Streptomyces strain C1 and application thereof in tobacco planting

Abstract

The invention discloses a Streptomyces sp. Strain C1 and application thereof in tobacco planting, and belongs to the technical field of biological control of plant diseases, the Streptomyces sp. Strain C1 has a preservation number of CGMCC NO.32161, is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 9, 2024, has a preservation number of CGMCC NO.32161, is preserved in China General Microbiological Culture Collection Center (CGMCC), and has the preservation number of CGMCC NO.32161 and the preservation time of CGMCC NO.32161. The streptomyces strain C1 and the fermentation liquor thereof can effectively prevent and treat tobacco black shank, test materials are provided for developing microbial pesticides for preventing and treating tobacco black shank, meanwhile, the strain also has a growth promoting effect, various agronomic character indexes are remarkably improved, and the effect is superior to that of commercially available microbial agents.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control of plant diseases, and specifically relates to a Streptomyces strain C1 and application thereof in tobacco planting. Background Art

[0002] Tobacco, as an economic crop with important economic significance worldwide, suffers from tobacco black shank caused by Phytophthora nicotianae. The infection of tobacco plants by Phytophthora nicotianae usually starts from the base of the stem and the root, and then quickly invades the pith of the stem, which causes the stem nodes to turn black and necrotize, and round or oval lesions appear on the stem. The color will gradually deepen from light gray to dark brown, with clear boundaries and sunken lesions. When the diseased stem is cut open longitudinally, the pith can be found to be a typical disc-shaped. Once the tobacco plant is severely infected, the whole plant will die. Tobacco black shank has become one of the most harmful root and stem diseases in my country's tobacco production, seriously restricting the improvement of tobacco yield and quality. In the prevention and control of tobacco black shank (TBS), there are currently some conventional prevention and control measures, such as non-host crop rotation, breeding resistant varieties and application of chemical fungicides.

[0003] As for non-host crop rotation, we have conducted extensive research on the effects of different crop combinations and different rotation / intercropping cycles on tobacco leaf yield and quality, soil properties, and the occurrence of tobacco root and stem diseases, and found favorable evidence that rotation, intercropping, and intercropping patterns of non-host crops such as wheat, sweet potatoes, and peanuts can overcome continuous cropping obstacles and prevent tobacco black shank. Unfortunately, no significant disease control effect has been achieved so far.

[0004] In terms of breeding of resistant varieties, there is currently a lack of efficient disease-resistant varieties suitable for promotion and planting. On the one hand, breeding resistant varieties is time-consuming and laborious. The entire process starts from screening, goes through multiple procedures until promotion and planting, and a lot of time is required during this period. This long cycle makes it difficult for resistant varieties to quickly meet the needs of tobacco planting; on the other hand, the evolution of tobacco phytophthora affects the effectiveness of resistant varieties. As tobacco phytophthora continues to evolve, its pathogenicity also changes. This change causes some resistant varieties to gradually lose their resistance to new pathogenic strains, thereby reducing the role of these varieties in disease prevention and control. In addition, the tobacco planting environment in different regions varies greatly. The resistant varieties selected may perform well in some areas, but in other areas due to the influence of soil, climate and other factors, their resistance effect may be greatly reduced. These factors have led to a gradual decrease in the effectiveness of resistant varieties in disease prevention and control.

[0005] Chemical fungicides have significant effects in controlling diseases. Some chemical fungicides also have plant health effects and are the preferred method for preventing and controlling plant diseases in the short term. According to my country's existing pesticide registration data, metalaxyl, mancozeb, oxazolidinone, sodium sulfone and other pesticides are generally used in production to prevent and control tobacco black shank disease. However, the long-term use of chemical fungicides faces environmental pollution, drug resistance and health risks, which is undoubtedly a major obstacle for the tobacco industry to move forward on the road of high-quality development.

[0006] As people pay more and more attention to environmental protection and sustainable development, exploring non-toxic, non-residue and environmentally friendly biological control strategies to control tobacco diseases has become a current research hotspot. Actinomycetes have excellent secondary metabolite production capabilities, among which Streptomyces spp. accounts for more than 7,600 of the more than 10,000 bioactive compounds produced by actinomycetes, making it the largest producer of bioactive microbial metabolites and possessing huge potential for biological control.

[0007] In the process of biological control, the application of biological control strains is easily interfered by complex field conditions. Endophytes benefit from their special ecological niche inside plants and have a safer and more stable living environment and resources, so they have become more advantageous candidate strains for biological control and have received widespread attention. However, although the use of endophytes to control plant diseases has become a hot topic in the field of biological control research, it is currently mostly focused on bacteria and fungi. There are few studies on endophytic actinomycetes to control plant diseases and promote plant growth, which is particularly prominent in the prevention and control of tobacco black shank. Summary of the invention

[0008] To solve the above problems, the first object of the present invention is to provide a Streptomyces sp. strain C1, which has been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on October 9, 2024, with a deposit number of CGMCC NO.32161, and the address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Streptomyces sp. strain C1 is isolated from the endogenous environment of tobacco plants, and due to its colonization characteristics in the endogenous environment of tobacco plants, it can stably function in different environments.

[0009] The second object of the present invention is to provide an application of Streptomyces strain C1 in tobacco cultivation. The fermentation liquid prepared by strain C1 can significantly inhibit the growth of tobacco blight, 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 object, the present invention provides a Streptomyces strain C1, the technical solution adopted is:

[0011] A strain of Streptomyces, named as Streptomyces sp. strain C1, with a preservation number of CGMCCNO.32161.

[0012] The present invention is a pioneering invention. The strain C1 of the present invention is a Streptomyces strain separated from the endogenous environment of a tobacco plant, has unique biological characteristics, and has a highly effective ability to prevent and treat tobacco black shank disease. It also has the functions of promoting seed germination, promoting faster and stronger growth of seed radicles, promoting the growth of roots, stems and leaves, supplementing auxins, increasing leaf chlorophyll and nitrogen content, and increasing tobacco leaf yield, and can stably play a role in preventing and treating tobacco black shank disease and promoting growth under different environmental conditions.

[0013] To achieve the above object, the present invention provides a technical solution for applying the Streptomyces strain C1 in tobacco planting:

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

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

[0016] More preferably, the application method is root irrigation using the fermentation liquid of the Streptomyces strain C1.

[0017] Further preferably, the C1 bacterial cake is fermented in LB culture medium to obtain an effective viable bacterial count of not less than 1×10 8 CFU / mL of fermentation broth.

[0018] More preferably, the dosage of the fermentation liquid is 10-15 mL / time, and the roots are irrigated twice in total, with an interval of 5-7 days between each irrigation.

[0019] More preferably, the fermentation liquid 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the plate inhibition effect of strain C1 on tobacco phytophthora in Example 2 of the present invention;

[0022] Figure 2 This is a diagram showing the plate inhibition effect of the aseptic fermentation liquid of strain C1 in Example 2 of the present invention on tobacco phytophthora;

[0023] Figure 3 The colony, conidia and spore morphology of strain C1 in Example 3 of the present invention are shown;

[0024] Figure 4The phylogenetic tree of strain C1 was constructed by the NJ method based on 16S rDNA and gyrB gene sequences in Example 3 of the present invention;

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

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

[0027] Figure 7 This is a diagram showing the qualitative determination 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 in a greenhouse test using specific primers in Example 7 of the present invention;

[0029] Fig. 9 This is a diagram showing the colonization results of strain C1 in tobacco plants in a field test using specific primers in Example 7 of the present invention. DETAILED DESCRIPTION

[0030] The present invention mainly aims at the many problems existing in the control methods of tobacco black shank in the prior art, such as poor effect of non-host crop rotation, reduced effectiveness of resistant varieties, and negative effects of chemical fungicides. The present invention provides a tobacco endophytic Streptomyces strain C1, which can effectively control tobacco black shank and significantly promote tobacco plant growth and increase tobacco leaf yield, and the effect is better than commercially available microbial agents.

[0031] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0032] Unless otherwise specified, the terms used in the present invention are commonly used terms 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 also commercially available.

[0033] Example 1

[0034] This example is about the isolation, cultivation and preservation of strain C1, and the specific implementation operations are as follows:

[0035] (1) Collection of test tobacco plants:

[0036] The tobacco plants were collected from 4 experimental fields in Sanmenxia area. The soil type was red soil and the cultivated variety was Yunyan 99. In 2023, field sampling was carried out in Sanmenxia City during the tobacco plant clumping period, vigorous growth period, and harvest period. Five strong and disease-free tobacco plants were randomly collected from each sample plot according to the five-point sampling method. They were quickly transported to the laboratory, washed with clean water, and dried for later use.

[0037] (2) Strain Isolation Steps:

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

[0039] ② Dilution and application: Use sterile water to dilute the tissue fluid to 10 -1 ~10 -3 Pipette 100 μL of each dilution onto a Gao's medium No. 1 plate and spread it evenly using a spreader.

[0040] ③ Cultivation and preliminary classification: Incubate the culture medium upside down at 28℃ for 7 days. After actinomycete colonies appear, perform preliminary classification based on the morphology and color of the colonies. Pick different colonies and culture them by drawing lines on new Gao's No. 1 culture medium.

[0041] (3) Purification of strains

[0042] Take 2 pieces of 6 mm diameter mushroom cakes, put them into a conical flask filled with 100 mL sterile water, and shake at 120 r / min for 30 min to prepare 10 -4 Up to 10 -6 Pipette 100 μL of each dilution onto a Gao's medium No. 1 plate and spread it evenly using a spreader. Repeat 3 times for each dilution and culture at 28°C in the dark for 4 to 7 days. Pick out single colonies based on colony morphology and color and culture repeatedly until pure culture is obtained.

[0043] (4) Cultivation of strains

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

[0045] Potato glucose medium is used for the cultivation of tobacco black shank pathogen Phytophthora nicotianae and the preparation of confrontation plates: 200g potato, 20g glucose, 15g agar powder, and distilled water to 1L.

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

[0047] (5) Preservation of strains

[0048] The strains were stored at 4°C for short term and stored in -80°C refrigerator for long term using 20% ​​glycerol cryovials.

[0049] The results showed that a total of 250 actinomycete strains were obtained after separation and purification. With the development of the growth period, the number of endophytic actinomycetes in tobacco plants showed a downward trend, and the isolation frequency was 118 strains in the cluster stage > vigorous growth stage > harvest stage, which were 106 strains and 26 strains respectively. The distribution of endophytic actinomycetes in tobacco plants was mainly concentrated in the roots, followed by the leaves, and finally the stems, and the isolation frequency was 172 strains in the roots > leaves > stems, which were 48 strains and 30 strains respectively.

[0050] Example 2

[0051] This example is the application of strain C1 in preventing and treating tobacco black shank disease, and the specific implementation operation is as follows:

[0052] Antagonistic effect of strains against tobacco phytophthora in vitro

[0053] The initial screening of antagonistic endophytic actinomycetes was carried out by the plate confrontation method. The test actinomycetes were inoculated into four 4mm×20mm areas 3cm away from the center of the plate using a sterilized toothpick. After incubation at 28℃ for 2 days, a 6mm diameter tobacco phytophthora cake was taken and inoculated into the center of the plate. The plate without actinomycete inoculation was used as the control. When the colony of the control group grew to 7-8cm, the colony diameter was measured by the cross method, and the inhibition rate was calculated. Each treatment was repeated 3 times.

[0054] The method for preparing the sterile fermentation broth for rescreening of antagonistic endophytic actinomycetes is as follows: 5 test actinomycete cakes with a diameter of 6 mm are taken, placed in a conical flask containing 200 mL of LB culture medium, and cultured in a constant temperature shaking incubator at 28°C and 150 r / min for 5 days to obtain the strain fermentation broth. A small amount of the fermentation broth is taken, centrifuged at 4722×g for 10 minutes, and the supernatant is filtered through a bacterial filter with a pore size of 0.22μm to remove the residual bacteria to obtain the sterile fermentation broth.

[0055] The mycelial growth rate method was used for the rescreening of antagonistic endophytic actinomycetes. The sterile fermentation liquid of the actinomycetes with an initial antibacterial rate of 60% was thoroughly mixed with PDA medium (about 40°C) at a ratio of 1:9, and poured into a culture dish to make a medium plate containing 10% sterile fermentation liquid. A 6mm diameter tobacco blight cake was taken and inoculated in the center of the plate. The plate without the addition of actinomycete sterile fermentation liquid was used as the control. When the colony of the control group grew to 7-8cm, the colony diameter was measured by the cross method, the antibacterial rate was calculated, and its antibacterial ability was evaluated. Each treatment was repeated 3 times.

[0056] The calculation formula of the antibacterial rate is:

[0057]

[0058] The results show:

[0059] After screening and identification, a strain of Streptomyces was found to have a significant antagonistic effect on tobacco phytophthora and a plant growth promoting effect. It was named C1. Strain C1 was isolated from sample materials during the tobacco harvest period, and its isolation site was located in the root tissue of the tobacco plant.

[0060] The 250 isolated endophytic actinomycetes were screened, and the results showed that strain C1 exhibited significant antagonism to tobacco phytophthora. Figure 1 As shown, the average antibacterial rate reached 98.53%.

[0061] The antagonistic activity of the fermentation liquid of 64 primary screened strains against Phytophthora nicotianae was determined, and the rescreening results showed that the fermentation liquid of strain C1 had the best antagonistic effect against Phytophthora nicotianae. Figure 2 As shown, the average diameter of the treated tobacco phytophthora is 6.00 mm, which indicates that tobacco phytophthora cannot grow at all on the 10% C1 fermentation liquid medium, and the inhibition rate of strain C1 on tobacco phytophthora reaches 100%.

[0062] Example 3

[0063] This example is the identification of strain C1, and the specific implementation operations are as follows:

[0064] Gram staining and morphological observation were performed on the screened strain C1, and the physiological and biochemical characteristics of the strain were determined with reference to the "Common Bacteria Identification Manual". The DNA of strain C1 was extracted using a bacterial genomic DNA rapid extraction kit, and their taxonomic status was determined using a multi-gene molecular biology technique based on 16S rDNA and gyrB genes. The primer information is shown in Table 1, the sequences of 16S rDNA primers are shown in SEQ ID NO: 1-2, and the sequences of gyrB primers are shown in SEQ ID NO: 3-4. The PCR amplification reaction system is: 2 μL DNA template; 1 μL of upstream and downstream primers; 12.5 μL of Taq PCR Mix premixed enzyme; ddH2O is supplemented to 25 μL. The PCR amplification program is: 95℃ pre-denaturation for 3min; 94℃ denaturation for 1min; primer specific temperature annealing for 30s; 72℃ extension for 90s; 30 cycles; 72℃ extension for 10min. The amplified product was detected by 1.5% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were submitted to the NCBI database for comparison and analysis, and ACOPTools and MEGA 7.0 software were used for sequence assembly and construction of a multi-gene phylogenetic tree.

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

[0066]

[0067] The results showed that the Gram staining results showed that strain C1 was a Gram-positive bacterium, and its colonies on Gao's medium No. 1 were dry and opaque. Figure 3 As shown, the front of the colony is silvery white ( Figure 3 B part), the back of the lotus seed is white ( Figure 3 The surface is rough with concentric circle patterns, the colony edges are neat and not easy to be picked up, the colonies are not easy to spread out, the hyphae in the base are well developed, the aerial hyphae are less, the spore hyphae are upright, and the spores are oblong.

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

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

[0070]

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

[0072] The 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 in the NCBI database, and a multi-gene phylogenetic tree was constructed based on the 16S rDNA and gyrB genes. Figure 4 As shown, strain C1 has the highest homology with Streptomyces sp.NA03103 and is located on the same branch in the phylogenetic tree. Based on the above analysis results, strain C1 was identified as Streptomyces sp.

[0073] Example 4

[0074] This example is the application of strain C1 in preventing and treating tobacco black shank disease, and the specific implementation operation is as follows:

[0075] Effect of strains on control of tobacco black shank in greenhouse

[0076] The experiment was conducted in the greenhouse of the Integrated Control Laboratory of Soil-borne Diseases of Crops, College of Horticulture and Plant Protection, Kaiyuan Campus, Henan University of Science and Technology (34°35′51″N, 112°25′08″E). The greenhouse conditions were controlled at 28°C, 65% humidity, and 14h / day of light. The tobacco variety tested was LY1306. Sterile substrate and sterile seeds were used to cultivate sterile tobacco seedlings. After the sterile tobacco seedlings grew to the cross stage, they were transplanted into nutrient pots (upper diameter × lower diameter × height = 7.3cm × 5.2cm × 7.7cm) to slow down the seedlings for subsequent experiments. The greenhouse control efficacy test used 1×10 4 CFU / mL tobacco Phytophthora zoospore suspension was inoculated by root irrigation to cause disease. First, each tobacco plant was inoculated with 10mL of tobacco Phytophthora zoospore suspension by root irrigation. After 4 days, each tobacco plant was inoculated with 10mL of strain C1 fermentation liquid. The strain C1 fermentation liquid was used for root irrigation twice, and once every 7 days. Sterile water treatment was used as a blank control, and commercially available Bacillus subtilis microcapsule granules (CG) were used as a control. The dosage was converted according to the product instructions and applied. Each treatment group had 15 tobacco plants, and the treatment was repeated 3 times. The water and fertilizer management was kept consistent during the period. The incidence of tobacco black shank was investigated and counted until the 21st day. The incidence rate and disease index were calculated according to the "Grading and Investigation Methods of Tobacco Pests and Diseases" (GB / T 23222-2008), and the greenhouse control effect of strain C1 on tobacco black shank was evaluated.

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

[0078] The results showed that the root irrigation treatment with the fermentation liquid of strain C1 could achieve a good disease prevention and control effect, as shown in Table 3 and Figure 5 As shown, the incidence of tobacco black shank in the strain C1 treatment group was only 4.44%, far lower than the 88.89% of the control group and significantly lower than the 20.00% of the Bacillus subtilis group; at the same time, its disease index was greatly reduced to 1.48, significantly better than the 61.23 of the control group and 15.56 of the Bacillus subtilis group. The greenhouse control effect of strain C1 on tobacco black shank reached 95.05%, and the disease index control effect reached 97.41%, which was significantly higher than the Bacillus subtilis group. These data show that compared with commercially available microbial agents, strain C1 can significantly reduce the infection and harm of tobacco black shank in greenhouse experiments.

[0079] Table 3 Control effect of strain C1 on tobacco black shank in greenhouse

[0080]

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

[0082] Example 5

[0083] This example is the application of strain C1 in preventing and treating tobacco black shank disease, and the specific implementation operation is as follows:

[0084] Field control effect of strains on tobacco black shank

[0085] The field prevention efficacy test adopted natural disease occurrence in the field. The experiment was designed with 3 treatment groups, each treatment was repeated 3 times, and there were 9 plots in total. The plots were arranged in random blocks. The row spacing and plant spacing of tobacco plants in the plots were 110 cm and 55 cm, respectively. Protection rows were set up around the plots. There were 100 tobacco plants in each plot, and 4 rows of tobacco were planted. During the period, planting measures required by local tobacco production technology were adopted, and water and fertilizer management was kept consistent. After the tobacco seedlings were transplanted and the seedlings were hardened, each tobacco plant in the C1 treatment group was inoculated with 10 mL of the fermentation liquid of strain C1 for a total of 2 root irrigations, with 1 irrigation once every 7 days. The sterile water treatment was used as the blank control group, and the commercially available Bacillus subtilis microcapsule granules (CG) were used as the drug control group. The fertilizer was applied after conversion according to the product instructions. The water and fertilizer management was kept consistent during the period. The incidence of tobacco black shank was investigated and counted until the tobacco plants reached their peak growth period. The incidence rate and disease index were calculated according to the "Grading and Investigation Methods of Tobacco Pests and Diseases" (GB / T23222-2008), and the field control effect of strain C1 on tobacco black shank was evaluated.

[0086] The results showed that the root irrigation treatment with the fermentation liquid of strain C1 could achieve 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 effect reached 96.47%, which was significantly higher than that of the Bacillus subtilis group. These data showed that compared with commercially available microbial agents, strain C1 could significantly reduce the occurrence and development of tobacco black shank when applied in the field.

[0087] Table 4 Field control effect of strain C1 on tobacco black shank

[0088]

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

[0090] Example 6

[0091] This example is the application of strain C1 in promoting tobacco growth, and the specific implementation operations are as follows:

[0092] Effects of strains on tobacco seed germination and its plant growth promotion effect

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

[0094] Healthy and full seeds of the same variety (LY1306) as the tested tobacco plants were selected. After surface disinfection and seedling induction treatment, the tobacco seeds were soaked in the fermentation liquid of strain C1 for 24 hours. After soaking, the seeds were taken out and the residual liquid was washed with sterile water, and the surface moisture was dried with sterile filter paper. In the control group, the tobacco seeds were soaked in an equal amount of sterile water instead of the fermentation liquid of strain C1 for 24 hours, and then the surface moisture was dried with sterile filter paper.

[0095] The seed germination test was carried out in a culture dish covered with moist filter paper. Twenty-five treated tobacco seeds were placed in each culture dish and cultured in a constant temperature incubator at 28°C with alternating light and dark.

[0096] From the time the seeds were placed in the culture dish, the germination of the seeds was observed and recorded on the 14th day. The germination standard was when the radicle broke through the seed coat and reached half the length of the seed. The number of germinated seeds was recorded and the seed germination rate was calculated.

[0097] The germination rate calculation formula is: germination rate = (number of germinated seeds / total number of test seeds) × 100%.

[0098] (2) Ability of strains to produce IAA

[0099] The IAA standard curve was drawn using the absorbance of the mixture of IAA standard solution and Salkowski colorimetric solution at a wavelength of 530 nm. The standard curve was obtained with IAA mass concentration as the horizontal axis and absorbance as the vertical axis: y = 0.0034x-0.0018R 2 =0.9990.

[0100] Qualitative detection of IAA: 2 mL of strain C1 fermentation broth was inoculated into a new conical flask containing 200 mL of LB culture medium, and 500 μg mL -1 2 mL of tryptophan solution was cultured at 28°C and 180 r / min for 3 days, and the culture was centrifuged at 7378 × g for 10 min. 0.1 mL of the supernatant was taken and mixed with an equal amount of Salkowski colorimetric solution in a 0.2 mL eight-row PCR tube for color development, and 50 μg mL -1 The indoleacetic acid standard solution was used as the positive control, and the LB culture medium was used as the 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 amount of Salkowski colorimetric solution in a quartz cuvette. Incubate it in the dark for 30 minutes and then measure the absorbance of the mixture at a wavelength of 530 nm. Do the same with LB culture medium for zero adjustment. Repeat each treatment 3 times.

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

[0103] The strain C1 was inoculated by root irrigation with fermentation liquid. In greenhouse potted plants, cross-stage tobacco plants with consistent growth were selected and root irrigation was performed with 10 mL of strain C1 fermentation liquid. The roots were irrigated twice, once every 7 days, and sterile water treatment was used as the control. There were 15 tobacco plants in each treatment group, and each treatment was repeated 3 times. 21 days after inoculation, the agronomic traits of tobacco plants were measured according to the "Methods for Survey and Measurement of Agronomic Traits of Tobacco" (YC / T142-2010) to evaluate the greenhouse growth promotion effect of the strain. Agronomic trait indicators include leaf length, leaf width, stem girth, root-crown ratio, chlorophyll content, and leaf nitrogen content.

[0104] (4) Field growth promotion effect determination

[0105] In the field test, after the tobacco seedlings were transplanted and the seedlings were slowed down, each tobacco plant was irrigated with 10 mL of the fermentation liquid of strain C1 for a total of 2 times, 7 days apart, and 1 time for irrigating the roots. The sterile water treatment was used as the control. The agronomic traits of tobacco plants were measured at the clustering stage and the vigorous growth stage according to the "Methods for Surveying and Measuring Agronomic Traits of Tobacco" (YC / T142-2010) to evaluate the field growth promotion effect of the strain. Agronomic trait indicators include leaf length, leaf width, plant height, stem girth, chlorophyll content and leaf nitrogen content.

[0106] The results show:

[0107] (1) Effect 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 on the 14th day was 81.00±3.83%, and the germination rate of tobacco seeds soaked in the fermentation liquid of strain C1 was 94.00±2.31%. Figure 6 As shown, the radicle of the tobacco seedlings in the treatment group after germination grew faster and was thicker than that in the control group.

[0109] Table 5 Effect of actinomycete fermentation liquid on tobacco seed germination

[0110]

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

[0112] (2) IAA qualitative and quantitative detection

[0113] like Figure 7 As shown, from left to right are water and colorimetric solution mixture, 50μg·mL -1 The mixture of IAA standard solution and colorimetric solution and the mixture of strain C1 fermentation solution and colorimetric solution were repeated three times. LB culture medium was used as negative control. Under the same color reaction and incubation conditions, no phenomenon of color deepening to pink was observed, indicating that LB culture medium itself would not produce a color reaction similar to IAA under this detection condition, thus eliminating the interference of the culture medium itself on the detection results. -1 The indoleacetic acid standard solution was used as a positive control. After a color reaction with the Salkowski colorimetric solution and incubation for 30 minutes in the dark, a pink color was observed. This indicates that the positive control system can develop color normally and provides an accurate reference standard for the subsequent detection of the test strains.

[0114] For strain C1, 2 mL of the test strain fermentation broth was inoculated into a new conical flask containing 200 mL of LB culture medium, and 500 μg mL -1 2 mL of tryptophan solution was subjected to specific conditions (28°C, 180 r·min -1 After shaking culture for 3 days, 0.1 mL of the supernatant was taken out after centrifugation and treated with an equal amount of Salkowski colorimetric solution (for color development). It was observed that the color of the culture medium deepened, indicating that the strain C1 was able to produce IAA.

[0115] Take 2 mL of the above supernatant and mix it with an equal amount of Salkowski colorimetric solution in a quartz cuvette. After incubation for 30 minutes in the dark, measure the absorbance of the mixture at a wavelength of 530 nm. Do the same treatment with LB culture medium for zero adjustment. Repeat each treatment 3 times. Substitute the measured absorbance value into the standard curve equation to calculate the IAA production of strain C1 to be 16.85 μg mL -1 .

[0116] (3) Greenhouse growth promotion

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

[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 by LSD test.

[0121] (4) Field growth promotion effect

[0122] The results of the field test are shown in Tables 7 and 8. The effects of strain C1 on leaf length, leaf width, stem girth and plant height of tobacco plants in the clustering stage and vigorous growth stage were consistent with those in the greenhouse test, and showed significant promotion effects compared with the control group. It significantly promoted the chlorophyll content and nitrogen content of tobacco leaves in the clustering stage, but this situation changed in the vigorous growth stage, and the chlorophyll content and nitrogen content of the leaves decreased. This shows 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 cluster stage in the field

[0124]

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

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

[0127]

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

[0129] Example 7

[0130] This example is the application of strain C1 in tobacco planting, and the specific implementation operations 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 was performed with specific primers to study the colonization of strain C1 in tobacco plants under greenhouse and field conditions.

[0133] Strain C1 was inoculated by root irrigation with fermentation liquid. In greenhouse potted plants, cross-stage tobacco plants with consistent growth were selected and root irrigation was performed with 10 mL of strain C1 fermentation liquid. The roots were irrigated twice, once every 7 days, with sterile water treatment as the control. There were 15 tobacco plants in each treatment group, and each treatment was repeated 3 times. Colonization was observed 21 days after inoculation. In the field test, after the tobacco seedlings were transplanted and the seedlings were slowed down, each tobacco plant was rooted with 10 mL of strain C1 fermentation liquid. The roots were irrigated twice, once every 7 days, with sterile water treatment as the control. After the tobacco plants grew to the cluster stage, samples were collected in the field to observe colonization.

[0134] Rinse the surface of the tobacco plant sample with water, dry it naturally, and randomly cut 8g of tissue blocks from the leaves, stems and roots of the tobacco plant. After disinfecting the surface of the tissue block, mix it with an appropriate amount of quartz sand and add 5mL of sterile water. Grind it thoroughly in a sterile mortar to obtain a tissue grinding liquid. Take part of the tissue liquid and use a bacterial DNA extraction kit to extract the tissue liquid DNA. The dilution plate method was used to re-isolate strain C1. The above tissue grinding liquid was diluted to 100 times with sterile water, and 100μL was evenly applied to the Gao's No. 1 medium plate using a spreader. Inverted culture was carried out in a constant temperature incubator at 28℃ for 7 days. After the actinomycete colonies appeared, different colonies were picked and propagated on a new Gao's No. 1 medium, and the bacterial DNA extraction kit was used to extract the colony DNA.

[0135] Based on the CHAT domain protein gene of strain C1, the specific primer C1T was 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 is: 2 μL DNA template; 1 μL of upstream and downstream primers; 12.5 μL of Taq PCR Mix premixed enzyme; ddH2O is added to 25 μL. The PCR amplification program is: 95℃ pre-denaturation for 3min; 94℃ denaturation for 1min; 55℃ annealing for 30s; 72℃ extension for 90s; 30 cycles; 72℃ extension for 10min, and the fragment length is 1245bp.

[0136] The specific primer C1T was used to amplify the DNA in the tissue fluid by PCR, and the amplified product was detected by 1.5% agarose gel electrophoresis. The colonization of strain C1 in the tobacco plant was determined by the presence or absence of the target fragment. The specific primer C1T was used to amplify the DNA of the re-isolated colonies by PCR. 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 test are as follows Figure 8 As shown: M is DM2000 DNA Marker; 1 is a positive control; 2-4 are negative controls of roots, stems and leaves of tobacco plants that were not inoculated with the strain; 5-7 are specific amplifications of roots, stems and leaves of tobacco plants that were inoculated with the strain. After PCR amplification of the leaf tissue fluid DNA of tobacco plants that were treated with strain C1, 1.5% agarose gel electrophoresis showed that the target fragment of 1245bp was not amplified in the leaf tissue samples of tobacco plants, indicating that strain C1 did not colonize in the leaves of tobacco plants; the PCR amplification results of stem tissue fluid DNA showed that the target fragment was amplified in the stem samples of tobacco plants, indicating that strain C1 colonized in the stems of tobacco plants; PCR detection of root tissue fluid DNA showed that the target fragment was amplified in the root samples of tobacco plants, proving that strain C1 colonized in the roots of tobacco plants. In the control group of tobacco plants treated with sterile water, no target fragment of 1245 bp appeared in the leaf, stem and root tissue fluid DNA after PCR amplification, and the target strain C1 was not detected in the re-isolated colonies, indicating that the strain C1 did not exist in the tobacco plants without inoculation.

[0138] The results are shown in Table 9. By counting the number of re-isolated colonies: the colonies obtained by diluting the grinding liquid of the root, stem and leaf tissues of the tobacco plants and then applying them on the plates and re-isolating them, and confirming the target strain by PCR amplification with specific primer C1T, it was calculated that the colony content of the tobacco root tissue was 2812.50 CFU / g, indicating that the strain C1 could colonize in the roots of the tobacco plants; the colony content of the stem tissue was 5312.50 CFU / g, indicating that the strain C1 could colonize in the stems of the tobacco plants; and the target strain C1 was not isolated from the leaf tissue, indicating that the strain C1 failed to colonize in the leaves of the tobacco plants.

[0139] In the greenhouse experiment, compared with the roots of tobacco plants, the colonization of strain C1 in the stems was relatively more active or more conducive to its growth and reproduction.

[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 by LSD test.

[0143] The results of the field trials are as follows Fig. 9 As shown, M is DM2000 DNA Marker; 1 is a positive control; 2-4 are negative controls of roots, stems and leaves of tobacco plants that were not inoculated with the strain; 5-7 are specific amplifications of roots, stems and leaves of tobacco plants inoculated with the strain. After PCR amplification of the leaf tissue fluid DNA of tobacco plants treated with strain C1, 1.5% agarose gel electrophoresis showed that the leaf tissue samples of tobacco plants amplified a target fragment of 1245bp, indicating that strain C1 colonized in the leaves of tobacco plants; the PCR amplification results of stem tissue fluid DNA showed that the stem samples of tobacco plants amplified the target fragment, indicating that strain C1 colonized in the stems of tobacco plants; PCR detection of root tissue fluid DNA showed that the root samples of tobacco plants amplified the target fragment, proving that strain C1 colonized in the roots of tobacco plants. In the control group of tobacco plants treated with sterile water, no target fragment of 1245 bp appeared in the leaf, stem and root tissue fluid DNA after PCR amplification, and the target strain C1 was not detected in the re-isolated colonies, indicating that the strain C1 did not exist in the tobacco plants without inoculation.

[0144] By counting the number of re-isolated colonies, the results are shown in Table 10: the colonies obtained by diluting the grinding liquid of the root, stem and leaf tissues of the tobacco plants and then applying them on the plates and re-isolating them, after PCR amplification with specific primers C1T to confirm the target strain, the colony content of the root tissue of the tobacco plants was calculated to be 5416.67±1094.49 CFU / g, indicating that the strain C1 can colonize in the roots of the tobacco plants; the colony content of the stem tissue was 4479.17±1076.50 CFU / g, indicating that the strain C1 can colonize in the stems of the tobacco plants; the colony content of the leaf tissue was 1041.67±1513.83 CFU / g, which indicates that the strain C1 can colonize in the leaves of the tobacco plants.

[0145] In the field test, the colonization of strain C1 in the roots, stems and leaves of tobacco plants showed a downward trend, and the 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 tobacco plants in the field

[0147]

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

[0149] Example 8

[0150] This example is the application of strain C1 in promoting tobacco growth, and the specific implementation operations are as follows:

[0151] Effects of strain C1 on tobacco leaf yield

[0152] After the tobacco seedlings were transplanted and the seedlings were slowed down, each tobacco plant was treated with 10 mL of the fermentation liquid of strain C1 for root irrigation, and the roots were irrigated twice, with an interval of 7 days. The sterile water treatment was used as a blank control, and the commercially available Bacillus subtilis microcapsule granules (CG) were used as a control. The tobacco leaves were harvested during the tobacco harvest period. Five tobacco plants with uniform growth were randomly selected by the five-point sampling method for each treatment, and 15 plants were picked for each treatment. After returning to the laboratory, each tobacco leaf was tied with a label, and the fresh weight of the leaves was immediately weighed and recorded. After that, the tobacco leaves were withered at 105°C for 30 minutes, dried at 65°C to constant weight, and their dry weight was measured. The dry weight of each tobacco leaf was weighed, and the tobacco leaf yield was calculated. The experiment was repeated 3 times.

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

[0154] Table 11 Effect of strain C1 on tobacco leaf yield

[0155]

[0156] Note: Different lowercase letters in the same column indicate significant differences at P < 0.05 by 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 strain C1 treatment group was significantly increased to 31981.25 kg / ha. The fresh weight was also increased compared with the Bacillus subtilis group, and the difference was significant. This shows that the strain 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, and the dry weight of the strain C1 treatment group was 3449.56 kg / ha. The dry weight of tobacco leaves in the strain C1 treatment group was significantly higher than that in the control group, and was improved to a certain extent compared with the Bacillus subtilis group, but there was no significant difference, which shows that strain C1 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, strain C1 has a significant effect on improving tobacco leaf yield in terms of both fresh weight and dry weight, and the effect is slightly better than that of commercially available microbial agents.

Claims

1. A strain of Streptomyces, characterized in that Named Streptomyces ( Streptomyces sp.) strain C1, with the deposit number being CGMCC NO.32161.

2. Use of the Streptomyces strain C1 as claimed in claim 1 in tobacco planting.

3. The use according to claim 2, characterized in that: The Streptomyces strain C1 is used to prevent and control tobacco black shank disease and / or promote tobacco growth.

4. The use according to claim 3, characterized in that: The application method is to use the fermentation liquid of the Streptomyces strain C1 for root irrigation.

5. The use according to claim 4, characterized in that: Take C1 bacterial cake and ferment it in LB culture medium until the effective viable count is not less than 1×10 8 CFU / mL of fermentation broth.

6. The use according to claim 5, characterized in that: The dosage of the fermentation liquid is 10-15 mL / time, and the roots are irrigated twice in total, with an interval of 5-7 days before irrigating the roots once.

7. The use according to claim 2, characterized in that: The fermentation liquid of the Streptomyces strain C1 is used to promote seed germination.

8. The use according to claim 7, characterized in that: The fermentation liquid is used to soak tobacco seeds for 18-24 hours.

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