Streptomyces isothiocyanate strain YC3071 and application thereof in prevention and treatment of tobacco diseases

Abstract

The invention provides a streptomyces isothiocyanate strain YC3071 and application of the streptomyces isothiocyanate strain YC3071 in tobacco disease prevention and control, and belongs to the technical field of biological prevention and control. The preservation name of the streptomyces isothiocyanate strain YC3071 is Streptomyces althioticus YC3071, the Latin name is Streptomyces althioticus, and the streptomyces isothiocyanate strain YC3071 is preserved in Guangdong Microbiological Culture Collection Center, and the preservation address is Institute of Microbiology, Guangdong Academy of Sciences, Floor 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou; the preservation date is July 4, 2024; the preservation number of the strain is GDMCC NO: 64829. The YC3071 strain has the characteristics of low use cost, good control effect, no residue and safety to human, livestock and environment, and the control effect of the microbial agent prepared from the YC3071 strain on tobacco black shank and tobacco brown spot is more than 70%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological control, in particular to a streptomyces ydostratus strain YC3071 and its application in tobacco disease control. BACKGROUND

[0002] Tobacco black shank caused by infection of Phytophthora nicotianae is one of the most serious diseases in tobacco planting (Ma Guosheng et al., 2001). The average incidence of tobacco black shank is 10%-15%, and the incidence of high incidence of tobacco black shank can reach 75%, and the incidence of serious tobacco black shank can even cause absolute yield. Tobacco brown spot is a kind of air-borne tobacco leaf disease caused by Alternaria alternata, which has the characteristics of short incubation period, easy prevalence and large damage area, mainly harms mature tobacco leaves, and seriously affects tobacco yield and quality (Zhang Shijie et al., 2024). At present, the prevention and control of tobacco black shank and brown spot mainly relies on chemical pesticides, which can easily cause environmental pollution, pathogen resistance and drug resistance and other problems. The biocontrol products developed by microorganisms have the advantages of environmental protection, safety and superimposed effect of prevention and control, and are favored by domestic and foreign people. Studies have shown that many microorganisms can antagonize the two pathogens and have great application value. Streptomyces is the main group of actinomycetes, which can produce various active metabolites antagonizing pathogenic fungi, and is an important microbial resource for developing biological pesticides. Streptomyces ydostratus is one of the members of Streptomyces, which is reported to produce industrial enzymes such as xylanase (Luo et al., 2016), but the research of the actinomycete in plant disease control is rarely reported. Phytophthora nicotianae Alternaria alternata Streptomyces S. althioticus SUMMARY

[0003] The purpose of the present application is to provide a streptomyces ydostratus strain YC3071 and its application in tobacco disease control, which has the characteristics of low cost, good effect, no residue, safety to human and environment, and the effect of the bacterial agent prepared by the YC3071 strain on tobacco black shank and tobacco brown spot is more than 70%.

[0004] The technical scheme of the present application is as follows: The present application provides a streptomyces ydostratus strain YC3071, the preservation name of the streptomyces ydostratus strain YC3071 is Streptomyces althioticus The YC3071 strain, the Latin name is Streptomyces althioticus, preserved in Guangdong Microbial Culture Collection Center, the preservation address is No. 59 Building, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100 Xianlie Road, Guangzhou; the preservation date is July 4, 2024; and the preservation number is GDMCC NO: 64829.

[0005] ​​​​The present invention further protects an isothiostreptomyces agent containing the above-mentioned isothiostreptomyces strain YC3071.

[0006] As a further improvement of the present invention, the effective viable bacteria count in the bacterial agent is above 100 million CFU / g.

[0007] As a further improvement of the present invention, it also contains lysate of Streptomyces isothioides strain YC3071 and fermentation metabolites of the strain.

[0008] This invention further protects the application of the above-mentioned Streptomyces isothioides strain YC3071 in the prevention and control of tobacco diseases.

[0009] As a further improvement of the present invention, the tobacco diseases include tobacco black shank disease and tobacco red star disease.

[0010] As a further improvement of the present invention, the tobacco black shank disease is caused by Phytophthora tobaccoii. Phytophthora nicotianae Caused by infection.

[0011] As a further improvement of the present invention, the tobacco red spot disease is caused by Alternaria. Alternaria alternata Caused by infection.

[0012] The method for preparing *Streptomyces isothioides* culture and its inoculum preparation according to the present invention includes the following steps: (1) Steriloma isothioides ( S. althioticus The YC3071 strain was inoculated onto a slant of solid culture medium in a test tube using conventional ISP4 medium and cultured at 28-37℃ for 4-8 days to obtain the slant culture. (2) Dispense ISP4 liquid culture medium into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, use an inoculation needle to pick up YC3071 cells from the slant of the test tube in a clean bench and inoculate them into the culture medium. After culturing at 24-37℃ and 120-180 rpm for 7-10 days, the fermentation culture of strain YC3071 is obtained. (3) The fermentation culture of strain YC3071 is concentrated to 1 / 5 of its original volume by rotary evaporation at 50°C. Then it is mixed evenly with an equal volume of diatomaceous earth and dried at 50°C-60°C until the moisture content is 10%-15%. Finally, it is pulverized to a particle size of less than 500 mesh. The effective live bacteria count of strain YC3071 in the YC3071 agent prepared by this method is more than 100 million CFU / g.

[0013] The present invention has the following beneficial effects: The inventors isolated an actinomycete strain YC3071 from the rhizosphere soil of tobacco in Fuquan City, Guizhou Province. Based on morphological, physiological, biochemical, and molecular biological characteristics, the actinomycete was identified as *Streptomyces isothioides*. S. althioticus Tests revealed that strain YC3071 exhibits strong antagonistic activity against the mycelial growth of the pathogens causing tobacco black shank and red spot disease. Microbial agents prepared using this strain demonstrate good control efficacy against both diseases. A literature search of domestic and international sources indicates that *Streptomyces isothioides* (…) S. althioticus No reports have been found on the biocontrol activity and application of this invention against tobacco black shank and red star disease, which highlights the innovation and potential application value of this invention.

[0014] (1) The YC3071 strain of the present invention is easy to ferment and culture. Its culture is resistant to both Phytophthora indicum and Alternaria alternata, and can be used as a fungicide to prevent and control tobacco black shank disease and red star disease.

[0015] (2) The YC3071 strain of the present invention has the advantages of high efficiency, low toxicity and low residue in the prevention and control of tobacco black shank disease and red star disease. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Based on the bacterial 16S rDNA sequence and a phylogenetic tree of strain YC3071 and its closely related species constructed using MEGA software, the taxonomic position of strain YC3071 in this invention is *Streptomyces isothioides*. S. althioticus ).

[0018] Figure 2 Morphological characteristics of strain YC3071 and its antagonistic effect on the mycelial growth of *Phytophthora indicum* and *Alternaria alternata*. A: Colony morphology of strain YC3071 cultured on ISP4 medium at 28ºC for 10 days; B: Morphology of spores and spore chains of strain YC3071; C: Antagonistic effect of strain YC3071 on the mycelial growth of *Phytophthora indicum*; D: Colonies of *Phytophthora indicum* on control plate; E: Antagonistic effect of strain YC3071 on the mycelial growth of *Alternaria alternata*; F: Colonies of *Alternaria alternata* on control plate. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The preservation name of the *Streptomyces isothioides* strain YC3071 described in this invention is: Streptomyces althioticus strain YC3071, Latin name is Streptomyces althioticus, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 4, 2024; accession number GDMCC NO:64829.

[0021] Example 1

[0022] The actinomycete of this invention is *Streptomyces isothioides* ( S. althioticus Strain YC3071. When strain YC3071 was cultured on ISP4 medium (soluble starch 10 g / L, peptone 1 g / L, yeast extract 0.5 g / L, (NH4)2SO4 2 g / L, MgSO4 1 g / L, K2HPO4 1 g / L, NaCl 1 g / L, CaCO3 2 g / L, agar 18 g / L, pH 7.2) at 28ºC for 10 days, the colonies were grayish-white and dry, exhibiting typical actinomycete colony characteristics. Figure 2 A); Spores are columnar and grow in chains on the hyphae ( Figure 2 B); combined with morphological, physiological and biochemical characteristics and phylogenetic analysis of the 16S rDNA sequence of strain YC3071 ( Figure 1 This indicates that the strain is *Streptomyces isothioides*. S. althioticus ).

[0023] Figure 1 The isothiostreptomyces of the present invention ( S. althioticus Phylogenetic analysis of strain YC3071, based on 16S rDNA sequence analysis, shows that strain YC3071 is *Streptomyces isothioides*. S. althioticus ).

[0024] This invention utilizes ISP4 liquid medium to culture strain YC3071 using conventional microbial fermentation methods at 28°C and 180 rpm for 10 days. The resulting culture exhibits resistance to *Phytophthora tobaccois* (…). Figure 2 C) and Alternaria ( Figure 2 E) Exhibits good antagonistic activity. Application of the YC3071 strain culture of the present invention in the preparation of fungicides for the control of tobacco black shank and red spot disease.

[0025] Figure 2 The images show the morphological characteristics of strain YC3071 in this invention and its antagonistic effect on the mycelial growth of *Phytophthora indicum* and *Alternaria alternata*. A: Colony morphology of strain YC3071 cultured on ISP4 medium at 28ºC for 10 days; B: Morphology of spores and spore chains of strain YC3071; C: Antagonistic effect of strain YC3071 on the mycelial growth of *Phytophthora indicum*; D: Colonies of *Phytophthora indicum* on a control plate; E: Antagonistic effect of strain YC3071 on the mycelial growth of *Alternaria alternata*; F: Colonies of *Alternaria alternata* on a control plate.

[0026] Example 2

[0027] 1. Fermentation of strain YC3071 and its inoculum production method The YC3071 strain was inoculated onto a slant of solid culture medium in test tubes using standard ISP4 medium. The culture was incubated at 28-37℃ for 4-8 days to obtain the slant culture. ISP4 liquid medium was dispensed into 500 mL Erlenmeyer flasks (300 mL per flask). After sterilization using standard microbiological methods, YC3071 cells were picked from the slant using an inoculation needle and inoculated into the medium in a clean bench. The culture was incubated at 24-37℃ and 120-180 rpm for 7-10 days to obtain the fermentation culture of the YC3071 strain. The fermentation culture of strain YC3071 was concentrated to 1 / 5 of its original volume by rotary evaporation at 50°C. Then, it was mixed evenly with an equal volume of diatomaceous earth and dried at 50°C-60°C until the moisture content was 10%-15%. Finally, it was pulverized to a particle size of less than 500 mesh. The effective viable count of strain YC3071 in the YC3071 inoculum prepared by this method is more than 100 million CFU / g.

[0028] 2. Method for determining the ability of strain YC3071 to inhibit the mycelial growth of Phytophthora tobaccois and Alternaria alterniflora. (1) Preparation of fermentation culture of strain YC3071: Dispense ISP4 liquid medium into 500 ml Erlenmeyer flasks, with a liquid volume of 300 ml / flask. After sterilization according to conventional microbial methods, use an inoculation needle to pick up YC3071 cells from the slant of the test tube in a clean bench and inoculate them into the culture medium. After culturing at 24-37℃ and 120-180 rpm for 7-10 days, the fermentation culture of strain YC3071 is obtained.

[0029] (2) Plate antagonism test: PDA medium plates were prepared according to conventional methods. The formula for PDA medium was: 200 g of peeled potato fragments, boiled for 30 minutes, filtered, and the filtrate was added to 20 g of glucose and 20 g of agar, and then diluted with water to 1000 ml. Figure 2As shown in CF, pathogenic mycelial blocks were inoculated in the center of PDA plates. 100 μL of YC3071 strain fermentation culture was then inoculated in a triangular pattern at a distance of 2 cm from the edge of the pathogenic mycelial blocks. An equal volume of ISP4 liquid medium was used as a control instead of the YC3071 strain fermentation culture. The plates were incubated at 28°C. When the colonies on the control plates fully covered the plates, the colony radius of each treatment was measured, and the inhibition rate was calculated using the following formula.

[0030] Inhibition rate (%) = (Control colony radius - Treated colony radius) / Control colony radius × 100% 3. Application method of YC3071 microbial agent for the prevention and control of tobacco black shank disease When using YC3071 inoculant to control tobacco black shank disease, dilute the inoculant with water 100-200 times before applying it to the tobacco plant. The application period is during the tobacco transplanting period and the vigorous growth period. The dosage for each application is 200 ml per plant, and the application method is root drenching.

[0031] 4. Application method of YC3071 inoculant for the prevention and control of tobacco red spot disease When using YC3071 inoculant to control tobacco red spot disease, dilute the inoculant with water 100-200 times before applying it to the tobacco red spot disease. The application period is 3-5 days before the initial outbreak of tobacco red spot disease in the local area and during the initial outbreak period. The dosage for each application is 50 ml / plant, and the application method is to spray it on both sides of the tobacco leaves.

[0032] 5. Determination method for the efficacy of YC3071 microbial agent against tobacco black shank disease The greenhouse pot experiment was conducted as follows: 2 kg of sterilized soil was placed in each pot, and one tobacco seedling (variety: Yun 87, Guiyan 20, Guiyan 36) was transplanted into each pot. Immediately after transplanting, the seedlings were treated with YC3071 inoculant prepared according to the above method (effective viable count = 1.5 × 10⁻⁶). 8 (CFU / g) was diluted 100, 200, and 300 times with water, and applied as a root drench once at transplanting, 200 ml / plant. Oospore suspensions of *Phytophthora tobaccois*, the pathogen of tobacco black shank, were prepared using conventional methods, with an ospore concentration of 500 ospores / ml. On the 3rd day after transplanting, 10 ml of the ospore suspension was inoculated per tobacco seedling. Forty-five days after transplanting, when the tobacco plants were in their vigorous growth stage, a second root drench was applied, 200 ml / plant. ISP4 liquid culture medium diluted as a control was used instead of the bacterial suspension. Each treatment had 15 replicates, randomly arranged. After 60 days of cultivation in a greenhouse, disease severity was classified, disease index was calculated, and control efficacy was determined according to the methods described in GB / T23222-2008 "Classification and Investigation Methods for Tobacco Diseases and Pests".

[0033] Disease index = (n1×1+ n2×2+ n3×3+ n4×4+ n5×5) / (S×5)×100, where n1-n5 represent the total number of plants at levels 1-5 respectively; and S represents the total number of plants surveyed.

[0034] Efficacy (%) = (1 - treated disease severity / control disease index) × 100% 6. Determination of the efficacy of YC3071 inoculant against tobacco red spot disease The greenhouse pot experiment was conducted as follows: 2 kg of sterilized soil was placed in each pot, and one tobacco seedling (variety: Yun 87, Guiyan 20, Guiyan 36) was transplanted into each pot. Immediately after transplanting, the seedlings were treated with YC3071 inoculant prepared according to the above method (effective viable count = 1.5 × 10⁻⁶). 8 (CFU / g) was diluted 100, 200, and 300 times with water, and sprayed on both sides of tobacco leaves 30 days after transplanting, 50 ml / plant. A conidial suspension of *Alternaria alternata*, the pathogen of tobacco star rot, was prepared using conventional methods, with a spore concentration of 1000 spores / ml. On the third day after spraying the bacterial suspension, 50 ml of the spore suspension was sprayed onto each tobacco plant. Sixty days after transplanting, the bacterial suspension was sprayed again using the same method and dosage. A diluted ISP4 liquid medium was used as a control. Each treatment had 15 replicates, randomly arranged. After 90 days of cultivation in a greenhouse, disease severity was classified, disease index was calculated, and control efficacy was determined according to the methods described in GB / T23222-2008 "Classification and Investigation Methods for Tobacco Diseases and Pests".

[0035] Disease index = (n1×1+ n2×2+ n3×3+ n4×4+ n5×5) / (S×5)×100, where n1-n5 represent the total number of plants at levels 1-5 respectively; and S represents the total number of plants surveyed.

[0036] Efficacy (%) = (1 - treated disease severity / control disease index) × 100% Experimental Example 1: Inhibitory Activity of YC3071 Strains on the Mycelial Growth of Phytophthora tobaccoii and Alternaria alterniflora

[0037] Cultures of strain YC3071 were prepared according to the above method, and the antibacterial activity of the strain YC3071 was determined using the above plate assay method. The inhibition zone was measured 4 days after treatment. The results (Table 1) showed that the culture of strain YC3071 inhibited the mycelial growth of *Phytophthora tobaccos* and *Alternaria alternata* by 39.68% and 72.35%, respectively, demonstrating good antibacterial activity.

[0038] Table 1. Inhibition rate of YC3071 strain culture on the mycelial growth of Phytophthora tobacco and Alternaria alternifolia

[0039] Experimental Example 2: Efficacy of YC3071 microbial agent in the prevention of tobacco black shank Prepare YC3071 bacterial agent (effective viable count = 1.5 × 10⁻⁶) using the method described above. 8 The efficacy was determined using the above-mentioned greenhouse pot experiment method (CFU / g). Among them, YC3071 inoculant was tested with 100-fold, 200-fold and 300-fold dilutions. The application periods were the transplanting period and the vigorous growth period 45 days after transplanting, respectively. The application method was root irrigation, 200 ml / plant each time. ISP4 liquid culture medium dilution was used as a control instead of inoculant solution. The results were investigated 60 days after transplanting.

[0040] The results (Table 2) show that the control efficacy of 100-fold dilution of YC3071 microbial agent against black shank disease in tobacco varieties Yun 87, Guiyan 20, and Guiyan 36 was 76.28%, 79.47%, and 75.92%, respectively; the control efficacy of 200-fold dilution was 71.26%, 74.62%, and 70.38%, respectively; and the control efficacy of 300-fold dilution was below 65%. Therefore, the appropriate dilution ratio for using YC3071 microbial agent to control black shank disease in tobacco is 100-200 times.

[0041] Table 2. Efficacy of YC3071 microbial agent against tobacco black shank.

[0042] Experimental Example 3: Efficacy of YC3071 microbial agent against tobacco scab Prepare YC3071 bacterial agent (effective viable count = 1.5 × 10⁻⁶) using the method described above. 8 The efficacy was determined using the above-mentioned greenhouse pot experiment method (CFU / g). Among them, YC3071 inoculant was tested with 100-fold, 200-fold and 300-fold dilutions. The application time was 30 days and 60 days after transplanting, respectively. The application method was spraying, 50 ml / plant each time. ISP4 liquid culture medium dilution was used as a control instead of inoculant solution. The results were investigated 90 days after transplanting.

[0043] The results (Table 3) show that the control efficacy of 100-fold dilution of YC3071 microbial agent against tobacco scab on Yun 87, Guiyan 20, and Guiyan 36 was 80.62%, 79.34%, and 81.05%, respectively; the control efficacy of 200-fold dilution was 77.62%, 74.33%, and 72.57%, respectively; and the control efficacy of 300-fold dilution was 73.92%, 70.28%, and 71.56%, respectively. Therefore, the appropriate dilution ratio for using YC3071 microbial agent to control tobacco scab is 100-300 times.

[0044] Table 3. Control efficacy of YC3071 inoculant against tobacco red spot disease.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Streptomyces isothioides strain YC3071, characterized in that, The preservation name of the *Streptomyces isothiocyanate* strain YC3071 is... Streptomyces althioticus strain YC3071, Latin name is Streptomyces althioticus It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 4, 2024; accession number GDMCC NO:64829.

2. A Streptomyces isothiocyanate inoculant, characterized in that, It contains the Streptomyces isothiocyanate strain YC3071 as described in claim 1.

3. The *Streptomyces isothiocyanate* inoculant according to claim 2, characterized in that, The effective viable bacteria count in the bacterial agent is above 100 million CFU / gram.

4. The *Streptomyces isothiocyanate* inoculant according to claim 2, characterized in that, It also contains lysates of Streptomyces isothioides strain YC3071 and fermentation metabolites of the strain.

5. The application of the Streptomyces isothioides strain YC3071 as described in claim 1 in the control of tobacco diseases.

6. The application according to claim 5, characterized in that, The tobacco diseases mentioned include tobacco black shank and tobacco red star disease.

7. The application according to claim 6, characterized in that, The tobacco black shank disease is caused by Phytophthora tobacco. Phytophthora nicotianae Caused by infection.

8. The application according to claim 6, characterized in that, The tobacco red spot disease is caused by Alternaria. Alternaria alternata Caused by infection.

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