong flue-cured tobacco

[0082] deal with Yield per mu (kg / mu) Output value per mu (yuan / mu) Average price (yuan / kg) High-quality tobacco percentage Medium smoke percentage CK 125.3b 4025.7b 32.1a 77.5b 22.5a WL-1 135.9a 4400.2a 32.4a 81.3a 18.7ab

[0083] Table 7 Effects of inoculation with strain WL-1 on chemical components of Wulong flue-cured tobacco

[0084]

[0085] As shown in Table 6, the per-acre yield, per-acre output value and proportion of high-quality tobacco in the flue-cured tobacco inoculated with WL-1 are significantly higher than those in the non-inoculated treatment (CK) group. In general, the total nitrogen content of flue-cured tobacco should be 1.5-3.5%, and the total potassium content should be 1.13-2.02%. The data in Table 7 show that compared with the non-inoculated (CK) group, the WL-1 treatment reduces the total nitrogen content of tobacco leaves and increases the total potassium content; in general, the nicotine content is about 2%, the total sugar is 22-28%, the reducing sugar is 15-20%, the sugar-alkali ratio is 6-10, and the nitrogen-alkali ratio is 0.8-0.9, which is considered to be high-quality flue-cured tobacco. As can be seen from Table 7, the WL-1 treatment can reduce the nicotine and total sugar content, increase the nitrogen-alkali ratio and sugar-alkali ratio of tobacco leaves, and increase the reducing sugar content of tobacco leaves, indicating that the WL-1 treatment can significantly improve the economic benefits of flue-cured tobacco and improve the coordination of the chemical composition of tobacco leaves.

Claims

1. A strain of Pseudomonas Pseudomonas sp .WL-1, its deposit number is CCTCC NO: M20242311.

2. Pseudomonas according to claim 1 Pseudomonas sp .Application of WL-1 in producing IAA.

3. A product containing IAA, characterized in that: Comprising the Pseudomonas described in claim 1 Pseudomonas sp .WL-1, Pseudomonas Pseudomonas sp .WL-1 secretion and / or Pseudomonas Pseudomonas sp .Fermentation broth of WL-1.

4. Pseudomonas according to claim 1 Pseudomonas sp .Application of WL-1 in promoting tobacco growth.

5. The application according to claim 4, characterized in that: The method for promoting tobacco growth is to promote the growth of the aboveground part of tobacco, promote the accumulation of dry matter in the middle leaves of tobacco, and increase plant height, stem girth, maximum leaf length, maximum leaf width and the number of effective leaves.

6. The application according to claim 4, characterized in that: The application is under low temperature conditions.

7. A microbial preparation for promoting tobacco growth, comprising the Pseudomonas sp. Pseudomonas sp .WL-1, Pseudomonas Pseudomonas sp .WL-1 secretion and / or Pseudomonas Pseudomonas sp .Fermentation broth of WL-1.

8. The Pseudomonas according to claim 1 Pseudomonas sp .Application of WL-1 in improving cold resistance or oxidation resistance of tobacco.

9. The Pseudomonas according to claim 1 Pseudomonas sp .Application of WL-1 in promoting the accumulation of NR, soluble sugar, soluble protein, CAT, POD or SOD in tobacco.

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