A Streptomyces strain C1 and its application in tobacco cultivation

Abstract

This invention discloses a Streptomyces strain C1 and its application in tobacco cultivation, belonging to the field of plant disease biological control technology. The Streptomyces strain of this invention (… Streptomyces Streptomyces strain C1, with accession number CGMCC NO.32161, deposited on October 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), can effectively control tobacco black shank disease. It provides experimental materials for the development of microbial pesticides for the control of tobacco black shank disease. At the same time, this strain also has a growth-promoting effect, significantly improving a number of agronomic traits, and its effect is better than that of commercially available microbial agents.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases, specifically relating to a Streptomyces strain C1 and its application in tobacco cultivation. Background Technology

[0002] Tobacco, a globally significant economic crop, is severely affected by tobacco black shank disease caused by *Phytophthora nicotianae*. *Phytophthora nicotianae* typically infects tobacco plants from the base of the stem and roots, then rapidly invades the pith, causing blackening and necrosis of the stem nodes. Circular or oval lesions appear on the stem, gradually deepening in color from light gray to dark brown, with clear borders and sunken lesions. When the diseased stem is longitudinally dissected, the pith is found to be typically disc-shaped. Severely infected tobacco plants will die completely. Tobacco black shank has become one of the most damaging root and stem diseases in tobacco production in my country, severely restricting the improvement of tobacco yield and quality. Currently, some conventional control methods exist for tobacco black shank (TBS), such as crop rotation with non-host crops, breeding resistant varieties, and applying chemical fungicides.

[0003] Regarding non-host crop rotation, extensive research has been conducted on the effects of different crop combinations and rotation / intercropping cycles on tobacco yield and quality, soil properties, and the occurrence of tobacco root and stem diseases. Evidence has been found that rotation, intercropping, and relay cropping patterns with non-host crops such as wheat, sweet potato, and peanuts are beneficial in overcoming continuous cropping obstacles and controlling tobacco black shank. However, regrettably, significant disease control effects have not yet been achieved.

[0004] In terms of breeding resistant varieties, there is currently a lack of highly effective disease-resistant varieties suitable for widespread planting. On the one hand, breeding resistant varieties is time-consuming and labor-intensive. The entire process, from screening to widespread planting, involves multiple steps and requires a significant investment of time. This lengthy cycle makes it difficult for resistant varieties to quickly meet the needs of tobacco cultivation. On the other hand, the evolution of *Phytophthora indicum* affects the effectiveness of resistant varieties. As *Phytophthora indicum* continues to evolve, its pathogenicity also changes. This change causes some resistant varieties to gradually lose their resistance to new pathogenic strains, thus reducing their effectiveness in disease control. Furthermore, the tobacco planting environment varies greatly across different regions. While resistant varieties may perform well in some areas, their resistance may be significantly reduced in others due to soil, climate, and other factors. These factors lead to a gradual decrease in the effectiveness of resistant varieties in disease control.

[0005] Chemical fungicides are highly effective in controlling plant diseases, and some even have beneficial effects on plant health, making them the preferred method for short-term disease control. According to existing pesticide registration data in my country, fungicides such as metalaxyl, mancozeb, dimethomorph, and sodium dichloroisocyanurate are commonly used in production to control tobacco black shank. However, the long-term use of chemical fungicides faces environmental pollution, resistance issues, and health risks, undoubtedly becoming a major obstacle to the tobacco industry's progress towards high-quality development.

[0006] Against the backdrop of increasing emphasis on environmental protection and sustainable development, exploring non-toxic, residue-free, and environmentally friendly biological control strategies for tobacco diseases has become a current research hotspot. Actinomycetes possess remarkable secondary metabolite production capabilities, with Streptomyces spp. accounting for over 7,600 of the more than 10,000 bioactive compounds produced by actinomycetes, making it the largest producer of bioactive microbial metabolites and possessing enormous potential for biological control.

[0007] In the process of biological control, the application of biological control strains is easily affected by complex field conditions. Endophytic bacteria, benefiting from their unique ecological niche within plants, possess a safer and more stable living environment and resources, thus becoming more advantageous candidate strains for biological control and attracting widespread attention. However, although the use of endophytic bacteria to control plant diseases has become a popular research direction in the field of biological control, current research focuses primarily on bacteria and fungi. Research on endophytic actinomycetes for controlling plant diseases and promoting plant growth is relatively limited, particularly in the control of tobacco black shank. Summary of the Invention

[0008] To address the aforementioned problems, the first objective of this invention is to provide a Streptomyces sp. strain C1, which was deposited on October 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.32161, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Streptomyces sp. strain C1 was isolated from the endophytic environment of tobacco plants. Due to its colonization characteristics within the endophytic environment of tobacco plants, it can function stably under various conditions.

[0009] The second objective of this invention is to provide the application of Streptomyces strain C1 in tobacco cultivation. The fermentation broth prepared using strain C1 can significantly inhibit the growth of Phytophthora tobaccois, thereby effectively preventing the occurrence and development of tobacco black shank disease. At the same time, it can also significantly improve multiple agronomic traits of tobacco plants and greatly increase tobacco leaf yield.

[0010] To achieve the above objectives, the present invention provides a Streptomyces strain C1 using the following technical solution:

[0011] A Streptomyces strain, named Streptomyces sp. strain C1, with accession number CGMCCNO.32161.

[0012] This invention is a pioneering invention. The strain C1 of this invention is a Streptomyces strain isolated from the endophytic environment of tobacco plants. It has unique biological characteristics and is highly effective in controlling tobacco black shank disease. It also promotes seed germination, promotes faster and stronger growth of seed radicles, promotes the growth of roots, stems and leaves, supplements auxin, increases leaf chlorophyll and nitrogen content, and increases tobacco yield. Moreover, it can stably exert its effects in controlling tobacco black shank disease and promoting growth under different environmental conditions.

[0013] To achieve the above objectives, the present invention provides a technical solution for the application of Streptomyces strain C1 in tobacco cultivation:

[0014] Application of a Streptomyces strain C1 in tobacco cultivation.

[0015] More preferably, Streptomyces strain C1 is used to control tobacco black shank disease and / or promote tobacco growth.

[0016] More preferably, the application method is to use the fermentation broth of Streptomyces strain C1 for root irrigation.

[0017] More preferably, the C1 bacterial cake is fermented in LB broth to obtain an effective viable count of not less than 1×10⁻⁶. 8 Fermentation broth at CFU / mL.

[0018] More preferably, the amount of fermentation broth used is 10-15 mL / time, and the roots are irrigated twice, with an interval of 5-7 days between irrigations.

[0019] More preferably, the fermentation broth of Streptomyces strain C1 is used to promote seed germination.

[0020] More preferably, the tobacco seeds are soaked in the fermentation liquid for 18-24 hours. Attached Figure Description

[0021] Figure 1 This is a plate inhibition effect diagram of strain C1 against Phytophthora in Example 2 of the present invention;

[0022] Figure 2 This is a plate inhibition effect diagram of the aseptic fermentation broth of strain C1 against Phytophthora in Example 2 of the present invention;

[0023] Figure 3 The images show the colony, sporulation hyphae, and spore morphology of strain C1 in Example 3 of this invention.

[0024] Figure 4In Example 3 of this invention, a phylogenetic tree of strain C1 was constructed using the NJ method based on the 16S rDNA and gyrB gene sequence.

[0025] Figure 5 This is a diagram illustrating the greenhouse control effect of strain C1 on tobacco black shank in Example 4 of the present invention.

[0026] Figure 6 This is a graph showing the effect of actinomycete fermentation broth on tobacco seed germination in Example 6 of the present invention;

[0027] Figure 7 This is a graph showing the qualitative measurement results of IAA in Example 6 of the present invention;

[0028] Figure 8 This is a diagram showing the colonization results of strain C1 in tobacco plants during a greenhouse experiment using specific primers, as described in Example 7 of this invention.

[0029] Figure 9 This is a diagram showing the colonization results of strain C1 in tobacco plants in a field experiment using specific primers, as described in Example 7 of this invention. Detailed Implementation

[0030] This invention addresses several problems with existing methods for controlling tobacco black shank, such as poor effectiveness of non-host crop rotation, reduced effectiveness of resistant varieties, and negative effects of chemical fungicides. This invention provides a strain C1 of *Streptomyces* endophytic fungi in tobacco, which effectively controls tobacco black shank, significantly promotes tobacco plant growth, increases tobacco leaf yield, and outperforms commercially available microbial inoculants.

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0032] Unless otherwise specified, the terminology used in this invention is a common term in the relevant field, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the reagents and products used are all commercially available.

[0033] Example 1

[0034] This example demonstrates the isolation, culture, and preservation of strain C1. The specific procedures are as follows:

[0035] (1) Collection of tobacco plants for testing:

[0036] Tobacco plants were collected from four experimental fields in the Sanmenxia area. The soil type was red soil, and the cultivated variety was Yunyan 99. In 2023, field sampling was conducted in Sanmenxia City at the tobacco plants' rosette stage, vigorous growth stage, and harvest stage. Five robust, disease-free tobacco plants were randomly collected from each plot using the five-point sampling method. The plants were quickly transported to the laboratory, washed with clean water, and dried for later use.

[0037] (2) Steps for isolating the strain:

[0038] ① Tissue processing: 8g tissue blocks were randomly cut from three parts of the tobacco plant: leaves, stems and roots. After disinfecting the surface of the tissue blocks, they were mixed with an appropriate amount of quartz sand and 5mL of sterile water were added. The mixture was then thoroughly ground in a sterile mortar to obtain tissue fluid.

[0039] ②Dilution and application: Dilute the tissue fluid to 10 with sterile water. -1 ~10 -3 The diluted solution was applied in batches of 100 μL onto Gao's No. 1 culture medium plates and spread evenly using a spreader.

[0040] ③Cultivation and preliminary classification: Incubate upside down in a constant temperature incubator at 28℃ for 7 days. After the appearance of actinomycetes, preliminarily classify them according to the colony morphology and color. Select different colonies and streak them on a new Gao's No. 1 medium for cultivation.

[0041] (3) Purification of strains

[0042] Take two 6mm diameter mycelium cakes and place them in an Erlenmeyer flask containing 100mL of sterile water. Shake at 120r / min for 30min to prepare 10. -4 Up to 10 -6 Take 100 μL of each dilution and apply it to a Gao's No. 1 agar plate. Spread the plate evenly using a spreader. Repeat each dilution 3 times. Incubate at 28°C in the dark for 4–7 days. Select single colonies based on colony morphology and color and repeat the culture to obtain a pure culture.

[0043] (4) Culture of strains

[0044] Gao's No. 1 medium was used for the isolation and propagation of the strain: 5g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 20g agar powder, and distilled water to a final volume of 1L, pH = 7.4–7.6.

[0045] Potato glucose medium for the culture and confrontation of Phytophthora indicum, the pathogen of tobacco black shank: 200g potato, 20g glucose, 15g agar powder, and distilled water to a final volume of 1L.

[0046] LB liquid medium was used to prepare the fermentation broth for the strain: 10g tryptone, 10g NaCl, 5g yeast extract, and distilled water to a final volume of 1L.

[0047] (5) Preservation of strains

[0048] The strain was preserved for a short period at 4°C and for a long period at -80°C using the 20% glycerol cryopreservation method.

[0049] The results showed that a total of 250 actinomycete strains were isolated and purified. The number of endophytic actinomycetes in the tobacco plant decreased with the growth stage, with isolation frequencies of 118 strains in the rosette stage, 106 strains in the vigorous growth stage, and 26 strains in the harvest stage. The distribution of endophytic actinomycetes within the tobacco plant was mainly concentrated in the roots, followed by the leaves, and finally the stems, with isolation frequencies of 172 strains in the roots, 48 ​​strains in the leaves, and 30 strains in the stems.

[0050] Example 2

[0051] This example demonstrates the application of strain C1 in the prevention and control of tobacco black shank disease. The specific implementation steps are as follows:

[0052] In vitro antagonistic effect of strains against Phytophthora intoxin

[0053] The initial screening for antagonistic endophytic actinomycetes was conducted using the plate confrontation method. Using sterile toothpicks, the tested actinomycetes were inoculated into four 4mm × 20mm areas equidistant from the center of the plate. After incubation at 28℃ upside down for 2 days, 6mm diameter *Phytophthora nicotineae* discs were inoculated into the center of the plate. Plates without actinomycete inoculation served as controls. When the colonies in the control group grew to 7–8cm, the colony diameter was measured using the cross-sectional method to calculate the inhibition rate. Each treatment was repeated three times.

[0054] The method for preparing sterile fermentation broth for secondary screening of antagonistic endophytic actinomycetes is as follows: Take five 6mm diameter mycelial cakes of the tested actinomycetes and place them in a conical flask containing 200mL LB culture medium. Incubate in a constant temperature shaking incubator at 28℃ and 150r / min for 5 days to obtain the strain fermentation broth. Take a small amount of fermentation broth, centrifuge at 4722×g for 10min, and filter the supernatant using a 0.22μm pore size bacterial filter to remove residual bacterial cells, obtaining sterile fermentation broth.

[0055] The secondary screening for antagonistic endophytic actinomycetes was conducted using the mycelial growth rate method. The sterile fermentation broth of actinomycetes with an initial inhibition rate of 60% was thoroughly mixed with PDA medium (approximately 40°C) at a 1:9 ratio and poured into Petri dishes to prepare agar plates containing 10% sterile fermentation broth. A 6mm diameter *Phytophthora nicotine* cake was inoculated into the center of the plate. Plates without added sterile fermentation broth served as a control. When the control colonies grew to 7–8 cm, the colony diameter was measured using the cross-sectional method to calculate the inhibition rate and assess the antibacterial ability. Each treatment was replicated three times.

[0056] The formula for calculating the antibacterial rate is:

[0057]

[0058] The results show:

[0059] After screening and identification, a Streptomyces strain with significant antagonistic effects against Phytophthora tobaccois and plant growth-promoting effects was discovered and named C1. Strain C1 was isolated from tobacco plant samples during the harvest period, and its isolation site was located in the root tissue of the tobacco plant.

[0060] A preliminary screening was conducted on 250 isolated endophytic actinomycetes. The results showed that strain C1 exhibited a significant antagonistic effect against *Phytophthora nicotineae*, with the antagonistic effect as follows: Figure 1 As shown, the average antibacterial rate reached 98.53%.

[0061] The antagonistic activity of the fermentation broth of 64 initially screened strains against *Phytophthora nicotineae* was determined. Secondary screening results showed that the fermentation broth of strain C1 had the best antagonistic effect against *Phytophthora nicotineae*. Figure 2 As shown, the average diameter of the treated Phytophthora indicum was 6.00 mm, indicating that Phytophthora indicum could not grow at all on the 10% C1 fermentation broth medium, and the inhibition rate of strain C1 against Phytophthora indicum reached 100%.

[0062] Example 3

[0063] This example demonstrates the identification of strain C1, and the specific procedures are as follows:

[0064] The selected strain C1 underwent Gram staining and morphological observation, and its physiological and biochemical characteristics were determined according to the "Handbook for Identification of Common Bacteria". DNA was extracted from strain C1 using a bacterial genomic DNA rapid extraction kit. Its taxonomic position was determined using multi-gene molecular biology techniques based on 16S rDNA and the gyrB gene. Primer information is shown in Table 1. The sequences of the 16S rDNA primers are shown in SEQ ID NO: 1-2, and the sequences of the gyrB primers are shown in SEQ ID NO: 3-4. The PCR amplification reaction system was as follows: 2 μL DNA template; 1 μL each of forward and reverse primers; 12.5 μL Taq PCR Mix premixed enzyme; ddH2O to a final volume of 25 μL. The PCR amplification program was: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 1 min; primer-specific annealing for 30 s; 72℃ extension for 90 s; 30 cycles; 72℃ extension for 10 min. The amplified products were detected by 1.5% agarose gel electrophoresis and sequenced by Sangon Biotech (Shanghai) Co., Ltd. Sequencing results were submitted to the NCBI database for comparison and analysis. The sequences were assembled and a multigene phylogenetic tree was constructed using ACOPTools and MEGA 7.0 software.

[0065] Table 1 Primers for 16S rDNA and gyrB gene

[0066]

[0067] The results showed that Gram staining indicated strain C1 was a Gram-positive bacterium, and its colonies on Gao's No. 1 medium were dry and opaque. Figure 3 As shown, the front of the colony is silvery-white ( Figure 3 Part B), the lotus seeds on the back are white ( Figure 3 Part A of the colony has a rough surface with concentric raised patterns. The colony edges are neat and not easy to pick up. The colony does not easily disperse. The mycelium inside the substrate is well-developed, with fewer aerial mycelium. The spore hyphae are erect and the spores are oblong.

[0068] The physiological and biochemical results are shown in Table 2. Strain C1 is a facultative anaerobe with a certain degree of motility. The results for citrate utilization, sugar alcohol fermentation, gelatin liquefaction, and nitrate reduction were positive. The results for malonate utilization, starch hydrolysis, acetylmethylethanol, methyl red, and phenylalanine deaminase reactions of strain C1 were negative.

[0069] Table 2. Physiological and biochemical test results of strain C1

[0070]

[0071] Note: + indicates a positive reaction; - indicates a negative reaction; ± indicates a facultative anaerobic.

[0072] Genomic DNA of strain C1 was amplified by PCR using 16S rDNA and gyrB primers. The 16S rDNA gene sequence is shown in SEQ ID NO: 5, and the gyrB gene sequence is shown in SEQ ID NO: 6. The gene sequences were compared with the NCBI database, and a multigene phylogenetic tree was constructed based on the 16S rDNA and gyrB genes. Figure 4 As shown, strain C1 has the highest homology with Streptomyces p. NA03103 and is located on the same branch in the phylogenetic tree. Based on the above analysis results, strain C1 is identified as Streptomyces sp.

[0073] Example 4

[0074] This example demonstrates the application of strain C1 in the prevention and control of tobacco black shank disease. The specific implementation steps are as follows:

[0075] Greenhouse control effect of strains on tobacco black shank disease

[0076] The experiment was conducted in the greenhouse of the Integrated Crop Disease Management Laboratory of the College of Horticulture and Plant Protection, Kaiyuan Campus, Henan University of Science and Technology (34°35′51″N, 112°25′08″E). Greenhouse conditions were controlled at 28℃, 65% humidity, and 14h / day of light. The tested tobacco variety was LY1306. Aseptic seedlings were cultivated using sterile substrate and sterile seeds. After reaching the cross-shaped stage, the seedlings were transplanted into nursery pots (top diameter × bottom diameter × height = 7.3cm × 5.2cm × 7.7cm) for acclimatization before subsequent experiments. The greenhouse efficacy test used a 1×10⁻⁶ control method. 4 Disease development was achieved through root drenching with a CFU / mL *Phytophthora tobaccois* zoospore suspension. First, each tobacco plant was drenched with 10 mL of *Phytophthora tobaccois* zoospore suspension. Four days later, each plant was drenched with 10 mL of strain C1 fermentation broth. This was repeated twice, with a 7-day interval between drenching sessions. Sterile water treatment served as a blank control, and commercially available *Bacillus subtilis* microcapsules (CG) were used as a control, with dosages adjusted according to the product instructions. Each treatment group consisted of 15 tobacco plants, with three replicates. Water and fertilizer management remained consistent throughout. On day 21, the incidence of tobacco black shank was assessed. Incidence and disease index were calculated according to the "Classification and Investigation Methods for Tobacco Diseases and Pests" (GB / T 23222—2008) to evaluate the greenhouse control efficacy of strain C1 against tobacco black shank.

[0077] The Bacillus subtilis microcapsule granules (CG) used were produced by Chengdu Tepu Biotechnology Co., Ltd., with pesticide registration certificate number PD20151514. The active ingredient and its content are: Bacillus subtilis 100 million CFU / g.

[0078] The results showed that root irrigation with the fermentation broth of strain C1 had a good disease control effect, as shown in Table 3. Figure 5 As shown, the incidence of tobacco black shank in the C1 treatment group was only 4.44%, far lower than the 88.89% in the control group and significantly lower than the 20.00% in the Bacillus subtilis group. Simultaneously, its disease index was greatly reduced to 1.48, significantly better than the 61.23 in the control group and the 15.56 in the Bacillus subtilis group. The greenhouse control efficacy of the C1 treatment against tobacco black shank reached 95.05%, and the disease index control efficacy reached 97.41%, significantly higher than the Bacillus subtilis group. These data indicate that compared with commercially available microbial agents, the C1 strain can significantly reduce the infection and damage of tobacco black shank in greenhouse experiments.

[0079] Table 3. Greenhouse control efficacy of strain C1 against tobacco black shank.

[0080]

[0081] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0082] Example 5

[0083] This example demonstrates the application of strain C1 in the prevention and control of tobacco black shank disease. The specific implementation steps are as follows:

[0084] Field control efficacy of the strain against tobacco black shank disease

[0085] The field efficacy trial used natural disease occurrence in the field. The trial design included 3 treatment groups, with 3 replicates for each treatment, for a total of 9 plots. The plots were arranged in a randomized block design. The row spacing of tobacco plants in the plot was 110 cm and the plant spacing was 55 cm. Protective rows were set up around the perimeter. Each plot had 100 tobacco plants planted in 4 rows. During the trial, the planting measures required by local tobacco production technology were adopted, and water and fertilizer management were kept consistent. After the tobacco seedlings recovered from transplanting, each plant in the C1 treatment group was inoculated with 10 mL of the C1 fermentation broth by root irrigation, for a total of 2 irrigations, with an interval of 7 days between irrigations. The sterile water treatment served as the blank control group, and the commercially available Bacillus subtilis microcapsule granules (CG) served as the agent control group. The granules were applied according to the product instructions. Water and fertilizer management were kept consistent until the tobacco plants were in full growth. The incidence of tobacco black shank was investigated and statistically analyzed. The incidence rate and disease index were calculated according to the "Classification and Investigation Methods for Tobacco Diseases and Pests" (GB / T23222—2008) to evaluate the field control effect of strain C1 on tobacco black shank.

[0086] The results showed that root irrigation with the fermentation broth of strain C1 had a good disease control effect. As shown in Table 4, the incidence of tobacco black shank in the strain C1 treatment group was only 4.48%, significantly lower than 69.36% in the control group and 19.14% in the Bacillus subtilis group. Its disease index was also greatly reduced to only 1.25, significantly better than 34.60 in the control group and 12.14 in the Bacillus subtilis group. The field control effect of strain C1 on tobacco black shank reached 93.41%, and the disease index control efficacy reached 96.47%, significantly higher than the Bacillus subtilis group. These data indicate that compared with commercially available microbial agents, strain C1 can significantly reduce the occurrence and development of tobacco black shank when applied in the field.

[0087] Table 4. Field control efficacy of strain C1 against tobacco black shank.

[0088]

[0089] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0090] Example 6

[0091] This example demonstrates the application of strain C1 in promoting tobacco growth. The specific implementation steps are as follows:

[0092] Effects of strains on tobacco seed germination and their plant growth promoting effects

[0093] (1) Effects of strains on tobacco seed germination

[0094] Healthy, plump seeds of the same variety (LY1306) as the tested tobacco plants were selected. After surface disinfection and germination-inducing treatment, the tobacco seeds were soaked in the fermentation broth of strain C1 for 24 hours. After soaking, the seeds were removed, rinsed with sterile water to remove residual liquid, and then dried with sterile filter paper. The control group used an equal amount of sterile water instead of the fermentation broth of strain C1 to soak the tobacco seeds for the same 24 hours, and then dried with sterile filter paper.

[0095] Seed germination experiments were conducted in petri dishes lined with moistened filter paper. Each petri dish contained 25 treated tobacco seeds, which were cultured in an incubator at 28°C under alternating light and dark conditions.

[0096] Starting from the time the seeds are placed in the petri dish, the germination status of the seeds is observed and recorded on the 14th day. The germination standard is defined as the length of the radicle breaking through the seed coat to reach half the length of the seed. The number of germinated seeds is recorded and the germination rate is calculated.

[0097] Germination rate calculation formula: Germination rate = (Number of germinated seeds / Total number of seeds tested) × 100%.

[0098] (2) The ability of the strain to produce auxin IAA

[0099] An IAA standard curve was plotted using the absorbance of a mixture of IAA standard solution and Salkowski colorimetric solution at a wavelength of 530 nm. The standard curve, with IAA mass concentration on the x-axis and absorbance on the y-axis, is: y = 0.0034x - 0.0018R. 2 =0.9990.

[0100] Qualitative detection of IAA: Inoculate 2 mL of strain C1 fermentation broth into a new Erlenmeyer flask containing 200 mL of LB medium, and add 500 μg / mL of the solution. -1 2 mL of tryptophan solution was added and cultured at 28℃ and 180 rpm for 3 days with shaking. The culture was then centrifuged at 7378 × g for 10 min. 0.1 mL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution in a 0.2 mL eight-tube PCR tube for colorimetric reaction, using 50 μg·mL⁻¹. -1 Indoleacetic acid (IAA) standard solution was used as a positive control, and LB culture medium was used as a negative control. After incubation in the dark for 30 minutes, the color change was observed. If the color deepened to pink, IAA was produced.

[0101] Quantitative detection of IAA: Take 2 mL of the above supernatant and mix it with an equal volume of Salkowski colorimetric solution in a quartz cuvette. After incubating in the dark for 30 min, measure the absorbance of the mixture at a wavelength of 530 nm. LB culture medium is treated in the same way for zeroing. Each treatment is repeated 3 times.

[0102] (3) Determination of greenhouse growth-promoting effect

[0103] Tobacco strain C1 was inoculated using a root drenching method with fermentation broth. In greenhouse potted plants, tobacco plants at the cruciferous stage with uniform growth were selected, and 10 mL of strain C1 fermentation broth was used for root drenching twice, with a 7-day interval between drenchings. Sterile water treatment served as a control. Each treatment group consisted of 15 tobacco plants, and each treatment was replicated three times. Twenty-one days after inoculation, agronomic traits of the tobacco plants were measured according to the "Methods for Surveying and Measuring Agronomic Traits of Tobacco" (YC / T142-2010) to evaluate the greenhouse growth-promoting effect of the strain. Agronomic trait indicators included leaf length, leaf width, stem circumference, root-to-shoot ratio, chlorophyll content, and leaf nitrogen content.

[0104] (4) Field measurement of growth-promoting effect

[0105] In the field trial, after the tobacco seedlings had recovered from transplanting, each tobacco plant was treated with 10 mL of the fermentation broth of strain C1 for root irrigation, twice in total, with an interval of 7 days between irrigations. Sterile water treatment served as a control. Agronomic traits of the tobacco plants were measured according to the "Methods for Surveying and Measuring Agronomic Traits of Tobacco" (YC / T142-2010) at both the rosette stage and the vigorous growth stage to evaluate the field growth-promoting effect of the strain. Agronomic trait indicators included leaf length, leaf width, plant height, stem circumference, chlorophyll content, and leaf nitrogen content.

[0106] The results show:

[0107] (1) Effects of strain C1 on tobacco seed germination

[0108] The results are shown in Table 5. The germination rate of tobacco seeds in the control group was 81.00±3.83% on day 14, while the germination rate of tobacco seeds soaked in the fermentation broth of strain C1 was 94.00±2.31%. Figure 6 As shown, the seedlings of the treated group showed faster and thicker radicle growth after germination compared to the control group.

[0109] Table 5. Effects of Actinomycete Fermentation Broth on Tobacco Seed Germination

[0110]

[0111] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0112] (2) Qualitative and quantitative detection of IAA

[0113] like Figure 7 As shown, from left to right, the solutions are a mixture of water and colorimetric solution, and 50 μg / mL solution. -1 A mixture of IAA standard solution and colorimetric solution, and three replicates of fermentation broth from strain C1 mixed with colorimetric solution. Using LB culture medium as a negative control, under the same colorimetric reaction and incubation conditions, no deepening of the color to pink was observed, indicating that LB culture medium itself does not produce a colorimetric reaction similar to IAA under these detection conditions, thus ruling out interference from the culture medium itself on the detection results. 50 μg / mL -1 Indoleacetic acid standard solution was used as a positive control. After reacting with Salkowski colorimetric solution and incubating in the dark for 30 minutes, the color turned pink. This indicates that the positive control system can develop color normally, providing an accurate reference standard for the subsequent detection of the tested strains.

[0114] For strain C1, 2 mL of the test strain's fermentation broth was inoculated into a new Erlenmeyer flask containing 200 mL of LB broth, and 500 μg·mL⁻¹ was added simultaneously. -1 2 mL of tryptophan solution was subjected to specific conditions (28 °C, 180 r·min) -1 After shaking culture for 3 days, centrifugation was performed, and 0.1 mL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution for color development. The color of the culture medium was observed to deepen, indicating that strain C1 can produce IAA.

[0115] Take 2 mL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution in a quartz cuvette. After incubating in the dark for 30 min, measure the absorbance of the mixture at a wavelength of 530 nm. Use LB medium for the same zeroing process. Each treatment is repeated three times. Substitute the measured absorbance values ​​into the standard curve equation to calculate the IAA yield of strain C1 as 16.85 μg·mL⁻¹. -1 .

[0116] (3) Greenhouse growth-promoting effect

[0117] The results are shown in Table 6. The average leaf length of the C1 treatment group reached 18.93±4.88 cm, while the average leaf length of the control group was only 13.13±2.81 cm. The average leaf width of the C1 treatment group was 9.65±1.94 cm, while the average leaf width of the control group was 7.55±1.57 cm, indicating that strain C1 can promote leaf growth. The average stem circumference of the C1 treatment group was 1.94±0.31 cm, while the average stem circumference of the control group was 1.57±0.22 cm, indicating that strain C1 helps to thicken the stem of tobacco plants. Strain C1 has a good promoting effect on the root growth of tobacco plants, and the aboveground growth is also promoted, but root growth is more significant. The aboveground fresh weight of the control group and the C1 treatment group were 15.35±0.60 g and 15.41±4.80 g, respectively, and the root fresh weight were 1.17±0.54 g and 2.39±0.83 g, respectively. The root-to-shoot ratio of the C1 treatment group was 0.16±0.06, higher than that of the control group (0.10±0.07), indicating that strain C1 plays a unique role in promoting root growth. The chlorophyll content of the C1 treatment group reached 33.04±3.87 SPAD, while the chlorophyll content of the control group was 27.13±2.82 SPAD. Higher chlorophyll content contributes to photosynthesis in tobacco plants, thereby promoting growth. The leaf nitrogen content of the C1 treatment group was 13.11±1.22 mg / g, while the leaf nitrogen content of the control group was 11.21±0.89 mg / g. Higher nitrogen content provides more nutrients for tobacco plant growth and promotes plant development.

[0118] Table 6 Effects of strain C1 on agronomic traits of greenhouse tobacco plants

[0119]

[0120] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0121] (4) Field growth-promoting effect

[0122] The results of the field trials are shown in Tables 7 and 8. The effects of strain C1 treatment on leaf length, leaf width, stem circumference, and plant height of tobacco plants during the rosette and vigorous growth stages were consistent with the greenhouse trials, showing a significant promoting effect compared to the control group. During the rosette stage, it significantly promoted the chlorophyll and nitrogen content of tobacco leaves, but this changed during the vigorous growth stage, with a decrease in both chlorophyll and nitrogen content. This indicates that strain C1 has a significant promoting effect on the growth of tobacco plants in the field.

[0123] Table 7 Effects of strain C1 on agronomic traits of tobacco plants at the field heading stage.

[0124]

[0125] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0126] Table 8 Effects of strain C1 on agronomic traits of tobacco plants in the field during the vigorous growth period.

[0127]

[0128] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0129] Example 7

[0130] This example demonstrates the application of strain C1 in tobacco cultivation. The specific implementation steps are as follows:

[0131] Colonization of strain C1 in tobacco plants

[0132] The experiment was designed based on the principles of Koch's postulates, and PCR detection using specific primers was used to study the colonization of strain C1 in tobacco plants under greenhouse and field conditions.

[0133] Tobacco strain C1 was inoculated using a root drenching method with fermentation broth. In greenhouse potted plants, tobacco plants at the cruciferous stage with uniform growth were selected, and each plant was treated with 10 mL of strain C1 fermentation broth for root drenching twice, with a 7-day interval between drenchings. Sterile water treatment served as a control. Each treatment group consisted of 15 tobacco plants, and each treatment was replicated three times. Establishment was observed 21 days after inoculation. In field trials, after the tobacco seedlings had recovered from transplanting, each plant was treated with 10 mL of strain C1 fermentation broth for root drenching twice, with a 7-day interval between drenchings. Sterile water treatment served as a control. Samples were collected in the field after the tobacco plants reached the rosette stage to observe establishment.

[0134] Rinse the surface of the tobacco samples with water, allow them to air dry, and then randomly cut 8g tissue blocks from the leaves, stems, and roots of the tobacco plants. After sterilizing the surface of the tissue blocks, mix them with an appropriate amount of quartz sand and add 5mL of sterile water. Grind thoroughly in a sterile mortar to obtain a tissue homogenate. Take a portion of the tissue homogenate and extract DNA from it using a bacterial DNA extraction kit. Re-isolate strain C1 using the dilution plating method. Dilute the tissue homogenate to 100-fold with sterile water, and apply 100μL to a Gao's No. 1 agar plate using a spreader. Incubate upside down in a 28℃ incubator for 7 days. After actinomycetes appear, pick different colonies and propagate them on fresh Gao's No. 1 agar. Extract colony DNA using a bacterial DNA extraction kit.

[0135] Based on the CHAT domain-containing protein gene of strain C1, specific primers C1T were designed. The C1T-F primer sequence is shown in SEQ ID NO: 7, and the C1T-R primer sequence is shown in SEQ ID NO: 8. The PCR amplification reaction system was as follows: 2 μL DNA template; 1 μL each of forward and reverse primers; 12.5 μL Taq PCR Mix premixed enzyme; ddH2O to a final volume of 25 μL. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 1 min; 55℃ annealing for 30 s; 72℃ extension for 90 s; 30 cycles; 72℃ extension for 10 min; the fragment length was 1245 bp.

[0136] PCR amplification of tissue fluid DNA was performed using specific primer C1T. The amplification products were detected by 1.5% agarose gel electrophoresis. The presence or absence of the target fragment determined the colonization of strain C1 within the tobacco plant. PCR amplification of re-isolated colony DNA was then performed using specific primer C1T. After confirming the target strain, the distribution of strain C1 in different parts of the tobacco plant was determined based on the number of re-isolated colonies.

[0137] The results of the greenhouse experiment are as follows Figure 8 As shown: M is the DM2000 DNA Marker; 1 is the positive control; 2-4 are negative controls from the root, stem, and leaf tissues of tobacco plants without the inoculated strain; 5-7 are specific amplifications from the root, stem, and leaf tissues of tobacco plants inoculated with the strain. After PCR amplification of DNA from the leaf tissue fluid of tobacco plants treated with strain C1 via root irrigation, 1.5% agarose gel electrophoresis revealed that the 1245bp target fragment was not amplified in the leaf tissue samples, indicating that strain C1 did not colonize the leaves of the tobacco plants. PCR amplification of DNA from the stem tissue fluid showed that the target fragment was amplified in the stem samples, indicating that strain C1 colonized the stems of the tobacco plants. PCR detection of DNA from the root tissue fluid showed that the target fragment was amplified in the root samples, proving that strain C1 colonized the roots of the tobacco plants. The control group of tobacco plants treated with sterile water did not show the 1245bp target fragment after PCR amplification of DNA from the leaf, stem and root tissue fluids. Furthermore, the target strain C1 was not detected in the isolated colonies, indicating that the strain was not present in the tobacco plants without inoculation with strain C1.

[0138] The results are shown in Table 9. By counting the number of re-isolated colonies: the colonies obtained by diluting the homogenate of tobacco root, stem and leaf tissues and then spreading it on plates, and then confirming the target strain by PCR amplification with specific primer C1T, the colony content of tobacco root tissue was calculated to be 2812.50 CFU / g, indicating that strain C1 can colonize in tobacco root tissue; the colony content of stem tissue was 5312.50 CFU / g, indicating that strain C1 can colonize in tobacco stem tissue; while no target strain C1 was isolated from leaf tissue, indicating that strain C1 failed to colonize tobacco leaves.

[0139] In greenhouse experiments, strain C1 showed more active colonization or was more conducive to growth and reproduction on the stem compared to the roots of tobacco plants.

[0140] Table 9. Colonization of strain C1 in greenhouse tobacco plants.

[0141]

[0142] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0143] Field trial results as follows Figure 9 As shown, M is the DM2000 DNA Marker; 1 is the positive control; 2-4 are negative controls from the root, stem, and leaf tissues of tobacco plants without the inoculated strain; 5-7 are specific amplifications from the root, stem, and leaf tissues of tobacco plants inoculated with the strain. After PCR amplification of DNA from the leaf tissue fluid of tobacco plants treated with strain C1 via root irrigation, 1.5% agarose gel electrophoresis revealed a 1245bp target fragment in the leaf tissue samples, indicating that strain C1 had colonized the leaves of the tobacco plants. PCR amplification of DNA from the stem tissue fluid showed that the target fragment was amplified in the stem samples, indicating that strain C1 had colonized the stems of the tobacco plants. PCR detection of DNA from the root tissue fluid showed that the target fragment was amplified in the root samples, proving that strain C1 had colonized the roots of the tobacco plants. The control group of tobacco plants treated with sterile water did not show the 1245bp target fragment after PCR amplification of DNA from the leaf, stem and root tissue fluids. Furthermore, the target strain C1 was not detected in the isolated colonies, indicating that the strain was not present in the tobacco plants without inoculation with strain C1.

[0144] The results of the re-isolation of bacterial colonies are shown in Table 10. Colonies obtained by diluting and plating the homogenates of tobacco root, stem, and leaf tissues were amplified by PCR using specific primer C1T to confirm the target strain. The calculated colony content in the root tissue was 5416.67±1094.49 CFU / g, indicating that strain C1 could colonize the roots of tobacco plants; the colony content in the stem tissue was 4479.17±1076.50 CFU / g, indicating that strain C1 could colonize the stems of tobacco plants; and the colony content in the leaf tissue was 1041.67±1513.83 CFU / g, indicating that strain C1 could colonize the leaves of tobacco plants.

[0145] In field trials, the colonization of strain C1 in the roots, stems, and leaves of tobacco plants showed a decreasing trend, while colonization in the roots and stems of tobacco plants was more active or more conducive to its growth and reproduction.

[0146] Table 10. Colonization of strain C1 in field tobacco plants.

[0147]

[0148] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0149] Example 8

[0150] This example demonstrates the application of strain C1 in promoting tobacco growth. The specific implementation steps are as follows:

[0151] Effects of strain C1 on tobacco yield

[0152] After the tobacco seedlings had recovered from transplanting, each seedling was treated with 10 mL of C1 fermentation broth for root irrigation, twice in total, with a 7-day interval between irrigations. Sterile water treatment served as a blank control, and commercially available Bacillus subtilis microcapsule granules (CG) served as a control. Tobacco leaves were harvested at the harvest time. For each treatment, five uniformly growing tobacco plants were randomly selected using a five-point sampling method, and 15 plants were harvested from each plant. Upon returning to the laboratory, each plant's leaves were tagged and tied, and their fresh weight was immediately weighed and recorded. The leaves were then blanched at 105℃ for 30 minutes and dried at 65℃ to constant weight. The dry weight of each plant's leaves was measured, and the tobacco yield was calculated. The experiment was repeated three times.

[0153] The tobacco leaf production data are shown in Table 11.

[0154] Table 11 Effects of strain C1 on tobacco yield

[0155]

[0156] Note: Different lowercase letters in the same column indicate significant differences at P<0.05 according to the LSD test.

[0157] The fresh weight of tobacco leaves in the control group was 7638.75 kg / ha, while the fresh weight of tobacco leaves in the C1 treatment group was significantly increased to 31981.25 kg / ha, showing a significant increase compared to the Bacillus subtilis group. This indicates that the C1 treatment has a significant promoting effect on the fresh weight of tobacco leaves. The dry weight of tobacco leaves in the control group was 1734.25 kg / ha, while the dry weight of the C1 treatment group was 3449.56 kg / ha. The dry weight of tobacco leaves in the C1 treatment group was significantly higher than that in the control group and showed a certain degree of increase compared to the Bacillus subtilis group, but the difference was not statistically significant. This indicates that the C1 strain not only affects the fresh weight of tobacco leaves but also has a positive promoting effect on the accumulation of dry matter in tobacco leaves. In summary, the C1 strain has a significant effect on improving both the fresh and dry weight of tobacco leaves, and its effect is slightly better than that of commercially available microbial inoculants.

Claims

1. An endophytic Streptomyces strain isolated from the roots of tobacco plants, characterized in that, Named as the genus Streptomyces ( Streptomyces strain C1 (sp.), with accession number CGMCC NO.32161.

2. The application of the endophytic Streptomyces isolated from the roots of tobacco plants as described in claim 1 in the prevention and control of tobacco black shank disease and the promotion of tobacco growth.

3. The application according to claim 2, characterized in that, The promotion of tobacco growth includes promoting the growth of stems and leaves, supplementing auxin IAA, increasing the chlorophyll and nitrogen content of leaves, and increasing tobacco yield.

4. The application according to claim 3, characterized in that, The application method involves drenching the roots with the fermentation broth of the Streptomyces strain C1.

5. The application according to claim 4, characterized in that, Fermentation culture of C1 bacterial cake in LB broth yielded an effective viable cell count of not less than 1 × 10⁻⁶. 8 Fermentation broth at CFU / mL.

6. The application according to claim 5, characterized in that, The amount of fermentation liquid used is 10-15 mL / time, and the roots are irrigated twice, with an interval of 5-7 days between irrigations.

7. The application of the fermentation broth of endophytic Streptomyces isolated from the roots of tobacco plants as described in claim 1 in promoting tobacco seed germination.

8. The application according to claim 7, characterized in that, Soak tobacco seeds in the fermentation broth for 18-24 hours.

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